Estudo dos fatores que afetam a eficiência do cultivo in vitro

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UNIVERSIDADE ESTADUAL DO CEARÁ
PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA
FACULDADE DE VETERINÁRIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS
VETERINÁRIAS
VALDEVANE ROCHA ARAÚJO
ESTUDO DOS FATORES QUE AFETAM A EFICIÊNCIA DO
CULTIVO IN VITRO DE FOLÍCULOS PRÉ-ANTRAIS CAPRINOS
E BOVINOS: EFEITO DO REGIME DE TROCA, MEIOS DE
CULTIVO DE BASE E SUPLEMENTOS
FORTALEZA-CE
2013
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VALDEVANE ROCHA ARAÚJO
ESTUDO DOS FATORES QUE AFETAM A EFICIÊNCIA DO
CULTIVO IN VITRO DE FOLÍCULOS PRÉ-ANTRAIS CAPRINOS E
BOVINOS: EFEITO DO REGIME DE TROCA, MEIOS DE CULTIVO
DE BASE E SUPLEMENTOS
Tese apresentada ao Programa de Pós-Graduação
em Ciências Veterinárias da Faculdade de
Veterinária da Universidade Estadual do Ceará,
como requisito parcial para a obtenção do título de
Doutor em Ciências Veterinárias.
Área de Concentração: Reprodução e Sanidade
Animal.
Linha de Pesquisa: Reprodução e Sanidade de
pequenos ruminantes.
Orientador: Prof. Dr. José Ricardo de Figueiredo.
FORTALEZA-CE
2013
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Dados Internacionais de Catalogação na Publicação
Universidade Estadual do Ceará
Biblioteca Central Prof. Antônio Martins Filho
Bibliotecário(a) Responsável – Thelma Marylanda Silva de Melo- CRB-3 / 623
A658e
Araújo, Valdevane Rocha
Estudo dos fatores que afetam a eficiência do cultivo in vitro
de folículos pré-antrais caprinos e bovinos: efeito do regime de troca,
meios de cultivo de base e suplementos / Valdevane Rocha Araújo. —
2013
CD-ROM. 290f.: il. (algumas color); 4 ¾ pol.
―CD-ROM contendo o arquivo no formato PDF do trabalho
acadêmico, acondicionado em caixa de DVD Slim (19 x 14 cm x 7
mm)‖.
Tese (doutorado) – Universidade Estadual do Ceará, Faculdade
de Veterinária, Programa de Pós-graduação em Ciências Veterinárias,
Fortaleza, 2013.
Área de concentração: Reprodução e Sanidade de Pequenos
Ruminantes.
Orientação: Prof. Dr. José Ricardo de Figueiredo.
Co-orientação: Prof. Dr. Eduardo Leite Gastal.
1. Bovino. 2. Caprino. 3. Competência oocitária. 4. Estradiol.
5. Folículos pré-antrais. I. Título.
CDD: 636.39
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Dedico esta tese em memória aos meus avós
queridos, Belchior Damião de Araújo e Daldete
Rocha Araújo, e José Muniz Rocha. Que Deus
permita confortar nossos corações da saudade
eterna.
Dedico também em memória do grande amigo da
família e meu segundo pai, Sr. Arnoldo Almeida
Catter. Alguém que dedicou sua vida a ajudar e
educar pessoas e que acreditou em mim.
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AGRADECIMENTOS
Deus e Nossa Senhora sabem o quão difícil foi toda essa jornada e por isso, a
Eles, agradeço.
À Universidade Estadual do Ceará, ao Programa de Pós-Graduação em
Ciências Veterinárias e a todos os seus funcionários (especialmente à Adriana
Albuquerque), coordenadores, ex-coordenadores e professores, agradeço.
A CAPES e ao CNPq pelos auxílios em forma de bolsa de estudos tanto no
Brasil quanto no exterior (Estados Unidos), agradeço.
Aos meus pais, Edite Araújo Rocha e Valdeci Rocha Araújo que são tudo para
mim. Em todos esses anos e em todos os momentos sempre estiveram ao meu lado.
Pessoas cujo apoio tive incondicional e que carregaram comigo todo o peso da
ausência de um ano. Moletas que me sustentaram durante a subida de cada degrau na
escada da vida. E que no topo da escada, receberam-me de braços abertos. A quem
devo tudo o que tenho e tudo o que sou. A quem amo muito e com todas as minhas
forças, agradeço.
Aos meus irmãos, Aurineide Rocha Araújo, Auricélio Rocha Araújo, Lázaro
Rocha Araújo, Jacób Rocha Araújo e Belchior José Rocha Araújo, que de uma maneira
ou de outra também me ajudaram, e continuam me ajudando. Aqueles que me apoiaram
na difícil tarefa de amar as diferenças e as semelhanças, os defeitos e as qualidades. As
minhas lindas irmãs que nem mesmo o prazer de conhecê-las tive, mas que sei que elas
estão olhando por nós junto a Deus, nosso pai. A toda minha família, meus sobrinhos e
afilhados, agradeço.
Ao Sr. Arnoldo, meu segundo pai, a quem dedico esta tese e a quem Deus
chamou para continuar sua missão junto ao Senhor dos senhores, agradeço.
A todos os amigos, especialmente minha amiga Maria Noeme da Silva, e
professores de graduação, especialmente aos Professores Lúcia de Fátima Lopes dos
Santos e Ricardo Toniolli, que tanto me apoiaram e acreditaram em mim, agradeço.
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A todos os amigos que fizeram do LAMOFOPA uma casa e me fizeram sentir em
família: Deborah Magalhães-Padilha, Anderson Almeida, Jamily Bruno, Juliana
Celestino, Viviane Saraiva, Ana Beatriz Duarte, Gerlane Modesto, Roberta Chaves,
Helena Matos, Isabel Lima-Verde, Rochele Falcão, Michelle Brasil, Valesca Luz,
Anelise Alves, Rebeca Rocha, Laritza Lima, Raphael Gonçalves, Fabrício Martins,
Ticiana Franco, Ana Kelen Lima, Edmara Costa, Franciele Lunardi, Mirlla Baracho,
Patrícia Andrade, Sra. Alzenira Andrade, Lindemara Rodrigues, Débora Sales,
Priscilla Campos, Marcelo Ricardo, Lidinane Sales, Francisco Léo Aguiar, Hudson
Correia, Denise Guerreiro, Andréa Moreira, Sr. Antônio Cézar Camelo, agradeço.
A duas amigas, que são como mães científicas que tenho, Jamily Bruno e
Juliana Celestino. Pessoas que mesmo nos momentos mais difíceis, estavam lá para me
ajudar e me confortar, especialmente nessa última etapa, agradeço.
A duas amigas que Deus colocou em meu caminho com o intuito de serem meus
anjos da guarda e que me fizeram acreditar que poderia ter minhas irmãs de volta,
Deborah Magalhães-Padilha e Ticiana Franco, agradeço.
A dois amigos que muito devo, Anderson Almeida e Cláudio Lopes, e a quem a
Deus peço toda a proteção e cuidado, agradeço.
As minhas duas filhotas científicas, Gerlane Modesto e Mirlla Baracho, que me
deram muitas alegrias e dividiram muitos ensinamentos, agradeço.
To my new Family, the American one, during my time out from Brazil. People
as Mike Isom, Casie and Jeremiah Bass, Christiane and Mike Bass, Cherie Watson,
Anja Meksem, Cindy McDaniel, Patrícia Krejcik, Joy Carter, Garcia and Brandon,
Ricot Saint-Aimé, Deb Sarvela, Douglas Gimeniz and Maíra Aranha, Walquiria and
David Adams, Shereen Hammad, Dra. Ana Migone, Áurea Wischral and Manoel
Adrião, Eduardo and Melba Gastal, Keith Haag, Gabriela Fonseca, Saulo Silva, thank
you very much.
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Mike Isom, you were the best American dad that I could have in my life. You
gave me Casie Bass as my sister and all that I needed in that time. For this and for
everything that you guys did to me, I really thank you.
A Walquíria e David Adams que foram meus amigos, pais e conselheiros.
Deram-me o apoio no momento em que mais precisei e foram um refúgio para mim,
agradeço. “Walquíria and David Adams were more than friends; they were my friends,
my parents and my advisors. They gave to me support when I needed it most and have
been my refuge, thank you.”
Ao Prof. Dr. Cláudio Cabral, à Profa. Dra. Liliam Tavares, à Profa. Dra. Melba
Gastal, à Profa. Dra. Áurea Wischral e ao Prof. Dr. Manuel Adrião pela amizade,
carinho e pelas tantas lições de vida que levarei comigo para sempre, agradeço.
Ao meu orientador, Prof. Dr. José Ricardo de Figueiredo, que me recebeu no
LAMOFOPA de braços abertos e que me deu votos de confiança desde o primeiro
momento. Alguém que demonstrou acreditar no meu trabalho e na minha capacidade. A
alguém que além de tudo, proferiu muitas palavras de conforto e aconcelhamento
fazendo-me acreditar cada vez mais na minha própria capacidade, agradeço.
À minha co-orientadora, Profa. Dra. Ana Paula Ribeiro Rodrigues, que foi
orientadora de mestrado e amiga, acima de tudo; e que nestes últimos momentos tem
me apoiado muito, adradeço.
Ao meu co-orientador, Prof. Dr. Eduardo Leite Gastal, que me deu a
oportunidade de testar meus conhecimentos e minha capacidade, agradeço.
Aos membros da banca examinadora, Prof. Dr. Cláudio Cabral Campello, Prof.
Dr. José Roberto Viana Silva, Profa. Dra. Áurea Wischral e Profa. Dra. Roberta
Nogueira Chaves, que tanto contribuíram com a melhora desse trabalho, agradeço.
Finalmente, obrigada, papai e mamãe do céu, por mais uma etapa ultrapassada
com sucesso em minha vida.
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RESUMO
Os objetivos do presente trabalho foram: 1) verificar o efeito do regime de troca de
meio (caprinos e bovinos) e do tipo de meio de cultivo de base (bovinos) sobre o
crescimento/viabilidade in vitro de folículos secundários isolados; 2) avaliar a influência
da proteína morfogenética óssea-6 (BMP-6) na ausência ou presença do hormônio
folículo estimulante (FSH) sobre o crescimento/viabilidade de folículos pré-antrais
caprinos cultivados in situ ou isolado; e quantificar os níveis de RNAm para BMPR1A/R-2 e Smads 1/4/5/6/7/8 antes e após o cultivo de folículos secundários caprinos
isolados; 3) investigar o efeito do fator de crescimento do endotélio vascular (VEGF)
sobre o desenvolvimento de folículos secundários caprinos isolados; 4) verificar a
influência da adição de VEGF, fator de crescimento semelhante à insulina-1 (IGF-1)
e/ou hormônio do crescimento (GH) ao meio de cultivo in vitro de folículos secundários
bovinos isolados utilizando sistemas de cultivo bi (2D) e tridimensional (3D). Para o
cultivo in situ, fragmentos de córtex ovariano foram cultivados por um ou sete dias em
MEM+ adicionado de BMP-6 (0, 1, 10, 50 ou 100 ng/mL). Folículos secundários foram
isolados por microdissecção e cultivados por 18 (caprino) ou 32 (bovino) dias em
αMEM+ (caprino e bovino) ou TCM-199+ (bovino) na presença ou ausência de FSH. O
meio foi ainda suplementado com BMP-6 (caprino: 1 ou 10 ng/mL), VEGF (caprino: 10
ou 100 ng/mL; bovino: 100 ng/mL), IGF-1 (bovino: 50 ng/mL), GH (bovino: 50 ng/mL)
ou VEGF+IGF-1+GH (bovino). Os resultados demonstraram que a adição periódica de
meio aumentou significativamente as percentagens de oócitos (≥ 100 µm) caprinos
destinados à maturação in vitro. O diâmetro, a taxa de crescimento, formação de antro e
as concentrações de estradiol de folículos secundários bovinos isolados foram maiores
(P<0,05) na adição periódica de meio utilizando α-MEM (MEM-S) quando comparada
ao controle (MEM-C). A BMP-6 aumentou significativamente o percentual de folículos
atrésicos e promoveu alterações ultraestruturais nos folículos primordiais caprinos após
cultivo in situ. Em caprinos, a BMP-6 (1 ng/mL) promoveu maiores taxas de folículos
antrais e completos níveis de expressão de RNAm da via de sinalização das BMPs; e o
VEGF (10 ou 100 ng/mL) aumentou o diâmetro, a taxa de crescimento folicular e de
recuperação oocitária, apresentando maiores percentagens de oócitos em metáfase II
(P<0,05). Em bovinos, o VEGF melhorou as taxas de crescimento e formação de antro e
o GH aumentou os níveis de estradiol após cultivo 2D e 3D, respectivamente (P<0,05).
Diante do exposto, pode-se concluir que: 1) a adição periódica de meio melhorou o
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cultivo de folículos secundários caprinos e bovinos isolados; 2) a BMP-6 induziu a
atresia em folículos primordiais caprinos, porém promoveu o desenvolvimento in vitro
de folículos secundários isolados; 3) a adição de VEGF melhorou as taxas de maturação
de oócitos oriundos de folículos pré-antrais caprinos isolados cultivados in vitro e
finalmente; 4) a adição de VEGF (sistema 2D) e GH (sistema 3D) melhoraram,
respectivamente, o desenvolvimento folicular (formação de antro e taxa de crescimento)
e a produção de estradiol em folículos secundários bovinos isolados cultivados in vitro.
Palavras-chave: Bovino. Caprino. Competência oocitária. Estradiol. Folículos préantrais.
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ABSTRACT
The aims of this study were to: 1) verify the effect of medium replacement methods
(caprine and bovine) and the type of culture media (bovine) in the in vitro
growth/viability of isolated secondary follicles; 2) evaluate the effect of bone
morphogenetic protein-6 (BMP-6) alone or associated with follicle-stimulating hormone
(FSH) in the in vitro growth/viability of caprine preantral follicles cultured in situ or
isolated; and to quantify the mRNA expression levels for BMPR-1A/R-2 and Smads
1/4/5/6/7/8 before and after in vitro culture of isolated caprine secondary follicles; 3)
verify the influence of vascular endothelial growth factor (VEGF) in the in vitro
development of isolated caprine secondary follicles; 4) investigate the effect of VEGF,
insulin-like growth factor-1 (IGF-1) and growth hormone (GH) alone or in combination
in the in vitro culture of isolated bovine secondary follicles using two- (2D) or threedimensional (3D) culture systems. To the in situ culture, fragments of ovarian cortex
were cultured in vitro for one or seven days in MEM+ supplemented with BMP-6 (0, 1,
10, 50 or 100 ng/mL). Secondary follicles were isolated by microdissection and cultured
for 18 (caprine) or 32 (bovine) days in α-MEM+ (caprine and bovine) or TCM-199+
(bovine) with or without FSH. The culture medium was supplemented also with BMP-6
(caprine: 1 or 10 ng/mL), VEGF (caprine: 10 ou 100 ng/mL; bovine: 100 ng/mL), IGF1 (bovine: 50 ng/mL) or GH (bovine: 50 ng/mL) or the combination of VEGF+IGF1+GH (bovine). The results demonstrated that the periodic addition of culture medium
increased (P<0.05) the percentage of caprine oocytes (≥ 100 µm) destined to in vitro
maturation. Follicular diameter, growth rate, antrum formation, and estradiol production
of isolated bovine secondary follicles were higher (P<0.05) using periodic addition
method and α-MEM culture medium (MEM-S) than the control (MEM-C). BMP-6
increased (P<0.05) the percentage of atretic follicles and promoted ultrastructual
alterations in caprine primordial follicles after in situ culture. In caprine, BMP-6 (1
ng/mL) improved the antrum formation rate (P<0.05) and allowed the complete mRNA
expression levels for BMP receptor and Smads; and VEGF (10 and 100 ng/mL)
increased (P<0.05) the follicular diameter and growth rate presenting higher percentage
of metaphase II oocytes. In bovine, VEGF improved the follicular growth and antrum
formation rate and GH increased the estradiol levels after 2D and 3D culture systems,
respectively (P<0.05). Thus, the main conclusions from this study are as follows: 1)
periodic addition of culture medium improved the follicular development of caprine and
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bovine isolated secondary follicles; 2) BMP-6 induced the atresia in caprine primordial
follicles, while improved the development of isolated secondary follicles; 3) the
addition of VEGF improved the in vitro maturation rate of oocytes from secondary
caprine follicles grown in vitro; and finally 4) the addition of VEGF (2D culture
system) and GH (3D culture system), respectively, improved the follicular development
(antral formation and growth rate) and increased the estradiol production in isolated
bovine secondary follicles cultured in vitro.
Keywords: Bovine. Caprine. Oocyte competence. Estradiol. Preantral follicles.
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LISTA DE FIGURAS
CAPÍTULO 1
Figure 1. Schematic sequence of complete follicular development. Preantral phase:
Formation and beginning of growth and activation of primordial follicles and growth
of primary and secondary follicles. Antral phase: Formation of tertiary follicle
(antral-filled follicular fluid cavity). Follicle growth continues through the phases of
recruitment, emergency, selection, dominance, and preovulatory stage of follicular
waves. Oogonia is a cell that arises from a primordial germ cell and differentiates
into an oocyte in the ovary. Primordial follicle has a single layer of flattened
granulosa cells. Primary follicle has a single layer of cuboidal granulosa cells.
Secondary follicle has two or more layers of cuboidal granulosa cells and a small
number of theca cells. All the preantral follicles have a primary oocyte. Tertiary
follicle has several granulosa cell layers, theca cells and primary oocyte and is
characterized by an antral cavity which containing follicular fluid. Preovulatory or
also called as Graafian follicle is the last stage of follicle development; these follicles
are larger, have more antral fluid and a secondary oocyte. Follicular fluid is a plasma
exudate conditioned by secretory products from the granulosa cells and oocyte.……..
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Figure 2. Isolated follicles (A) by tissue chopper and microdissection, and (B) in situ
follicles stained with PAS-hematoxilin. o: oocyte; n: oocyte nucleus; fgc: flattened
granulosa cells; cgc: cuboidal granulosa cells; tc: theca cells; zp: zona pellucida.
*Antral follicle grown in vitro...………………………………………………………..
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Figure 3. Schematic representation of the (A) two- and (B) three-dimensional culture
systems utilized for bovine preantral follicles..………………………………………
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CAPÍTULO 2
Figure 1. VEGF isoforms generated by alternative splicing. VEGF-A comprises
monomers designated according to the number of amino acids in the polypeptide
chain (VEGF110, VEGF111, VEGF121, VEGF145, VEGF148, VEGF162, VEGF165,
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VEGF165b, VEGF183, VEGF189 and VEGF206)…………………………….....................
Figure 2. Binding complex VEGF-heparin-receptor involved in biological responses
to VEGF in various cells and tissues. VEGF-A binds both to VEGFR-1 and VEGFR2, whilst PIGF and VEGF-B interact only with VEGFR-1. VEGF-C and VEGF-D
bind to receptors VEGFR-2 and VEGFR-3, and VEGF-E binds only to VEGFR-2…..
106
Figure 3. Biological activities of VEGF in the mammalian ovarian follicle. The
expansion of the vascular network during follicle development enhances oxygenation
and diffusion of several substances important for follicle cells, and leads to the
discussed biological responses…………………………………………………………
108
CAPÍTULO 3
Figure 1. Normal caprine preantral follicle before culture (A); antral follicle after 18
days of culture in T2 (periodic addition of medium). Note the chromatin
configuration of the oocytes in germinal vesicle (C) and telophase I (D - from T2
treatment)……………………………………………………………………………….
128
Figure 2. Percentage of isolated morphologically normal preantral follicles after 18
days of culture.
(a,b)
treatment (P<0.05).
Differs significantly among culture periods within the same
(A,B)
Differs significantly among treatments within the same
culture period (P<0.05).………………………………………………………………...
131
Figure 3. Follicular diameter after 18 days of culture with different protocols of
medium replacement. (a,b)Differs significantly among culture periods within the same
treatment (P<0.05).
(A,B)
Differs significantly among treatments within the same
culture period (P<0.05).………………………………………………………………...
132
Figure 4. Percentage of antral cavity formation in follicles cultured with different
protocols of medium replacement after 18 days.
(a,b)
culture periods within the same treatment (P<0.05).
(A,B)
Differs significantly among
Differs significantly among
treatments within the same culture period (P<0.05)........................................................
133
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CAPÍTULO 4
Figure 1. Morphologically normal (a) and degenerated (b) bovine follicles before and
after 32 days of in vitro culture, respectively. Bars = 20 µm (a) and 50 µm (b)……….
146
Figure 2. Viable and non-viable bovine follicles after 32 days of in vitro culture. (a,
b, i, j) Conventional and (c, d, k, l) Small supplementation methods using α-MEM+.
(e, f, m, n) Conventional and (g, h, o, p) Small supplementation methods using TCM199+. Note that viable follicles (a-h) had shiny granulosa cells arranged in several
layers, intact basal membrane, and antrum cavity. However, non-viable follicles (i-p)
had very dark granulosa cells, irregularities in the basal membrane, and no antral
cavity. Bars = 100 µm (a-p)…………………………………………………………….
150
Figure 3. Relative mRNA expression (mean±SEM) for FSHR, IGF1, VEGF, and
P450AROM at days 0 and 32 of in vitro culture.
A,B
Relative mRNA expression
differed (P<0.05) among groups. No expression of VEGF was detected in fresh,
MEM-S, and TCM-S groups. No difference (P>0.05) was observed for P450AROM
among all groups.………………………………………………………………………
154
CAPÍTULO 5
Figure 1. Percentages (means±S.E.M) of atretic preantral follicles in uncultured tissue
(fresh control) and tissue cultured for 1 and 7 days in MEM+ and MEM+
supplemented with 1, 10, 50, and 100 ng/mL BMP-6. For each treatment, 30 follicles
were evaluated in each of five replicates. *P<0.05, significantly different from
uncultured ovarian cortex tissue (control/D0).
(A, B)
Different letters denote significant
differences between culture periods within the same medium (P<0.05)….……………
172
Figure 2. Histological section of (A) normal follicles from uncultured tissue and, (B)
atretic follicles after culture in the presence of BMP-6 O: oocyte; NU: oocyte
nucleus; GC: granulosa cells. Staining with periodic acid Schiff-hematoxylin, 400x…
172
Figure 3. Electron micrograph of caprine preantral follicle from (A) an uncultured
control (5800x), (B) MEM+ alone, (C) 1 ng/ml of BMP-6, and (D) 50 ng/ml of BMP-
174
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6 cultured (8000x) for 7 days. Homogeneous cytoplasm with numerous rounded
mitochondria is characteristic of non-cultured follicles and cultures with only MEM+
(3A and 3B, respectively). Extreme vacuolization and great holes are present in the
cytoplasm, indicative of degeneration (3C and 3D; solid arrow). Note the empty
space in degenerated granulosa cells after in vitro culture with BMP-6 (3C and 3D;
open arrow). NU: oocyte nucleus, GC: granulosa cells, m: mitochondria, ser: smooth
endoplasmic reticulum, v: vesicle ……………………………………………………..
CAPÍTULO 6
Figure 1. (A) Morphologically normal preantral (day 0) and (B) antral follicles (day
6) using BMP-6 at 1 ng/mL withou rFSH®.....................................................................
192
Figure 2. Antrum formation rate (%) in follicles cultured for 18 days in αMEM+ or
medium supplemented with BMP-6 (1 or 10 ng/mL) in the absence or presence of
rFSH®.
A,B
Different letters denote significant differences among treatments in the
same period (P<0.05).…………………………………………………………………..
194
Figure 3. The oocytes from follicles grown in vitro in αMEM+ medium (A-C) or
under treatment with BMP-6 at 1 ng/mL without FSH® (D-F). Note the presence of
the intact germinal vesicle (GV) in the MEM treatment and the metaphase II (MII)
stage indicated in blue after Hoechst 33342 staining in BMP-6 treatment…………….
196
Figure 4. Relative expression of mRNA (means±SD) of (A) bmpr2; (B) smad1; (C)
smad5; (D) smad8; (E) smad6; and (F) smad7 in the non-cultured control (D0) and
after 18 days of culture in αMEM+ medium or BMP-6 at 1 ng/mL without rFSH®
(BMP1).
A,B
Different letters denote significant differences among treatments
(P<0.05)………………………………………………………………………………...
197
CAPÍTULO 7
Figure 1. Oocytes from goat follicles, grown in vitro, at the end of the culture period
(after 18 days) with various treatments: control (a, d, g, j), with 10 ng/ml VEGF (b, e,
h k), or with 100 ng/ml VEGF (c, f, i, l). Oocytes are marked in green by Calceina-
215
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AM in d-f and in red by ethidium homodimer in j-l for all treatments. Bars 50 μm…...
Figure 2. Percentages of goat preantral follicles with normal morphology (healthy
follicles) cultured for 18 days (D0, D6, D12, D18) in αMEM+ (Control) and αMEM+
supplemented with 10 ng/ml VEGF (VEGF10) or 100 ng/ml VEGF (VEGF100).
Different lowercase letters denote significant differences among culture periods
within the same medium (P<0.05)……………………………………………………...
216
Figure 3. Diameter of goat follicles cultured for 18 days (D0, D6, D12, D18) in
αMEM+ (Control) and αMEM+ supplemented with 10 ng/ml VEGF (VEGF10) or 100
ng/ml VEGF (VEGF100). Different lowercase letters denote significant differences
among culture periods within the same medium (P<0.05). Different uppercase letters
denote significant differences among treatments in the same period (P<0.05)………...
217
Figure 4. Antrum formation in goat follicles cultured for 18 days (D0, D6, D12, D18)
in αMEM+ (Control) and αMEM+ supplemented with 10 ng/ml VEGF (VEGF10) or
100 ng/ml VEGF (VEGF100). Different lowercase letters denote significant
differences among culture periods within the same medium (P<0.05). Different
uppercase letters denote significant differences among treatments in the same period
(P<0.05)………………………………………………………………………………...
218
Figure 5. Oocytes from goat follicles grown in vivo (a-c) and in vitro under control
conditions (d-f) and after treatment with 10 ng/ml VEGF (g-i) or 100 ng/ml VEGF (jl). b, e, h, k Viable oocytes marked in green by Calcein-AM for all the treatments.
Note the presence of the germinal vesicle in the controls (f) and metaphase II in
oocytes in vivo (c) and after treatment with 10 ng/ml VEGF (i) or 100 ng/ml VEGF
(l), marked in blue by Hoechst 33342. Bars 50 μm…………………………………….
220
CAPÍTULO 8
Figure 1. Bovine follicles before (A; day 0) and after in vitro culture (B-D; day 32) in
medium containing only α-MEM+ (B), or α-MEM+ plus VEGF (C), or α-MEM+ plus
GH (D). Normal in vitro grown preantral (A) and antral follicles (B-D) using 2D (BC) or 3D (D) culture systems, respectively. o: oocyte; gc: granulosa cells; tc: theca
236
18
cells; a: antral cavity formation. Scale bars = 50 µm. Images were captured at 32X
(A) and 10X (B-D)..…………………………….............................................................
19
LISTA DE TABELAS
CAPÍTULO 1
Table 1. Chronological advances in in situ culture system of early bovine preantral
follicles.*…………………….………………………………………………………...
74
Table 2. Chronological advances in two and three dimensional (2D and 3D) in vitro
culture systems for isolated bovine preantral follicles.*………………………………
76
CAPÍTULO 3
Table 1. Meiotic stages of goat oocytes from preantral follicles cultured for 18 days
with three different protocols for medium exchange.………………………………….
134
CAPÍTULO 4
Table 1. Oligonucleotide primers used for real-time polymerase chain reaction
analysis of bovine follicles before and after in vitro culture.…………………………..
148
Table 2. Morphological normal follicles (%), follicular viability (%), follicular
diameter (µm) and growth rate (µm/day), antrum formation (%), and estradiol
concentration (ng/ml) of bovine follicles after 32 days of in vitro culture in α-MEM+
and TCM-199+ using two medium replacement methods (Conventional-C or Small
Supplementation-S)………………………………………………………………….....
151
Table 3. Frequency of slow (<1 µm/day), medium (1 to 4.9 µm/day), and fast (≥5
µm/day) growth rates of bovine follicles after 32 days of in vitro culture in two media
(α-MEM+ or TCM-199+) using two medium replacement methods (Conventional-C
or Small Supplementation-S)…………………………………………………………..
152
Table 4. Mean (±SEM) estradiol concentrations (ng/ml) produced by bovine follicles
in α-MEM+ or TCM-199+ using two medium replacement methods (Conventional-C
or Small Supplementation-S) according to the speed of the growth rate after 32 days
153
20
of in vitro culture……………………………………………………………………….
Table 5. Mean (±SEM) estradiol concentrations (ng/ml) produced by antral versus no
antral bovine follicles in α-MEM+ or TCM-199+ using two medium replacement
methods (Conventional-C or Small Supplementation-S) after 32 days of in vitro
culture…………………………………………………………………………………..
153
CAPÍTULO 5
Table 1. Percentages (mean±S.E.M.) of primordial and growing follicles (primary
and secondary) in uncultured tissues and tissues cultured for 1 or 7 days in MEM +
(control medium) and MEM+ supplemented with various concentrations of BMP-6….
170
Table 2. Follicle and oocyte diameters (mean±S.E.M.) in uncultured tissues and
tissues cultured for 1 or 7 days in MEM+ (control medium) and MEM+ supplemented
with various concentrations of BMP-6. For each treatment, 20 follicles were
evaluated………………………………………………………………………………..
171
CAPÍTULO 6
Table 1. Oligonucleotide primers used for the real-time polymerase chain reaction
analysis of caprine follicles before (Day 0) and after in vitro culture (Day 18)……….
191
Table 2. Percentage of morphological normal follicles, means±SEM of follicular
diameter (µm) and overall growth rate (µm/day) of caprine follicles after long-term
culture (18 days) in αMEM+ or medium supplemented with BMP-6 at 1 or 10 ng/mL
in the absence or presence of rFSH®…………………………………………………...
193
Table 3. Oocyte viability (%) and diameter (µm), recovery rate of oocytes cultured in
vitro (%), and meiotic stages (%) of caprine oocytes from preantral follicles after
long-term culture (18 days) in αMEM+ or medium supplemented with BMP-6 (1 or
10 ng/mL) in the absence or presence of rFSH®……………………………………….
195
21
CAPÍTULO 7
Table 1. Recovery rate of oocytes (≥110 μm) grown in vitro and meiotic stages of
goat oocytes from preantral follicles cultured for 18 days in αMEM+ (Control) and
αMEM+ supplemented with 10 ng/ml VEGF (VEGF10) or 100 ng/ml VEGF
(VEGF100). Significant differences between treatments in the same column are
indicated by uppercase letters (P<0.05)………………………………………………...
219
CAPÍTULO 8
Table 1. Morphologically normal follicles (%), antrum formation (%), growth rate
(µm/day), and estradiol concentration (ng/ml) of bovine follicles after 32 days of in
vitro culture in two- (2D: Experiment 1) and three-dimensional (3D using alginate:
Experiment 2) culture systems in the absence (Control group: only α-MEM+) or
presence of VEGF, IGF-I, or GH alone, or a combination of all (VEGF+IGF+GH)….
237
22
LISTA DE ABREVIATURAS E SIGLAS
18S:
18 unidades Svedberg de parte do RNA ribossomal
2D:
Two-dimensional culture system (Sistema de cultivo
bidimensional)
3D:
Three-dimensional culture system (Sistema de cultivo
tridimensional)
A1:
B-cell leukemia/lymphoma 2 related protein A1
ActR-I/Alk2
Activin type I receptor/activin receptor-like kinase-2/ (Receptor
tipo 1 da ativina/ Receptor de ativina semelhante à quinase-2)
ActR-IIA
Activin type IIA receptor (Receptor tipo 2A da ativina)
ActR-IIB
Activin type IIB receptor (Receptor tipo 2B da ativina)
ANOVA:
Analysis of variance (Análise de variância)
Bcl-2:
B-cell leukemia/lymphoma protein 2
BMP:
Bone Morphogenetic Proteins (Proteínas morfogenética óssea)
BMP-2/4/6/7/8/15:
Bone Morphogenetic Protein (Proteína morfogenética óssea)2/4/6/7/8/15
BMPR-IA/Alk-3:
Bone Morphogenetic Protein type IA receptor/ activin receptorlike kinase-3 (Receptor tipo 1A das proteínas morfogenéticas
ósseas/ Receptor de ativina semelhante à quinase-3)
BMPR-IB/Alk-6:
Bone Morphogenetic Protein type IB receptor/ activin receptorlike kinase-6 (Receptor tipo 1B das proteínas morfogenéticas
ósseas/ Receptor de ativina semelhante à quinase-6)
BMPR-II:
Bone Morphogenetic Protein type II receptor (Receptor tipo 2 das
proteínas morfogenéticas ósseas)
BrdU:
5-bromo-2'-deoxyuridine
BSA:
Bovine serum albumin (Albumina sérica bovina)
-C:
Conventional method for medium replacement
CaCl2:
Cloreto de cálcio
Calceína-AM:
Calceína acetoximetil
CAPES:
Coordenação de aperfeiçoamento de pessoal de nível superior
cDNA:
Complementary deoxyribonucleic acid (Ácido
desoxirribonucleico completar)
cgc:
Cuboidal granulosa cells (Células da granulosa cuboides)
23
CGP:
Células germinativas primordiais
CNPq:
Conselho Nacional de Desenvolvimento Científico e Tecnológico
CO2:
Dióxido de carbono
COC:
Cumulus oocyte complexes (Complexos cúmulos oócito)
Co-Smad:
Common-mediator Smad (Smad mediadora comum)-Smad4
Ct:
Cycle threshold (Ciclo de threshold)
CXCL12:
Chemokine (C-X-C motif) ligand 12 (Quimiocina CXCL12)
CYC-A:
Cyclophilin-A (Ciclofilina-A)
D:
Dia
D0:
Day zero/fresh control/non-cultured control (Dia 0/ controle não
cultivado)
e.g.:
For example (por exemplo)
E2:
Estradiol
EGF:
Epidermal Gorwth Factor (Fator de crescimento epidermal)
ELISA:
Enzyme-Linked ImmunoSorbent Assay (Ensaio de
imunoadsorção enzimática)
FAVET:
Faculdade de Veterinária
fgc:
Flattened granulosa cells (Células da granulosa pavimentosas);
FGFb:
Fibroblast Growth Factor (Fator de Crescimento Fibroblástico) b
Fig.
Figure (Figura)
FSH:
Follicle Stimulating Hormone (Hormônio folículo estimnulante)
FSHR:
Receptor do hormônio folículo estimulante
FUNCAP:
Fundação Cearense de Apoio ao Desenvolvimento Científico e
Tecnológico
G:
Gauge (calibre)
GADPH:
Glyceraldehyde-3-phosphate-dehydrogenase (Gliceraldeído
trifosfato desidrogenase)
GC:
Granulosa cells (Células da granulosa)
GDF-9:
Growth and differentiation factor (Fator de crescimento e
diferenciação)-9
GH:
Gowth hormone (Hormônio do crescimento)
GHR:
Growth hormone receptor (Receptor de hormônio do crescimento)
h:
Hora
HAS:
Hyaluronan synthase (Hialuronona sintetase)
24
hCG:
Human chorionic gonadotrophin (Gonadotrofina coriônica
humana)
HE:
Hematoxylin-eosin (Hematoxilina-eosina)
HEPES:
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (4-(2hidroxietil)-1-piperazina ethanesulfonic de ácido)
i.e.:
This is (Isto é)
IAA:
Indole-3-acetic acid (Ácido 3-indol Acético)
IBGE:
Instituto Brasileiro de Geografia e Estatística
IGF-1/2:
Insulin like growth factor (Fator de crescimento semelhante à
insulina)-2
IGFBP:
Insulin-like growth factor-binding proteins (Proteínas ligantes
transportadoras de fator de crescimento semelhante à insulina)
ISABR:
International Symposium on Animal Biology of Reproduction
I-Smad:
Inhibitory or antagonistic Smads (Smads inibitórias ou
antagonistas) -Smad6, Smad7
ITS:
Insulin, transferrin and selenium (Insulina, transferrina e selênio)
IVC:
In vitro culture (Cultivo in vitro)
JAK2:
Janus kinase 2
kb:
Kilo base
KCl:
Cloreto de potássio
kD:
Kilo Dalton
KL:
Kit ligand
LAMOFOPA:
Laboratório de manipulação de oócitos e folículos pré-antrais
LH:
Luteinizing hormone (Hormônio luteinizante)
m:
Mitochondria (mitocôndria)
M:
Molar
MAP:
Mitogen-activated protein kinases (Proteínas quinases ativadas
por mitógeno)
MEM/HEPES:
Meio essencial mínimo adicionado de tampão HEPES
MEM:
Minimum essential medium (Meio essencial mínimo)
MEM+:
Minimum essential medium supplemented (Meio essencial
mínimo suplementado)
MII:
Metaphase (Metáfase)- II
MIV/IVM:
Maturação in vitro/In vitro maturation
25
ml/mL:
Mililitro
mm:
Milímetro
mM:
Milimolar
mm3:
Milímetro cúbico
MMP-9:
Matrix metalloproteinases-9 (Metalopreinase da matriz-9)
MOIFOPA:
Manipulação de oócitos inclusos em folículos ovarianos préantrais
NaCl:
Cloreto de sódio
ng:
Nanograma
nm:
Namômetro
NU/n:
Oocyte nucleus (Núcleo do oócito)
Nubis:
Núcleo de Biotecnologia de Sobral
O/o:
Oocyte (Oócito)
Oct4/POU5F1:
Octamer-binding transcription factor 4 (Fator de transcrição de
ligação ao octâmero 4)
OPU:
Ovum pick-up (colheita de oócitos)
P < 0.05:
Probabilidade de erro menor do que 5%
P > 0.05:
Probabilidade de erro maior do que 5%
P4:
Progesterona
P450AROM:
P450aromatase
PAS:
Periodic acid Shiff (Ácido periódico de Shiff)
PBS:
Phosphate buffered saline (Tampão fosfato salina)
PCNA:
Proliferation marker proliferating cell nuclear antigen (Antígeno
nuclear de proliferação celular)
PDGF:
Platelet-derived growth factor (Fator de crescimento derivado de
placenta)
PI3K/AKT:
Phosphatidylinositide 3-kinases (Fosfatidil inositol 3-quinase)
PIGF:
Placental growth factor (fator de crescimento placentário)
PIV:
Produção in vitro de embriões
PPGCV:
Programa de Pós-Graduação em Ciências Veterinárias
qPCR:
Quantitative polimerase chain reaction (Reação em cadeia
polimerase quantitativa)
RENORBIO:
Rede Nordeste de Biotecnologia
26
rFSH:
Recombinant follicle stimulating hormone (hormônio folículo
estimulante recombinante)
RNAm/mRNA:
Ribonucleic acid messenger (Ácido ribonucléico mensageiro)
R-Smad:
Receptor-regulated Smads (Smads reguladoras-receptoras)Smad1, Smad2, Smad3, Smad5 e Smad8
RT-qPCR:
Real time reverse transcription quantitative polimerase chain
reaction (Transcrição reversa da reação em cadeia polimerase
quantitativa em tempo real)
S.E.M./SEM:
Standard error of means (Erro padrão da média)
s/sec:
Seconds (Segundos)
-S:
Small Supplementation for medium replacement (Pequena
suplementação para troca de meio)
SAS:
Statistical Analysis System (Sistema de análise estatística)
SCF:
Stem cell factor (Fator de células-tronco)
SEM:
Standard error of the mean (Erro padrão da média)
ser:
Smooth endoplasmic reticulum (retículo endoplasmático liso)
SIU:
Southern Illinois University (Universidade do Sul de Illinois)
Smads:
small-mothers against dpp gene Drosophila
SNK test:
Student-Newman-Keels test
SSR:
Annual Meeting of the Society for the Study of Reproduction
(Reunião annual da Sociedade para o estudo da reprodução)
STAT-1/3/5:
Signal transducters and activators of transcription (Transdutores
de sinal e ativação de transcrição)-1/3/5
T:
Treatment (Tratamento)
TC/tc:
Theca cells (Células da teca)
TCM-199:
Tissue medium culture (Meio de cultivo de tecido)-199
TE:
Transferência de embriões
TEM/MET:
Transmission eletron microscopy (Microscopia eletrônica de
transmissão)
TGF-β:
Transforming growth factor (Fator de crescimento transformante)
-β
TI:
Telophase (Telófase)-1
TMB:
Tetramethylbenzidine (Tetrametilbenzidina)
TUNEL:
Terminal deoxynucleotidyl transferase dUTP nick end labeling
27
UECE:
Universidade Estadual do Ceará
UFC:
Universidade Federal do Ceará
UnB:
Universidade de Brasília
v/v:
Volume/volume
v:
Vesicle (vesícula)
VEGF:
Vascular endothelial growth factor (Fator de crescimento do
endotélio vascular)
VEGF10:
VEGF treatment at 10 ng/mL (Tratamento com VEGF na
concentração de 10 ng/mL)
VEGF100:
VEGF treatment at 100 ng/mL (Tratamento com VEGF na
concentração de 100 ng/mL)
VEGF110-206:
Monômeros de VEGF de acordo com o número de aminoácidos
(110, 111, 121, 145, 148, 162, 165, 165b, 183, 189, 206) da
cadeia polipeptídica
VEGF-A-E:
Isoformas de VEGF dos tipos A, B, C, D, e E
VEGFR-1/Flt-1:
Fms-like tyrosine kinase-1 (Tirosina quinase 1 semelhante a Fms)
VEGFR-2/KDR:
kinase domain receptor (Receptor de domínio quinase)
VEGFR-3/Flt-4:
Fms-like tyrosine kinase-4 (Tirosina quinase 4 semelhante a Fms)
VPF:
Vascular permeability factor (Fator de permeabilidade vascular)
w/v:
Massa/volume
x:
Eixo das abicissas
y:
Eixo das ordenadas
ZP/zp:
Zona pelúcida (Zona pelúcida)
α-MEM:
Minimum essential medium alpha (Meio essencial mínimo alfa)
α-MEM+:
Supplemented minimum essential medium alpha (Meio essencial
mínimo alfa suplementado)
α-MEM-HEPES:
Meio essencial mínimo alfa tamponado com HEPES
%:
Percentagem
≥:
Maior ou igual a
°C:
Graus Celsius
µg:
Micrograma
µl/µL:
Microlitro
28
SUMÁRIO
1 INTRODUÇÃO...................................................................................................
30
2 REVISÃO DE LITERATURA…………………………………………………
32
2.1 FOLICULOGÊNESE OVARIANA E CARACTERIZAÇÃO FOLICULAR
(ASPECTOS BÁSICOS DA FOLICULOGÊNESE OVARIANA)......................
32
2.1.1 Formação e início do crescimento de folículos primordiais.........................
32
2.1.2 Crescimento de folículos primários e secundários........................................
33
2.2 POPULAÇÃO E ATRESIA FOLICULAR.....................................................
34
2.3 NEOFOLICULOGÊNESE...............................................................................
35
2.4 BIOTÉCNICA DE MOIFOPA (Ovário Artificial)..........................................
37
2.5 ESTADO ATUAL DO CULTIVO IN VITRO DE FOLÍCULOS PRÉANTRAIS...............................................................................................................
37
2.6 SISTEMAS DE CULTIVO IN VITRO DE FOLÍCULOS PRÉ-ANTRAIS...
38
2.7 IMPORTÂNCIA DA COMPOSIÇÃO DO MEIO DE CULTIVO DE BASE
39
2.8 HORMÔNIO FOLÍCULO ESTIMULANTE (FSH).......................................
40
2.9 FATOR DE CRESCIMENTO DO ENDOTÉLIO VASCULAR (VEGF)......
41
2.10 PROTEÍNA MORFOGENÉTICA ÓSSEA-6 (BMP-6).................................
42
2.11 FATOR DE CRESCIMENTO SEMELHANTE À INSULINA-1 (IGF-1)...
45
2.12 HORMÔNIO DO CRESCIMENTO (GH).....................................................
46
2.13 TÉCNICAS PARA AVALIAÇÃO DA EFICIÊNCIA DO CULTIVO IN
VITRO....................................................................................................................
47
2.13.1 Histologia clássica......................................................................................
47
2.13.2 Microscopia eletrônica de transmissão.....................................................
48
2.13.3. Microscopia de fluorescência e microscopia confocal..............................
48
2.13.4. Análise de esteroides..................................................................................
49
2.13.5. Reação em cadeia da polimerase em tempo real (PCR em tempo real)....
50
3 JUSTIFICATIVA.................................................................................................
52
4 HIPÓTESES CIENTÍFICAS..............................................................................
54
5 OBJETIVOS.........................................................................................................
55
5.1 OBJETIVOS GERAIS.....................................................................................
55
5.2 OBJETIVOS ESPECÍFICOS...........................................................................
55
29
6 CAPÍTULO 1
Cultivo in vitro de folículos pré-antrais bovinos: Uma revisão..............................
57
7 CAPÍTULO 2
Importância do fator de crescimento do endotélio vascular (VEGF) na fisiologia
ovariana de mamíferos............................................................................................
98
8 CAPÍTULO 3
Efeito do protocolo de troca de meio sobre o desenvolvimento in vitro de
folículos pré-antrais caprinos isolados...................................................................
122
9 CAPÍTULO 4
Crescimento in vitro, produção de estradiol e expressão gênica de folículos préantrais bovinos isolados: Efeito do meio de base e método de troca de meio........
139
10 CAPÍTULO 5
Proteína morfogenética óssea-6 (BMP-6) induz atresia em folículos primordiais
caprinos cultivados in vitro.....................................................................................
162
11 CAPÍTULO 6
Efeito da proteína morfogenética óssea-6 (BMP-6) e do hormônio folículo
estimulante (FSH) durante o desenvolvimento in vitro de folículos pré-antrais
ovarianos caprinos, e expressão relativa de RNAm para os receptores de BMP e
Smads em folículos cultivados...............................................................................
181
12 CAPÍTULO 7
Fator de crescimento do endotélio vascular-A165 (VEGF-A165) estimula o
desenvolvimento in vitro e a competência oocitaria de folículos pré-antrais
caprinos...................................................................................................................
205
13 CAPÍTULO 8
Desenvolvimento in vitro de folículos secundários bovinos em sistemas bi e
tridimensional utilizando fator de crescimento do endotélio vascular (VEGF),
fator de crescimento semelhante à insulina-1 (IGF-1) e hormônio do
crescimento (GH)...................................................................................................
226
CONCLUSÕES..............................................................................................................
245
PERSPECTIVAS...........................................................................................................
246
REFERÊNCIAS BIBLIOGRÁFICAS........................................................................
247
30
1 INTRODUÇÃO
As pesquisas realizadas nos últimos anos em reprodução assistida têm
possibilitado oportunidades extraordinárias para a reprodução animal (TROUNSON et
al., 1998), visto que visam aumentar o potencial reprodutivo e a produtividade dos
rebanhos, proporcionando uma grande revolução na multiplicação de animais de
elevado potencial econômico. Considerando a expressividade dos rebanhos caprino (9
milhões de cabeças) e bovino (209,5 milhões de cabeças) para o Brasil (IBGE, 2010),
diversas biotécnicas reprodutivas, tais como a inseminação artificial, sincronização de
estro, produção in vitro (PIV) e transferência de embriões (TE), têm sido utilizadas.
Outra biotécnica bastante estudada é a manipulação de oócitos inclusos em folículos
ovarianos
pré-antrais
(MOIFOPA),
também
denominada
ovário
artificial
(FIGUEIREDO et al., 2008). A biotécnica de MOIFOPA compreende as etapas de
isolamento e cultivo in vitro, bem como a criopreservação dos oócitos inclusos em
folículos pré-antrais. Em associação a outras tecnologias reprodutivas, como a
fecundação in vitro e a transferência de embriões, a MOIFOPA poderá, no futuro, não
somente otimizar, como também conservar o material genético de animais valiosos e de
espécies em vias de extinção.
Tendo em vista que o ovário dos mamíferos contém milhares de folículos préantrais, e que a sua grande maioria se tornará atrésica naturalmente durante seu
desenvolvimento (CARROLL et al., 1990), deve-se buscar técnicas que visem otimizar
o potencial reprodutivo das fêmeas prevenindo a ocorrência da atresia folicular. Desta
forma, o desenvolvimento de técnicas para recuperação e crescimento in vitro de
folículos pré-antrais seria uma alternativa aos métodos já disponíveis para reprodução
animal, uma vez que forneceria uma população grande e uniforme de oócitos de animais
geneticamente superiores (BETTERIDGE et al., 1989). Neste sentido, a identificação de
fatores produzidos localmente em folículos ovarianos caprinos e bovinos, bem como a
avaliação do efeito destes fatores sobre o crescimento e maturação oocitária poderão
contribuir para uma melhor compreensão da foliculogênese, otimizando a produção de
embriões a partir de oócitos inclusos em folículos pré-antrais crescidos in vitro.
Diversos grupos de fatores de crescimento produzidos localmente no ovário já
foram identificados em animais de laboratório, primatas e ruminantes. Além disso, tem
sido demonstrado que fatores como a proteína morfogenética óssea-6 (BMP-6 –
OTSUKA et al., 2001a), o fator de crescimento do endotélio vascular (VEGF – YANG;
31
FORTUNE, 2007; BRUNO et al., 2009) e o fator de crescimento semelhante à insulina
1 (IGF-1: THOMAS et al., 2007) podem exercer importantes funções no controle do
crescimento folicular e posterior maturação oocitária. No tocante à utilização de
hormônios, os hormônios folículo estimulante (FSH; MATOS et al., 2007a;
MAGALHÃES et al., 2009) e hormônio do crescimento (GH; LANGHOUT et al.,
1991) têm sido considerados importantes
na regulação do crescimento e
desenvolvimento folicular. Contudo, ainda são poucos os estudos sobre os fatores de
crescimento que controlam o desenvolvimento folicular inicial e a maturação oocitária,
sendo uma prioridade a análise do efeito destes fatores sobre a foliculogênese pré-antral
de mamíferos, visando o desenvolvimento de meios de cultivo in vitro.
Para um maior esclarecimento da importância deste projeto, a revisão de
literatura a seguir abordará aspectos relacionados à foliculogênese ovariana e
caracterização folicular, população e atresia folicular, neofoliculogênse, biotécnica de
MOIFOPA (ovário artificial), estado atual do cultivo in vitro, sistemas de cutlivo in
vitro de folículos pré-antrais, importância da composição do meio de cultivo de base e
substâncias relacionadas à foliculogênese com ênfase para o FSH, BMP-6, VEGF, IGF1 e GH, bem como para as técnicas de avaliação da eficiência do cultivo in vitro de
folículos pré-antrais.
32
2 REVISÃO DE LITERATURA
2.1 FOLICULOGÊNESE OVARIANA E CARACTERIZAÇÃO FOLICULAR
(ASPECTOS BÁSICOS DA FOLICULOGÊNESE OVARIANA)
A foliculogênese pode ser definida como o processo de formação e
desenvolvimento (crescimento e maturação) dos folículos. É um evento iniciado ainda
na vida pré-natal, na maioria das espécies, com a formação do folículo primordial e
culminando com a formação do folículo de De Graaf ou pré-ovulatório (van den
HURK; ZHAO, 2005). Ocorre simultaneamente à oogênese na maioria das espécies
quando o oócito está entre as fases de prófase I e metáfase II. A foliculogênese pode ser
dividida em duas fases, considerando o grau de evolução dos folículos: 1) fase préantral, composta pelos folículos não cavitários em que ocorre a ativação dos folículos
primordiais e crescimento dos folículos primários e secundários; e 2) fase antral, na qual
ocorre o crescimento inicial e terminal dos folículos terciários dando origem aos
folículos pré-ovulatórios.
2.1.1 Formação e início do crescimento de folículos primordiais
Na espécie caprina, os folículos primordiais são formados por volta do 62° dia
de vida fetal. Estes folículos apresentam um oócito circundado por uma camada de
células somáticas planas, conhecidas como células da pré-granulosa, originárias do
epitélio celômico (BEZERRA et al., 1998). Os folículos primordiais representam cerca
de 95% do total de folículos pré-antrais presentes no ovário (ERICKSON, 1986). Estes
folículos possuem diâmetro médio de 35, 22 e 20 µm em bovinos (HULSHOF et al.,
1994), ovinos (AMORIM et al., 2000) e caprinos (LUCCI et al., 1999),
respectivamente. Após a formação dos folículos primordiais, as células da pré-granulosa
param de se multiplicar e entram num período de quiescência.
Com a evolução folicular e após a ativação, os folículos pré-antrais iniciam uma
série de mudanças morfofisiológicas que envolvem o crescimento e a diferenciação do
oócito, bem como a proliferação e a diferenciação das células da granulosa, além do
desenvolvimento das células da teca (SUH et al., 2002). No início do crescimento
folicular, fase conhecida como ativação, os folículos primordiais passam do pool de
reserva ou folículos quiescentes para o pool de folículos em crescimento (primário,
33
secundário, terciário e/ou pré-ovulatório; RÜSSE, 1983). Os sinais de ativação dos
folículos primordiais incluem a retomada da proliferação das células da granulosa (van
den HURK; BEVERS; BECKERS, 1997) e a mudança na morfologia dessas células de
pavimentosas para cúbicas. No entanto, os fatores e mecanismos responsáveis pela
ativação de folículos primordiais, bem como os mecanismos envolvidos no início do
crescimento folicular, são ainda enigmáticos e representam uma das maiores questões
relacionadas com a biologia ovariana.
Vários fatores de crescimento produzidos pelas células foliculares podem estar
relacionados com a ativação dos folículos primordiais. O kit ligand (KL), também
conhecido como fator de células-tronco (Stem cell factor – SCF), e o fator de
crescimento fibroblástico básico (bFGF), por exemplo, foram relatados como fatores
necessários à ativação de folículos primordiais caprinos (bFGF: MATOS et al., 2007b;
KL: CELESTINO et al., 2010a) e bovinos (bFGF: TANG et al., 2012). Além disso,
gonadotrofinas, como o hormônio folículo estimulante (FSH), promovem a ativação de
folículos primordiais, bem como o crescimento dos folículos ativados, uma vez que o
FSH parece estar envolvido na proliferação e diferenciação das células da granulosa in
vitro (MATOS et al., 2007a; MAGALHÃES et al., 2009; TANG et al., 2012).
2.1.2 Crescimento de folículos primários e secundários
Após a ativação, os folículos primordiais gradualmente adquirem células da
granulosa de formato cúbico, tornando-se folículos de transição. Em seguida, quando
todas as células que circundam o oócito tornam-se cúbicas, aumentando em número e
volume, os folículos são chamados folículos primários (van den HURK; BEVERS;
BECKERS, 1997). Em caprinos, o aparecimento de folículos primários e secundários
ocorre aos 71 e 80 dias de gestação, respectivamente (BEZERRA et al., 1998). Os
folículos primários possuem diâmetro médio de 34,7 µm e apresentam-se com uma
camada completa de células da granulosa. Com a multiplicação destas células, ocorre a
formação de várias camadas de células ao redor do oócito formando os folículos
secundários, cujo diâmetro médio é de 58,94 µm (LUCCI et al., 1999). Nestas
categorias foliculares (folículos primários e secundários), a zona pelúcida (ZP) começa
a ser formada circundando o oócito (RANKIN et al., 2001). No entanto, na espécie
humana (GOOK et al., 2008) e ovina (MATOS et al., 2004), foram verificadas a
presença de proteínas, bem como de pequenas quantidades visíveis de material de ZP
34
em folículos desde o estágio primordial. Tais resultados sugerem que estas proteínas
estão presentes desde o início da foliculogênese.
O crescimento folicular após o estágio de folículo primário é, também
caracterizado pelo aparecimento das células da teca recrutadas de seus precursores
presentes no tecido circundante do estroma (PARROTT; SKINNER, 2000) além de ser
dependente de fatores de crescimento como o fator de crescimento e diferenciação-9
(GDF-9). Martins et al. (2008) verificaram que a adição de GDF-9 ao meio de cultivo
de folículos pré-antrais caprinos permitiu o desenvolvimento folicular in vitro com
aumento no número de folículos secundários. Além disso, outros fatores também são
considerados como estimuladores do desenvolvimento folicular como o KL
(CELESTINO et al., 2010a) e o fator de crescimento do endotélio vascular (VEGF –
YANG; FORTUNE, 2007; BRUNO et al., 2009).
À medida que ocorre o crescimento dos folículos secundários e a organização
das células da granulosa em várias camadas, inicia-se a formação do antro folicular,
definido como sendo uma cavidade repleta de líquido folicular entre as células da
granulosa. A partir deste estágio, os folículos passam a ser denominados terciários ou
antrais, os quais aparecem na fase fetal aos 110 dias de gestação (RÜSSE, 1983). Em
bovinos, a formação do antro inicia-se em folículos com diâmetro em torno de 130 µm
(LUSSIER et al., 1987). Em caprinos, o menor diâmetro observado no folículo terciário
de fetos foi de 130 µm (BEZERRA et al., 1998). Contudo, já foram observados
diâmetros superiores a 200 µm em folículos secundários em várias espécies, tais como:
caprinos (SARAIVA et al., 2010, 2011; CELESTINO et al., 2011; MAGALHÃES et
al., 2011; 2012), ovinos (ARUNAKUMARI; SHANMUGASUNDARAM; RAO, 2010;
LUZ et al., 2012), bovinos (McLAUGHLIN et al., 2010; McLAUGHLIN; TELFER,
2010; ROSSETTO et al., 2012, 2013) e caninos (SERAFIM et al., 2010, 2012).
2.2 POPULAÇÃO E ATRESIA FOLICULAR
A população folicular preantral já foi estimada em diferentes espécies, sendo de
285.000 em bovinos (SILVA-SANTOS et al., 2011), 33.000 em ovinos (AMORIM et
al., 2000), 45.000 em caprinos (LUCCI et al., 1999) e 599.000 em suínos (ALVES et
al., 2012) por ovário. Além da variação individual, vários fatores podem afetar o
número de folículos presentes no ovário, tais como: a raça (CAHILL; MARIANA;
MAULÉON, 1979), a idade (ERICKSON, 1966a; RÜSSE, 1983), os níveis hormonais
35
(PETERS, 1976), a genética (ERICKSON, 1966b), bem como o status reprodutivo
(ERICKSON;
REYNOLDS;
MURPHREE,
1976)
e
nutricional
do
animal
(SCARAMUZZI et al., 1993).
Apesar do grande pool de reserva ovariana, é sabido que aproximadamente
99,9% dos folículos são eliminados pelo processo fisiológico conhecido como atresia
folicular, tornando o ovário um órgão de baixíssima produtividade. A atresia ocorre por
um processo de morte celular programada conhecido por apoptose (TSAFIRI; BRAW,
1984). Em folículos pré-antrais, as primeiras alterações indicativas de atresia ocorrem
no oócito, como por exemplo, retração da cromatina nuclear e fragmentação oocitária
(MORITA; TILLY, 1999). As alterações nas células da granulosa são raras, uma vez
que essas células são mais resistentes à degeneração que os oócitos (SILVA et al.,
2002).
Ao longo do desenvolvimento folicular, o folículo adquire a cavidade antral.
Neste momento, o oócito torna-se altamente resistente e as primeiras alterações
indicativas de atresia são observadas nas células da granulosa pela sensibilidade
adquirida ao longo de seu desenvolvimento (SILVA et al., 2002). O destino final dos
folículos ovarianos, (i) ovulação ou (ii) atresia, é dependente de um balanço entre
diferentes fatores endócrinos, parácrinos e autócrinos que promovem a sobrevivência e
aqueles que induzem a apoptose (HSU; HSUEH, 2000).
Diante disso, pesquisas têm desenvolvido vários modelos in vitro que
possibilitam o estudo dos fatores que controlam a atresia e favorecem o
desenvolvimento folicular (CELESTINO et al., 2010; MATOS et al., 2007a,b), evitando
assim as perdas foliculares que ocorrem naturalmente in vivo.
2.3 NEOFOLICULOGÊNESE
A continuidade da oogênese e foliculogênese no período pós-natal, pela atuação
de células-tronco, caracteriza e define o processo de neofoliculogênese (JOHNSON et
al., 2004, 2005). Desde a apresentação desse conceito, a neofoliculogênese tem sido,
portanto, motivo de muitas discussões na comunidade científica, uma vez que os
estudos ainda são escassos e imprecisos.
Até pouco tempo atrás, se acreditava no paradigma de que mulheres e as demais
fêmeas (exceto as murinas) perdiam sua capacidade de produzir células germinativas
primordiais (CGP) durante o desenvolvimento da vida fetal e nasciam com um número
36
finito de oócitos inclusos em folículos, dos quais apenas um pequeno número iria ovular
após a puberdade (WOODRUFF, 2008). Esse conceito foi considerado uma premissa
básica da fisiologia da reprodução por mais de 150 anos (BYSKOV et al., 2005).
Entretanto, estudos recentes, da equipe do Dr. Jonathan Tilly da Universidade de
Harvard nos Estados Unidos, apresentaram evidências de que uma fêmea teria a
capacidade de produzir novos folículos durante a vida adulta a partir de células-tronco
de linhagem germinativa extra e intraovariana (JOHNSON et al., 2004, 2005). A equipe
do Dr. Tilly então postulou que, pelo menos em camundongos, a morte folicular ocorre
rapidamente e que a fertilidade normal de um indivíduo adulto não poderia ser mantida
se dependesse exclusivamente do pool de folículos presentes no ovário ao nascimento.
Resultados semelhantes foram observados em humanos quando células do epitélio
germinativo ovariano foram cultivadas e permitiram o desenvolvimento de células da
granulosa e oócitos (BUKOVSKY; SVETLIKOVA; CAUDLE, 2005). Recentes
achados ainda propuseram que na medula óssea, há células semelhantes às célulastronco embrionárias originárias do epiblasto, as quais podem persistir também durante a
vida adulta para regenerar tecidos e órgãos (SHIN et al., 2010).
Tendo em vista que a definição clássica das células-tronco é a de que estas
células perpetuadamente se renovam e geram progênies diferenciadas, sua presença no
ovário levaria a formação de novos oócitos. Sendo assim, se a população folicular está
sendo reestabelecida
continuamente durante
a vida
adulta,
como
sugerido
anteriormente, as células-tronco germinativas deveriam progressivamente perder sua
habilidade de se multiplicar para permitir o declínio da fertilidade (BYSKOV et al.,
2005). Neste contexto, outros estudos foram realizados e, de fato, não encontraram
evidências de que células progenitoras de origem extragonadal poderiam renovar as
células foliculares no ovário adulto (KERR et al., 2006; BEGUM; PAPAIOANNOU;
GOSDEN, 2008).
Na tentativa de entender o comportamento das CGP no ovário, algumas
citocinas e fatores de crescimento pleiotrópicos tais como as BMPs (LAWSON et al.,
1999; YING et al., 2000), o Chemokine (C-X-C motif) ligand 12 (CXCL12), o KL
(MOLYNEAUX; WYLIE, 2004; KUNWAR; SIEKHAUS; LEHMANN, 2006) e o
Oct4 ou POU5F1 (PANGAS; RAJKOVIC, 2006) têm sido estudados. Tais moléculas
foram, portanto, consideradas como importantes para controlar o processo de formação
dos folículos primordiais durante a colonização da gônada pelas CGP. A ausência das
BMP-4 (LAWSON et al., 1999) e BMP-8 (YING et al., 2000), em embriões de
37
camundongas, por exemplo, promoveu falha no desenvolvimento das CGP sugerindo
que as BMPs podem de alguma maneira estar envolvidas na formação e no
desenvolvimento dos folículos primordiais. Além disso, a ausência de Oct4 em CGP
provocou apoptose prematura dessas células antes da colonização da gônada (KEHLER
et al., 2004). Neste contexto, o estudo da foliculogênese pré-antral abre oportunidadades
de esclarecer os mecanismos envolvidos na formação dos folículos primordiais.
2.4 BIOTÉCNICA DE MOIFOPA (Ovário Artificial)
A biotécnica de manipulação de oócitos inclusos em folículos ovarianos préantrais (MOIFOPA) também conhecida como ―ovário artificial‖, é uma biotécnica da
reprodução que vem sendo aprimorada nos últimos anos e consiste numa das principais
ferramentas utilizadas atualmente para a elucidação da foliculogênese inicial. Tal
biotécnica possibilita a padronização de populações de oócitos para utilização em outras
biotécnicas como a produção in vitro de embriões (PIV), a transgênese e a clonagem. A
MOIFOPA tem como principal objetivo resgatar oócitos oriundos de folículos préantrais, a partir do ambiente ovariano, e posteriormente cultivá-los in vitro até sua
completa maturação, prevenindo-os da atresia. Para alcançar esse objetivo, diversos
estudos têm sido realizados com o intuito de desenvolver um sistema de cultivo in vitro
ideal para cada etapa do desenvolvimento folicular (FIGUEIREDO et al., 2008).
2.5 ESTADO ATUAL DO CULTIVO IN VITRO DE FOLÍCULOS PRÉ-ANTRAIS
Notável progresso tem sido observado no cultivo in vitro de folículos pré-antrais
em diferentes espécies animais. Nas espécies bovina (GUTIERREZ et al., 2000;
McLAUGHLIN; TELFER 2010; ROSSETTO et al., 2012, 2013) e humana (ROY;
TREACY, 1993; TELFER et al., 2008), folículos pré-antrais isolados foram cultivados
in vitro e se desenvolveram até o estágio antral. Em suínos (WU et al., 2001), bubalinos
(GUPTA et al., 2008) e mais recentemente em caprinos (SARAIVA et al., 2010;
MAGALHÃES et al., 2011), ovinos (ARUNAKUMARI et al., 2010; LUZ et al., 2012)
e primatas não-humanos (XU et al., 2011), folículos secundários crescidos in vitro
tiveram seus oócitos fecundados in vitro, com posterior desenvolvimento embrionário.
Contudo, os resultados mais satisfatórios até o presente momento foram observados em
38
animais de laboratório. Eppig; Schroeder (1989) obtiveram o primeiro nascimento a
partir de folículos primordiais crescidos, maturados e fecundados in vitro. Anos mais
tarde, essa mesma equipe, utilizando um protocolo revisado e melhorado, conseguiu
aumentar o número de crias nascidas vivas (O’BRIEN et al., 2003). Carroll et al. (1990)
também obtiveram o nascimento de camundongos após congelação e descongelação,
crescimento, maturação e fecundação in vitro de oócitos oriundos de folículos
primários. Apesar desses resultados, o rendimento referente à produção de oócitos
maturos a partir de folículos pré-antrais ainda é extremamente baixo e variável devido à
inadequação dos meios de cultivo disponíveis.
Diversos fatores podem afetar a eficiência do cultivo in vitro de folículos préantrais. Dentre eles pode-se destacar a espécie animal, pH, temperatura, tensão de
oxigênio, tipo de sistema de cultivo (bi ou tridimensional) e composição do meio, sendo
esses dois últimos, objetos de estudo da presente tese.
2.6 SISTEMAS DE CULTIVO IN VITRO DE FOLÍCULOS PRÉ-ANTRAIS
Basicamente, o cultivo de folículos pré-antrais pode ser realizado utilizando
fragmentos do córtex ovariano (in situ) ou estruturas foliculares na forma isolada. O
cultivo in situ tem como vantagens proporcionar a manutenção da integridade
tridimensional dos folículos e os mantém interagindo com as células do estroma,
assemelhando-se às condições in vivo. Melhor perfusão do meio e ainda, o
monitoramento individual dos folículos são as principais vantagens do cultivo de
folículos isolados, o qual é atualmente o mais utilizado (FIGUEIREDO et al., 2008).
O cultivo de folículos isolados permite o crescimento e desenvolvimento de
oócitos imaturos e favorece a elucidação dos mecanismos envolvidos no
desenvolvimento oocitário, na diferenciação das células da granulosa e na regulação dos
fatores autócrinos/parácrinos que controlam a foliculogênese (THOMAS et al., 2003).
Esses folículos podem ser cultivados de maneira bidimensional (2D) ou tridimensional
(3D). No sistema 2D, os folículos são cultivados diretamente sobre uma superfície
plástica ou mesmo sobre uma matriz extracelular. Vários estudos tem utilizado esse
sistema com grande sucesso, inclusive com a obtenção de embriões produzidos a partir
de folículos pré-antrais crescidos in vitro (GUPTA et al., 2008; ARUNAKUMARI;
SHANMUGASUNDARAM; RAO, 2010; SARAIVA et al., 2010; MAGALHÃES et
al., 2011; LUZ et al., 2012). Já no sistema 3D, os folículos são cultivados
39
completamente inclusos em uma matriz extracelular (XU et al., 2010, 2011), ou mesmo
na ausência de matriz (WYCHERLEY et al., 2004; NATION; SELWOOD, 2009;
WANG et al, 2012). O princípio do cultivo 3D é o de que os folículos são cultivados de
maneira que não haja contato e, consequentemente, aderência da estrutura folicular a
superfície de cultivo. Esse sistema, portanto, permite a manutenção da estrutura
folicular sem deformações durante o cultivo in vitro. Maiores detalhes serão abordados
na revisão de literatura apresentada no Capítulo 1, a qual descreverá mais
especificamente sobre os sistemas de cultivo de folículos pré-antrais.
2.7 IMPORTÂNCIA DA COMPOSIÇÃO DO MEIO DE CULTIVO DE BASE
Diversos meios de cultivo vêm sendo utilizados para o cultivo de células
ovarianas. Dentre esses meios podemos destacar o meio essencial mínimo (MEM) e
suas modificações (α-MEM, MEM Glutamax, etc.), o meio de cultivo de tecido (TCM199), além de outros meios como o McCoy’s, Waymouth, Leibowitz e Menezo B2.
Cada meio de cultivo difere em sua composição de sais, vitaminas, minerais e
principalmente,
nas
concentrações
de
aminoácidos,
ribonucleosídeos
e
desoxiribonucleosídeos. De uma maneira geral, os meios de cultivo são utilizados de
acordo com as espécies estudadas ou mesmo, de acordo com a equipe de pesquisadores.
Contudo, o principal objetivo desses meios é o de manter a sobrevivência das células
em cultivo, bem como promover e/ou melhorar o desenvolvimento celular. O meio
MEM, por exemplo, tem sido utilizado com sucesso para as espécies caprina (MATOS
et al., 2007a,b; BRUNO et al., 2009; ARAÚJO et al., 2010a; SARAIVA et al., 2010,
2011; MAGALHÃES et al., 2011, 2012), bovina (FIGUEIREDO et al., 1994a;
ROSSETTO et al., 2012, 2013), canina (SERAFIM et al., 2010, 2012), murina (JIN et
al., 2010; JEE et al., 2012), bem como em primatas não-humanos (XU et al., 2010,
2011). Outro meio amplamente utilizado tem sido o TCM-199, que além do cultivo de
folículos ovarianos (KATSKA; RYNSKA, 1998; ITOH; HOSHI, 2000; SAHA et al.,
2000, 2002; ROSSETTO et al., 2012), tem sido utilizado para estudos de maturação e
fertilização em diferentes espécies (ARLOTTO et al., 1996; ALM et al., 2006;
ABEDELAHI et al., 2010; ANTOSIK et al., 2010). Além dos meios de cultivo de base,
outras substâncias como fatores de crescimento e/ou hormônios vêm sendo testados no
cultivo folicular in vitro. Tendo em vista a necessidade da definição de um meio padrão
para a espécie bovina, por exemplo, dois meios de cultivo de base (αMEM e TCM-199)
40
foram objetos de estudo da presente tese, bem como as diferentes formas de troca de
meio durante o cultivo in vitro (Capítulo 4).
2.8 HORMÔNIO FOLÍCULO ESTIMULANTE (FSH)
Na foliculogênese, o hormônio folículo estimulante (FSH) destaca-se por ser
considerado um dos reguladores primários da foliculogênese. A aquisição de receptores
para este hormônio (FSHR) é fundamental para uma variedade de reações que incluem
proliferação e diferenciação das células da granulosa, maturação folicular (ADASHI,
1994), síntese de esteroides e expressão de receptores para o fator de crescimento
epidermal (EGF), hormônio luteinizante (LH), dentre várias outras substâncias
(FORTUNE, 2003).
O FSHR é um receptor do tipo acoplado à proteína G, que é dividido em três
domínios: um extracelular, um transmembranário, composto por 7 hélices hidrofóbicas
que ancoram
o receptor no
citoplasma, e um
domínio intramembranário
(GUDERMANN et al., 1995). O domínio intracelular do receptor do FSH (C-terminal)
é acoplado a uma proteína G e, após a ativação do receptor pela interação hormonal com
o domínio extracelular (N-terminal), inicia-se uma cascata de eventos que culmina com
efeitos biológicos específicos da gonadotrofina (SIMONI et al., 1997).
Sabe-se que as gonadotrofinas são necessárias para o desenvolvimento de
folículos antrais, mas ainda não está claro de que maneira o FSH afeta o
desenvolvimento de pequenos folículos pré-antrais. A expressão de FSHR já foi
observada nas células da granulosa de folículos primários, secundários e antrais bovinos
(XU et al., 1995). Méduri et al. (2002) observaram o RNAm e a proteína para o FSHR
em oócitos suínos, começando nos folículos primários seguindo até o estágio préovulatório. Recentemente, Durlej et al. (2011) reforçaram esses achados e ainda
verificaram a presença de FSHR em oócitos de folículos primordiais e primários, bem
como em células da granulosa de folículos primários suínos. In vivo, a administração de
um neutralizador do FSH no útero de hamsters, reduziu o número de folículos
primordiais (ROY; ALBEE 2000). Tendo em vista que o pré-requisito para a atuação do
FSH é a expressão de seu receptor, os resultados apresentados por Durlej et al. (2011),
nos quais além de folículos primordiais, os cordões de oócitos apresentavam marcação
para o FSHR, claramente demonstram o envolvimento do FSH na formação dos
folículos primordiais e na sua ativação levando-os para o pool de folículos em
41
desenvolvimento.
In vitro, o FSH promoveu um aumento do diâmetro de folículos primários e
secundários isolados bovinos e a manutenção da sobrevivência folicular, bem como a
secreção de progesterona e estradiol (WANDJI et al., 1996a). Além do crescimento
folicular, Gutierrez et al. (2000) observaram que o FSH permite que folículos
secundários isolados bovinos adquiram cavidade antral após cultivo in vitro. O FSH
também está envolvido na proliferação e diferenciação de células da granulosa de
folículos pré-antrais suínos (HIRAO et al., 1994). Em caprinos, a adição de 50 ng/mL
de FSH ao meio de cultivo de folículos pré-antrais inclusos em tecido ovariano foi
responsável pela manutenção da sobrevivência e ultraestrutura dos folículos, bem como
pelo aumento dos diâmetros folicular e oocitário (MATOS et al., 2007a; MAGALHÃES
et al., 2009). Por outro lado, o FSH não afetou a ativação, bem como o diâmetro
folicular e oocitário e o número de células da granulosa durante cultivo de fragmentos
ovarianos bovinos (BRAW-TAL; YOSSEFI, 1997; FORTUNE et al., 1998).
2.9 FATOR DE CRESCIMENTO DO ENDOTÉLIO VASCULAR (VEGF)
Outra substância estudada na presente tese foi o fator de crescimento do
endotélio vascular (VEGF). Durante as fases de crescimento e atresia folicular ocorre
uma reorganização dos capilares sanguíneos a fim de suprir as necessidades teciduais.
Neste caso, o VEGF, um importante fator angiogênico (BARBONI et al., 2000;
SHIMIZU et al., 2003), é responsável pela angiogênese folicular, pois atua estimulando
a mitose de células endoteliais expandindo-as, e aumentando a permeabilidade vascular
(REDMER; REYNOLDS, 1996). Além disso, tem sido sugerido que o VEGF pode
desempenhar um importante papel no crescimento e formação do antro folicular,
seleção do folículo dominante, maturação do oócito, ovulação e formação do corpo
lúteo (KACZMAREK; SCHAMS; ZIECIK, 2005).
Em folículos ovarianos, o VEGF é produzido pelas células da teca e da
granulosa (YAMAMOTO et al., 1997), e seus receptores, VEGFR-1 (Flt-1) e VEGFR-2
(Flk-1/KDR), também são expressos nesses mesmos locais. A produção de VEGF
aumenta progressivamente a partir do folículo primário até o folículo pré-ovulatório
(SHARMA; SUDAN, 2010) variando de acordo com a fase da dinâmica folicular.
Resultados semelhantes foram observados por Bruno et al. (2009) em relação ao
receptor do tipo 2 (VEGFR-2/KDR), em que foi demonstrada a presença desta proteína
42
em todas as categorias foliculares. Além disso, a presença desse receptor em oócitos de
folículos
primordiais
indica o envolvimento do VEGF no crescimento
e
desenvolvimento destas células (BRUNO et al., 2009). De fato, o VEGF desempenha
um papel importante na regulação do crescimento e sobrevivência de folículos
primordiais (ROBERTS et al., 2007; YANG; FORTUNE, 2007).
Diante de sua relevância, três dos capítulos desta tese apresentarão aspectos mais
específicos acerca do VEGF; sendo uma revisão de literatura mostrando sua
importância para a foliculogênese em mamíferos, apresentada no Capítulo 2, bem como
dois artigos científicos em que foi estudado o efeito do VEGF sobre o desenvolvimento
de folículos secundários caprinos (Capítulo 7) e bovinos (Capítulo 8) isolados.
2.10 PROTEÍNA MORFOGENÉTICA ÓSSEA-6 (BMP-6)
As proteínas morfogenéticas ósseas (BMPs) são membros da superfamília do
fator de crescimento transformante-β (TGF-β). Originalmente as BMPs foram
associadas a suas habilidade de formar tecido ósseos e cartilagens. Dentre as várias
BMPs descritas até o momento, as BMP-2 (ZHU et al., 2013), BMP-4 (LAWSON et al.,
1999; GLISTER; KEMP; KNIGHT, 2004; JUENGEL et al., 2006; ZHU et al., 2013),
BMP-6 (OTSUKA et al., 2001a; GLISTER; KEMP; KNIGHT, 2004; JUENGEL et al.,
2006; ZHU et al., 2013), BMP-7 (GLISTER; KEMP; KNIGHT, 2004; JUENGEL et al.,
2006; ARAÚJO et al., 2010a; ZHU et al., 2013), BMP-8 (YING et al., 2000) e BMP-15
(OTSUKA et al., 2001b) têm sido relacionadas com funções na foliculogênese e
ovulação. Como objeto dessa tese, mais detalhes serão abordados em relação à BMP-6.
O RNAm que codifica a BMP-6 foi identificado em ovários de várias espécies
mamíferas (SHIMASAKI et al., 1999; SHIMIZU et al., 2004). Foram detectadas
proteínas e/ou RNAm da BMP-6 em ovários de ratas (OTSUKA
et al., 2001a;
ERICKSON; SHIMASAKI, 2003) e porcas (SHIMIZU et al., 2004; BRANKIN et al.;
2005a). A BMP-6 é uma proteína expressa no oócito (ovelha: JUENGEL et al., 2006;
camundongo: ELVIN et al. 2000; rata: OTSUKA et al., 2001a; vaca: GLISTER;
KEMP; KNIGHT, 2004; porca: BRANKIN et al., 2005a), células da granulosa (rata:
ERICKSON; SHIMASAKI, 2003; vaca: GLISTER; KEMP; KNIGHT, 2004 e porca:
BRANKIN et al., 2005a) e células da teca (ovelha: CAMPBELL et al., 2004; vaca:
GLISTER; KEMP; KNIGHT, 2004) de folículos em diferentes estágios de
desenvolvimento. A expressão da proteína da BMP-6 foi demonstrada no líquido
43
folicular de grandes folículos suínos (BRANKIN et al., 2005a). Recentemente, o RNAm
para a BMP-6 foi verificado em folículos secundários avançados e antrais iniciais
caprinos antes e após o cultivo in vitro (FROTA et al., 2011; COSTA et al., 2012).
Além disso, a imunomarcação da BMP-6 foi observada em oócitos caprinos de todas as
categorias foliculares (primordial, primário, secundário, pequenos e grandes folículos
antrais), enquanto que em células da granulosa a marcação foi observada apenas a partir
do estágio primário (FROTA et al., 2011).
As BMPs interagem com duas classes de receptores transmembranários do tipo
serina-treonina quinase, receptores de BMP tipo I e tipo II. Nos mamíferos, três
receptores tipo I (BMPR-IA/Alk3, BMPR-IB/Alk6 e ActR-I/Alk2) e três receptores do
tipo II (BMPR-II, ActR-IIA, e ActR-IIB) foram identificados (KNIGHT; GLISTER,
2003). Estudos verificaram que o RNAm para os receptores da BMP-6 (BMPR-IA, -IB
e II) são expressos nos oócitos e nas células da granulosa de folículos caprinos (SILVA
et al., 2004; COSTA et al., 2012) e de várias outras espécies mamíferas: camundongo
(ELVIN et al., 2000), rata (SHIMASAKI et al., 1999; ERICKSON; SHIMASAKI,
2003), ovelha (SOUZA et al., 2002) e vaca (GLISTER; KNIGHT, 2002). Na superfície
do epitélio ovariano de ovelhas (WILSON et al., 2001; SOUZA et al., 2002), a
expressão da proteína para estes receptores é observada apenas nas células da granulosa.
Além disso, em vacas foi detectada a expressão de RNAm do BMPR-IA, BMPR-IB,
ActR-I, ActR-IIB e
BMPR-II em todos os compartimentos de folículos antrais,
enquanto que a proteína BMPR-II foi encontrada somente nos oócitos destes folículos
(FATEHI et al., 2005). A expressão da proteína dos receptores de BMP (BMPR-IA,
BMPR-IB e BMPR-II) foi demonstrada em células da granulosa, células da teca e
oócitos suínos (QUINN et al., 2004), indicando possíveis efeitos autócrinos e/ou
parácrinos.
Para a atuação das BMPs, os receptores de BMP fosforilam proteínas
sinalizadoras intracelulares chamadas Smads, as quais convertem o sinal para o núcleo
modificando a expressão gênica. As Smads constituem três subfamílias: Smads
receptoras (R-Smads: Smad-1, Smad-5 e Smad-8); Smad mediadora comum (Co-Smad:
Smad4); e Smads inibidoras (I-Smads: Smad-6 e Smad-7). A via Smad é regulada pelo
co-mediador Smad-4 e pelas Smads inibidoras (Smad-6 e Smad-7; ten DIJKE; HILL,
2004), sendo as BMPs ativadoras das Smad-1, Smad-5 e Smad-8 (MIYAZAWA et al.,
2002). As R-Smads são fosforiladas pelo BMPR-I, enquanto que as I-Smads exercem
um feedback negativo, uma vez que competem com as R-Smads pelas interações com os
44
receptores levando-os à degradação (MOUSTAKAS et al., 2001). As R-Smads que
forem fosforiladas interagem com a Smad-4 e são translocados ao núcleo para ativar a
maquinaria transcricional e modular a transcrição dos genes de BMP (WANG et al.
2010). Desta forma, a formação do complexo BMP, receptores e Smads regulará vários
processos biológicos (COSTELLO et al. 2009), determinando os possíveis efeitos
autócrinos e parácrinos da BMP-6 durante o crescimento folicular. Recentemente, Costa
et al. (2012) verificaram a expressão do RNAm para as Smad-1, Smad-5 e Smad-8 em
folículos pré-antrais e antrais caprinos. Em vacas, a expressão da proteína para Smad-1
foi verificada em folículos antrais e sua expressão foi ativada pela BMP-6 em células da
granulosa cultivadas in vitro (GLISTER; KEMP; KNIGHT, 2004). Além disso, a BMP6 induziu a fosforilação das Smad-1 e Smad-5, mas não da Smad-8 em osteoblastos
(EBISAWA et al. 1999; AOKI et al. 2001).
Modelos knockout, têm sido utilizados para investigar os efeitos in vivo e in vitro
das BMPs e Smads no ovário. A ausência da Smad-4 no ovário impediu o
desenvolvimento de folículos antrais (PANGAS et al., 2006), aumentou a apoptose
folicular (PANGAS et al., 2006) e celular (WANG et al., 2010), além de ter inibido a
proliferação e a esteroidogênese das células da granulosa (WANG et al., 2010). Ratas
knockout para o gene da BMP-6 parecem normais no que diz respeito à fertilidade e
tamanho da ninhada, sugerindo que esta proteína pode não ser essencial para a
fertilidade de murinos (SOLLOWAY et al., 1998). Ao contrário da BMP-15, a BMP-6
não estimula a proliferação de células da granulosa (ratas: OTSUKA et al., 2001a,b;
ovelhas: JUENGEL et al., 2006), além de inibir sua diferenciação (ovelhas: JUENGEL
et al., 2006). Entretanto, em bovinos, GLISTER; KEMP; KNIGHT (2004) verificaram
um pequeno, porém significativo, aumento no número de células da granulosa viáveis
após cultivo in vitro em meio adicionado de BMP-6. Além disso, esta proteína foi
demonstrada também ser importante na proliferação de células da granulosa
(BRANKIN et al., 2005a) e células da teca em suínos (BRANKIN et al., 2005b). Em
caprinos, o cultivo de folículos secundários por seis dias com adição de BMP-6,
promoveu aumento no diâmetro folicular e no percentual de folículos antrais após
cultivo in vitro (FROTA et al., 2011). Neste contexto, as diferentes respostas celulares
às BMPs podem ser devido a múltiplos fatores, incluindo o estágio de maturação ou
número de células da granulosa dos folículos, às diferenças nas composições dos meios
e os períodos de cultivo, ou ainda a adição de outros hormônios e/ou fatores de
crescimento. Tendo em vista essas difereças entre as categorias foliculares, dois dos
45
capítulos da presente tese objetivaram verificar os efeitos da BMP-6 sobre o cultivo de
duas categorias foliculares diferentes, folículos primordiais (Capítulo 5) e folículos
secundários avançados (Capítulo 6).
2.11 FATOR DE CRESCIMENTO SEMELHANTE À INSULINA-1 (IGF-1)
O sistema dos fatores de crescimento semelhantes à insulina (IGF) é composto
pelos ligantes IGF-1 e IGF-2, os quais apresentam elevado grau de homologia estrutural
com a pró-insulina, além de receptores 1 (IGFR-1) e 2 (IGFR-2) e seis proteínas
transportadoras denominadas proteínas de ligação (IGFBP-1, IGFBP-2, IGFBP-3,
IGFBP-4, IGFBP-5, e IGFBP-6). O ligante IGF-1 tem origem ontogênica (WANDJI et
al., 1992) e embora possa ser produzido pela maioria dos órgãos e tecidos do
organismo, o IGF-1 é produzido predominantemente no fígado, exercendo a função de
um hormônio endócrino. Por não existir um órgão de armazenamento, o IGF-1 é
secretado à medida que é produzido (YAKAR et al., 2002), podendo sua produção ser
estimulada pelo GH. Além disso, no ovário, as IGFBPs intrafoliculares desempenham
uma função-chave na regulação do desenvolvimento folicular por modularem os IGFs,
apresentando seus níveis alterados no líquido folicular durante a foliculogênese
(MONGET; MONNIAUX; DURAND, 1989).
A maioria das ações conhecidas dos IGFs é mediada via IGFR-1, não sendo
ainda claro o papel fisiológico do IGFR-2. O IGFR-1 apresenta alta afinidade, tanto
pelo IGF-1 quanto pelo IGF-2, entretanto, a afinidade do IGFR-2 pelo IGF-2 é
aproximadamente 500 vezes maior que pelo IGF-1 (LeROITH et al., 1995). O IGFR-1,
apresenta estrutura similar à do receptor da insulina, sendo composto por duas
subunidades α (região extracelular) e duas subunidades β (apresenta uma parte
extracelular, um seguimento transmembranário e uma parte intracelular). Cada
subunidade α é ligada a uma subunidade β por uma ponte dissulfídrica, formando um αβ hemi-receptor que, por sua vez, se liga a outro hemi-receptor por ponte dissulfídrica
entre as subunidades α para formar o receptor completo (JONES; CLEMMONS, 1995;
LeROITH et al., 1995). Uma vez ligante e o receptor acoplados, múltiplas vias de
sinalização são ativadas, tais como a via da fosfatidil inositol 3-quinase (PI3K) e das
proteínas quinases ativadas por mitógenos (MAP) (JONES; CLEMMONS, 1995).
No ovário bovino, o RNAm para o IGFR-1 foi demonstrado em oócitos e células
da granulosa e da teca de folículos pré-antrais (ARMSTRONG et al., 2000)
46
apresentando como principal função estimular o desenvolvimento folicular pré-antral e
antral (ARMSTRONG; BENOIT, 1996). O IGF-1 estimulou o crescimento folicular e
oocitário, bem como a formação de antro durante cultivo de longa duração em bovinos
(GUTIERREZ et al., 2000; ITOH et al., 2002) e caprinos (MAGALHÃES et al., 2012).
Após 18 dias de cultivo, o IGF-1 foi capaz de manter a viabilidade folicular e aumentar
a taxa de retomada da meiose de oócitos oriundos de folículos secundários caprinos
crescidos in vitro (MAGALHÃES et al., 2012). Além disso, foi observada a produção
de estradiol por folículos bovinos após cultivo in vitro em meio contendo IGF-1
(THOMAS et al., 2007). Sinergicamente com o FSH, o IGF-1 aumentou a expressão do
RNAm para o ligante IGF-1 e para o FSHR, enquanto que o FSH sozinho aumentou a
expressão do RNAm para o IGFR-1 após cultivo in vitro de folículos pré-antrais
caprinos (BRITO et al., 2012). Resultados semelhantes foram observados em estudos
nos quais o FSH induziu a expressão do RNAm para o IGFR-1 após cultivo in vitro de
células da granulosa bovinas (SUDO et al., 2007). Neste contexto, dois dos artigos da
presente tese apresentam maiores detalhes sobre o IGF-1 no ovário (Capítulo 1 –
revisão de literatura), bem como sua ação sobre o desenvolvimento de folículos
secundários bovinos isolados (Capítulo 8).
2.12 HORMÔNIO DO CRESCIMENTO (GH)
O hormônio do crescimento (GH) é um hormônio somatotrófico secretado pelo
lobo anterior da hipófise na circulação. Este peptídeo se liga a receptores nos tecidosalvo com o objetivo de estimular o crescimento (HERRINGTON; CARTER-SU, 2001).
O receptor de GH (GHR) é uma citocina que apresenta um domínio extracelular, uma
porção transmembranária e um domínio citoplasmático (SJÖGREN et al., 1999). São
necessárias duas moléculas de GHR para que a molécula de GH se ligue, uma vez que
esta apresenta dois sítios de ligação na sua estrutura (CARTER-SU et al., 1996). A
transmissão do sinal ocorre mediante a ativação e a fosforilação da enzima Janus kinase
2 (JAK2) e de resíduos do domínio intracelular do GHR, o que resulta no engajamento
de diversas proteínas de sinalização intracelular, incluindo os transdutores de sinal e
ativação de transcrição (STAT-1, STAT-3 e STAT-5), e componentes da via das MAP
quinases. A fosforilação do STAT-5 é importante nas ações somatotróficas do GH, pois
participa da regulação da secreção do IGF-I e da IGFBP-3 (KOFOED et al., 2003).
47
Estudos in vitro e in vivo têm revelado a importância deste hormônio durante o
desenvolvimento folicular (HUTCHINSON et al., 1988; GONG et al., 1997;
SIROTKIN; MAKAREVICH, 2002) através de seus efeitos diretos ou indiretos. Os
efeitos indiretos estão relacionados ao fato de o GH estimular a liberação do IGF-1. Já
os efeitos diretos estão relacionados com a expressão do GHR, os quais foram
detectados em ovários humanos (SHARARA; NIEMAN, 1994) e bovinos (SHIMIZU et
al., 2008). Em bovinos, o transcrito do GHR está presente em oócitos de folículos
primordiais e primários, bem como o RNAm para o GHR está presente em folículos
secundários e terciários (SHIMIZU et al., 2008). O artigo de revisão de literatura
apresentado no Capítulo 1, bem como os resultados do artigo técnico apresentado no
Capítulo 8, a seguir, descrevem maiores detalhes acerca da importância do GH para a
foliculogênese in vitro.
2.13 TÉCNICAS PARA AVALIAÇÃO DA EFICIÊNCIA DO CULTIVO IN VITRO
O monitoramento da eficiência do cultivo in vitro de folículos ovarianos préantrais pode ser realizado através da avaliação da ativação folicular, mudanças
morfológicas no oócito e nas células somáticas foliculares durante o crescimento in
vitro, surgimento da cavidade antral, além da produção de hormônios e esteroides pelos
folículos durante o cultivo in vitro. Cada um desses parâmetros necessita de uma técnica
específica para sua avaliação. Dentre as principais técnicas utilizadas para avaliar os
sistemas de cultivo in vitro destacam-se: a análise histológica e ultraestrutural (MATOS
et al., 2006; 2007a,b; MAGALHÃES et al., 2009; ARAÚJO et al., 2010a), expressão
gênica através dos níveis de RNAm (HAYASHI et al., 1999; CELESTINO et al., 2010,
2011) e a dosagem hormonal para mensuração, por exemplo, dos níveis de estradiol
(KOBAYASHI et al., 2000; SILVA et al., 2013).
2.13.1 Histologia clássica
A microscopia de luz tem sido utilizada para avaliar o crescimento folicular e a
qualidade das células em diferentes espécies (caprino: MATOS et al., 2007a,b;
MAGALHÃES et al., 2009; ARAÚJO et al., 2010a; bovino: AERTS; OSTE; BOLS,
2005; AERTS et al., 2008; CELESTINO et al., 2008; equino: HAAG et al., 2013a,b,c;
humano: ABIR et al., 2010). Os processos de ativação folicular e as mudanças
48
morfológicas nos oócitos e nas células da granulosa e da teca durante o crescimento
folicular podem ser avaliadas usando técnicas histológicas. Além disso, as alterações
pelo processo de degeneração do folículo, como retração citoplasmática do oócito,
presença ou ausência de corpos picnóticos nucleares, desorganização das células da
granulosa e baixa densidade celular também podem ser avaliadas sob microscopia de
luz.
2.13.2 Microscopia eletrônica de transmissão
De maneira geral, as análises utilizando microscopia eletrônica de transmissão
são realizadas após avaliação sob microscopia de luz. A microscopia eletrônica é
utilizada de maneira qualitativa, observando detalhes como o formato e as dimensões do
núcleo, a dispersão da cromatina nuclear, presença de citoplasma intacto, qualidade das
organelas, vacuolização citoplasmática, preservação do contato entre as células da
granulosa e o oócito, bem como a aparência das células do estroma (OSKAM et al.,
2010).
Folículos pré-antrais de várias espécies têm sido avaliados com sucesso
utilizando o microscópio eletrônico de transmissão, sendo observados tanto folículos
apresentando ultraestrutura normal (FAIR et al., 1997; van den HURK et al., 1998;
MATOS et al., 2007a; CELESTINO et al., 2008; ARAÚJO et al., 2010a), como aqueles
com ultraestrutura alterada (SILVA et al., 2002; MATOS et al., 2006). Folículos préantrais normais apresentam um oócito com núcleo bem delimitado, mitocôndrias
arredondadas com cristas irregulares, membrana mitocrondrial contínua e mitocôndrias
alongadas com cristas paralelas (CELESTINO et al., 2008), bem como gotas lipídicas e
ambos retículo endoplasmático liso e rugoso e material de ZP (FAIR et al., 1997).
2.13.3. Microscopia de fluorescência e microscopia confocal
Em geral, a viabilidade celular é avaliada pelas diferentes marcações de células
viáveis e não-viáveis, e a determinação dos percentuais de viabilidade celular pela
microscopia de fluorescência, a qual é mais sensível que a microscopia de luz. Folículos
de diferentes espécies vêm sendo avaliados com sucesso utilizando esta técnica
(THOMAS et al., 2001; AERTS et al., 2008; BRUNO et al., 2009). Para a marcação,
células viáveis necessitam apresentar membrana intacta, enquanto que células não-
49
viáveis apresentam integridade da membrana comprometida. Dentre os vários
marcadores disponíveis para avaliação da viabilidade celular, a Calceína-AM e o Etídeo
homodímero-1 são os mais comumente utilizados. Além dos marcadores fluorescentes,
utilizando o microscópio confocal a laser, a detecção de outros aspectos do
desenvolvimento folicular in vitro também podem ser avaliados, tais como: a produção
das proteínas da ZP, a dinâmica das junções gap, a atividade mitocondrial, a detecção
do núcleo das células da granulosa em proliferação (SCHOTANUS et al., 1997), bem
como a distribuição de filamentos de actina e da proteína conexina (McLAUGHLIN et
al., 2010).
2.13.4. Análise de esteroides
Os folículos ovarianos são responsáveis principalmente por permitir a liberação
de oócitos e a produção de esteroides e hormônios proteicos (CORTVRINDT; SMITZ,
2002). Cada estágio do desenvolvimento folicular exibe um padrão único de
sensibilidade às gonadotrofinas, produção de esteroides e vias de feedback hormonal
(RICHARDS, 2001). Além disso, cada categoria folicular passa por mudanças
estruturais, nas quais diferentes células foliculares adquirem a maquinaria intracelular
requerida para sintetizar e secretar esteroides e hormônios proteicos, e expressar
receptores estágio-específicos para responder a estímulos endócrinos (CORTVRINDT;
SMITZ, 2002).
Quando os folículos crescem in vitro, alguns indicadores da saúde folicular são
requeridos. Portanto, o meio de cultivo pode ser monitorado e quaisquer fatores
secretados que possa indicar a saúde do folículo e do oócito podem ser facilmente
mensurados (TELFER et al., 2000). Hormônios esteróides, tais como a progesterona,
androstenediona e o estradiol podem ser mensurados em meio de descarte, ou seja,
meios retirados durante as trocas, podendo então, serem avaliados utilizando
radioimunoensaio ou ensaio imunoenzimático (enzyme-linked immunosorbent assay –
ELISA). Entretanto, dependendo do estágio folicular, os níveis de esteroides podem não
ser detectados, ou mesmo serem alterados (WANDJI et al., 1996a). A ausência de uma
camada de células da teca completamente definida em folículos pré-antrais durante o
cultivo in vitro, contribui para a inabilidade (bovinos: WANDJI et al., 1996a) ou pouca
habilidade (murinos: KOBAYASHI et al., 2000) de se detectar níveis mensuráveis de
androstenediona. Além disso, a inibina e o estradiol foram significativamente reduzidos
50
quando folículos pré-antrais murinos foram cultivados sem células da teca
(KOBAYASHI et al., 2000). A habilidade de folículos pré-antrais aumentarem os níveis
de estradiol e progesterona indicam o desenvolvimento folicular e a diferenciação das
células da granulosa. A conversão de testosterona exógena à estradiol sob estímulo do
FSH induziu a atividade da enzima P450aromatese em folículos pré-antrais cultivados
in vitro (WANDJI et al., 1996a).
2.13.5. Reação em cadeia da polimerase em tempo real (PCR em tempo real)
A localização de proteínas, RNAm e seus receptores auxilia a compreensão do
papel de cada hormônio e fator de crescimento em cada fase do desenvolvimento
folicular, uma vez que além da localização, esta técnica possibilita quantificar cada uma
das substâncias produzidas no ovário. Muitas atuações das células foliculares ovarianas
relacionadas à sobrevivência, ao crescimento e à diferenciação, são refletidas na
alteração dos padrões da expressão gênica. Desta forma, a capacidade de quantificar os
níveis de transcrição de genes específicos é fundamental para qualquer investigação das
funções foliculares (ZAMORANO et al. 1996). Após a extração do RNAm, a partir de
folículos ovarianos, e da sua conversão em um DNA complementar (DNAc), a PCR
realiza a síntese de milhões de cópias deste DNAc na presença da enzima DNA
polimerase, caracterizando o princípio fundamental da técnica que é a amplificação do
DNA. A reação de PCR em tempo real, uma variante da PCR convencional, permite
uma análise precisa da quantificação da expressão gênica em determinado tecido ou
amostra biológica. Esse método utiliza um sistema fluorescente em plataforma, capaz de
detectar a luz oriunda da reação de amplificação de um determinado gene no momento
real da amplificação (BUSTIN, 2002).
Vários estudos demonstraram que a PCR é uma ferramenta adequada para o
estudo mais detalhado do complexo e misterioso processo da foliculogênese. Os RNAm
para receptores e ligantes para várias substâncias já foram observados em folículos préantrais de várias espécies como caprinos, bovinos, ovinos, suínos, murinos e humanos.
Alguns hormônios e fatores de crescimento já tiveram o RNAm para o ligante e seus
receptores detectados em folículos pré-antrais, tais como: BMP-6 (caprino: FROTA et
al 2011; COSTA et al., 2012) e BMPRs (caprino: SILVA et al., 2004; COSTA et al.,
2012), FSHR (caprino: SARAIVA et al., 2011; FROTA et al., 2011; BRITO et al.,
2012; humano; OKTAY; BRIGGS; GOSDEN, 1997), VEGF e VEGFRs (bovino:
51
YANG; FORTUNE, 2007; humano: ABIR et al., 2010); e IGF-1 e IGFR-1 (caprino:
BRITO et al., 2012; MAGALHÃES et al., 2012).
52
3 JUSTIFICATIVA
Como visto anteriormente, os folículos pré-antrais
representam a quase
totalidade da população folicular ovariana, cerca de 90%, entretanto, a grande maioria
(99,9%) dos folículos não chegam à fase ovulatória, sendo eliminados por atresia. Neste
contexto, a biotécnica de MOIFOPA visa resgatá-los do ovário antes que se tornem
atrésicos e cultivá-los in vitro até sua completa maturação. Os oócitos oriundos destes
folículos crescidos in vitro representam uma população homogênea de um mesmo
animal e poderiam ser utilizados em programas de produção in vitro e transferência de
embriões e/ou criopreservação. Além disso, o desenvolvimento de um sistema de
cultivo eficiente poderá fornecer subsídios para uma melhor compreensão sobre os
fatores que regulam o crescimento e a maturação folicular.
No que se refere a espécie caprina, a maturação de oócitos oriundos de folículos
pré-antrais crescidos in vitro ainda é considerada um fator limitante para o sucesso da
biotécnica de MOIFOPA. Além disso, na espécie bovina, os resultados ainda são
inconsistentes em relação aos meios de base utilizados, limitado apenas à produção de
folículos antrais após cultivo in vitro. Portanto, o desafio atual dos pesquisadores em
todo mundo, e que justifica a elaboração da presente tese, tem sido a definição de um
meio de cultivo de base e do regime de troca de meio mais eficientes, bem como o
desenvolvimento de um sistema de cultivo que permita a manutenção da viabilidade, a
ativação, o crescimento e a completa maturação folicular, otimizando o aproveitamento
do potencial oocitário dos animais e incrementando a eficiência da reprodução animal.
Desta forma, além da determinação de um meio de cultivo de base para a
manutenção da viabilidade e promoção do crescimento de folículos secundários bovinos
isolados, outro aspecto considerado importante foi o conhecimento acerca dos efeitos
dos fatores de crescimento (exemplos: VEGF, BMP-6 e IGF-1) e/ou hormônios
(exemplos: FSH e GH) sobre o cultivo in vitro dos folículos pré-antrais bovinos e/ou
caprinos. Esses estudos foram extremamente necessários para que estratégias de cultivo
pudessem ser desenvolvidas e buscassem otimizar o crescimento e a maturação folicular
in vitro. No tocante as substâncias adicionadas aos meios de cultivo, tornou-se
necessária a determinação das melhores concentrações da BMP-6 em um cultivo in situ
de curta duração, para posteriormente utilizá-las em um cultivo de longa duração de
folículos secundários caprinos isolados. Adicionalmente, o VEGF foi utilizado com o
objetivo principal de melhorar as taxas de maturação dos oócitos oriundos de folículos
53
pré-antrais caprinos crescidos in vitro. Tendo em vista os resultados limitados
apresentados anteriormente para a espécie bovina, e na tentativa de melhorá-los, os
fatores de crescimento VEGF e IGF-1, bem como o hormônio GH, foram utilizados
baseados nos melhores resultados presentes na literatura para a espécie caprina.
Para melhor avaliar a eficiência dos sistemas de cultivo empregados no presente
estudo, além da histologia clássica, foi utilizada a microscopia eletrônica de transmissão
e/ou de fluorescência, bem como a análise dos níveis de estradiol (ELISA) para
determinar a qualidade de folículos pré-antrais caprinos e/ou bovinos cultivados in
vitro. Na tentativa de melhor desvendar a foliculogênese caprina e bovina, foi ainda
realizado um estudo de quantificação da expressão gênica do RNAm para os BMPR1A, BMPR-2, Smad-1, Smad-3, Smad-4, Smad-5, Smad-6, Smad-7, Smad-8 sob a
influência de BMP-6 em folículos secundários caprinos, bem como o RNAm para o
VEGF, IGF-1, P450aromatase, FSHR sob a influência de diferentes meios de cultivo de
base e protocolos para troca de meio em folículos secundários bovinos por meio da
técnica de transcrição reversa da PCR quantitativa em tempo real (RT-qPCR).
54
4 HIPÓTESES CIENTÍFICAS
Diante do exposto, formularam-se as seguintes hipóteses científicas:
a) O regime de troca de meio influencia positivamente o desenvolvimento e a
maturação in vitro de folículos secundários caprinos isolados após cultivo de longa
duração;
b) Os meios de cultivo de base αMEM e TCM-199, bem como o regime de troca de
meio influenciam positivamente o desenvolvimento, a produção de estradiol e a
expressão gênica de folículos secundários bovinos isolados após cultivo in vitro de
longa duração;
c) A BMP-6 mantém a sobrevivência folicular e influencia positivamente a ativação e o
crescimento in vitro de folículos pré-antrais caprinos inclusos no tecido ovariano
após cultivo de curta duração;
d) A BMP-6 e/ou FSH afetam positivamente o desenvolvimento folicular e modulam a
expressão gênica dos receptores para a BMP-6, bem como seus sinalizadores
intracelulares (Smads) de maneira diferenciada durante o cultivo in vitro de folículos
secundários caprinos isolados;
e) O VEGF mantém a sobrevivência folicular e estimula a maturação de oócitos
oriundos de folículos pré-antrais caprinos isolados, após cultivo in vitro de longa
duração;
f) Os fatores de crescimento VEGF e IGF-1 e o hormônio GH utilizados
individualmente, ou em combinação, promovem o desenvolvimento folicular e a
produção de estradiol após cultivo in vitro de folículos secundários bovinos isolados.
55
5 OBJETIVOS
5.1 OBJETIVOS GERAIS
a) Verificar o efeito do regime de troca de meio sobre o cultivo in vitro de folículos
secundários caprinos isolados;
b) Avaliar o efeito do meio de cultivo de base (αMEM ou TCM-199) e do regime de
troca sobre o cultivo de folículos secundários bovinos isolados;
c) Investigar o efeito de diferentes concentrações de BMP-6 no cultivo de tecido
ovariano (in situ) caprino;
d) Estudar o efeito da BMP-6 isoladamente ou em associação com FSH sobre o cultivo
in vitro de folículos secundários caprinos isolados;
e) Verificar o efeito do VEGF sobre o cultivo in vitro de folículos secundários caprinos
isolados;
f) Verificar a influência da adição de VEGF, IGF-1 e GH isoladamente ou em
associação ao meio de cultivo in vitro de folículos secundários bovinos isolados
utilizando sistemas de cultivo bi (2D) e tridimensional (3D).
5.2 OBJETIVOS ESPECÍFICOS
a) Verificar as taxas de sobrevivência, crescimento e formação de antro, bem como as
taxas de maturação de oócitos oriundos de folículos secundários caprinos crescidos
in vitro utilizando diferentes protocolos para troca de meio;
b) Avaliar as taxas de crescimento, formação de antro, produção de estradiol, bem como
expressão gênica (RNAm para FSHR, VEGF, IGF-1 e P450arom) de folículos
secundários bovinos isolados utilizando dois diferentes meios de base (αMEM ou
TCM-199) e dois protocolos para troca de meio;
c) Verificar o efeito de diferentes concentrações de BMP-6 (1, 10, 50 e 100 ng/mL)
sobre as taxas de
sobrevivência, ativação, crescimento e características
ultraestruturais de folículos pré-antrais inclusos em tecido ovariano caprino após
cultivo in vitro;
d) Avaliar o efeito da BMP-6 (concentrações determinadas pelos resultados do cultivo
in situ) associada ou não ao FSH, sobre a sobrevivência, formação de antro, taxas de
56
crescimento folicular e maturação de oócitos oriundos de folículos secundários
caprinos isolados crescidos in vitro;
e) Verificar a influência da BMP-6 sobre os níveis de expressão do RNAm para BMPR1A, BMPR-2, Smad-1, Smad-4, Smad-5, Smad-6, Smad-7 e Smad-8 antes e após o
cultivo in vitro de folículos secundários caprinos isolados;
f) Avaliar o efeito de duas concentrações de VEGF (10 e 100 ng/ml), sobre as taxas de
sobrevivência, formação de antro, crescimento folicular e maturação de oócitos
oriundos de folículos secundários caprinos isolados crescidos in vitro;
g) Verificar o efeito do VEGF, IGF-1 ou GH, isoladamente ou em associação sobre as
taxas de crescimento, formação de antro e produção de estradiol de folículos
secundários bovinos isolados em sistemas de cultivo 2D (superfície plástica) ou 3D
(matriz de alginato).
57
6 CAPÍTULO 1
Cultivo in vitro de folículos pré-antrais bovinos: Uma revisão
“In vitro culture of bovine preantral follicles: A review”
Periódico: Molecular Reproduction and Development (Submetido em: 1 de agosto de
2013)
58
RESUMO
Os folículos pré-antrais constituem a maioria da população folicular ovariana e sua
utilização como fonte de oócitos homogêneos levaria a um grande avanço das
tecnologias de reprodução assistida em bovinos. Entretanto, quando comparada às
outras espécies, na espécie bovina, os resultados têm sido limitados à ativação de
folículos primordiais e a formação de folículos antrais iniciais a partir de grandes
folículos secundários cultivados in vitro. Portanto, esta revisão abordará os aspectos
básicos da foliculogênese em bovinos com foco nos folículos pré-antrais, os métodos
para o isolamento folicular, e os principais resultados obtidos nos últimos 20 anos, bem
como as potenciais substâncias candidatas (suplementos básicos, fatores de crescimento
e hormônios) para melhorar a eficiência do cultivo in vitro de folículos pré-antrais.
Palavras-chave: Vaca. Foliculogênese. Oócito. Folículos ovarianos.
59
In vitro Culture of Bovine Preantral Follicles: A Review
V.R. Araújo1,2,3, M.O. Gastal1, J.R. Figueiredo2 and E.L. Gastal1,4
1
Department of Animal Science, Food and Nutrition, Southern Illinois University,
1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
2
Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA),
Veterinary Faculty, State University of Ceará, Av. Paranjana 1700, Campus do Itaperi,
Fortaleza, CE, 60740-903, Brazil.
Running head: In vitro bovine folliculogenesis
Keywords: Cow; Folliculogenesis; Oocyte; Ovarian follicles.
3
Araújo VR is the recipient of a PhD scholarship from CNPq, Brazil.
4
Correspondence: Eduardo Gastal, Department of Animal Science, Food and Nutrition,
Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901,
USA. FAX: 618-453-5231; e-mail: egastal@siu.edu
Abbreviations
ZP, Zona pellucida;
VEGF, Vascular Endothelial Growth Factor;
DNAse, Deoxyribonuclease;
OPU, Ovum pick-up technique;
2D, Two-dimensional system;
3D, Three-dimensional system;
α-MEM, Minimum essential medium alpha modification;
TCM-199, Tissue culture medium -199;
MMP-9, Matrix metalloproteinases-9;
IGFs, Insulin-like growth factors;
FGFs, Fibroblast growth factors;
BMPs, Bone morphogenetic proteins;
GDFs, Growth and differentiation factors;
60
IGFBP-2, IGF binding protein-2;
LH, Luteinizing hormone;
TGF-β, Transforming growth factor β;
FSH, Follicle-stimulating hormone;
flt-1 or VEGFR-1, Fms-like tyrosine kinase-1 (VEGF receptor 1);
flk-1 or VEGFR-2, Kinase domain receptor (VEGF receptor 2);
BrdU-label, Bromodeoxyuridine;
GH, Growth hormone;
GHR, GH receptor
SUMMARY
Preantral follicles are the majority of the ovarian follicle population and their use as a
source of homogeneous oocytes for bovine reproductive biotechnologies could result in
a substantial advance in this field. However, while in other species, in bovine species
the results have been limited to the follicular activation of small (primordial) preantral
follicles and formation of early antral follicles from large (secondary) preantral follicles
after in vitro culture. Therefore, this review will highlight the basic aspects of bovine
folliculogenesis by focusing on preantral follicles, the methods of harvesting preantral
follicles, the main results from in vitro follicular culture during the last 20 years, and the
potential candidate substances (basic supplements, growth factors and hormones) for
improving the efficiency of in vitro culture.
TABLE OF CONTENTS
INTRODUCTION
BASIC ASPECTS OF EARLY BOVINE FOLLICULOGENESIS
Formation and Initiation of Primordial Follicle Growth
Growth of Primary and Secondary Follicles
HARVESTING BOVINE PREANTRAL FOLLICLES
Mechanical Isolation Using Tissue Chopper or Microdissection
Enzymatic Isolation
Ovarian Biopsy In Vivo
IN VITRO CULTURE SYSTEMS FOR BOVINE PREANTRAL FOLLICLES
61
In Vitro Culture of Preantral Follicles Enclosed in Ovarian Tissue (In Situ)
In Vitro Culture of Isolated Preantral Follicles
IMPROVING IN VITRO GROWTH OF BOVINE PREANTRAL FOLLICLES
Culture Media
Basic Supplements
Growth Factors
Insulin-like Growth Factor 1 (IGF-1)
Basic Fibroblast Growth Factor (bFGF)
Vascular Endothelial Growth Factor (VEGF)
Hormones
Activin
Follicle-Stimulating Hormone (FSH)
Growth Hormone (GH)
Insulin
FINAL CONSIDERATIONS
62
INTRODUCTION
The mammalian ovary is responsible for the development, maturation, and
release of mature oocytes for fertilization, as well as the synthesis and secretion of
hormones that are essential for follicular development, menstrual/estrous cyclicity, and
maintenance of the reproductive tract and its function. In each ovarian cycle, many
follicles are activated to enter the growth phase, which is characterized by both
proliferation of the granulosa cells and an increase in the oocyte size (Gougeon, 2003).
However, most of these follicles gradually become atretic during in vivo growth phase;
this fact awakes a great interest in the development of a culture system that might be
able to maintain follicular growth and avoid this loss of follicles.
Considering that primordial follicles constitute the supreme starting material for
in vitro culture due to their large number when compared with mature follicles
(Cortvrindt and Smitz, 2001), it would be of remarkable help to possess a renewable
source of primordial follicles from high-yielding animals for culture in order to
maximize offspring from these animals (Aerts et al., 2005). Moreover, the elucidation
of the yet incomprehensible mechanisms of primordial follicle activation would
constitute an important leap forward in the understanding of follicular dynamics (Aerts
et al., 2008).
Preantral follicles for research are usually obtained from ovaries from
slaughterhouses or through laparotomy or ovarian biopsies. Studies using ovarian
biopsy have shown minimum or no disturbance to ovarian function in several species,
including bovine (Bols et al., 1995; Aerts et al., 2005; 2008), equine (Haag et al.,
2013a,b,c), and human (Cortvrindt and Smitz, 2001; Abir et al., 2010). This technique
shall be of great value for experimental or diagnostic purposes. Profound similarities in
the dynamics of follicle development exist between the menstrual cycle in women and
the estrous cycle in cow and mares (Ginther et al., 2004; Gastal et al., 2011). In this
regard, research using animal models for studying human ovarian function is important
to provide a hypothetical basis for further studies in women, which will ultimately lead
to the development of safer and more efficacious infertility and contraceptive therapies
(Baerwald, 2009). Therefore, if preantral follicles could be efficiently isolated from
ovaries, a large potential source of oocytes (genetic material) could be obtained to reach
meiotic competence in vitro. Moreover, immature oocytes from preantral follicles could
63
be used in other assisted reproductive technologies, such as in vitro maturation and
embryo production, transgenesis, and conservation of endangered species.
An in vitro culture system that allows complete growth of oocytes from preantral
or early antral follicles has been studied. However, besides the differences among
species, in vitro culture success depends on initial oocyte size, as well as follicle
categories used. In regards to large animals, the production of embryos from buffalo
(Gupta et al., 2008), sheep (Arunakumari et al., 2011; Luz et al., 2012), goats
(Magalhães et al., 2011), and monkeys (Xu et al., 2011) have been obtained only from
large (advanced secondary follicle) preantral follicles, while in mouse, embryos and live
offspring have been obtained from primordial follicles (Eppig and O’Brien, 1996;
O’Brien et al., 2003). However, in bovine species the best results have been only with
antrum cavity formation after in vitro culture of advanced secondary follicles (Gutierrez
et al., 2000).
Ovarian follicular development and oocyte growth depend on a bidirectional
communication between oocytes and somatic cells. Oocytes have an essential role in
controlling the proliferation and differentiation of granulosa cells during follicular
development (Eppig et al., 2002). The ability to sustain preantral follicle growth in vitro
to support the acquisition of oocyte competence could represent a breakthrough in the
reproduction field since this source of oocytes could be beneficial for assisted
reproductive technologies. Additionally, research aiming at further understanding of
somatic cell-oocyte interactions in species with prolonged follicular growth, such as the
bovine species (McLaughlin et al., 2010), would be of great significance for human
reproduction. Therefore, in vitro culture systems have to allow for these conditions and
properly maintain cell interactions during follicle development.
This review aims to describe and discuss the advancements in and current status
of the emerging research of bovine preantral follicles. Firstly, we summarize current
knowledge of achievements in the development of in vitro systems for culture of bovine
preantral follicles. Secondly, we address the methods of harvesting preantral follicles,
culture media, and systems used. Finally, we describe the most common growth factors
and hormones utilized to culture bovine preantral follicles.
64
BASIC ASPECTS OF BOVINE EARLY FOLLICULOGENESIS
Development of bovine oocytes and follicles begins in the fetal phase (Rüsse,
1983) and takes around 6 months to be completed (Lussier et al., 1987). The follicle
development is comprised of two distinct and consecutive phases (Fig. 1): the first
phase, characterized by the formation and beginning of growth of primordial follicles,
and the second phase, in which the growth of primary and secondary follicles occurs as
granulosa cells transform from a flattened to a cuboidal shape and proliferate, while the
oocyte experiences a rapid increase in size. It has been reported that the critical point of
follicle growth is when the follicle has about 40 granulosa cells and the oocyte
undergoes the first significant change in diameter (Braw-Tal and Yossefi, 1997).
65
Figure 1. Schematic sequence of complete follicular development. Preantral phase: Formation and beginning of growth and activation of
primordial follicles and growth of primary and secondary follicles. Antral phase: Formation of tertiary follicle (antral-filled follicular fluid
cavity). Follicle growth continues through the phases of recruitment, emergency, selection, dominance, and preovulatory stage of follicular
waves. Oogonia is a cell that arises from a primordial germ cell and differentiates into an oocyte in the ovary. Primordial follicle has a single
layer of flattened granulosa cells. Primary follicle has a single layer of cuboidal granulosa cells. Secondary follicle has two or more layers of
cuboidal granulosa cells and a small number of theca cells. All the preantral follicles have a primary oocyte. Tertiary follicle has several
granulosa cell layers, theca cells and primary oocyte and is characterized by an antral cavity which containing follicular fluid. Preovulatory or
also called as Graafian follicle is the last stage of follicle development; these follicles are larger, have more antral fluid and a secondary oocyte.
Follicular fluid is a plasma exudate conditioned by secretory products from the granulosa cells and oocyte.
66
Formation and Initiation of Primordial Follicle Growth
Primordial germ cells proliferate by mitosis to form primary oocytes and the
first meiotic prophase starts between days 75 and 80 of pregnancy in the cattle
(Erickson, 1966). The formation of the primordial follicles occurs at the diplotene stage
of meiosis, at approximately day 130 of pregnancy (Erickson, 1966). At this point, the
oocyte is surrounded by a single layer of six pre-granulosa (flattened) cells which is in
turn surrounded by a basal membrane; these are the first generation of follicle cells
(Braw-Tal and Yossefi, 1997) and are originated from the celomic epithelium. From
day 170 on, the ovigerous cords are absent and there are only primordial follicles
present (Erickson, 1966). After the formation of primordial follicles, the pre-granulosa
cells stop multiplying and remain in the resting phase until they are stimulated to grow
(Erickson, 1966). In bovine species, primordial follicles have a mean diameter of 35.2
µm and oocyte growth is initiated only during the fourth generation of follicle cells,
compared with the second or third generation in rodents and humans, respectively
(Braw-Tal and Yossefi, 1997).
During the initiation of follicular growth, in a phase known as primordial
follicular activation, some primordial follicles leave the reserve pool (quiescent
follicles) to enter into the growing pool (primary, secondary, tertiary, and preovulatory;
Rüsse, 1983). The activation of primordial follicles is a nonreversible process; therefore
it is important in regulating the size of the resting primordial follicle pool, which will
affect the reproductive lifespan and fertility (Yang and Fortune, 2008). Follicular
activation is characterized by the morphological modifications of granulosa cell from
flattened to cuboidal, as well as the resumption of cell proliferation (van den Hurk et al.,
1997) and the initiation of oocyte growth. However, the factors and mechanisms
responsible for the control of early folliculogenesis are still poorly known and represent
one of the major questions related to ovarian biology.
Growth of Primary and Secondary Follicles
After activation, bovine primordial follicles gradually acquire cuboidal
granulosa cells and become transitional and primary follicles; the latter with one
complete layer of 11-40 cuboidal granulosa cells around the oocyte (Hulshof et al.,
1994; Braw-Tal and Yossefi, 1997). Secondary follicles are characterized by the
addition of a second complete layer of granulosa cells, the initial deposition of zona
pellucida (ZP) material, formation of cortical granules within the oocyte cytoplasm
67
(using transmission electron microscopy, Fair et al., 1997), mRNA synthesis in the
oocyte (McLaughlin et al., 2010), and gonadotropin responsiveness (Fair, 2003).
Primary and secondary follicles appear in the bovine fetal ovary around days
140 and 210 (Rüsse, 1983), and have a mean diameter of 46.1 µm (Hulshof et al., 1994)
and 81.0 µm (Braw-tal and Yossefi, 1997), respectively. Unlike primordial follicles, at
these follicular stages the ZP begins to form, surrounding the oocyte (Fair et al., 1997;
Rankin et al., 2001). Braw-tal and Yossefi (1997) verified that the ZP first appeared in
small secondary follicles (range, 81-130 µm in diameter), but formed a complete ring
around the oocyte during the late secondary stage (range, 131-250 µm in diameter).
The growth of preantral follicles after the primary stage also depends on
important events that include the expression of growth and differentiation factors such
as vascular endothelial growth factor (VEGF) and growth and differentiation factor-9
(GDF-9). VEGF, in particular, has been considered as a stimulator of bovine follicular
development because it provides support for the transition from the primary to the
secondary follicle stage (Yang and Fortune, 2007).
During the growth of secondary follicles and organization of the granulosa cells
in several layers, a cavity is formed among these cells which is filled with follicular
fluid and is called the antral cavity (Fair et al., 1997). From this stage on, the follicles
are called tertiary or early antral follicles and are observed during the bovine fetal phase
at days 140 (Rüsse, 1983) or 210 (Carambula et al., 1999) of gestation. The transition
from secondary to tertiary stage includes the development of the internal and external
theca cell layers and the beginning of cumulus cell formation (Fair et al., 1997) in
follicles around 120 µm of diameter (Lussier et al., 1987).
Although antral cavities are usually established when the follicles reach at least
200 µm in diameter (Lussier et al., 1987; McNatty et al., 2000), as we mentioned
previously, large secondary follicles (greater than 190 µm in diameter) have been
mechanically isolated from bovine ovaries (Araújo et al., 2012a,b; Rossetto et al., 2012,
2013), as well as from the ovaries of other species such as caprine (Araújo et al.,
2011a,b; Magalhães et al., 2011) and ovine (Arunakumari et al., 2011; Luz et al., 2012).
68
HARVESTING BOVINE PREANTRAL FOLLICLES
Mechanical Isolation Using Tissue Chopper or Microdissection
The first studies using mechanical isolation techniques were developed during
the early 1990s and represented major advances in the isolation of morphologically
normal preantral follicles. Early preantral follicles were mechanically isolated using a
machine called a tissue chopper (Figueiredo et al., 1993), a homogenizer (Nuttinck et
al., 1993), or a cell dissociation sieve (Jewgenow and Goritz, 1995; Jewgenow, 1998).
Furthermore, isolation of later stage preantral follicles via microdissection was reported
using insulin needles (van den Hurk et al., 1998).
Bovine preantral follicles have been successfully isolated utilizing tissue
chopper and microdissection. Both follicular isolation methods have shown no
detrimental effect on the tridimensional structure of the small follicles, because follicles
have been recovered without damage to the basal membrane (Figueiredo et al., 1993,
1994a; Fig. 2A). The preservation of the follicular basal membrane may prevent the
spreading of granulosa cells during in vitro culture (Figueiredo et al., 1994a), preserving
follicular morphology by maintaining follicular adhesion to extracellular compounds.
Additionally, the basement membrane contains proteoheparansulfate (Woodley et al,
1983), which binds to a variety of growth factors (Gospodarowicz et al., 1978).
Therefore, the presence of a basement membrane around the follicles might optimize
the effects of growth factors and hormones added to the culture medium (Figueiredo et
al., 1995).
69
Figure 2. Isolated follicles (A) by tissue chopper and microdissection, and (B) in situ
follicles stained with PAS-hematoxilin. o: oocyte; n: oocyte nucleus; fgc: flattened
granulosa cells; cgc: cuboidal granulosa cells; tc: theca cells; zp: zona pellucida.
*Antral follicle grown in vitro.
The number of follicles isolated by tissue chopper differs according to the
species (Lucci et al., 2002) and even among breeds. In goats and sheep, the best results
were obtained with the intervals of 75 and 87.5 µm, respectively (Lucci et al., 1999;
Amorim et al., 2000). The best interval for sectioning ovarian tissue varies from 50 µm
for European cattle (Bos Taurus, Figueiredo et al., 1993) and 125 µm for Zebu cattle
(Bos Indicus, Lucci et al., 2002). These differences regarding the most suitable cut
interval to isolate preantral follicles may be explained by differences in quantity of
ovarian tissue and variation in its composition, as in corpora lutea and corpora albicans
(Figueiredo et al., 1993). Follicles embedded in a more fibrous stroma can be more
difficult to isolate and smaller cut intervals would be necessary (Lucci et al., 2002).
70
The microdissection method has been used to isolate large bovine follicles using
fine needles under stereomicroscopy. This method maintains the theca cell layers, which
ensures follicle quality (Katska and Rynska, 1998; Saha et al., 2002). The presence of
theca cells is a crucial condition for normal follicular growth, preservation of estrogen
production (Gougeon, 1996), maintenance of follicular health, and remodeling of the
basement membrane (McCaffery et al., 2000). In this regard, maintaining
communication among the oocyte, the surrounding somatic cells, and the extracellular
matrix is vital to the achievement of normal folliculogenesis, and to sustain follicular
growth and viability (McCaffery et al., 2000). This technique allows the isolation of
several morphologically normal and intact follicles from ovarian tissue. Large bovine
preantral follicles have been successfully isolated and cultured in vitro until antrum
formation after short- (McCaffery et al., 2000; McLaughlin et al., 2010) and long-term
(Gutierrez et al., 2000, Itoh et al., 2002; Araújo et al., 2012a,b; Rossetto et al., 2012)
culture.
Enzymatic Isolation
The fibrous nature of the ovaries of most domestic species complicates follicular
isolation (Telfer, 1996). Therefore, some studies have been conducted using different
types of enzymes to recover preantral follicles in different species. In this regard,
collagenase (from Clostridium histolyticum) has been used to isolate numerous
preantral follicles from murine (Eppig and Downs, 1987), swine (Greenwald and Moor,
1989), and bovine (Figueiredo et al., 1993) ovaries. In addition, an enzymatic method
using deoxyribonuclease (DNAse), has been developed to isolate human follicles (Roy
and Treacy, 1993). However, this latter process requires a lengthy cooling time and
consequently reduces the viability of the follicles by causing damage to cell membranes.
The degree of enzymatic damage depends on the duration of treatment, the
concentration of the enzyme(s), and the type of tissue (Figueiredo et al., 1993; Roy and
Greenwald, 1985). Morphologically normal follicles have been isolated from bovine
ovaries using a combination of collagenase and DNAse treatment (Wandji et al.,
1996a). However, it was reported that although the oocytes from freshly isolated
preantral follicles appeared healthy under an inverted microscope, histological
examinations revealed that the enzymatic process could have damaged the oocytes,
especially in smaller preantral follicles.
71
Ovarian Biopsy In Vivo
A new method for the repeated collection of ovarian biopsies from living donors
through transvaginal, ultrasound-guided puncture of the ovary has been successfully
developed and tested in cows (Aerts et al., 2005), women (Schmidt et al., 2003), and
recently in mares (Haag et al., 2013a,b,c). This procedure can be seen as a modified
version of a commercial ovum pick-up (OPU) technique. Using ovarian biopsies, Aerts
et al. (2005) had a recovery a rate of 68% small preantral follicles per biopsy session.
Although rather small, these follicles were suitable for both histological (Fig. 2B) and
immunohistochemical evaluation and revealed the presence of morphologically normal
primordial and preantral follicles. In addition, in a later study, the restoration of ovarian
tissue morphology (using light microscopy) and the preservation of follicle viability
(using fluorescence microscopy) in the majority of preantral follicles after multiple
ovarian biopsy sessions was reported (Aerts et al., 2008).
IN VITRO CULTURE SYSTEMS FOR BOVINE PREANTRAL FOLLICLES
Basically, there are two ways to culture bovine preantral follicles: 1) enclosed in
ovarian tissue fragments (slices or strips), also called ―in situ‖; or 2) using isolated
follicles. The isolated follicles have been cultured either in a two-dimensional (2D)
system (Fig. 3A), i.e. the follicle is placed on the surface, which may be a plastic or
extracellular matrix (e.g., collagen gel, matrigel, etc), or in a three-dimensional (3D)
system (Fig. 3B), in which the follicles are cultured within an extracellular matrix.
Currently, the major use of isolated follicles for culture is to support the growth
and development of immature oocytes and allow the understanding of the mechanisms
involved in oocyte development, granulosa cell differentiation, and regulation of
autocrine/paracrine factors that control early stages of folliculogenesis (Thomas et al.,
2003).
72
Figure 3. Schematic representation of the (A) two- and (B) three-dimensional culture
systems utilized for bovine preantral follicles.
In Vitro Culture of Preantral Follicles Enclosed in Ovarian Tissue (In Situ)
In the in situ culture system, follicles are cultured with the surrounding ovarian
tissue, including the stromal cells. This culture system allows the interaction between
the follicles and their adjacent cells, such as stromal/theca cells and granulosa cells,
which may influence their growth (Peluso and Hirschel, 1988). This is a very practical
method and prevents prolonged exposure of the cells to the external environment.
The spontaneous activation of primordial follicles has been known to occur in
vitro using the in situ culture system in several species, including murine (Eppig and
O’Brien, 1996; Nilsson et al., 2001), bovine (Wandji et al., 1996b; Fortune et al., 1998;
Braw-Tal and Yossefi, 1997; Derrar et al., 2000; Gigli et al., 2006; Tang et al., 2012;
Table 1), equine (Haag et al., 2013c), caprine (Silva et al., 2004; Matos et al., 2007),
and primates (Wandji et al., 1997; Hovatta et al., 1999). The majority of the bovine
primordial follicles cultured in situ may activate within 2 days of culture (Wandji et al.,
1996b; Braw-Tal and Yossefi, 1997; Yang and Fortune, 2008) and reach the secondary
stage in 6 (McLaughlin and Telfer, 2010), 10 (Yang and Fortune, 2006; 2007; 2008), or
22 days (Tang et al., 2012).
Despite the fact that in vitro culture of ovarian tissue is able to develop
primordial follicles until primary and secondary stages (Yang and Fortune, 2006; 2007;
2008; Tang et al., 2012), this technique has not been very effective for follicle
maturation. A two-step culture system for bovine (McLaughlin and Telfer, 2010) and
73
human (Telfer et al., 2008) preantral follicles has been tested recently. The aim of this
system was to determine whether in situ-grown bovine and human follicles could be
isolated at the secondary stage and cultured to late preantral/early antral stages.
However, in both species, only a few antral follicles were obtained after 4 (Telfer et al.,
2008) or 15 days (McLaughlin and Telfer, 2010) of culture of secondary follicles.
74
Table 1. Chronological advances in in situ culture system of early bovine preantral follicles.*
Authors
Duration of
Type of
Maintenance of
Follicular activation (from
culture
medium
follicular survival primordial to transitional
(days)
utilized
and/or viability
or primary stage)
Peluso and Hirschel, 1988
2
TCM-199
Yes
Yes
Wandji et al., 1996a
0, 2, 4 or 7 Waymouth
Yes
Yes
Braw-Tal and Yossefi, 1997
4
α-MEM
Fortune et al., 1998
7 or 14
Waymouth
Yes
Yes
Derrar et al., 2000
8
Waymouth,
Yes
Yes
α-MEM
Gigli et al., 2006
7
Waymouth
Yes
Yang and Fortune, 2006
10
Waymouth
Yes
Yang and Fortune, 2007
10
Waymouth
Yes
Yang and Fortune, 2008
2 or 10
Waymouth
Yes
McLaughlin and Telfer, 2010
6
McCoy’s
Yes
Yes
Andrade et al., 2012
8
α-MEM
Yes
Yes
Tang et al., 2012
22
α-MEM
Yes
*All the results were compared with the fresh control group (Day 0).
Increase of
follicular and/or
oocyte diameter
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
75
In Vitro Culture of Isolated Preantral Follicles
Although primordial and primary follicles can easily be isolated from bovine
ovaries using mechanical or enzymatic methods, mostly small (diameter ≤150 µm) and
large (diameter >150 µm) secondary follicles have commonly been used for this in vitro
culture system (McLaughlin and Telfer, 2010; Araújo et al., 2012a,b; Rossetto et al.,
2012, 2013). In the bovine species, several studies have used in vitro culture of isolated
follicles (Table 2). The best results produced so far have been obtained from culture of
large secondary follicles (McLaughlin et al., 2010; Araújo et al., 2012a,b; Rossetto et
al., 2012, 2013). It has been reported that small secondary follicles (75 to 125 µm in
diameter) attached to the culture wells and created a monolayer, which resulted in
flattened and damaged follicular structures (Katska and Rynska, 1998). Conversely, the
culture of isolated large secondary follicles was able to maintain follicular viability, and
increase follicular diameter, and foster estradiol and progesterone production (Wandji et
al., 1996a).
76
Table 2. Chronological advances in two and three dimensional (2D and 3D) in vitro culture systems for isolated bovine preantral follicles.*
Authors
Duration
of culture
(days)
Follicular
diameter
(µm)†
Type of
medium
utilized
Culture system
description
Figueiredo et al., 1994a
5
30-70
α-MEM
Figueiredo et al., 1994b
Figueiredo et al., 1995
5
1
30-70
30-70
α-MEM
α-MEM
Wandji et al., 1996b
6
60-220
Waymouth
Hulshof et al., 1997
Schotanus et al., 1997
5
8
30-70
30-80
α-MEM
TCM-199
Katska and Rynska, 1998
Gutierrez et al., 2000
23
28
75-195
166 ± 2.2
TCM-199
McCoy’s
Itoh and Hoshi, 2000
30
30-70
TCM-199
6
10
12
13
7
6
8
12-15
16
100-200
40-100
146 ± 1.7
145-170
120
145 ± 0.6
157 ± 3
111 ± 1.5
>150
18
>150
McCoy’s
TCM-199
McCoy’s
TCM-199
TCM-199
McCoy’s
McCoy’s
McCoy’s
α-MEM,
TCM-199,
McCoy’s
α-MEM
2D-plastic substrate (3
follicles/drop)
3D-collagen (4 follicles/well)
2D-uncoated plastic or coated
with BSA, Laminin,
Fibronectin, Matrigel or
Collagen and 3D-Collagen
3D-agar gel (30-40
follicles/drop)
3D-collagen (5 follicles/drop)
3D-collagen (5-10
follicles/drop)
2D-under mineral oil
2D-plastic substrate (1
follicle/well)
2D-somatic cells (15-20
follicles/well)
2D-plastic substrate
2D (1-3 follicles/well)
2D-plastic substrate
3D-collagen
2D
2D
2D
2D
2D-plastic substrate
McCaffery et al., 2000
Saha et al., 2000
Thomas et al., 2001
Itoh et al., 2002
Saha et al., 2002
Thomas et al., 2007
McLaughlin et al., 2010
McLaughlin and Telfer, 2010
Rossetto et al., 2012
Rossetto et al., 2013
2D-plastic substrate
Maintenance of
follicular
survival and/or
viability
Yes
Increase of
follicular
and/or oocyte
diameter
Yes
Antrum
formation
Steroid
secretion
-
-
Yes
-
Yes
-
-
-
Yes
Yes
-
E2 and P4
Yes
Yes
Yes
Yes
-
-
Yes
-
Yes
Yes
Yes
-
Yes
Yes
-
-
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
E2
E2
E2
E2
-
Yes
Yes
Yes
-
*All the results were compared with the fresh control group (Day 0). Estradiol (E2) and Progesterone (P4) hormones. †At Day 0 of culture.
77
In general, isolated bovine preantral follicles have been cultured using well
plates without mineral oil in a 2D culture system. In vitro culture without mineral oil
has maintained follicular viability and increased antrum formation (Gutierrez et al.,
2000; McLaughlin et al., 2010; Rossetto et al., 2012, 2013) and estradiol production
(McLaughlin et al., 2010) in isolated bovine preantral follicles. Similarly, studies using
mineral oil in a 2D culture system also have shown high follicular viability and antrum
formation (Katsa and Rynsk, 1998; Araújo et al., 2012a,b), as well as an increase in
estradiol production after in vitro culture (Araújo et al., 2012b).
A 3D culture system has been developed to culture isolated preantral follicles in
mice (Oktem and Oktay, 2007; Desai et al., 2012), buffalo (Sharma et al., 2009), cows
(Figueiredo et al., 1994b; Wandji et al., 1996a; Schotanus et al., 1997; Araújo et al.;
unpublished observations), and human (Hovatta et al., 1997) and nonhuman (Xu et al.,
2011) primates. In this method, the follicles are cultured inside an extracellular matrix,
which mimics the ovary and maintains the spherical morphology of the ovarian follicle
and preserves the cell-cell and cell-matrix connections important for regulating follicle
development in vivo (Pangas et al., 2003; Kreeger et al., 2005; West et al., 2007). The
3D culture system maintained high follicular viability and increased follicular diameter
(Figueiredo et al., 1994b; Wandji et al., 1996a) and steroid production after in vitro
culture (Wandji et al., 1996a). It is believed that 3D systems more effectively simulate
physiological conditions because many cellular processes in organogenesis occur
exclusively in 3 dimensions (Xu et al., 2006).
IMPROVING IN VITRO GROWTH OF BOVINE PREANTRAL FOLLICLES
Oocyte-secreted paracrine factors promote the proliferation, differentiation, and
function of granulosa cells. Moreover, the development of the oocyte in vitro to a stage
where normal embryonic development can be supported is dependent on the oocyte
reaching the appropriate stage of development to respond to the endocrine and paracrine
signals responsible for the induction of maturation (Telfer et al., 2000). Therefore, an
elucidation of the bidirectional interplay between these two cell types is also important
for the development of a successful culture system (Aerts et al., 2008).
Culture Media
Different commercial media are used to culture bovine preantral follicles in vitro
(Tables 1 and 2). However, based on literature reports there is no standard, reliable
78
culture medium for bovine preantral follicles. The most commonly used culture media
are: minimum essential medium alpha modification (α-MEM, Figueiredo et al., 1994a;
Araújo et al., 2012a,b; Rossetto et al., 2012; 2013), tissue culture medium-199 (TCM199, Katska and Rynska, 1998; Itoh and Hoshi, 2000; Saha et al., 2000; 2002; Rossetto
et al., 2012), and McCoy (Gutierrez et al., 2000; McCaffery et al., 2000; Thomas et al.,
2001; 2007; McLaughlin et al., 2010; McLaughlin and Telfer, 2010; Rossetto et al.,
2012). The lack of standardized protocols may affect in vitro follicle culture and can
also explain the different results from several research groups. Among the commercial
culture media, TCM-199 and α-MEM have been the most commonly used to maintain
follicular survival and viability and to improve the development of bovine follicles. A
recent study compared TCM-199, McCoy, and α-MEM under the same experimental
conditions and demonstrated that TCM-199 was the best medium to culture bovine
secondary follicles, based on the high percentage of viable follicles after in vitro culture
(Rossetto et al., 2012). However, this study also revealed that follicles cultured in αMEM or TCM-199 preserved at the ultrastructural level the cytoplasmic membrane and
oocyte nucleus, and normal and uniformly distributed mitochondria and endoplasmic
reticulum. Comparing α-MEM with TCM-199 we found recently that α-MEM can be
used to replace the TCM-199 for bovine preantral follicle culture if progressive addition
of medium is used for medium change (Araújo et al.; unpublished observations). These
results provide new perspectives in order to identify the best culture system for each
species, taking into consideration the base culture medium, supplements (hormones and
growth factors), and medium replacement methods.
Basic Supplements
Substances such as pyruvate, glutamine, hypoxantine (Figueiredo et al., 1994a),
and ascorbic acid (Thomas et al., 2001) have been used with success for the culture of
bovine ovarian preantral follicles. Therefore, these substances have become part of the
base medium used to culture ovarian follicles of several species. Pyruvate and
glutamine are energy substrates and the addition of both substances to the culture
medium increased the percentage of intact follicles (Saha et al., 2000). Pyruvate was
shown as a predominant substrate used by immature bovine oocytes (Khurana and
Niemann, 2000) and isolated growing mouse oocytes (Eppig, 1976). Glutamine is an
efficient energy substrate required for biomass synthesis (Genzel et al., 2005) by bovine
79
preantral follicles (Figueiredo et al., 1994a). Hypoxantine is a substance that has
increased the number of morphologically normal oocytes (Figueiredo et al., 1994a),
maintained oocyte-granulosa cell communication during the culture of mouse preantral
follicles (Eppig and Downs, 1987) via gap junctions, and promoted oocyte growth in
vitro (Buccione et al., 1990). It has been suggested that hypoxanthine improves the
utilization of additional energy substrates by maintaining interactions between the
oocyte and the surrounding granulosa cells (Figueiredo et al., 1994a).
Another important substance that has been used in culture media is ascorbic
acid. Ascorbic acid is a vitamin that acts as an antioxidant and it is involved in
processes of hormone secretion, gonadal tissue remodeling and apoptosis (Luck et al.,
1995). It has also been associated with several processes during follicular and luteal
development (Thomas et al., 2001) because it accumulates in granulosa cells, theca
interna cells, luteal cells, and oocytes (Kramer et al., 1933; Hoch-Ligeti and Bourne,
1948; Deane, 1952). Moreover, it was observed in vitro that ascorbic acid maintained
follicle integrity in the absence of serum, reduced the incidence of cell death, and may
participate in the regulation of extracellular matrix remodeling by increasing matrix
metalloproteinases-9 (MMP-9) activity (Thomas et al., 2001). Additionally, the addition
of ascorbic acid to the culture medium stimulates the activation of in vitro cultured
primordial follicles in cattle and subsequent growth of activated follicles (Andrade et
al., 2012). Therefore, the use of ascorbic acid is crucial for culture of isolated follicles
because it ensures the integrity of the basement membrane of the follicles.
Growth Factors
Ovarian follicular growth is controlled by complex interactions between the
oocyte and the surrounding granulosa and theca cells, as well as by locally produced
growth factors and hormones. In addition, the balance of stimulatory and suppressive
factors dramatically affects the growth of granulosa cells of small preantral follicles in
vitro (Wandji et al., 1996a). Among the known major growth factors present in bovine
ovarian cells are insulin-like growth factors (IGFs, Thomas et al., 2007), fibroblast
growth factors (FGFs, Buratini et al., 2005; 2007), vascular endothelial growth factors
(VEGFs, Yang and Fortune, 2007), bone morphogenetic proteins (BMPs), and growth
and differentiation factors (GDFs, Bodensteiner et al., 1999; Tang et al., 2012). The
following sections will describe only the growth factors which have been used in in
vitro culture systems for bovine preantral follicles.
80
Insulin-like Growth Factor-1 (IGF-1)
The IGF-1 binding ontogeny (Wandji et al., 1992) and its type 1 receptor mRNA
have been demonstrated in the oocytes and granulosa and theca cells of bovine preantral
follicles (Armstrong et al., 2000). In addition, IGF binding protein-2 (IGFBP-2) mRNA
has been detected in granulosa cells and oocytes, and IGFBP-3 mRNA in oocytes from
bovine preantral follicles (Armstrong et al., 2002). During the stages of antral and
preovulatory follicles, IGF-1 mRNA has been detected in bovine granulosa (Schams et
al., 1999) and theca cells (Armstrong et al., 2000), indicating that IGF-1 is important
during the later stages of folliculogenesis (e.g., in relation to luteinizing hormone (LH)
responsiveness, Ginther et al., 2004).
IGF-1 has been identified as a stimulatory growth factor for bovine follicular
and oocyte growth, as well as antrum formation during prolonged culture (Itoh et al.,
2002; Gutierrez et al., 2000). Long-term in vitro culture may allow the differentiation of
granulosa cells by IGF-1, which acts as a stimulator of follicular development.
Follicular growth (Thomas et al., 2007), antral cavity formation (McCaffery et al., 2000;
Thomas et al., 2007; Rossetto et al., 2013), and estradiol production (Thomas et al.,
2007) were observed after using IGF-1 in the in vitro culture of bovine preantral
follicles. Conversely, McCaffery et al. (2000) observed that treatment of immature
follicles with IGF-1 resulted in precocious differentiation, which might have retarded
follicular growth and cell proliferation. Recently, Rossetto et al. (2013) showed that
addition of IGF-1 to the culture medium of bovine preantral follicles had no effect on
the follicular morphology and antrum formation. Similarly, we observed that IGF-1 did
not interfere in any end point, including the estradiol concentrations, evaluated during
the in vitro culture of bovine secondary follicles (Araújo et al.; unpublished
observations). Therefore, the action of IGF-1 in bovine oocyte and follicular
development is strictly regulated by the developmental stage, period of culture, and
concentration of IGF-1 used (Walters et al., 2006).
Basic Fibroblast Growth Factor (bFGF)
Immunoreactivity, bioactivity, and mRNA of bFGF are present in bovine
granulosa cells (Neufeld et al., 1987). Both bFGF alone and bFGF in combination with
follicle-stimulating hormone (FSH) allowed the maintenance of follicular survival,
promoted in vitro growth of granulosa cells, and increased the diameter of bovine
81
preantral follicles. However, when bFGF was combined with transforming growth
factor β (TGF-β), there was an inhibition of the stimulatory effect of bFGF on follicular
diameter and a decrease in follicular survival (Neufeld et al., 1987). Although bFGF
alone stimulated estradiol and progesterone production during in vitro culture of bovine
preantral follicles, it suppressed FSH-stimulated progesterone production (Wandji et al.,
1996a). These results suggest that bFGF antagonizes, at least in some aspects, the FSHmediated cytodifferentiation of cultured bovine preantral follicles.
Vascular Endothelial Growth Factor (VEGF)
VEGF has been known as a regulator of the various phases of follicle
development (Araújo et al., 2013). Yang and Fortune (2007) demonstrated that the
mRNA for both VEGF receptors (flt-1 or VEGFR-1 and flk-1 or VEGFR-2), as well as
for the VEGF ligand, were expressed in the fetal bovine ovary at day 90 of gestation.
However, mRNA expression for the VEGF ligand increased when the first secondary
follicles were observed at day 210 of gestation (Yang and Fortune, 2007). As the
follicle grows and the antral cavity becomes filled with follicular fluid, VEGF
production increases and the follicular fluid becomes rich in VEGF (Barboni et al.,
2000; Ferrari et al., 2006). These aspects have been confirmed by increasing of the
VEGF ligand (mRNA and protein) with the proliferation of microvessels, progression
of gestation, and ovarian development (Yang and Fortune, 2007).
The role of VEGF in promoting the primary to secondary follicle transition has
been demonstrated in vitro during culture of fetal bovine ovarian tissue (Yang and
Fortune, 2007). In caprine, VEGF has been shown to be crucial to the in vitro growth of
preantral follicles and their oocytes enclosed in ovarian tissue (Bruno et al., 2009), and
to meiosis progression during the maturation of oocytes grown from secondary follicles
cultured in vitro (Araújo et al., 2011b). Recently, we have demonstrated that VEGF
increases antrum formation and follicular growth rate after in vitro culture of bovine
preantral follicles (Araújo et al.; unpublished observations). Taken together these results
lead us to believe that VEGF may be an excellent constituent for the in vitro culture
media for bovine secondary follicles.
Hormones
There seems to exist an overall consensus that preantral follicles can develop in
the absence of gonadotropins. However, the use of gonadotropins for in vitro culture has
82
been important for obtaining optimal development of preantral follicles. Treatment of
large, isolated preantral follicles with FSH stimulated granulosa cell proliferation and
antrum formation (Wandji et al., 1996a; McLaughlin and Telfer, 2010; Araújo et al.,
2012a,b; Rossetto et al., 2012). Moreover, hormones such as FSH (McLaughlin and
Telfer, 2010; Araújo et al., 2012b; Rossetto et al., 2012), and activin stimulated
steroidogenesis in bovine isolated preantral follicles (McLaughlin et al., 2010;
McLaughlin and Telfer, 2010; Rossetto et al., 2012). The following sections will
describe only the hormones which have been used in in vitro culture systems for bovine
preantral follicles and ovarian cells.
Activin
Activin, a proteic hormone that enhances FSH biosynthesis and secretion, is
expressed by its receptor on theca and granulosa cells, and oocytes of bovine preantral
follicles (Hulshof et al., 1997). It is composed of two beta subunits, A and B, and exists
as a homo- (A and B) or heterodimer (AB) with activin-A as the predominant isoform.
Activins are involved in primordial follicle activation in vitro (Fortune et al., 2000),
preantral follicle development (Hulshof et al., 1997; Knight and Glister, 2001; Ethier
and Findlay, 2001; Findlay et al., 2002), granulosa cell proliferation, antral cavity
formation, maintenance of normal oocyte morphology, and interactions between the
oocyte and granulosa cells (McLaughlin et al., 2010). Polarized expression of cell
contact interactions promoted by activin supports ongoing folliculogenesis, which is
characterized by increased peripheral granulosa cell adhesion to the basement
membrane and retention of adhesion at the surface of the ZP (McLaughlin et al., 2010).
Although activin has caused a significant increase in the size of follicles and
estradiol concentrations in immature mice, in adult mice it did not change the follicle
diameter and completely blocked the action of FSH on both follicle diameter and
estradiol concentration (Liu et al., 1999). In ovine, the activin promoted preantral
follicle and oocyte growth in vitro, but did not accelerate follicle differentiation nor had
any effect on antrum formation or follicle survival. Moreover, activin and FSH
interacted positively to stimulate the follicle growth and granulosa proliferation of
bovine preantral follicles (Hulshof et al., 1997). All these results indicate that activin
has a paracrine role through proliferative and cytodifferentiative action on granulosa
cells and that its action is age and FSH dependent.
83
Follicle-Stimulating Hormone (FSH)
Gonadotropins seem to be important for the optimal development of preantral
follicles in vitro. FSH in particular is considered a critical hormone for the survival of
large secondary follicles (Xu et al., 2010) and has been observed in granulosa cells of
bovine preantral follicles (primary and secondary, Wandji et al., 1992). In vitro culture
of bovine isolated preantral follicles with FSH stimulated an increase in follicular
diameter (Wandji et al., 1996a; Hulshof et al., 1997), granulosa cell proliferation by
bromodeoxyuridine (BrdU-label, Hulshof et al., 1997), and progesterone (Wandji et al.,
1996a) and estradiol secretion (McLaughlin et al., 2010). Moreover, FSH maintained
normal oocyte morphology and interactions between the oocyte and granulosa cells
after in vitro culture of bovine secondary follicles (McLaughlin et al., 2010). Wandji et
al. (1996a) observed that large preantral follicles (150 to 220 µm) produced more
progesterone in response to FSH than smaller (60 to 179 µm) preantral follicles. These
findings indicated that the responsiveness to FSH increases as the bovine follicles
develop.
Growth Hormone (GH)
Among metabolism-related endocrine factors, GH has been shown to be a
crucial factor for follicular development in the mammalian ovary (Shimizu et al., 2008).
Both fetal and adult bovine ovaries revealed distinct amounts of GH receptor (GHR)
and its transcript in the oocytes of primordial and primary follicles, as well as mRNA
for GHR in secondary and tertiary follicles. These results support the concept that GHR
is involved in the development and differentiation of primordial follicles in both
prenatal and postnatal life (Kolle et al., 1998).
In vivo, GH may stimulate specific follicle populations selectively. GH inhibited
the development of preovulatory follicles and stimulated the growth of the secondlargest follicle in heifers (Lucy et al., 1994). GHR immunoreactivity and mRNA
encoding GHR in granulosa cells, theca cells and luteal cells of the bovine ovary (Kolle
et al., 1998) suggests the GH action by means of the detection of GH binding activity.
Moreover, GHR expression increases in the granulosa cells when the follicles become
estrogen-active, even when compared to preovulatory follicles (Kolle et al., 1998).
Thus, the increase of GHR expression in these follicles may be regulated by estradiol
(Kolle et al., 1998). The negative interaction between GH and estradiol during later
84
folliculogenesis seems to be true; however this effect seems to be positive during early
folliculogenesis, since the addition of GH to the culture medium of bovine preantral
follicle increased the estradiol concentrations (Araújo et al.; unpublished observations).
In addition, bovine granulosa cells' expression of mRNA for GHR was stimulated in
vitro by FSH (Shimizu et al., 2008). Also, GH has been shown to enhance cell
proliferation and steroidogenesis of cultured granulosa cells in cattle (Langhout et al.,
1991), suggesting an important role for GH in the regulation of granulosa cell
proliferation and follicular growth.
Insulin
Among the endocrine factors, insulin is a crucial hormone for follicular
development (Shimizu et al., 2008), granulosa cell function, and ovulation (Bossaert et
al., 2010). Additionally, insulin may regulate various intracellular processes in the
follicle such as amino acid transport, lipid metabolism, gene transcription, and protein
synthesis (Louhio et al., 2000). Insulin acts through its own receptor, which first appears
in the granulosa cells of small bovine antral follicles (Bossaert et al., 2010). Insulin
receptor is widely distributed throughout all ovarian compartments, including granulosa
and theca cells and stromal tissue (Shimizu et al., 2008; Bossaert et al., 2010). In
addition, the concentrations of insulin in follicular fluid are constant at all follicular
developmental stages (Shimizu et al., 2008).
Infusion of insulin in beef heifers increased the diameter of the dominant follicle
(Simpson et al., 1994). High levels of insulin receptor mRNA expression in granulosa
cells of preovulatory follicles seem to be necessary for development of the ovulatory
stage (Shimizu et al., 2008). The insulin receptor presence in small antral follicles,
together with the absence of the insulin receptor in preantral follicles, indicates the
involvement of insulin, and the acquisition of its receptor, during early follicular growth
in bovine (Bossaert et al., 2010). This hypothesis has been supported by the fact that
increased dietary intake of insulin was associated with recruitment of small follicles (<4
mm), but did not affect follicle selection (medium: 4-8 mm) or dominance (large: >8
mm) follicles (Gutierrez et al., 1997).
In vitro, insulin has been shown to be essential for follicle culture. Absence of
insulin in the culture medium resulted in follicle degeneration (Saha et al., 2000).
Gutierrez et al. (2000) demonstrated that bovine preantral follicles grew for a long
period in culture, even in the absence of tropic hormones, but in the presence of insulin.
85
Considering that type 1 IGF-1 receptor is present in oocytes and granulosa and theca
cells of bovine preantral follicles (Armstrong et al., 2000), and that insulin competes
with the IGF receptors, the follicular growth effect was probably promoted by
interaction of insulin and the type 1 IGF-1 receptor.
FINAL CONSIDERATIONS
Several studies of bovine folliculogenesis have examined the aspects of in vitro
follicular development. However, it is still not clear which culture medium needs to be
used to culture bovine preantral follicles, as well as which growth factors and hormones
could influence follicular development. Moreover, it will be important to have an
optimum and standard culture system for bovine follicles, either using two- or threedimensional approaches. A culture system to be selected needs to affect positively
follicular morphology, survival, proliferation, steroidogenesis, and gene expression.
Furthermore, it is important to reevaluate the effect of growth factors and/or hormones
on follicular growth. The follicle microenvironment must be considered, as well as the
role of growth factors and hormones and their respective signaling pathways during in
vitro follicular development. Additionally, factors such as age (immature or adult) of
the animals, follicular category (early or late preantral follicles, or antral follicles) to be
used, and system of in vitro follicular culture (two- or three-dimensional) should be
considered very important sources of data variation.
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7 CAPÍTULO 2
Importância do fator de crescimento do endotélio vascular (VEGF) na fisiologia
ovariana de mamíferos
“Importance of vascular endothelial growth factor (VEGF) in ovarian physiology of
mammals”
Periódico: Zygote, v. 21, p. 295-304, 2011
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RESUMO
A foliculogênese ovariana em mamíferos é um processo complexo. Várias substâncias
vêm sendo testadas no cultivo in vitro de células foliculares para um melhor
entendimento acerca dos mecanismos e fatores relacionados à foliculogênese ovariana
em mamíferos. Dentre essas substâncias pode-se destacar o fator de crescimento do
endotélio vascular (VEGF), o qual está fortemente relacionado com a angiogênese, e
nos últimos anos, sua presença em células ovarianas tem sido extensivamente
investigada. Estudos prévios têm demonstrado que a presença da proteína do VEGF,
bem como, a expressão do RNAm do seu receptor 2 (VEGFR-2) aumenta durante o
desenvolvimento folicular. Desta forma, é provável que a interação entre o VEGF e o
VEGFR-2 seja crucial para promover o desenvolvimento folicular. Contudo, poucos
estudos sobre a influência deste fator no desenvolvimento folicular têm sido reportados.
Portanto, a presente revisão abordará aspectos relacionados à caracterização estrutural e
mecanismo de ação do VEGF e seus receptores, bem como sua importância biológica
no ovário de mamíferos.
Palavras-chave: Angiogênese. Foliculogênese. Cultivo in vitro. Maturação. Tirosina
quinase.
100
Importance of vascular endothelial growth factor (VEGF) in ovarian physiology of
mammals
Running: VEGF in mammalian ovary physiology
Valdevane Rocha Araújo*, Ana Beatriz Graça Duarte, Jamily Bezerra Bruno, Cláudio
Afonso Pinho Lopes, José Ricardo de Figueiredo
Laboratory of Manipulation of Oocytes and Preantral Follicles,
State University of Ceará, 60740-930, Fortaleza, Ceará, Brazil
*
Corresponding author, val_exclusiva@yahoo.com.br
SUMMARY
Ovarian folliculogenesis in mammals is a complex process. Several compounds have
been tested during in vitro culture of follicular cells for a better understanding of the
mechanisms and factors related to ovarian folliculogenesis in mammals. From these
compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is
strongly associated with angiogenesis and, in recent years, its presence in ovarian cells
has been investigated extensively. Previous studies have shown that the presence of
VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases
during follicular development. Therefore, it is likely that the interaction between VEGF
and VEGFR-2 is crucial to promote follicular development. However, few studies on
the influence of this factor on follicular development have been reported. This review
addresses aspects related to the structural characterization and mechanism of action of
VEGF and its receptors, and their biological importance in the ovary of mammals.
Keywords: Angiogenesis, Folliculogenesis, In vitro culture, Maturation, Tyrosine
kinase
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INTRODUCTION
Ovarian folliculogenesis in mammals is a complex process that is comprised of
interactions between several autocrine, paracrine and endocrine factors. With respect to
paracrine factors, the role of vascular endothelial growth factor (VEGF) is noteworthy.
VEGF was initially identified and named vascular permeability factor (VPF).
Subsequently, its angiogenic activity was described, and the renamed VEGF is now
considered possibly the most potent angiogenic agent ever described. VEGF also
stimulates the survival of endothelial cells in vessels through the inhibition of apoptosis,
as well as promoting their proliferation, migration and differentiation, and causing
changes in gene expression patterns and inhibition of senescence (Dvorak, 2000).
The VEGF family is comprised of several members: VEGF-A, placental growth
factor (PIGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. VEGF-A is the most studied
subtype and has been detected in preantral follicles from several mammalian species
such as humans (Otani et al., 1999; Harata et al., 2006), rats (Celik-Ozenci et al., 2003),
pigs (Barboni et al., 2000), goats (Sharma and Sudan, 2010) and cows (Greenaway et
al., 2005). Moreover, regulatory effects of VEGF on mammalian folliculogenesis and
luteogenesis have been observed (Quintana et al., 2004; Roberts et al., 2007; Yang et
al., 2008).
For preantral folliculogenesis, the importance of VEGF for the survival and
growth of early (Bruno et al., 2009) and advanced (Fisher et al., 2009) follicles has been
reported. Based on the observation of a positive correlation between follicle diameter
and VEGF production, Fisher et al. (2009) demonstrated that this compound might play
an important role during in vitro follicle development. The involvement of VEGF in the
regulation of the various phases of follicle development has been shown, however more
studies are necessary for a better understanding of the mechanisms by which this factor
(ligand and receptors) acts in mammalian ovarian folliculogenesis.
FEATURES OF THE OVARIAN VASCULAR SYSTEM AND FOLLICULAR
ANGIOGENESIS
The ovary in mammalian species is comprised of two distinct portions: (1) the
cortex, which is the outermost part with a stroma of conjunctive tissue, and follicles and
corpora lutea at several developmental stages; and (2) the medulla, the inner region,
102
which contains loose conjunctive tissue highly vascularized and originating from
ovarian arteries. Histologically, the limits between these two regions are not well
defined.
The folliculogenesis process takes place within the cortex, from the formation of
the primordial follicle to the development to the preovulatory stage, which comprises
the preantral (primordial, primary and secondary follicles) and antral (tertiary and
preovulatory follicles) phases. Despite the fact that preantral follicles do not possess
their own vascular supply, the formation of the capillary network that surrounds the
follicle is critical for growth beyond this phase. Angiogenesis begins within the stroma
during early follicular development (Suzuki et al., 1998). Up to this point, nutrition and
oxygenation of primordial and primary follicles rely on passive diffusion from stromal
blood vessels, which are thin and single layered at this time. At the secondary stage or
later, stromal cells that surround the follicles become organized in thecal layers, in
which the innermost part (theca internal) contains many blood vessels, whilst the outer
layer (theca external) is composed mainly of fibrous conjunctive tissue. Thereafter,
during the appearance of the antral cavity full of follicular fluid, follicles become
surrounded by a capillary network, which promotes the nutrition of both these cells and
granulosa cells. This vascular system is divided into two distinct parts that enters either
the external and internal thecal cells layers (Stouffer et al., 2001), and both contribute to
the production of follicular fluid (van den Hurk & Zhao, 2005), which is rich in VEGF
(Ferrari et al., 2006). The number and diameter of blood vessels increase as the follicle
develops, but these never penetrate the basement membrane that separates theca interna
and granulosa cells layers.
STRUCTURAL CHARACTERIZATION OF VEGF AND ITS RECEPTORS
VEGF is a cytokine and is a homodimeric glycoprotein that is expressed in
several tissues as various types, with a molecular weight of about 45 kD (Ferrara &
Henzel, 1989). Its structure forms an antiparallel homodimer that is linked covalently by
two disulphide bridges between cystine residues. The cystine knot motif consists of an
eight-residue ring formed by the disulphide bridges and is conserved in the same
position by a third disulphide bond (Muller et al., 1997). VEGF-A is formed by two
monomers that contain a cystine knot motif determined by three intrachain disulphide
bridges, whilst the homodimer is assembled by two interchain disulphide bridges
103
linking the monomers. Overall, the VEGF monomer resembles that of other cystine
knot growth factors such as platelet-derived growth factor (PDGF), but its N-terminal
segment is helical rather than extended. The dimerization mode of VEGF is similar to
that of PDGF and is very different from that of transforming growth factor (TGF)-β.
Mutational analysis of VEGF reveals that symmetrical binding sites for the receptor
kinase domain receptor (KDR) are located at each pole of the VEGF homodimer
(Muller et al., 1997).
In humans, the gene that encodes VEGF is comprised of eight exons that are
separated by seven introns, and the coding region is approximately 14 kb (Tischer et al.,
1991; Houck et al., 1991). VEGF mRNA undergoes alternative splicing events that lead
to the production of mature homodimeric proteins. Each monomer is designated in
accordance with the number of amino acids along their chains (VEGF110, VEGF111,
VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189 and
VEGF206; Fig. 1). The isoforms VEGF110 (Keyt et al., 1996) and VEGF121 (Park et al.,
1993) do not bind to heparin as the carboxy-terminal domain located between amino
acids 111 and 165 is not present, which makes both molecules freely diffusible within
cells. In contrast, VEGF165 and VEGF189 bind to heparin with greater affinity. The use
of heparinase either in vivo (Sasisekharan et al., 1994) or in vitro (Rathjen et al., 1990)
indicates the potential of heparin molecules to be an important element of the binding
complex VEGF receptor. In both cases, cell proliferation and neovascularization were
inhibited. The absence of binding may not be due to a loss of VEGF receptors (GitayGoren et al., 1992), as this activity could be recovered by the use of exogenous heparin
(Rathjen et al., 1990). Therefore, it was observed that successful signal transduction
depends on the formation of a complex of VEGF, its receptors and heparin (VEGFheparin-receptor) (Gitay-Goren et al., 1992). These data suggest that the stability of
VEGF-heparin-receptor complexes probably contributes to effective signal transduction
and stimulation of endothelial cell proliferation (Keyt et al., 1996).
104
Figure 1. VEGF isoforms generated by alternative splicing. VEGF-A comprises
monomers designated according to the number of amino acids in the polypeptide chain
(VEGF110, VEGF111, VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b,
VEGF183, VEGF189 and VEGF206).
All transcripts contain exons one to five and exon eight, with diversity generated
through the alternative splicing of exons six and seven, except for VEGF-A165b, which
contains an alternative exon eight (Holmes & Zachary, 2005). This variant is an
endogenous inhibitory VEGF molecule that does not contain exon six, but possesses an
105
alternative exon (eight) that encodes a new carboxy terminus that increases the chances
of the occurrence of a family of isoforms with this novel carboxy-terminal end (Bates et
al., 2002).
In relation to the necessity of the above complex for the activity of VEGF, it is
known that this molecule binds directly to three receptor types: VEGFR-1/Flt-1 (Fmslike tyrosine kinase-1; De Vries et al., 1992), VEGFR-2/KDR (kinase insert domain
containing region; Terman et al., 1992) and VEGFR-3/Flt-4 (Fms-like tyrosine kinase4; Kaipainen et al., 1995; Karkkainen et al., 2002) (Fig. 2). These receptors are
members of the tyrosine kinase family, and have as common features the presence of
seven
immunoglobulin-like
domains
in
the
extracellular
portion,
a
single
transmembrane region and a tyrosine kinase sequence interrupted by the kinase
insertion domain in its intracellular portion (Shibuya et al., 1990). Nevertheless, VEGF
binds with high affinity to only two of these three receptors (VEGFR-1/Flt-1 and
VEGFR-2/KDR), whilst VEGFR-3/Flt-4 is involved in interactions with other VEGF
forms (VEGF-C and VEGF-D; Neufeld et al., 1999). The cleavage of VEGF165 by
plasmin, which is an important process in the angiogenesis cascade (Mignatti et al.,
1989), releases an N-terminal fragment that is comprised of amino acids 111-165. This
polypeptide binds to two of the VEGF receptors, VEGFR-1/Flt-1 and VEGFR-2/KDR,
in the absence of heparin (Keyt et al., 1996). Once VEGF has bound, VEGFR-2
dimerizes and autophosphorylates, which in turn activates several signal transduction
cascades (Byrne et al., 2005).
106
Figure 2. Binding complex VEGF-heparin-receptor involved in biological responses to
VEGF in various cells and tissues. VEGF-A binds both to VEGFR-1 and VEGFR-2,
whilst PIGF and VEGF-B interact only with VEGFR-1. VEGF-C and VEGF-D bind to
receptors VEGFR-2 and VEGFR-3, and VEGF-E binds only to VEGFR-2.
EXPRESSION, IMMUNOLOCALIZATION AND MECHANISM OF ACTION
OF VEGF AND ITS RECEPTORS
VEGF and its receptors VEGFR-1/Flt-1 and VEGFR-2/KDR are expressed in
the ovary of mammals, and have been identified in several reproductive tissues such as
the ovarian follicle, corpus luteum, endometrial vessels and at embryo implantation sites
(Jakeman et al., 1993; Shweiki et al., 1993; Gordon et al., 1996; Neufeld et al., 1999;
Krussel et al., 2001; Al-zi’abi et al., 2003). Previous studies have demonstrated the
presence of VEGF and its respective mRNA expression in the endometrium of fertile
women with normal uteri (Shifren et al., 1996). Both the protein and mRNA
corresponding to VEGF and its receptors were also detected in the granulosa and in
thecal cells of bovine secondary ovarian follicles (Yang & Fortune, 2007). In sows,
expression of mRNA for VEGF was observed in granulosa cells of antral follicles
107
(Shimizu et al., 2002, 2003) during the early and mid-luteal phases (Kaczmarek et al.,
2007), whilst mRNA for receptors VEGFR-1 and VEGFR-2 were expressed especially
within the layers of thecal cells (Shimizu et al., 2002, 2003). VEGF expression was also
reported in granulosa and thecal cells of secondary follicles in rats, and could be
enhanced in response to the gonadotropins follicle stimulating hormone (FSH),
luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) (Koos, 1995;
Yang et al., 2008). Conversely, at the end of the growth phase in porcine
folliculogenesis, a progressive decrease in the production of VEGF is observed in
response to the LH surge or to the administration of hCG (Barboni et al., 2000).
Immunolocalization of VEGF in caprine ovarian tissue reveals the presence of
VEGF in follicles at all developmental stages, with a progressive increase from the
primary to the preovulatory stage, as well as in surrounding stroma cells (Sharma &
Sudan, 2010). With regard to the immunoreactivity of goat ovaries in relation to
receptor 2 (VEGFR-2/KDR), Bruno et al. (2009) observed the expression of this
receptor in all follicle categories, but antral follicles displayed weak positive reactions.
Furthermore, their study demonstrated the presence of this receptor in oocytes of
primordial follicles, which indicated the involvement of VEGF in the growth and
development of these cells (Bruno et al., 2009). Immunolocalization reactions for
VEGF are stronger in cells of the theca interna in comparison with granulosa cells and
stromal vascular tissue (Sharma & Sudan, 2010). As VEGF levels rise whilst the
respective receptors decline throughout follicle development, the role of this factor may
rely more probably on its function of promoting cellular permeability.
VEGF and its receptors VEGFR-1/Flt-1 and VEGFR-2/KDR are detected in
cells from the endothelium and during pregnancy corpora lutea in sows (Kaczmarek et
al., 2007), and also in endothelial cells of bovine ovaries (Berisha et al., 2000). In
humans, mRNA and proteins corresponding to these receptors, as well as to VEGF-A
(protein), were expressed in oocytes, granulosa and stroma cells (Abir et al., 2010). In
addition, VEGF was also immunolocalized in granulosa and theca interna cells of
healthy follicles from rats (Koos, 1995) and cows (Berisha et al., 2000), as well as in
luteinized granulosa cells in buffalos (Papa et al., 2007) and mares (Al-zi’abi et al.,
2003).
108
BIOLOGICAL ACTIVITY AND ROLE OF VEGF IN MAMMALIAN
FOLLICULOGENESIS
The selective activation of each of the VEGF receptor types results in distinct
biological responses. Binding to VEGFR-1/Flt-1 leads to organizational effects on
vascular structures, which are important for the interaction of endothelial cells and for
blood vessels formation. In contrast, activation of VEGFR-2/KDR induces the
formation, migration and proliferation of vascular endothelial cells (Neufeld et al.,
1999; Ho & Kuo, 2007), as well as contributing to cellular survival. Binding to
VEGFR-3/Flt-4, predominantly expressed in lymphatic vessels, resulted in lymphatic
angiogenesis (Ho & Kuo, 2007). Some biological responses to VEGF binding to its
receptors important for follicle development are described in more detail below (Fig. 3).
Figure 3. Biological activities of VEGF in the mammalian ovarian follicle. The
expansion of the vascular network during follicle development enhances oxygenation
and diffusion of several substances important for follicle cells, and leads to the
discussed biological responses. GC, granulosa cell; TC, theca cell; VEGF, vascular
endothelial growth factor; ZP, zona pellucida.
109
Angiogenic action of VEGF
VEGF was discovered originally as a compound that was capable of enhancing
the permeability of vessels, thus enabling proteins and other molecules to exit blood
vessels and enter perfused tissues (Senger et al., 1983; Dvorak et al., 1995). With regard
to the mammalian ovary, VEGF properties were demonstrated first in the bovine corpus
luteum (Tischer et al., 1989), and later in the same tissue from ewes (Redmer et al.,
1996). The cyclic changes during formation and regression of the corpus luteum
comprise the formation of new blood vessels (Redmer & Reynolds, 1996; Wulff et al.,
2001) from pre-existing vessels, and is named angiogenesis.
Later studies have revealed that this factor was also involved in other processes
such as the promotion of growth of vascular cells derived from arteries, veins and
lymphatic vessels (Ferrara & Davis-Smyth, 1997; Ferrara & Alitalo, 1999). Moreover,
VEGF was found to induce a potent angiogenic response in a wide range of in vivo
(Leung et al., 1989) and in vitro (Pepper et al., 1992; 1994) models.
In the ovary, angiogenesis facilitates oxygenation and nutrition of target cells,
and secures an increasing supply of gonadotropins, growth factors, oxygen, steroid
precursors, as well as other substances to the growing follicle (Kaczmarek et al., 2005).
Such rise in the delivery of nutrients can be a decisive factor for the selection of the
dominant follicle (Zimmermann et al., 2001). Therefore, there is evidence that thecal
angiogenesis plays a pivotal role in follicle development (Tamanini & De Ambrogi,
2004). Furthermore, granulosa cells are important for the angiogenic process, as these
cells secrete several angiogenic factors that act on thecal cells.
VEGF and cell permeability
VEGF can also act indirectly through reorganization or formation of a primitive
capillary plexus for supply of tissue needs, increase in vascular permeability and
enabling a higher availability of growth factors, gonadotropins, steroids and oxygen,
which are important for follicle growth. This fact was confirmed in vivo by Danforth et
al. (2003) and Quintana et al. (2004) through direct injection of VEGF into the ovarian
bursa in mice that enhanced neovascularization, increased the numbers of primary and
secondary follicles and vascular permeability for developing follicles, and, as a
110
consequence, reduced apoptosis. In vitro, Mattioli et al. (2001) observed that VEGF
production raised blood supply and activated primordial follicles.
The cellular permeability induced by VEGF is attributed to the appearance of
fenestrations that, through a not well defined mechanism, enables a rise in the efflux of
small solutes (Roberts & Palade, 1995). Dvorak (2000) observed that the interaction
between VEGF and its receptors VEGFR-1 and VEGFR-2 triggers a cascade of events
that includes an increase in microvascular permeability, leading to deposition of proangiogenic fibrin in the extracellular matrix and formation of new vessels. Furthermore,
VEGF induces an increase in calcium influx, as well as a rise in the concentration of this
ion within endothelial cells (Bates & Curry, 1997).
In the ovarian follicle, the promotion of vascular permeability, vasodilation and
development of endocrine function by theca cells resulted in a gradual rise in ovarian
blood flux, and supported antrum formation and functional adaptation events for
ovulation, which led to follicle rupture (Jiang et al., 2003; Tamanini & De Ambrogi,
2004). Thus, the establishment of an adequate vascular supply is possibly a limiting step
in the selection and maturation of the one dominant follicle that will ovulate (Stouffer et
al., 2001).
The formation of the antral cavity is a spontaneous event during the in vitro
culture of advanced preantral follicles, however mitogenic factors such as VEGF may
enhance rates of occurrence of this process (Araújo et al., unpublished data). One study
showed that VEGF secretion is stage dependent and increases as the follicle grows,
which reflects in the amounts of VEGF in the follicular fluid (Barboni et al., 2000).
VEGF is also produced by cells of preovulatory follicles, as well as by luteinized cells
(Taylor et al., 2004).
VEGF and cell survival
The role of VEGF as a survival factor was observed either in vitro or in vivo
with endothelial cells (Alon et al., 1995; Yuan et al., 1996), as well as with other cell
types. VEGF inhibits apoptosis induced by absence of serum in culture medium (Gerber
et al., 1998a) or by injuries that result from cryopreservation (Shin et al., 2006). This
property may be mediated via PI3kinase/Akt (Gerber et al., 1998a), which is a
signalling pathway fundamental for regulation of cell proliferation, survival, migration
and metabolism, and also plays an important role in the activation of primordial follicles
111
(Cantley, 2002). Moreover, VEGF induces the expression of anti-apoptotic proteins
such as Bcl-2 and A1 in endothelial cells (Gerber et al., 1998a). The addition of VEGF
to in vitro culture supported the maintenance of viability and ultrastructure of goat early
preantral follicles (Bruno et al., 2009).
Mitogenic action of VEGF
In addition to its angiogenic properties, VEGF is also a potent mitogenic factor
that is secreted by many differentiated cells in response to several stimuli such as, for
instance, hypoxia. Nonetheless, the loss of its carboxy-terminal domain reduces
significantly the potency for induction of proliferation in endothelial cells (Keyt et al.,
1996). VEGF exerts direct mitogenic effects on granulosa cells, and then acts on follicle
growth in human ovaries (Otani et al., 1999). The presence of VEGF-A receptors,
especially in granulosa cells, suggests that this factor may be involved in proliferation
events, as well as in the onset of development of primordial follicles in humans (Abir et
al., 2010). Furthermore, during the transition of these follicles to the primary stage, an
increase in VEGF and its mRNA takes place in rats (Kezele et al. 2005). Yang &
Fortune (2007) observed the transition of primary follicles to the secondary stage, and
also the increase in follicle diameter, through the in vitro culture of ovarian tissue
retrieved from bovine fetuses in medium supplemented with VEGF. Similarly, in
addition to follicular growth, an increase in oocyte diameter could also be seen in early
(Bruno et al., 2009) and advanced (Araújo et al., unpublished data) goat preantral
follicles.
Role of VEGF on oocyte maturation
As VEGF expression increases progressively from the primary to the
preovulatory stage, which is directly correlated to the expansion of vascularization and
oxygenation of follicles (Sharma & Sudan, 2010), selection of the dominant follicle
depends on the formation and the differentiation of a rich vascular supply with an
increment in the permeability of the respective vessels (Kawano et al., 2003). Such
conditions are very important because hypoxia may reduce oocyte metabolism and
cause changes in intracellular pH, which in turn affects organization and stability of the
meiotic spindle (Gaulden, 1992). Such an effect can result in chromosomal disorders
112
(non-disjunction of chromosomes) (Van Blerkom et al., 1997). Moreover, deficiencies
in blood supply impair the delivery of substances that are essential for the development
of follicles to the preovulatory phase (Zimmermann et al., 2003). Therefore, VEGF is
an important factor for the development of mammalian oocytes, and contributes to
making these gametes competent for fertilization, embryo development and pregnancy.
The incomplete cytoplasmic maturation commonly observed after in vitro
maturation of oocytes (First & Barnes, 1989) may explain the low rates of fertilization
and extrusion of the first polar body (Trounson et al., 1977). The use of VEGF in
culture of bovine cumulus-oocyte complexes promoted nuclear (Einspanier et al., 2002;
Luo et al., 2002) and cytoplasmic (Luo et al., 2002) maturation of the oocytes, and
enhanced normal fertilization rates and the subsequent embryo development to the
blastocyst stage. Moreover, Iijima et al. (2005) observed that treatment of rats with
VEGF promoted ovarian follicular angiogenesis, stimulated follicle development and
increased the number of ovulated oocytes, which showed normal fertilization and
developmental competence to term.
Despite the evidence that VEGF can contribute to oocyte maturation, the
mechanisms by which this factor acts in this process are still unclear. It has been
postulated that VEGF may exert its main paracrine effects directly on oocytes or
indirectly via cumulus cells that express VEGF receptors type 2 (VEGFR-2/KDR)
(Bruno et al., 2009) and are expanded in bovine (Einspanier et al., 2002; Luo et al.,
2002) and caprine (Araújo et al., unpublished data) cumulus-oocytes complexes
cultured with VEGF.
CONCLUSIONS
A full understanding of the role of VEGF on the modulation of ovarian
physiology is very important as this growth factor controls vascularization and therefore
the availability of oxygen and nutrients for the follicles. Studies have demonstrated that
VEGF influences cell survival, proliferation and thus follicular development positively,
along with the stimulation of secretion of some steroid hormones such as, for instance,
progesterone. In spite of the recognized potential of VEGF for enhancing follicle and
oocyte developmental processes, studies on the functions of this factor in
folliculogenesis are still scarce. Therefore, more investigation is necessary in order to
explore the various biological properties of VEGF and its receptors.
113
Acknowledgements
The authors thank Dr Anderson P. Almeida for the creation and editing of the
images of this work. Valdevane R. Araújo is a recipient of a grant from the
Coordination for the Improvement of Higher Education Personnel (CAPES-Brazil).
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8 CAPÍTULO 3
Efeito do protocolo de troca de meio sobre o desenvolvimento in vitro de folículos
pré-antrais caprinos isolados
“Effect of culture medium replacement protocol on the in vitro development of
isolated caprine secondary follicles”
Periódico: Small Ruminant Research, v. 95, p. 139-143, 2011.
123
RESUMO
O objetivo deste trabalho foi verificar a influência de três diferentes protocolos de troca
de meio sobre o cultivo de folículos pré-antrais caprinos isolados. Independentemente
do protocolo, folículos pré-antrais foram individualmente cultivados por 18 dias (D18)
com volume de meio inicial de 25 µl e o intervalo para renovação do meio de cultivo
(trocas) foi a cada dois dias. Os protocolos testados foram: T1 (Controle) - Troca parcial
de 15 μl (retirada de 15 µl de meio de cultivo seguido da adição do mesmo volume de
meio fresco), mantendo o volume final de 25 μl; T2 - Somente adição de 5 μl de meio
fresco a cada troca (o volume do meio aumentou 5 µl a cada troca sendo o volume final
de 65 μl no dia 18) e T3 - Retirada inicial na primeira troca de 15 μl de meio com
adição de 20 μl de meio fresco (aumento de 5 μl no volume final do meio a cada troca).
Nas trocas subsequentes do T3, a quantidade de meio adicionado na troca anterior foi
removida, seguida pela adição do mesmo volume mais 5 μl de meio fresco (semelhante
ao T2, o volume final no dia 18 foi de 65 μl). As análise de sobrevivência, diâmetro e
taxa de formação de antro folicular, bem como taxa de crescimento diária foram
realizadas a cada 6 dias. No final do perído de cultivo, oócitos (≥ 110 µm) que
apresentavam-se normais foram destinados à maturação in vitro (MIV). Os resultados
demonstraram que apenas o T2 (Adição sem remoção de meio) foi capaz de manter a
sobrevivência folicular até o final do período de cultivo. No D18, ambos o diâmetro
folicular e a taxa de crescimento diário foram similares entre o T2 e T3
(Remoção+Adição), e ambos foram significativamente superiores ao T1 (Troca parcial).
Além disso, o T2 obteve uma alta percentagem de oócitos ≥ 110 µm destinados à MIV e
foi o único tratamento que obteve um oócito em telófase-I. Desta forma, pode-se
concluir que a adição periódica de meio é recomendada em função de sua praticidade e
manutenção da sobrevivência, além de permitir o desenvolvimento in vitro de folículos
pré-antrais caprinos.
Palavras-chave: Folículos ovarianos. Cabras. Troca de meio. Oócitos competentes.
124
Effect of culture medium replacement protocol on the in vitro development of
isolated caprine secondary follicles
Valdevane Rocha Araújo*,a, Roberta Nogueira Chavesa, Ana Beatriz Graça Duartea,
Juliana Jales de Hollanda Celestinoa, Gerlane Modesto da Silvaa, Diego Diógenes
Fernandesa, Maria Helena Tavares de Matosb, Cláudio Cabral Campelloa, José Ricardo
de Figueiredoa
a
Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and Preantral
Follicles (LAMOFOPA), State University of Ceara, Fortaleza, CE, Brazil
b
Nucleus of Biotechnology Applied to Ovarian Follicle Development, Federal
University of São Francisco Valley, Petrolina, PE, Brazil
*Corresponding address:
Programa de Pós-Graduação em Ciências Veterinárias (PPGCV)
Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais (LAMOFOPA)
Universidade Estadual do Ceará (UECE)
Av. Paranjana, 1700, Campus do Itaperi
Fortaleza – CE – Brasil. CEP: 60740-000
Tel.: +55.85.3101.9852, Fax: +55.85.3101.9840
E-mail address: val_exclusiva@yahoo.com.br (Valdevane R. Araújo)
125
ABSTRACT
The aim of this study was to verify the influence of three different protocols for medium
refreshing on the in vitro culture of isolated caprine preantral follicles. Independently of
the protocol, preantral follicles were individually cultured for 18 days, the initial
volume of medium was 25 μl, and the interval of medium refreshing was every two
days. The protocols tested were: T1 (Control) – refreshing of 15 μl (removal of 15 µl of
culture medium followed by the addition of the same volume of fresh medium),
maintaining a final volume of 25 μl, T2 – only the addition of 5 μl of fresh medium
every two days (the medium volume increases 5 μl for each change up to a final volume
of 65 μl at day 18), and T3 – initial removal of 15 μl of medium in the first change, with
addition of 20 μl of fresh medium (net increase of 5 μl in the final volume at each
change). In the subsequent changes for T3, the amount of medium added in the previous
change was removed, followed by the addition of the same volume plus 5 μl fresh
medium (as occurred for T2 the final volume at day 18 is also 65 μl). Analyses of
survival, diameter and antrum formation, as well as the rate of daily follicular growth
were performed every 6 days. At the end of the culture period, normal oocytes ≥ 110
µm were destined for in vitro maturation (IVM). The results showed that only T2
(addition without removal of medium) maintained follicular survival until the end of the
culture period. In day 18, both follicular diameter and the rate of daily growth was
similar in T2 and T3 (removal + addition of medium), which were both higher than in
T1 (partial change). Moreover, T2 obtained a greater percentage of oocytes >110 µm
destined for IVM and was the only treatment that achieved an oocyte in the telophase-I
stage. In conclusion, periodic addition of medium is recommended because it is more
practical, maintains survival and promotes the development of caprine preantral follicles
in vitro.
Keywords: ovarian follicles, goat, medium change, competent oocytes.
126
1. Introduction
The ovaries of mammals contain a large number of oocytes enclosed in preantral
follicles. In vivo, follicles develop through primordial, primary and secondary stages
(preantral follicle phase) before acquiring an antral cavity. In primordial and primary
follicles, the granulosa cells in beginning of proliferation are more resistant to degenera
tion than oocytes (Braw-tal and Yossefi, 1997). However, in secondary follicles, both
oocyte and granulosa cells are equally affected. The increase in oocyte sensitivity in
advanced follicles maybe due to the significant morphological alterations in this
compartment or the need for greater nutritional support (Silva et al., 2002). The in vitro
culture of secondary follicles up to antral stage is very important to provide information
about follicular requirements during the late preantral follicle phase as well as during
the antral phase. Due to these differences among follicular categories, the development
of culture systems that support complete follicular growth in vitro is necessary. Such
culture systems depend on the physical and biochemical conditions used, which may
limit the diffusion of nutritional gradients, thus providing an adequate environment for
the growth and development of competent oocytes (Hastshorne, 1997).
The development of protocols for the periodic replacement of culture medium
(change of medium) is extremely important for in vitro culture because cultured cells
produce substances that are favorable and/or harmful to their survival. For instance, the
amino acids present in culture medium can be metabolized at 37oC and thus, stimulate
the production of ammonia, which may be deleterious for the cells in culture. To avoid
the accumulation of this compound and other toxic products, some authors have
suggested that for the culture of embryos, medium replacement should occur at least
every 72 h (Gardner et al., 1994; Trounson et al., 1994).
For advanced preantral follicles, partial replacement of the medium is most
commonly used for growth in culture. This procedure provided satisfactory results, such
as the production of embryos in swine (Wu et al., 2001), mouse (Demeestere et al.,
2002) and buffalo (Gupta et al., 2008). In caprine, the partial change of medium allowed
the formation of an antral cavity in follicles cultured in a group (Huanmin and Yong,
2000) and meiosis resumption, reaching metaphase I in isolated follicles cultured in an
atmosphere of 20% oxygen (Silva et al., 2010). Other studies cultured mouse preantral
follicles in 10 µl drops wherein the replacement of the medium was performed by the
addition of 10 µl in day 1 of culture and subsequent replacement of half of the medium
127
(partial change). This protocol promoted follicular growth and production of matured
oocytes in vitro (Mousset-Siméon et al., 2005; Lee et al., 2007).
Despite the encouraging results obtained with the culture of isolated follicles, the
establishment of an efficient protocol of medium replacement is still necessary to
produce a greater number of caprine oocytes competent to resume meiosis in vitro.
Thus, the aim of the present study was to observe the influence of different protocols of
partial replacement and/or periodic addition of medium in the in vitro culture of
advanced isolated preantral follicles from caprine ovaries.
2. Materials and methods
2.1. Source of chemicals and ovaries
Unless mentioned otherwise, the culture media and other chemicals used in the
present study were purchased from Sigma Chemical Co. (St. Louis, USA). Ovaries (n =
24) were collected at a local slaughterhouse from 12 adult (ages 1-3 years) mixed-breed
goats. Immediately postmortem, the ovaries were washed in 70% alcohol for
approximately 10 s, followed by two rinses in Minimum Essential Medium with HEPES
(MEM HEPES) supplemented with 100 µg/ml penicillin and 100 µg/ml streptomycin.
The ovaries were transported within 1 h to the laboratory in 15 ml tubes containing
washing medium at 4oC (Chaves et al., 2008).
2.2. Isolation and selection of caprine preantral follicles
In the laboratory, the fat tissue and ligaments surrounding the ovaries were
stripped off, and caprine ovarian cortical slices (1mm thick) were cut from the ovarian
surface using a surgical blade under sterile conditions. Then the ovarian fragment was
placed in a fragmentation medium consisting of MEM HEPES. Caprine preantral
follicles that were approximately ≥150 µm in diameter were visualized under a
stereomicroscope (SMZ 645 Nikon, Tokyo, Japan) and manually dissected from strips
of the ovarian cortex using 27.5 gauge (27.5 G) needles. After isolation, follicles were
transferred to 100 µl drops containing fresh medium to further evaluate the follicular
quality. Follicles with a visible oocyte that were surrounded by two or more layers of
128
granulosa cells, with an intact basement membrane and no antral cavity were selected
for culture (Fig. 1A).
Fig. 1. Normal caprine preantral follicle before culture (A); antral follicle after 18 days
of culture in T2 (periodic addition of medium). Note the chromatin configuration of the
oocytes in germinal vesicle (C) and telophase I (D - from T2 treatment).
2.3. Culture of caprine preantral follicles
After selection, follicles were individually cultured in 25 µl drops (initial volume
for all protocols tested) of culture medium under mineral oil in Petri dishes
(60mm×15mm, Corning, USA). The basic culture medium consisted of α-MEM (pH
7.2-7.4) supplemented with 1.25 mg/ml bovine serum albumin (BSA), ITS (insulin 10
µg/ml, transferrin 5.5 µg/ml and selenium 5 ng/ml), 2mM glutamine, 2mM
hypoxantine, 50 µg/ml ascorbic acid, and 1000 ng/ml recombinant Follicle Stimulating
Hormone (rFSH®, Nanocore, Brazil). Preantral follicles were individually distributed in
microdrops according to the protocol of medium replacement used (treatments): T1
(Control) – Partial change of 15 µl (removal of 15 µl of culture medium followed by the
129
addition of the same volume of fresh medium, maintaining a final volume of 25 µl, T2 –
only addition of 5 µl of fresh medium at each change (final volume of 65 µl), and T3 –
initial removal of 15 µl of medium in the first change, with addition of 20 µl of fresh
medium (increase in 5 µl in the final volume at each change). In the subsequent changes
for T3, the amount of medium added in the previous change was removed, followed by
the addition of the same volume plus 5 µl fresh medium (final volume of 65 µl).
Independent of the treatment, addition and/or removal of the culture medium occurred
every two days with the medium being incubated for 1 h prior to use. Incubation was
carried out at 39oC, in 5% CO2 in air for 18 days after which follicles were recovered
for in vitro maturation. The experiment was replicated four times, and at least 30
follicles were used for each treatment.
2.4. Morphological evaluation of follicle morphology and development
During and after culture, follicles were classified according to their
morphological characteristics, and those showing morphological signs of degeneration,
such as darkness of the oocytes and the surrounding granulosa cells, misshapen oocytes,
rupture of the basement membrane and/or oocyte extrusion were classified as
degenerated. Analyses of follicular viability as well as the medium replacement were
performed every two days of culture. The rate of daily follicular growth was calculated
by the variation of follicular diameter (diameter of viable follicles after 18 days of
culture minus diameter on day 0) divided by the period of culture. Follicular diameter
was measured only in healthy follicles from the basement membrane, in the x and y
dimensions (90o), using an ocular micrometer (100x magnification) inserted into a
stereomicroscope (SMZ 645 Nikon, Tokyo, Japan) every six days of culture (at days 0,
6, 12 and 18 of culture). Antral cavity formation was defined as a visible translucent
cavity within the granulosa cell layers (Fig. 1B).
2.5. Oocyte recovery rate (≥110 µm in diameter) from in vitro grown caprine preantral
follicles
After 18 days of culture, all of the healthy follicles were carefully and
mechanically opened with 27.5 G needles under a stereomicroscope for oocyte
recovery. Only oocytes ≥110 µm, with a homogeneous cytoplasm that were surrounded
130
by at least one compact layer of cumulus cells were selected for in vitro maturation
(IVM). The recovery rate was calculated by the relation between the number of oocytes
≥110 µm and the number of viable follicles after the culture period. The selected
cumulus oocyte complexes were washed three times in a maturation medium composed
of TCM 199 supplemented with 10% fetal calf serum, 100 µg/ml of rFSH, 100 µg/ml
recombinant Luteinizing Hormone (rLH), Epidermal growth factor (10 ng/ml) and 17βestradiol (1 µg/ml). After washing, the oocytes from each treatment were transferred to
50 µl drops of maturation medium under mineral oil and then incubated for 26 h at 39oC
with 5% CO2 in air. At the end of the maturation period, oocytes were fixed in acetic
acid:methanol (1:3, v/v) for 12-24 h and then analyzed by chromatin configuration after
staining with lacmoid.
2.6. Statistical analysis
Percentages of surviving follicles, antrum formation and oocytes selected for
IVM were analyzed as dispersion of frequency by a Chisquared test. Data from
follicular diameters and growing rate did not show homoscedasticity, even after
transformation, and were analyzed by a Kruskal-Wallis non-parametric test. The results
are expressed as the mean±standard error of the mean (SEM) and differences are
considered to be significant when P < 0.05.
3. Results
3.1. Survival of caprine preantral follicles cultured in vitro
A total of 93 caprine preantral follicles were selected for culture. When the
different culture periods were analyzed, there was a progressive reduction in follicular
survival during the culture periods, except for T2 (addition of medium). Only T2, i.e.,
periodic addition of 5 µl of fresh medium during replacement, maintained follicular
survival similar to day 0 (100%), even after 18 days of culture (88.89%). Moreover,
after culture, T2 showed a significantly higher percentage of morphologically normal
follicles when compared to the other treatments (P < 0.05; Fig. 2).
131
Fig. 2. Percentage of isolated morphologically normal preantral follicles after 18 days of
culture.
0.05).
(a,b)
Differs significantly among culture periods within the same treatment (P <
(A,B)
Differs significantly among treatments within the same culture period (P <
0.05).
3.2. Rate of follicular growth
With the progression of the culture from day 0 to day 18, there was a significant
increase in follicular diameter in all treatments (P < 0.05). However, at 18 days of
culture, follicular diameter was significantly larger in T2 (Addition) and T3 (Removal +
Addition), when compared to T1 (Partial change; P < 0.05; Fig. 3). Similar results were
observed in the rate of daily follicular growth, in which the higher rates were obtained
in treatments T2 (16.25±0.09 µm/day: Addition) and T3 (15. 52±0.13 µm/day: Removal
+ Addition), when compared to T1 (9.55±0.12 µm/day: Partial change; P < 0.05).
132
Fig. 3. Follicular diameter after 18 days of culture with different protocols of medium
replacement.
(P < 0.05).
(a,b)
Differs significantly among culture periods within the same treatment
(A,B)
Differs significantly among treatments within the same culture period (P
< 0.05).
3.3. Antral cavity formation
When compared at each day of culture, all treatments significantly increased the
percentage of antrum formation from day 0 to day 6 (Fig. 4, P < 0.05), and there were
no significant changes in these percentages until the end of the culture (P > 0.05).
Furthermore, independent of the culture period, T2 (addition) showed a significantly
higher rate of antrum formation when compared to other treatments (P < 0.05).
133
Fig. 4. Percentage of antral cavity formation in follicles cultured with different
protocols of medium replacement after 18 days.
periods within the same treatment (P < 0.05).
(a,b)
Differs significantly among culture
(A,B)
Differs significantly among treatments
within the same culture period (P < 0.05).
3.4. Ability of in vitro grown oocytes in resume meiosis
For IVM, only oocytes with a diameter ≥110 µm were recovered for all
treatments after 18 days of culture of preantral follicles (Table 1). However, only T2
(addition) showed a significantly higher percentage of oocytes destined for IVM when
compared to the other treatments (T1 – Partial change and T3 – Removal + Addition; P
< 0.05). Moreover, only this same treatment obtained an oocyte competent to resume
meiosis, showing a nucleus in telophase I (Fig. 1D), while the other oocytes remained in
the germinal vesicle stage (Fig. 1C).
134
Table 1. Meiotic stages of goat oocytes from preantral follicles cultured for 18 days with three different protocols for medium exchange.
Treatments
Number of follicles (%)
Number of oocytes (%)
Cultured (n)
Fully grown* n (%)
B
GV
TI
n (%)
n (%)
T1 – Partial change
34
2/34 (5.9)
2/2 (100.0)
0/2 (0.00)
T2 – Addition
27
15/27 (55.7)A
14/15(93.3)
1/15(6.7)
T3 – Removal + Addition
32
5/32 (15.6)B
5/5(100.0)
0/15(0.0)
A,B
*
indicates significant differences among treatments. GV: germinal vesicle; TI: Telophase I
Only oocytes ≥ 110 μm were selected for the in vitro maturation procedure.
135
4. Discussion
This study demonstrated for the first time that a low volume of medium and/or
an abrupt renewal in the medium affects the in vitro development of caprine preantral
follicles. In this study, after 18 days of culture, only T2, i.e., addition of 5 µl of medium
every two days, maintained the percentage of morphologically normal follicles similar
to day 0. In a shorter culture period of 10 days, Calongos et al. (2008) showed that a
protocol of partial change of medium also contributes to the maintenance of mouse
follicle survival. However, in the present study, progressive addition of the medium
may be the most viable option, because it is more practical, fast and promotes a
reduction in the time for which follicles were exposed to the external environment
during manipulation (outside the incubator). Consequently, the stress on the follicular
cells and the release of reactive oxygen species would be reduced, as would cell
degeneration (Correa et al., 2007). In addition, others factors secreted by the follicles
may have a positive influence in the culture, promoting the maintenance of follicular
survival, such as EGF, insulin like growth factor-1 (IGF-1) and transforming growth
factor-β (TGF-β), which are expressed in granulosa and theca cells (Gutierrez et al.,
2000).
After the maintenance of follicular survival, it is necessary that the culture
system enables the growth of follicles. In the present work, the rate of daily growth was
16.25±7.02 µmin T2 (Addition), which was higher than in T1 (Partial change of 15 µl
ofmedium). Some authors have also demonstrated that it is possible to obtain greater
rates of follicular growth after culturing secondary preantral follicles in drops of
medium under mineral oil (Mousset-Siméon et al., 2005; Lee et al., 2007; Calongos et
al., 2008). The satisfactory results obtained with T2 (Addition) may be due to the
substances produced by follicular compartments during their in vitro culture, because
these substances will be diluted and will remain in the medium to act directly or
indirectly to promote follicle growth. Moreover, the worst results obtained with T1
(Partial change) suggest that the removal of a great volume of medium from the culture
drop would also remove these locally produced substances. Furthermore, after removal,
addition of fresh medium may cause stress to the follicles because their metabolism
must increase to produce all the substances necessary for their development.
Regarding antrum formation, all treatments showed follicles with an antral
cavity. However, only T2 (Addition) reached higher percentages of antrum formation
136
when compared to the other treatments from day 6 onwards. Similar results were
observed by our team after 6 days (goat: Silva et al., 2010) and by other authors after 6
and 7 days of preantral follicle culture (mouse: Lee et al., 2007; Calongos et al., 2008).
Nevertheless, Mousset-Siméon et al. (2005) observed the beginning of antral cavity
formation only after 11 days of mouse preantral follicle culture. These differences in the
results may be due to the different protocols used in addition to the species tested.
The best results obtained for T2 (addition) in relation to the percentage of
oocytes destined for IVM (≥110 µm) suggest that locally produced factors may
positively influence oocyte growth. Orisaka et al. (2006) demonstrated that the
production of survival factors by the oocyte, such as growth and differentiation factor-9
(GDF-9), may support these results through the suppression of granulosa cell apoptosis.
These observations are important because for the caprine species, oocytes with a
diameter less than 110 µm do not resume meiosis (Crozet et al., 2000). More recently,
Silva et al. (2010) showed that after culturing goat preantral follicles, a fully in vitro
grown oocyte reached metaphase I.
In general for T2, the gradual increase in the culture medium volume through the
addition of fresh medium would provide new nutrients and also maintain the medium
with substances produced in the different compartments of the follicle. Dilution of these
substances (removal of the medium followed by a progressive increase in its volume),
which occurred in T3, was harmful to follicle survival throughout the 18-days of
culture. Nevertheless, the increase in medium volume and the addition of new nutrients
are essential to support the growth and development of the follicles.
Thus, it can be concluded that periodic addition of medium (T2) is
recommended as the most practical and effective medium replacement protocol.
Furthermore, this treatment maintains the survival and promotes the development of
caprine preantral follicles in vitro. However, the rate of oocytes resuming meiosis from
caprine preantral follicles grown in vitro is still low and requires more research to
overcome this problem.
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137
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Lee, S.T., Choi, M.H., Han, J.Y., Lim, J.M., 2007. Establishment of a basic method for
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preantral follicles and intrafollicular oocytes. Zygote 15, 109-116.
Huanmin, Z., Yong, Z., 2000. In vitro development of caprine ovarian Preantral
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9 CAPÍTULO 4
Crescimento in vitro, produção de estradiol e expressão gênica de folículos préantrais bovinos isolados: Efeito do meio de base e método de troca de meio
“In vitro growth, estradiol production, and gene expression of isolated bovine
preantral follicles: Effect of base medium and medium replacement method”
Periódico: Molecular Reproduction and Development (Submetido em: 26 de maio de
2013).
140
RESUMO
Dois diferentes métodos para troca de meio foram comparados durante cultivo in vitro
de longa duração de folículos secundários bovinos utilizando α-MEM+ ou TCM-199+
como meio de base. Os métodos de troca de meio testados foram: Convencional –
remoção e subsequente adição da mesma quantidade de meio (60 µl) em um gota de 100
µl (MEM-C e TCM-C), e Suplementação de pequenas quantidades – adição de 5 µl de
meio fresco à uma gota inicial de 50 µl, resultando num volume final de 125 µl no
último dia de cultivo (MEM-S e TCM-S). Um total de 207 folículos secundários foram
cultivados individualmente durante 32 dias à 38,5oC e 5% de CO2 e a mudança do meio
foi realizada a cada dois dias. MEM-S apresentou diâmetro folicular (P<0.01), taxa de
crescimento (P<0.02) e formação de antro (P<0.02), bem como concentrações de
estradiol (P<0.0001) significativamente superiores quando comparado ao MEM-C. Em
relação ao meio TCM-199+, nenhum dos parâmetros avaliados foi afetado pelo método
de troca (P>0.05). A expressão para o FSH-R foi maior (P<0.03) no TCM-C que no
TCM-S, enquanto que a expressão do RNAm para o IGF1 foi maior (P<0.02) no MEMS que no TCM-S e para o VEGF foi maior (P<0.02) no MEM-C que no TCM-C. Em
conclusão, o tipo de meio de base e o efeito da adição peródica de meio afetou
diferentemente o desenvolvimento folicular, a produção de estradiol e a expressão
gênica. Além disso, o α-MEM+ pode ser usado em substituição ao TCM-199+, se a
adição periódica de meio for usada como método de troca de meio.
Palavras-chave: Vaca. Foliculogênse. Folículo secundário.
141
In Vitro Development, Estradiol Production, and Gene Expression of
Isolated Bovine Preantral Follicles: Effect of Base Medium and
Medium Replacement Method1
V. R. Araújo3,4, M. O. Gastal3, A. Wischral3, J. R. Figueiredo4, E. L. Gastal2,3
3
Department of Animal Science, Food and Nutrition, Southern Illinois University,
1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
4
Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA),
Veterinary Faculty, State University of Ceará, Av. Paranjana 1700, Campus do Itaperi,
Fortaleza, 60740-903, CE, Brazil.
Short title: In vitro growth of bovine secondary follicles
1
Supported by a start-up package (Gastal EL) from SIU. Araújo VR is the recipient of a
PhD scholarship from CNPq, Brazil. Results of this study have been partially presented
as abstracts at the 45th Annual Meeting of the Society for the Study of Reproduction
(SSR), August 12-15, 2012, State College, Pennsylvania, USA and IV International
Symposium on Animal Biology of Reproduction (ISABR), October 17-20, 2012,
Campinas, SP, Brazil.
2
Correspondence: Eduardo Gastal, Department of Animal Science, Food and Nutrition,
Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901,
USA. FAX: 618 4535231; e-mail: egastal@siu.edu
Abbreviations:
α-MEM, minimum essential medium;
TCM-199, tissue culture medium-199;
FSHR, follicle stimulating-hormone receptor gene;
IGF1, insulin-like growth factor 1 gene;
VEGF, vascular endothelial growth factor gene;
P450AROM, p450 aromatase gene;
HEPES, buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
BSA, bovine serum albumin;
142
ITS, insulin, transferrin, and sodium selenite commercial compound;
ELISA, Enzyme-Linked ImmunoSorbent Assay;
CYC-A, Cyclophilin-A housekeep gene;
rFSH, recombinant follicle stimulating hormone
ABSTRACT
Two culture methods were compared during long-term in vitro culture of bovine
secondary follicles using α-MEM+ (minimum essential medium supplemented) or
TCM-199+ (tissue culture medium-199 supplemented) as base media. The medium
replacement methods tested were: Conventional – removal and subsequent addition of
the same amount (60 µl) in a 100 µl drop (called MEM-C and TCM-C), and Small
Supplementation – addition of 5 µl of fresh medium to an initial small drop (50 µl),
resulting in a final volume of 125 µl on the last day of culture (called MEM-S and
TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at
38.5°C in 5% CO2 and medium replacement was performed every other day. MEM-S
resulted in larger (P<0.01) follicular diameter, higher (P<0.02) growth rate, greater
(P<0.02) antrum formation, as well as higher (P<0.0001) estradiol concentrations when
compared with MEM-C. In addition, a highest concentration of estradiol was observed
in the MEM-S. The medium change methods did not affect (P>0.05) any of the studied
end points for TCM-199+. The expression for FSHR was greater (P<0.03) in TCM-C
than TCM-S, while the mRNA expression for IGF1 was higher (P<0.02) in MEM-S
than TCM-S and for VEGF was higher (P<0.02) in MEM-C than TCM-C. In
conclusion, the type of base medium and the effect of periodic addition of medium
affected differently follicle development, estradiol production, and gene expression.
Furthermore, α-MEM+ can be used to replace the TCM-199+ for bovine preantral
follicle culture if progressive addition of medium is used for medium change.
Keywords: Cattle, Folliculogenesis, Secondary follicle
143
INTRODUCTION
Development of preantral follicles to a stage where the oocyte can mature in
vitro requires a long-term culture period and involves several follicular changes during
its development (Thomas et al., 2007). However, a long-term culture period can impair
the steroidogenic capability of the follicle and affect the oocyte competency
(McLaughlin and Telfer, 2010). Therefore, to successfully culture bovine preantral
follicles it is necessary to appropriately define in vitro culture conditions which would
allow the oocyte to survive, grow, and induce the differentiation of the somatic cell
compartment for a prolonged period.
Development of an ideal culture system for in vitro culture of preantral follicles
has been the focus of different research groups. However, to our knowledge, in vitro
experiments with bovine preantral follicles are still scarce and with limited success
compared to other species such as caprine (Saraiva et al., 2010; Magalhães et al., 2011),
ovine (Arunakumari et al., 2010; Luz et al., 2012), and murine (Eppig and O’Brien,
1996; O'Brien et al., 2003). The in vitro follicular culture performance may be affected
by different factors such as follicle size (Katska and Rynska, 1998), type of base culture
medium (Rossetto et al., 2012), addition of supplements (Figueiredo et al., 1994), gas
concentration (Gigli et al., 2006), method of medium replacement (i.e., interval,
removal, and addition of culture medium; Araújo et al., 2011), and presence or absence
of mineral oil (Fukui et al., 1996).
Several studies have evaluated the developmental characteristics of large (≥150
µm) bovine preantral follicles in culture media, which have a larger growth rate than
small preantral follicles in culture (Katska and Rynska, 1998). In this regard, culture
media such as α-MEM (Braw-Tal and Yossefi, 1997; Rossetto et al., 2012), TCM-199
(Katska and Rynsk,a 1998; Itoh and Hoshi, 2000; Saha et al., 2000; 2002; Rossetto et
al., 2012) and McCoy (Gutierrez et al., 2000; McCaffery et al., 2000; Thomas et al.,
2001; 2007; McLaughlin et al., 2010; McLaughlin and Telfer, 2012; Rossetto et al.,
2012) have been used to maintain viability and improve in vitro development of bovine
follicles. Recently, a study suggested that TCM-199 was the best medium to culture
isolated bovine secondary follicles, based on the high percentage of viable follicles after
16 days of in vitro culture (Rossetto et al., 2012). In regard to medium replacement
method, a study performed by our group using isolated caprine secondary follicles
showed that periodic addition of medium maintained survival and promoted in vitro
follicular development, and was the only treatment to produce a competent oocyte
144
which resumed meiosis (Araújo et al., 2011). However, it is not known if this medium
addition method could also work in the culture of isolated bovine preantral follicles.
Therefore, the aim of this study was to compare two different culture methods
using a partial removal and replacement of medium versus the addition of a small
amount of medium every other day during long-term in vitro culture. For this, the end
points evaluated were follicle viability, development, antrum formation, estradiol
production, and mRNA expression for FSHR, IGF1, VEGF, and P450AROM in large
bovine secondary follicles using α-MEM+ and TCM-199+ after long-term (32 days) in
vitro culture.
MATERIALS AND METHODS
Chemicals and media
Unless otherwise indicated, the culture media, and chemicals used in the present
study were purchased from Sigma Chemical Co. (St. Louis, MO).
Source of ovaries
Ovaries (n = 68) from 34 adult crossbred Aberdeen Angus beef cows were
collected at a local slaughterhouse and 234 were isolated and randomly distributed
among the non-cultured and cultured treatments. The ovaries were washed in 70%
alcohol for 10 sec, followed by two washes in minimum essential medium alpha (αMEM). The ovaries were placed into tubes containing 20 ml of α-MEM plus HEPES (αMEM-HEPES), supplemented with 100 µg/ml penicillin and 100 µg/ml streptomycin,
and then transported to the laboratory at 4°C within 1.5 h.
Isolation and selection of secondary follicles
In the laboratory, the surrounding fat tissue and ligaments were stripped off from
the ovaries. Ovarian cortical slices (1 mm thick) were cut from the ovarian surface using
a surgical blade under sterile conditions. Ovarian cortex fragments were placed and
washed in manipulation medium, which consisted of α-MEM-HEPES. Preantral
follicles ≥190 m in diameter were selected under a stereomicroscope (SMZ 645 Nikon,
Tokyo, Japan), manually dissected from the strips of ovarian cortex using 25 gauge
needles, and transferred to the culture medium for further evaluation of the follicular
quality. Preantral follicles with a visible central oocyte, surrounded by two or more
granulosa cell layers, an intact basement membrane and no antral cavity, were
145
considered normal secondary follicles (Gutierrez et al., 2000) and selected for in vitro
culture.
Culture of secondary follicles
Follicles were cultured at 38.5°C in 5% CO2 in air for 32 days. Fresh medium
was prepared immediately before use and incubated for at least 1 h. The medium
replacement was done every other day, independently of the method utilized.
The media used were α-MEM and TCM-199 (pH 7.2 - 7.4; CellGro Mediatech,
Inc., Manassas, VA) supplemented with 3 mg/ml bovine serum albumin (BSA), ITS
(insulin 10 µg/ml, transferrin 5.5 µg/ml, and sodium selenite 6.7 ng/ml; CellGro
Mediatech, Inc., Manassas, VA), 2 mM glutamine, 2 mM hypoxanthine, 50 μg/ml of
ascorbic acid, and 100 ng/ml of recombinant follicle stimulating hormone (rFSH;
BioVision, Inc., Milpitas, CA). The concentration of rFSH was chosen based on a
previous study (Rossetto et al., 2012). After these additions media were named α-MEM+
and TCM-199+.
Experimental design
Selected follicles were individually distributed in drops in Petri dishes (60 x 15
mm, Corning, USA) and randomly assigned to different basic culture media (α-MEM+
and TCM-199+) and two different medium replacement methods: Conventional (C) –
removal and subsequent addition of the same amount (60 µl) in a 100 µl drop,
maintaining the initial volume of 100 µl; and Small Supplementation (S) – addition of 5
µl of fresh medium to an initial small drop (50 µl), resulting in a final volume of 125 µl
on the last day of culture. The four treatments were named MEM-C, MEM-S, TCM-C,
and TCM-S, and each treatment was repeated four times with 6 to 16 follicles per
repetitioan totalizing 36-68 secondary follicles cultured per treatment.
Morphological evaluation of follicle development
Follicular features such as the integrity of the basement membrane, the morphological
aspects of the oocyte, and the surrounding granulosa cells (Fig. 1a) were evaluated
during culture. In addition, morphological signs of degeneration, such as darkness or
abnormality of oocytes and surrounding granulosa cells (Fig. 1b) were also evaluated as
previously reported (Gutierrez et al., 2000). Follicular diameter was measured every 8
days only in morphologically normal follicles with the aid of an ocular micrometer
146
inserted into a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan; 75X magnification).
Two perpendicular measures were recorded for each follicle and the average of the two
values was reported as follicular diameter. In regard to follicular growth, the mean
increase in follicular diameter (µm/day) was calculated as follows: the diameter of
normal follicles at day 32 minus the diameter of normal follicles at day 0, divided by
32. Antral cavity formation was defined as a visible translucent cavity within the layers
of granulosa cells.
Figure 1. Morphologically normal (a) and degenerated (b) bovine follicles before and
after 32 days of in vitro culture, respectively. Bars = 20 µm (a) and 50 µm (b).
Viability assessment of follicles cultured in vitro
For a more accurate evaluation of follicular integrity after 32 days of culture,
live/dead fluorescent labeling (Rossetto et al., 2012) was performed. Follicles were
placed in droplets of α-MEM-HEPES with 4 μM calcein-AM and 2 μM ethidium
homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany), followed by
incubation at 38.5°C for 15 min. Finally, the follicles were examined using a
fluorescence microscope (Zeiss, Axiovert 10, NY, USA). The emitted fluorescent
signals of calcein-AM and ethidium homodimer were collected at 450-490 nm. The
probe detected the intracellular esterase activity of viable cells first, and then the nucleic
acids of non-viable cells by plasma membrane disruption. The follicles were considered
147
live if the cytoplasm was labeled positively with calcein-AM (green) and dead if
cellular chromatin was labeled with ethidium homodimer (red).
Estradiol concentration measured by enzyme immunoassay
To evaluate follicular steroidogenesis in vitro, concentrations of estradiol were
measured in reserved culture media against standard dilutions using estradiol EnzymeLinked ImmunoSorbent Assay (ELISA) kit (Neogen, Lexington, KY, USA). Media
were removed from all treatments on days 0 and 32 of in vitro culture and stored at 80 C until assay. Briefly, the reserved media were diluted with EIA buffer (1:10),
placed in microplate wells coated with polyclonal (rabbit) antibody raised against the
estradiol antigenic site, mixed with estradiol enzyme conjugate, and incubated for 60
min. After incubation, the unbound conjugate was washed three times with diluted EIA
buffer, and a substrate solution of tetramethylbenzidine (TMB) was added to allow
development of color. After 30 min the absorbance of the plate was read at 650 nm
using a microplate reader (Synergy 2 Multi-Mode Microplate Reader, Winooski, VT).
Results were obtained using the 4 parameter logistic (4PL) curve with the Readerfit ©
program (Hitachi Solutions America, Ltd., 2012). All samples were run in double assay.
The intra-assay coefficient of variation and sensitivity of the assay were 5.1% and 0.02
ng/ml, respectively.
Bovine follicle RNA extraction
Bovine follicles (n=237) were pooled according to each the treatments before
(day 0; n=4) and after (day 32; n=12-14) in vitro culture, and total RNA was extracted
using the RNeasy Mini Kit (Qiagen Inc.,Valencia, CA). The elutes of total follicle RNA
were treated with 1 l DNase (1 u/µl; Fisher Scientific, Pittsburgh, PA) for 10 min at
37oC to remove genomic DNA and 2 min at 60oC to terminate DNase reaction. Quantity
and purity of the extracted RNA was checked by spectrophotometer (NanoDrop 1000,
Wilmington, DE). The entire total mRNA was intact with high quality, i.e. optical
density (O.D.) 260/280 and 260/230 ratios were between 1.08-1.89 and 0.03-1.29,
respectively; and 2 l of the elutes were used for quantitative real time-PCR analysis
(RT-qPCR). The total RNA was reverse transcribed into first-strand cDNA using an
iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA) with random
primers.
148
Quantitative PCR for FSHR, IGF1, VEGF, and P450AROM genes
The primers for the genes evaluated (Table 1) have been reported (Armstrong et
al., 2000; Marsters et al., 2003; Yang and Fortune, 2007; Caixeta et al., 2009) and were
ordered from Eurofins MWG Operon (Huntsville, AL). The relative expression of
mRNA was determined by qPCR. Aliquots of 2 µl of cDNA (100 ng) as a template
were used in 5 µl of SYBR Green Master Mix (PE Applied Biosystems, Foster City,
CA), 2 µl of ultra-pure water, and 1 µl of each primer. The 18S and Cyclophilin-A
(CYC-A) primers were used as endogenous controls for normalization of steady-state
levels of mRNA of tested genes. The amplifications were carried out by one initial
denaturation and activation of the polymerase for 30 sec at 95oC, followed by 40 cycles
of 2 sec each at 95oC, and 5 sec at 60oC. All reactions were performed in duplicate
using a RT-qPCR Master cycler (CFX 384 - Bio-Rad Laboratories Inc., Hercules, CA).
The delta-delta-CT method (Livak and Schmittgen 2001) was used to transform CT
values into normalized relative steady-state levels of mRNA.
Table 1. Oligonucleotide primers used for real-time polymerase chain reaction analysis
of bovine follicles before and after in vitro culture.
Target gene
VEGF
IGF1
P450AROM
FSHR
18S
CYC-A
Primer sequence (5´→ 3´)
Sense
CCTGATGCGGTGCGGGGGCT
Forward
TGGTGGTGGCGGCGGCTATG
Reverse
CCTCTGCGGGGCTGAGTTGGT
Forward
CGACTTGGCGGGCTTGAGAGGC
Reverse
CGCAAAGCCTTAGAGGATGA
Forward
ACCATGGCGATGTACTTTCC
Reverse
GCCAAGTCAACTTACCGCTT
Forward
TGACCCCTAGCCTGAGTCAT
Reverse
GCTCGCTCCTCTCCTACTTG
Forward
GATCGGCCCGAGGTTATCTA
Reverse
GCCATGGAGCGCTTTGG
Forward
CCACAGTCAGCAATGGTGATCT
Reverse
Statistical analyses
Reference
Yang and Fortune 2007
Armstrong et al. 2000
Marsters et al. 2003
Marsters et al. 2003
Yang and Fortune 2007
Caixeta et al. 2009
149
Follicle, estradiol, and RT-qPCR data were challenged for extreme values with
the Dixon outlier test (Zar 1984). Data for end points that were not normally distributed,
according to Shapiro-Wilk test, were transformed to logarithms or ranks. Single-point
data were analyzed by one-way ANOVA. If a main effect of group was significant, the
differences between groups were examined by Duncan’s multiple range tests.
Frequency data were analyzed by chi-square test. A probability of P<0.05 indicated that
a difference was significant. Data are given as the mean ± SEM unless otherwise stated.
RESULTS
Follicular morphology, viability, diameter, growth rate, antrum formation, and
estradiol concentrations during in vitro culture
A total of 207 bovine secondary follicles were cultured individually for 32 days
being 66, 68, 37 e 36 for MEM-C, MEM-S, TCM-C, and TCM-S, respectively. Viable
(a-b, c-d, e-f, g-h) and non-viable (i-j, k-l, m-n, o-p) follicles after culture are shown
(Fig. 2).
150
Figure 2. Viable and non-viable bovine follicles after 32 days of in vitro culture. (a, b, i,
j) Conventional and (c, d, k, l) Small supplementation methods using α-MEM+. (e, f, m,
n) Conventional and (g, h, o, p) Small supplementation methods using TCM-199+. Note
that viable follicles (a-h) had shiny granulosa cells arranged in several layers, intact
basal membrane, and antrum cavity. However, non-viable follicles (i-p) had very dark
granulosa cells, irregularities in the basal membrane, and no antral cavity. Bars = 100
µm (a-p).
By the end of the culture period (day 32), the morphology and viability analyses
using fluorescent probes demonstrated high morphological normal follicles and
follicular viability rates for all treatments, respectively. The follicular viability rate was
higher (P<0.01) in the MEM-C than TCM-S, but it was similar (P>0.05) to the other
151
treatments (MEM-S and TCM-C; Table 2). When the TCM-199+ was used as base
medium the replacement medium method did not affect (P>0.05) any studied end
points. However, for α-MEM+ base medium, the progressive addition of small amounts
of medium (MEM-S) resulted in larger (P<0.01) follicular diameter, higher (P<0.02)
growth rate, greater (P<0.02) antrum formation, as well as higher (P<0.0001) estradiol
concentrations when compared to conventional medium change (MEM-C). When
conventional medium change was used, TCM-C had larger (P<0.01) follicular diameter
and higher (P<0.02) percentage of antrum formation, but lower (P<0.0001) estradiol
concentration than MEM-C. Except for the higher (P<0.0001) levels of estradiol
concentrations in the MEM-S, MEM-S and TCM-S were similar (P>0.05) for all end
points tested. A highest level of estradiol was obtained when medium was added to αMEM+ (MEM-S). However, regardless of the medium replacement method (TCM-C
and TCM-S), except for the estradiol concentrations, both TCM-199+ treated groups
were similar (P>0.05) to MEM-S.
Table 2. Morphological normal follicles (%), follicular viability (%), follicular diameter
(µm) and growth rate (µm/day), antrum formation (%), and estradiol concentration
(ng/ml) of bovine follicles after 32 days of in vitro culture in α-MEM+ and TCM-199+
using two medium replacement methods (Conventional-C or Small Supplementation-S).
End points
MEM-C
MEM-S
TCM-C
TCM-S
n=66
n=68
n=37
n=36
56 (84.8)A
53 (77.9)A
30 (81.1)A
29 (80.6)A
41 (91.1)A
37 (80.4)AB
29 (78.4)AB
25 (69.4)B
281.7 ± 16.4B
364.2 ± 22.6A
355.2 ± 27.2A
366.6 ± 26.2A
Growth rate (µm/day)
2.7 ± 0.4B
4.7 ± 0.5A
4.0 ± 0.6AB
4.2 ± 0.7A
Antrum formation (%)
11 (16.7)B
23 (33.8)A
14 (37.8)A
16 (44.4)A
Estradiol concentration
3.3 ± 0.5B
12.6 ± 2.3A
1.5 ± 0.3C
Morphological normal
follicles (%)
Follicular viability (%)
Follicular diameter (µm)
2.5 ± 0.4AC
(ng/ml)
A-C
Within same end point, no common superscripts means that treatments were different
(P<0.05).
Growth rate (Table 3) and estradiol concentrations (Table 4) were further
analyzed by separating the follicles into subgroups based on the frequency of growth
152
rate during 32 days of culture, i.e., follicles that grew <1 m/day (slow growth), 1-4.9
m/day (medium growth) and ≥5
m/day (fast growth). The percentage of follicles
cultured in vitro that grew ≥5 µm/day was greater (P<0.004) in α-MEM+ using small
supplementation (MEM-S) than in the conventional (MEM-C) method (Table 3), but it
was similar (P>0.05) to TCM-199+ treated groups. Follicles with a growth rate that
ranged from 1 to 4.9 m had higher (P<0.02) estradiol concentration in the α-MEM+
medium than in the TCM-C treatment regardless of the method for medium change
used, but the concentration was similar (P>0.05) to the TCM-S (Table 4). Moreover,
follicles that grew ≥5 µm produced more (P<0.0001) estradiol in the MEM-S than in all
other treatments. Regardless of the type of treatment, the growth rate categories did not
affect (P>0.05) the estradiol concentrations.
Table 3. Frequency of slow (<1 µm/day), medium (1 to 4.9 µm/day), and fast ( 5
µm/day) growth rates of bovine follicles after 32 days of in vitro culture in two media
(α-MEM+ or TCM-199+) using two medium replacement methods (Conventional-C or
Small Supplementation-S).
Frequency1 (%)
Growth
rate
MEM-C
MEM-S
TCM-C
TCM-S
(µm/day)
n=66
n=68
n=37
n=36
<1
19 (28.8)Aa
13 (19.1)Aa
7 (18.9)Aa
5 (13.9)Aa
1 to 4.9
33 (50.0)Ab
24 (35.3)Ab
16 (43.2)Ab
17 (47.2)Ab
≥5
14 (21.2)Ba
31 (45.6)Ab
14 (37.8)ABab
14 (38.9)ABb
1
Frequency of all the four repetitions.
A,B
a,b
Within rows, frequencies of a growth rate differed (P<0.004) among treatments.
Within columns, the frequency of growth rates differed (P<0.05).
153
Table 4. Mean (± SEM) estradiol concentrations (ng/ml) produced by bovine follicles in
α-MEM+ or TCM-199+ using two medium replacement methods (Conventional-C or
Small Supplementation-S) according to the speed of the growth rate after 32 days of in
vitro culture.
Growth
Estradiol concentration (ng/ml)
rate
MEM-C
MEM-S
TCM-C
TCM-S
(µm/day)
n=13
n=14
n=12
n=14
<1
2.0 ± 1.2Aa
11.5 ± 6.2Aa
0.9 ± 0.3Aa
3.1 ± 1.5Aa
1 to 4.9
3.6 ± 0.4Aa
9.4 ± 2.4Aa
1.1 ± 0.3Ba
2.1 ± 0.5ABa
≥5
3.8 ± 0.9Ba
15.8 ± 4.0Aa
2.1 ± 0.5Ba
2.6 ± 0.2Ba
A,B
a
Within rows, estradiol concentrations differed (P<0.02) among treatments.
Within columns, estradiol concentrations did not differ (P>0.05).
When evaluating subgroups of follicles that formed antrum in vitro (Table 5),
higher (P<0.0001) estradiol concentration was observed for both α-MEM+ treatments
than the TCM-199+ and in the MEM-S was higher (P<0.0001) than in the MEM-C. In
addition, in those follicles that did not form antrum in vitro the MEM-S produced more
(P<0.04) estradiol than the TCM-C treatment.
Table 5. Mean (± SEM) estradiol concentrations (ng/ml) produced by antral versus no
antral bovine follicles in α-MEM+ or TCM-199+ using two medium replacement
methods (Conventional-C or Small Supplementation-S) after 32 days of in vitro culture.
Estradiol concentrations (ng/ml)
Antrum cavity
MEM-C
MEM-S
n=13
n=14
Ba
Yes
4.4 ± 0.5
No
2.7 ± 0.6ABb
A-C
a-b
TCM-C
16.2 ± 3.5
TCM-S
n=12
Aa
9.9 ± 2.8Aa
2.1 ± 0.5
n=14
Ca
1.2 ± 0.2Ba
2.3 ± 0.5Ca
2.6 ± 0.5ABa
Within rows, estradiol concentrations differed (P<0.04) among treatments.
Within columns, estradiol concentrations differed (P<0.03).
154
Expression of mRNA for FSHR, IGF1, VEGF, and P450AROM on bovine
secondary follicles cultured in vitro
Expression of mRNA for FSHR, IGF1, VEGF, and P450AROM before (noncultured group) and after culture period in all the treatments is shown in Figure 3. The
expression for FSHR was greater (P<0.03) in the non-cultured control and TCM-C
treatments than in the TCM-S. The expression for IGF1 was higher (P<0.02) in MEM-S
than in non-cultured control and TCM-S. The mRNA expression for VEGF was higher
(P<0.02) in MEM-C than in TCM-C and was only observed in these two treatments. No
significant (P>0.05) difference in mRNA expression for P450AROM was observed
among treatments.
Figure 3. Relative mRNA expression (mean ± SEM) for FSHR, IGF1, VEGF, and
P450AROM at days 0 and 32 of in vitro culture.
A,B
Relative mRNA expression differed (P<0.05) among groups.
No expression of VEGF was detected in fresh, MEM-S, and TCM-S groups.
No difference (P>0.05) was observed for P450AROM among all groups.
155
DISCUSSION
The data presented in this study demonstrate for the first time that the base
medium (TCM-199+ vs. α-MEM+), as well as the method to renew the medium during
the culture period affect the follicular development, estradiol concentrations, and gene
expression in bovine secondary follicles. In the conventional method of medium
replacement, both TCM-C and MEM-C had equivalent efficiency for the percentage of
morphological normal follicles and follicular growth rate. Although estradiol
concentration was higher in MEM-C than TCM-C, a larger follicular diameter and
higher percentage of antrum formation was observed in TCM-C when compared with
MEM-C. These results clearly show that the type of base culture medium differently
affect the most common parameters used to evaluate the efficiency of in vitro culture of
bovine preantral follicles. It is known that both α-MEM and TCM-199 are culture media
with rich composition, including aminoacids, vitamins, salt, and precursors of DNA
(ribonucleosides and deoxynucleosides). Moreover, the addition of FSH and insulin or
ITS to the culture medium greatly improved the development of bovine secondary
follicles cultured in α-MEM (Hulshof et al., 1995), TCM-199 (Katska and Rynska,
1998), and McCoy's (McLaughlin and Telfer, 2010) media, respectively. The TCM-199
has been used as a culture medium for ovarian cells in several species, such as mice
(Abedelahi et al., 2010), cattle (Rossetto et al., 2012), goat (Zhou et al., 2008), sheep
(Arunakumari et al., 2010), and gilts (Antosik et al., 2010). A recent study (Rossetto et
al., 2012), demonstrated that TCM-199+ was the best option in oil-free culture system of
bovine secondary follicles, characterized by the maintenance of follicular viability and
higher rates of antrum formation. However, the basement membrane integrity,
uniformly distributed organelles (especially mitochondria and endoplasmic reticulum),
and intact oocyte nucleus did not show any difference after ultrastructural analysis
between follicles cultured in TCM-199+ and α-MEM+.
An important finding of the current study was related to the medium change
approach. In this regard, it was demonstrated that a progressive addition of medium
(small supplementation) was advantageous when α-MEM+ base medium was used.
Follicular diameter and the percentage of antral formation increased in MEM-S when
compared to MEM-C. Using the small supplementation method, the MEM-S became
equivalent to both TCM-C and TCM-S treatments, except in the case of estradiol
concentration, which remained higher in α-MEM+. The difference between
supplementation and conventional medium change methods for estradiol production in
156
the α-MEM+ base medium may suggest a certain degree of dilution, as well as the
removal of content by discard of the culture media when the conventional media was
used, which could be considered harmful to follicle development throughout the 32 days
of culture. The renewal of medium throughout the in vitro culture of preantral follicles
has been used with success, leading to embryo production in goats (Saraiva et al., 2010;
Magalhães et al., 2011), sheep (Arunakumari et al., 2010; Luz et al., 2011), buffalo
(Gupta et al., 2008), and monkeys (Xu et al., 2011). However, the periodic renewal of
culture medium is laborious and may be detrimental to embryonic development by
removing embryo-derived growth factors, changing pH and gas atmosphere, and
lowering the temperature of culture medium (Fukui et al., 1996). The increase in
medium volume and the addition of new nutrients have been shown to be essential to
support the growth and development of bovine preantral follicles. Recently, using αMEM+ as a base culture medium, our team demonstrated that periodic addition of
culture medium, without renewal of medium, during the in vitro culture of caprine
secondary follicles improved the follicular and oocyte development followed by higher
fully grown oocyte rate and meiotic resumption (Araújo et al., 2011). These results
suggested that locally produced factors accumulated during the in vitro culture may
positively influence follicular and oocyte growth.
The composition of base medium interfered in the gene expression, because the
mRNA expression for IGF1 was higher in MEM-S than in non-cultured control and
TCM-S and for VEGF was higher in MEM-C than TCM-C. In this regard, the increase
of estradiol concentration in α-MEM+ was associated with increase of mRNA
expression for IGF1 and VEGF. In case of IGF1 mRNA expression, while there were
no difference in antral formation between MEM-S and TCM-S, it was observed that
MEM-S produced more estradiol than TCM-S. Considering that IGF-1 stimulated the
antrum formation and steroidogenesis after in vitro culture of buffalo preantral follicles
(Sharma et al., 2010) and that bovine antral follicles expressed more IGF binding than
secondary follicles (Wandji et al., 1992), we believe that the higher mRNA expression
of IGF1 in the MEM-S may have been associated to the presence of antral follicles and
the higher estradiol concentrations after in vitro culture. However, the accumulation of
estradiol in the small supplementation medium replacement method (MEM-S and
TCM-S) may have inhibited the VEGF production because VEGF expression was not
detected. In swine, VEGF increased and inhibited estradiol output by granulosa cells
from small and large follicles, in vitro, respectively (Grasselli et al., 2002; 2003). In this
157
regard, it could be speculated that in the present study the in vitro grown antral follicles
behaved as in vivo conditions, and that the increase of estradiol concentrations, by
adding medium during the culture, could have been associated with an inhibition of
VEGF production in both MEM-S and TCM-S.
In conclusion, the base medium, as well as periodic addition of medium (small
supplementation method) differently affected follicle development, estradiol production,
and gene expression. Furthermore, α-MEM+ can be used as alternative to TCM-199+ if a
progressive addition of medium change is used during in vitro culture. Therefore, these
results suggest that studies on comparisons among different preantral follicle culture
systems should not only focus on medium supplements (e.g., hormones and growth
factors) but also on the differences among the types of base media and medium
replacement methods.
Acknowledgments
The authors thank Mr. Alvin Kasten for providing the ovaries at the
slaughterhouse.
Conflict of interest
There is no conflict of interest that could be perceived as prejudicing the
impartiality of the research reported.
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10 CAPÍTULO 5
Proteína morfogenética óssea-6 (BMP-6) induz atresia em folículos primordiais
caprinos cultivados in vitro
“Bone Morphogenetic Protein-6 (BMP-6) induces atresia in goat primordial follicles
cultured in vitro”
Periódico: Pesquisa Veterinária Brasileira, v. 30, p. 770-776, 2010.
163
RESUMO
O presente estudo investigou os efeitos da proteína morfogenética óssea-6 (BMP-6) no
desenvolvimento in vitro de folículos primordiais caprinos. Amostras de córtex
ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (meio
controle) suplementado com diferentes concentrações de BMP-6. As taxas de
sobrevivência, ativação e crescimento foram avaliadas por histologia clássica e
microscopia eletrônica de transmissão (MET). Após 7 dias de cultivo, a análise
histológica demonstrou que a BMP-6 aumentou o percentual de folículos primordiais
degenerados no dia 7 quando comparados ao controle fresco (D0). Além disso, houve
um aumento significativo do diâmetro folicular e oocitário em ambos os períodos de
cultivo em todos os tratamentos na presença de BMP-6. Com a progressão do cultivo do
dia 1 para o dia 7, nos tratamentos com 1 ou 50 ng/ml de BMP-6, foi observado um
aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio
controle, a MET revelou que os folículos cultivados nesses tratamentos apresentavam
sinais evidentes de atresia. Em conclusão, esse estudo demonstrou que a BMP-6 afetou
negativamente a sobrevivência e a ultraestrutura de folículos primordiais caprinos.
Palavras-chave: Superfamília TGF-ß. Foliculogênese. Célula germinativa. Fatores de
crescimento. Caprinos.
164
Bone Morphogenetic Protein-6 (BMP-6) induces atresia in
goat primordial follicles cultured in vitro1
Valdevane Rocha Araújo2*, Isabel Bezerra Lima-Verde2, Khessler Patrícia Olazia
Name3, Sônia Nair Báo3, Cláudio Cabral Campello2, José Roberto Viana Silva4, Ana
Paula Ribeiro Rodrigues2 and José Ricardo de Figueiredo2
ABSTRACT.- Araújo V.R., Lima-Verde I.B., Name K.P.O., Báo S.N., Campelo C.C.,
Silva J.R.V., Rodrigues A.P.R. & Figueiredo J.R. 2010. Bone Morphogenetic Protein6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro. Pesquisa
Veterinária Brasileira 30(9):770-776. Programa de Pós-Graduação em Ciências
Veterinárias, Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais,
Universidade Estadual do Ceará, Av. Paranjana 1700, Campus do Itaperi, Fortaleza, CE
60740-000, Brasil. *Corresponding author: val_exclusiva@yahoo.com.br
This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on
in vitro primordial follicle development in goats. Samples of goat ovarian cortex were
cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium)
supplemented with different concentrations of BMP-6. Follicle survival, activation and
growth were evaluated through histology and transmission electron microscopy (TEM).
After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the
percentages of atretic primordial follicles when compared to fresh control (day 0).
Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture
periods. As the culture period progressed from day 1 to day 7, a significant increase in
follicle diameter was observed with 1 or 50 ng/ml BMP-6. However, on the contrary to
that observed with the control medium TEM revealed that follicles cultured for up to 7
days with 1 or 50 ng/ml BMP-6 had evident signs of atresia. In conclusion, this study
demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat
primordial follicles.
INDEX TERMS: TGF-ß superfamily, folliculogeneis, germ cell, growth factors, caprine.
___________________
1
Received on May 19 and September 22, 2009.
165
Accepted for publication on May 5, 2010.
2
Programa de Pós-Graduação em Ciências Veterinárias (PPGCV), Laboratório de Manipulação de
Oócitos e Folículos Pré-Antrais (Lamofopa), Universidade Estadual do Ceará (UECE), Av. Paranjana
1700,
Campus
do
Itaperi,
Fortaleza,
CE
60740-000,
Brasil.
*Corresponding
author:
val_exclusiva@yahoo.com.br
3
Laboratório de Microscopia Eletrônica, Departamento de Biologia Celular, Instituto de Ciências
Biológicas, Universidade de Brasília, 70919-970 Brasilia, DF, Brasil.
4
Núcleo de Biotecnologia de Sobral (Nubis), Faculdade de Medicina de Sobral, Universidade Federal
do Ceará, Av. Geraldo Rangel 100/186, Sobral, CE 60041-040, Brasil.
166
INTRODUCTION
Folliculogenesis results from a complex balance among proliferation,
differentiation, and cell death of both the somatic and germ cell compartments of the
follicle (Hussein et al. 2005). This process is controlled by gonadrotophins and locally
produced growth factors, such as bone morphogenetic proteins (BMPs). The BMP
family is the largest within the TGF-ß superfamily of growth factors and several studies
have demonstrated that the BMPs regulate growth, differentiation, and apoptosis in a
wide variety of tissues, including the ovary (Shimasaki et al. 2004, Araújo et al. 2010).
The BMP-6 protein is expressed in oocytes (ovine: Juengel et al. 2006, murine:
Otsuka et al. 2001; bovine: Glister et al. 2004; porcine: Brankin et al. 2005a), granulosa
(murine: Erickson & Shimasaki 2003; bovine: Glister et al. 2004; porcine: Brankin et al.
2005a), and theca cells (ovine: Campbell et al. 2004; bovine: Glister et al. 2004) of
ovarian follicles during different stages of development. As part of its biological
function, BMP-6 forms heteromeric complexes with a type I and type II receptor. The
mRNAs for BMP receptors (BMPR-IA, -IB, and II) are expressed in oocytes and
granulosa cells of goat ovarian follicles (Silva et al. 2004) as well as in follicles of other
mammalian species (murine: Elvin et al. 2000, Shimasaki et al. 1999, Erickson &
Shimasaki 2003; ovine: Souza et al. 2002, McNatty et al. 2005; bovine: Glister &
Knight 2002), which is indicative of possible autocrine and paracrine effects during
follicle growth.
In vitro studies have demonstrated that BMP-6 controls steroidogenesis as well
as granulosa and theca cell proliferation in ovine (Juengel et al. 2006) and porcine
(Brankin et al. 2005a). Juengel et al. (2006) established that BMP-6 inhibits ovine
granulosa cell differentiation. In addition, BMP-6 is effective at inhibiting FSH-induced
progesterone synthesis by murine granulosa cells, without affecting estradiol production
(Otsuka et al. 2001). However, the involvement of BMP-6 in the control of ovarian
function in goats and in vitro effects on the development of primordial follicles are still
unknown. The present study was performed to determine the possible role of BMP-6 in
the growth and survival of primordial follicles during the culture of goat ovarian cortical
slices.
167
MATERIALS AND METHODS
The culture media, BMP-6 and other chemicals used in the present study were
purchased from Sigma Chemical Co. (St Louis, MO), unless otherwise indicated.
Source of ovaries
Ovarian cortical tissues were obtained from six mixed-breed goats (n=6)
collected at a local slaughterhouse. Immediately postmortem, the ovaries were washed
in 70% alcohol for 10 seconds followed by two washes in Minimum Essential Medium
(MEM) plus HEPES (MEM HEPES) supplemented with 100 μg/ml penicillin and 100
μg/ml streptomycin. The ovary pairs were transported within 1 hour to the laboratory in
MEM at 4°C (Chaves et al. 2008).
Experimental protocol
The culture system has been described in our previous research (Matos et al.
2007). Ovarian cortical tissue from the same ovarian pair was cut into 11 slices
(3x3x1mm) using a scissor and scalpel under sterile conditions. The tissue pieces were
then either directly fixed for histological and ultrastructural analysis (fresh tissue,
control) or placed into culture medium for 1 or 7 days. Caprine tissues were transferred
to 24-well culture dishes containing 1 ml of culture medium. In vitro culture was
performed at 39°C in a humidified incubator with 5% CO2. The basic culture medium
consisted of MEM (pH 7.2-7.4) supplemented with ITS (10 μg/ml insulin, 5.5 μg/ml
transferrin, and 5 ng/ml selenium), 0.23 mM pyruvate, 2 mM glutamine, 2 mM
hypoxantine, and 1.25 mg/ml of bovine serum albumin BSA. This supplemented
medium was called MEM+. Different concentrations of BMP-6 (0, 1, 10, 50 or 100
ng/ml) were added to the MEM+ to test the effects of this growth factor. Each treatment
was repeated six times and the culture medium was replenished every other day.
Morphological analysis and assessment of in vitro folicular growth
Before culture (fresh control) and after 1 or 7 days in culture, samples were fixed
in Carnoy solution for 12 h and then dehydrated in increasing concentrations of ethanol.
After paraffin (Synth, São Paulo, Brazil) embedding, the ovarian pieces were cut into 7
μm sections and stained by Periodic Acid Schiff-hematoxylin. Follicle stage and
168
survival were assessed microscopically on serial sections. Coded slides were examined
via microscopy (Nikon, Japan) under 400x magnification.
The follicles were classified as primordial (one layer of flattened granulosa cells
around the oocyte) or growing follicle, i.e., primary (a single layer of cuboidal
granulosa cells around the oocyte), or secondary (oocyte surrounded by two or more
layers of cuboidal granulosa cells), as described by Hulshof et al. (1994). These follicles
were classified individually as histologically normal when an intact oocyte was present,
i.e., an oocyte without a pyknotic nucleus or cytoplasmic retraction that is surrounded
by granulosa cells, which are well organized in one or more layers and have no pyknotic
nucleus. Atretic follicles were defined as those with a retracted oocyte, pyknotic
nucleus, and/or disorganized granulosa cells detached from the basement membrane.
Overall, 180 follicles were evaluated for each treatment (30 follicles per treatment x 6
repetitions = 180 follicles).
The percentages of healthy primordial and growing follicles were calculated
before (fresh control) and after culture in each medium. In addition, follicle and oocyte
diameters were only measured in healthy follicles. Follicle diameter was recorded from
one edge of the granulosa cell membrane to the other edge, or from the outside edge of
the theca cell layer when present. Oocyte diameter was recorded from edge to edge of
the oocyte membrane. Two perpendicular diameters were recorded for each and the
average was reported as the follicle and oocyte diameters, respectively. Each follicle
was carefully counted only once, as previously described (Matos et al. 2007).
Ultrastructural analysis of caprine preantral follicles
For better evaluation of the follicular morphology, ultrastructural studies were
performed on fragments of fresh controls (Day 0) and treatments (Day 7) that
maintained follicular morphology during the histological analysis. Small pieces (1 mm3)
of caprine ovarian tissues were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde
in 0.1 M sodium cacodylate buffer (pH 7.2) for 4 h at room temperature. After fixation,
fragments were post-fixed in 1% osmium tetroxide, 0.8% potassium ferricyanide, and 5
M calcium chloride in 0.1 M sodium cacodylate buffer for 1 h. Subsequently, samples
were dehydrated through a gradient of acetone and the tissues were embedded in Spurr.
Semi-thin sections (3 μm) were cut on an ultramicrotome (Reichert Supernova,
Heidelberg, Germany) for light microscopy studies and stained with toluidine blue.
Uranyl acetate and lead citrate were added to the ultra-thin sections (60-70 nm) for
169
contrast, and sections were examined under a Jeol 1011 (Jeol, Tokyo, Japan)
transmission electron microscope. Parameters, such as density and integrity of the
ooplasmic and granulosa cell organelles, vacuolization, and basement membrane
integrity, were evaluated.
Statistical analysis
Means of surviving follicles at all stages, as well as of primordial and
developing follicles (primary or secondary) obtained after 1 or 7 days in the various
treatments, were subjected to analysis of variance (ANOVA), using the GLM procedure
of SAS (1999) and Dunnett’s test applied to compare BMP-6 treated groups against
control and MEM+. The Student-Newman-Keels (SNK test) and t-student was used to
compare differences among BMP-6 concentrations and to compare means between
cultures at 1 and 7 days, respectively. The diameters of oocytes and follicles under the
various treatments were subjected to ANOVA followed by SNK. Differences were
considered to be significant when P<0.05 and data were expressed as mean ± standard
error of means (S.E.M.) (Steel et al. 1997).
RESULTS
Goat primordial follicle activation and growth during in vitro culture
In the present study, a total of 1,980 preantral follicles were analyzed. The
percentages of primordial and growing follicles in non-cultured cortex (fresh control)
were 80.27% and 19.73%, respectively (Table 1). In all treatments, after 1 or 7 days of
culture, there was no significant effect of BMP-6 on follicular activation, as indicated
by the percentage of primordial and growing follicles (P>0.05).
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Table 1. Percentages (mean
S.E.M.) of primordial and growing follicles (primary
and secondary) in uncultured tissues and tissues cultured for 1 or 7 days in MEM+
(control medium) and MEM+ supplemented with various concentrations of BMP-6
Treatments
Primordial follicles
Growing follicles
Uncultured (Day 0)
80.27 ± 10.42
19.73 ± 10.42
Cultured
Day 1
Day 7
Day 1
Day 7
MEM+
81.03
9.70
74.44
14.62
18.97
9.70
25.56
14.62
BMP-6 (1)
76.18
18.96
75.04
9.73
23.81
18.96
24.96
9.73
BMP-6 (10)
84.91
8.12
75.12
10.07
15.09
8.12
24.88
10.07
BMP-6 (50)
73.04
18.58
76.57
18.75
26.96
18.58
23.42
18.75
BMP-6 (100)
76.89
14.31
78.61
15.06
23.11
14.31
21.39
15.06
Follicle and oocyte diameters before and after in vitro culture are shown in Table
2. In comparison to uncultured control values (fresh control), a significant increase in
follicle diameter was observed after culturing ovarian tissue in all treatments (P<0.05),
except in the control medium (MEM+) alone or medium supplemented with 1 ng/ml
BMP-6 after culturing for 1 day. No significant differences (P>0.05) in follicle and
oocyte diameters were found when treatments were compared with control medium
(MEM+), except for 50 ng/ml BMP-6 at day 1 of culture in regards to oocyte diameter.
As the culture period progressed from 1 to 7 days, a significant increase in follicle
diameter was observed in ovarian tissues cultured in the presence of 1 or 50 ng/ml
BMP-6 (P<0.05). Similar results were observed for oocyte diameter in ovarian tissues
cultured in medium supplemented with only BMP-6 (1 ng/ml). After 1 or 7 days of
culture, the addition of BMP-6 significantly increased oocyte diameter compared to
uncultured control (P<0.05). With the increase in culture period from 1 to 7 days,
tissues cultured in medium supplemented with 1 ng/ml BMP-6 had a significant
increase in oocyte diameter (P<0.05).
171
Table 2. Follicle and oocyte diameters (mean
S.E.M.) in uncultured tissues and
tissues cultured for 1 or 7 days in MEM+ (control medium) and MEM+
supplemented with various concentrations of BMP-6. For each treatment, 20
follicles were evaluated
Treatments
Follicle diameter (μm)
Oocyte diameter (μm)
Uncultured (Day 0)
31.08±5.44
20.82±2.00
Cultured
Day 1
Day 7
Day 1
Day 7
MEM+
34.33±6.04A
36.79±5.91*,A
22.48±1.75A
23.86±2.58*,A
BMP-6 (1)
34.87±4.53B
40.22±6.68*,A
23.41±2.10*,B
25.56±2.50*,A
BMP-6 (10)
38.23±7.88*,A
37.93±6.74*,A
24.05±2.62*,A
23.87±2.47*,A
BMP-6 (50)
36.43 ± 5.04*,B
39.86±5.05*,A
24.41±1.91*,,A
25.19±1.26*,A
BMP-6 (100)
36.25 ± 4.64*,A
37.99±4.14*,A
23.98±2.04*,A
24.77±2.12*,A
* P<0.05, significantly different from uncultured ovarian cortical tissues (control/Day 0)
 P<0.05, significantly different from cultured ovarian cortex tissue in MEM.
(A, B) Different letters in the same row denote significant differences between culture
periods within the same medium (P<0.05).
Effect of BMP-6 and culture periods on follicle atresia
180 follicles were evaluated per treatment, resulting in a total of 1,451
primordial, 459 primary, and 70 secondary follicles analyzed. Figure 1 shows the effects
of diferente concentrations of BMP-6 (1, 10, 50, or 100 ng/ml) on the percentages of
morphologically atretic follicles after 1 or 7 days of culture. After 1 day, cultured
ovarian tissue in all medium maintained percentages of healthy follicles similar to
uncultured tissue (fresh control). However, a significant increase in the percentages of
atretic follicles was only observed in follicles cultured in all medium supplemented with
BMP-6 (P<0.05) after 7 days. With the increase in culture period from 1 to7 days, a
significant increase in the percentage of atretic follicles was observed in tissue cultured
in medium supplemented with 10, 50, or 100 ng/ml BMP-6. Figure 2 shows normal
follicles before culture (Fig. 2A), and degenerated follicles after 7 days of culture in the
presence of 50 ng/ml BMP-6 (Fig. 2B).
172
Fig.1. Percentages (means
S.E.M) of atretic preantral follicles in uncultured tissue
(fresh control) and tissue cultured for 1 and 7 days in MEM+ and MEM+ supplemented
with 1, 10, 50, and 100 ng/mL BMP-6. For each treatment, 30 follicles were evaluated
in each of five replicates. *P<0.05, significantly different from uncultured ovarian cortex
tissue (control/D0). (A, B) Different letters denote significant differences between culture
periods within the same medium (P<0.05).
Fig.2. Histological section of (A) normal follicles from uncultured tissue and, (B) atretic
follicles after culture in the presence of BMP-6 O: oocyte; NU: oocyte nucleus; GC:
granulosa cells. Staining with periodic acid Schiff-hematoxylin, 400x.
173
Ultrastructural analysis of goat preantral follicles
To better evaluate follicle quality before and after culture, TEM was performed
to study the ultrastructure of follicles that were considered morphologically normal
during histological analysis of the uncultured control and cells cultured for 7 days with
MEM+
alone and supplemented with 1 or 50 ng/ml BMP-6. Figures 3A and 3B
illustrate morphologically normal follicles from the uncultured control and cells
cultured only in MEM+, with intact nuclear and cytoplasmic membranes, as well as a
small number of vacuoles and various organelles, including mitochondria and
endoplasmic reticulum without degenerative signs. Figures 3C and 3D show follicles
cultured up to 7 days with 1 or 50 ng/ml BMP-6, respectively. BMP-6 negatively
affected the ultrastructure of follicles cultured for 7 days, since evident signs of
degeneration were observed. In these follicles, a large number of vacuoles and a low
density of organelles were observed in the ooplasm. In addition, granulosa cells lost gap
junctions, reducing the contact with the oocyte membrane. Moreover, follicles cultured
in medium supplemented with 1 ng/ml BMP-6 for 7 days that were morphologically
normal at the histological level had ultrastructural signs of degeneration, including
irregularly shaped nuclear and cytoplasmic membranes. As the culture period
progressed from 1 to 7 days, a large increase in the number of vacuoles and broken
nuclear membranes were occasionally observed. Fragmented granulosa cells associated
with empty areas between oocyte and granulosa cells were also observed, which is
indicative of lost cellular communication.
174
Fig.3. Electron micrograph of caprine preantral follicle from (A) uncultured control
(5800x), (B) MEM+ alone, (C) 1 ng/ml of BMP-6, and (D) 50 ng/ml of BMP-6 cultured
(8000x) for 7 days. Homogeneous cytoplasm with numerous rounded mitochondria is
characteristic of non-cultured follicles and cultures with only MEM+ (3A and 3B,
respectively). Extreme vacuolization and great holes are present in the cytoplasm,
indicative of degeneration (3C and 3D; solid arrow). Note the empty space in
degenerated granulosa cells after in vitro culture with BMP-6 (3C and 3D; open arrow).
NU: oocyte nucleus, GC: granulosa cells, m: mitochondria, ser: smooth endoplasmic
reticulum, v: vesicle.
175
DISCUSSION
This study demonstrates for the first time that BMP-6 is not involved in the
initiation of the growth in vitro of goat primordial follicles. The concentrations of BMP6 used in this experiment (1, 10, 50 and 100 ng/ml) were based on previous works on
ovarian somatic cells culture (thecal and granulosa cells, Brankin et al. 2005b, Glister et
al. 2005), since, to our knowledge, no report regarding physiological levels of this factor
within the ovary or in plasma has been published.
Ovarian follicle development is regulated by gonadotropins and local growth
factors that interact and promote oocyte growth and granulosa cell proliferation and
differentiation (Krysko et al. 2008). Various growth factors are well known to be
involved in primordial follicle activation (Kit Ligand: Parrott & Skinner 1999; BMP-7:
Lee et al. 2001, 2004; FGF-2: Nilsson et al. 2001), but several results have indicated
that the early stage of folliculogenesis is controlled by a balance between stimulatory
and inhibitory factors. Despite the expression of mRNA for BMP receptors by goat
primordial follicles (Silva et al. 2004), the translation of BMP receptor mRNAs into
proteins is still uncharacterized. Proliferation of granulosa cells is critical for primordial
follicle growth, but according to Otsuka et al. (2001), BMP-6 did not promote granulosa
cell proliferation in murine cells. BMP-6 inhibits steroidogenesis and differentiation of
ovine granulosa cells, but did not affect proliferation (Juengel et al. 2006). Conversely,
in bovine cells, Glister et al. (2004) demonstrated an increase in granulosa cell number
after a 6 day culture period in medium supplemented with BMP-6. The differential
response of granulosa cells to BMP-6 may be a result of these cells being obtained from
large antral follicles with differentiated granulosa cells, while granulosa cells that come
from primordial follicles are still quiescent.
Histological analysis demonstrated that BMP-6 promoted an increase in
follicular and oocyte diameter in surviving follicles, but TEM did not confirm the
ultrastructural integrity of these follicles, since oocyte and granulosa cells had evident
signs of atresia. The increase in follicle diameter was most likely caused by swollen
oocyte and granulosa cells. The different concentrations of BMP-6 utilized in this study
were toxic for preantral follicles cultured in vitro up to 7 days, since culture using only
MEM+ presented follicles with preserved ultrastructural integrity. Several reports have
emphasized the importance of ultrastructural analysis after culturing preantral follicles
in vitro (cow: Van Den Hurk et al. 1998; rat: Zhao et al. 2000; mouse: Salehnia et al.
176
2002; goat: Matos et al. 2007, Araújo et al. 2010). In the presente study, cultured
follicles were characterized by a large number of vacuoles, the absence of organelles in
the ooplasm, as well as irregular or fragmented nuclear and cytoplasmic membranes. In
addition, fragmented granulosa cells with no oocyte contact were observed. Similar
results were observed after culturing goat preantral follicles in medium containing
indole-3-acetic acid (IAA: Matos et al. 2006).
This study demonstrates for the first time that BMP-6 promotes atresia in goat
primordial follicles during the in vitro culture of ovarian cortical tissues for 7 days. In
contrast, Brankin et al. (2005a) determined that BMP-6 maintained the pig theca cell
viability during a culture period of 6 days. However, differences in cell types and
animal species must be considered to understand the modes of BMP-6 action. Fortune et
al. (2004) established that supplementation of culture medium with IGF-I increased
atresia in bovine primordial follicles cultured in vitro. The presence of growth factor in
culture medium can induce atresia in primordial follicles depending on the species,
follicle stage, and culture system. Several reports have suggested that preantral follicle
(primordial, primary, and secondary) viability is determined by the secretion of growth
factors by oocyte and/or granulosa cells (GDF-9: Dong et al. 1996; KL, EGF and FGFb:
Reynaud & Driancourt 2000). According to Silva et al. (2002), 8.5% of primordial
follicles within goat ovaries are atretic. Utilizing the TUNEL technique to detect
apoptosis in goat follicles after the culture of ovarian tissue, these results indicated that
apoptosis in early follicles could be provoked by reduced oxygen and nutrient diffusion
for preantral follicles within ovarian cortex (Silva et al. 2006).
In conclusion, BMP-6 promotes primordial follicle atresia during the culture of
goat ovarian cortical tissue. In addition, these results verified that morpho-functional
analysis by TEM allows for a marked improvement in the evaluation of caprine ovarian
tissue integrity. Further studies are necessary to determine the appropriate culture
system conditions that promote the activation of primordial follicles and the further
growth of caprine oocytes by preservation of ultrastructural integrity.
Acknowledgements.- To Cleidson M.G. Silva, Deborah M. Magalhães, Gerlane M.
Silva and Liliam M.T. Tavares. This study was supported by Fundação Cearense de
Apoio à Pesquisa (Funcap) and the International Foundation for Science. Valdevane R.
Araújo is a recipient of a grant from CAPES (Brazil).
177
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11 CAPÍTULO 6
Efeito da proteína morfogenética óssea-6 (BMP-6) e do hormônio folículo
estimulante (FSH) durante o desenvolvimento in vitro de folículos pré-antrais
ovarianos caprinos, e expressão relativa de RNAm para os receptores de BMP e
Smads em folículos cultivados
“The Effects of Bone Morphogenetic Protein-6 (BMP-6) and Follicle-Stimulating
Hormone (FSH) During In Vitro Development of Ovarian Caprine Preantral
Follicles, and the Relative mRNA Expression of BMP Receptors and Smads in
Cultured Follicles”
Periódico: Molecular and Cellular Endocrinology (Submetido em: 1 de maio de 2013).
182
RESUMO
O presente estudo investigou o efeito da proteína morfogenética óssea-6 (BMP-6) e do
hormônio folículo estimulante recombinante (rFSH) sozinho ou em combinação durante
o cultivo in vitro (CIV) de folículos secundários caprinos isolados e os níveis de RNAm
para a via de sinalização BMP/Smad signaling (bmpr1A, bmpr2, smad1, smad4, smad5,
smad6, smad7 e smad8) antes e após o CIV. Folículos secundários foram cultivados em
αMEM+ sozinho (controle) ou suplementado com BMP-6 nas concentrações de 1 e 10
ng/mL. A formação de antro foi significativamente superior no tratamento com adição
de 1 ng/mL de BMP-6 quando comparado ao αMEM+. A expressão de RNAm para
bmpr2, smad1, smad5 e smad6 foi detectada antes (controle não cultivado) e após o
CIV (αMEM+ e 1 ng/mL de BMP-6), enquanto que smads7 e smad8 foram detectadas
apenas após CIV e a Smad4 foi detectada apenas no tratamento com adição de BMP-6.
Em conclusão, baixas concentrações de BMP-6, sem adição de FSH, influenciaram
positivamente a formação de antro e asseguraram um completo padrão de expressão
para os receptores de BMP e Smads após CIV de folículos secundários caprinos.
Palavras-chave: Formação de antro. BMP. Cabra. Competência meiótica. Smad.
183
The Effects of Bone Morphogenetic Protein-6 (BMP-6) and FollicleStimulating Hormone (FSH) During In Vitro Development of Ovarian
Caprine Preantral Follicles, and the Relative mRNA Expression of
BMP Receptors and Smads in Cultured Follicles
V.R. AraújoA,D, G.M. SilvaA, A.B.G. DuarteA, D.M. Magalhães-PadilhaA, A.P.
AlmeidaB, F.O. LunardiA, M.K.B. SerafimA, A.A.A. MouraC, C.C. CampelloA, A.P.R.
RodriguesA and J.R. FigueiredoA
A
Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and Preantral
Follicles (LAMOFOPA), State University of Ceará, Fortaleza, 60740-903, CE, Brazil.
B
Laboratory of Molecular Biology and Development, University of Fortaleza, 60811-
905, CE, Brazil.
C
Laboratory of Animal Physiology, Department of Animal Science, Federal University
of Ceará, Fortaleza, 60440-900, CE, Brazil.
D
Corresponding author. E-mail: val_exclusiva@yahoo.com.br (Valdevane R. Araújo)
Highlights: BMP receptors/Smad signaling in caprine preantral follicles
184
ABSTRACT
This study investigated the effect of bone morphogenetic protein (BMP-6) and
recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in
vitro culture (IVC) of isolated caprine secondary follicles and the mRNA levels for
BMP receptors/Smad signaling (bmpr1A, bmpr2, smad1, smad4, smad5, smad6, smad7
and smad8) before and after IVC. Secondary follicles were cultured in αMEM+ alone
(control) or supplemented with BMP-6 at 1 or 10 ng/mL and rFSH alone or in
combination. The antrum formation was higher in the BMP-6 at 1 ng/mL (P<0.05) than
in MEM. The mRNA expression for bmpr2, smad1, smad5 and smad6 was detected
before (non-cultured control) and after IVC (MEM and 1 ng/mL BMP-6), while smads7
and smad8 mRNA expression was only detected after IVC and smad4 was only detected
in the BMP-6 treatment. In conclusion, the low BMP-6 concentration positively
influenced antrum formation and ensured complete mRNA expression for BMP
receptor and Smads after IVC of secondary follicles.
Keywords: Antrum formation; BMP; Goat; Meiotic competence; Smad
185
1. Introduction
Somatic cell proliferation, differentiation and cellular death occur in all follicular
compartments, including oocytes, granulosa and theca cells, during folliculogenesis, and
these processes are controlled through gonadotropins and growth factors locally
produced in the ovary. Oocyte-secreted factors, such as bone morphogenetic proteins-6
(BMP-6; Juengel et al., 2006) and BMP-15 (Guéripel et al., 2006), are essential to
normal follicular growth and fertility in sheep (Galloway et al., 2000; Juengel et al.,
2002; Hanrahan et al., 2004; Juengel et al., 2006) and humans (Di Pasquale et al.,
2004). In addition, gonadotropins such as FSH have been implicated in the regulation of
apoptosis in follicular cells and in the ultrastructure maintenance of preantral follicles in
goats (Matos et al., 2007; Magalhães et al., 2009) and cows (Wandji et al., 1996;
Gutierrez et al., 2000).
The signaling of BMP-6 occurs in response to the formation of specific
heteromeric complexes of type I (BMPR-IA, also known as ALK-3, and BMPR-IB, also
known as ALK-6) and type II (BMPR-II) serine/threonine kinase receptors. In the
ovary, the mRNAs for BMP receptors are present in oocytes and the granulosa cells of
caprine ovarian follicles (Silva et al., 2004) and in murine (Elvin et al., 2000, Shimasaki
et al., 1999, Erickson and Shimasaki, 2003), ovine (Souza et al., 2002, McNatty et al.,
2005) and bovine (Glister and Knight, 2002) follicles. The BMP receptors
phosphorylate intracellular effectors, called Smads. Functionally, the Smads fall into
three subfamilies: receptor-activated Smads (R-Smads: Smad1, Smad5, and Smad8),
which are phosphorylated through BMPR-I; the common mediator Smad (Co-Smad:
Smad4), which oligomerizes with activated R-Smads; and inhibitory Smads (I-Smads:
Smad6 and Smad7), which exert a negative feedback effect through competition with RSmads for receptor interactions, which indicate the receptors for degradation
(Moustakas et al., 2001). As part of its biological function, BMP-6 complexes with type
I and type II BMP receptors and Smads, which determine its possible autocrine and
paracrine effects during follicle growth.
The BMP-6 has been implicated in steroidogenesis, as this factor reduces
progesterone production in granulosa cells through the inhibition of the premature
luteinization of the dominant follicles in cows (Glister et al., 2004) and rats (Otsuka et
al., 2001). In addition, the production of others steroids, such as androstenedione and
estradiol, was inhibited after the addition of BMP-6 in the culture medium of swine
186
granulosa and theca cells (Brankin et al., 2005). However, the combination of BMP-6
and FSH increases the action of gonadotropin, as there is no change in the estradiol
production in murine cells in vitro (Otsuka et al., 2001).
Recently, Frota et al. (2011) showed that the addition of FSH to the culture
medium increased the mRNA expression for bmp6 gene in isolated caprine preantral
follicles. In other study, Costa et al. (2012) studied the mRNA expression for smad1,
smad5, smad8, and bmp2, bmp4, bmp6, bmp7, and bmp15, and bmpr1A, bmpr1B and
bmpr2 genes in a basic culture medium containing FSH. However, to our knowledge
there are no reports in goats regarding the effect of BMP-6 on the mRNA expression for
BMP receptors/Smad signaling (bmpr1A, bmpr2, smad1, smad4, smad5, smad6, smad7,
and smad8) in the in vitro culture of caprine isolated secondary follicles. Indeed, we
reported the first use of BMP-6 in the culture medium of primordial follicles (Araújo et
al., 2010), in which the follicles were cultured enclosed in the caprine ovarian cortex,
i.e., in situ.
Thus, considering the differences between the isolated and in situ culture
systems, the aims of the present study were 1) to investigate the effect of BMP-6 and
FSH alone or in combination on the in vitro culture of caprine isolated secondary
follicles and 2) to verify the mRNA levels for BMP receptors/Smad signaling (bmpr1A,
bmpr2, smad1, smad4, smad5, smad6, smad7, and smad8) before and after in vitro
culture.
2. Materials and Methods
2.1. Chemicals and media
Unless mentioned otherwise, the culture media, ascorbic acid and other
chemicals used in the present study were purchased from Sigma Chemical Co. (St
Louis, MO, USA).
2.2. Source of ovaries
The ovaries (n = 50) from 25 adult mixed-breed goats (one to three years old)
were collected at a local slaughterhouse (Fortaleza, State of Ceará, Brazil). The ovaries
were washed in 70% alcohol for 10 seconds, and then washed twice in minimum
187
essential medium (MEM) supplemented with 100 µg/mL penicillin and 100 µg/mL
streptomycin and buffered using HEPES (MEM-HEPES). The ovaries were placed into
tubes containing 15 mL of MEM-HEPES and transported to the laboratory at 4°C
within one hour.
2.3. Isolation and selection of caprine secondary follicles
In the laboratory, the surrounding fat tissue and ligaments were stripped from the
ovaries. Ovarian cortical slices (1 mm thick) were cut from the ovarian surface using a
surgical blade under sterile conditions. Subsequently, the ovarian cortex tissues were
washed in fragmentation medium comprising MEM-HEPES. Preantral follicles ≥ 200
m in diameter were visualized under a stereomicroscope (SMZ 645 Nikon, Tokyo,
Japan) and manually dissected from the ovarian cortex using 27.5 gauge (27.5 G)
needles and the microdissection technique. During the evaluation of follicular quality,
only secondary follicles with a visible central oocyte, surrounded by two or more
granulosa cells layers, an intact basement membrane and no antral cavity, were selected
for culture.
2.4. Culture of isolated caprine secondary follicles
The isolated secondary follicles were cultured for 18 days at 39°C in 5% CO2 in
air. The fresh media were incubated for at least one hour prior to use. Every other day,
60 µL of medium was replenished in each drop, and at days 6 and 12 of culture, all
medium (100 µL) was replenished with fresh medium. αMEM medium (pH 7.2 - 7.4)
was supplemented with 3 mg/mL bovine serum albumin (BSA), ITS (10 µg/mL insulin,
5.5 µg/mL transferrin and 5 ng/mL selenium), 2 mM glutamine, 2 mM hypoxanthine
and 50 μg/mL of ascorbic acid to generate αMEM+, was used.
2.5. Experimental design
Selected follicles were individually cultured in 100-µL drops of culture medium
in Petri dishes (60 x 15 mm, Corning, USA) and randomly assigned to six different
treatments. Follicles cultured in only αMEM+ (control medium) or control medium
supplemented with BMP-6 at 1 ng/mL or 10 ng/mL and recombinant follicle-
188
stimulating hormone (rFSH) alone or in combination with both BMP-6 concentrations
(1 ng/mL or 10 ng/mL) associated with FSH.
The rFSH® (Nanocore, Campinas, São Paulo, Brazil) was used in increasing
concentrations throughout the culture period, corresponding to sequential FSH (100
ng/mL from day 0 to day 6, 500 ng/mL from day 6 to day 12 and 1000 ng/mL from day
12 to day 18). The concentrations of rFSH® (Saraiva et al., 2011), ascorbic acid (Silva
et al., 2011) and BMP-6 (Araújo et al., 2010) were chosen based on previous studies
performed in our laboratory. The culture was replicated five times, and at least 40
follicles were used per treatment.
2.6. Morphological evaluation of follicle development
The follicles were classified according to morphology; abnormal follicular
morphology was characterized as a rupture of the basement membrane, extrusion of the
oocyte from the follicle, darkness of the oocyte and the surrounding cumulus cells or
opacity of GCs. The follicular diameter was measured only in morphologically normal
follicles every 6 days with an ocular micrometer inserted into a stereomicroscope (SMZ
645 Nikon, Tokyo, Japan; 100X magnification). Two perpendicular measures were
recorded for each follicle, and the averages of the two values were reported as the
follicular diameter. The daily mean increase in the follicular diameter (follicular growth
rate) was calculated as the diameter of morphologically normal follicles at day 18 minus
the diameter of the same follicle at day 0, divided by the total number of days in culture.
Antral cavity formation was defined as a visible translucent cavity within the granulosa
cell layers.
2.7. In vitro maturation (IVM) of caprine oocytes from in vitro cultured secondary
follicles
At the end of the 18-day culture period, the morphologically normal follicles
were carefully and mechanically opened with 27.5 G needles under a stereomicroscope
for oocyte recovery. Only oocytes that were ≥ 110 µm with homogeneous cytoplasm
and that were surrounded by at least one compact layer of cumulus cells were selected
for IVM. The recovery rate was calculated as the number of oocytes ≥ 110 µm divided
by the number of cultured follicles multiplied by 100. The selected cumulus oocyte
189
complexes (COC) were washed three times in maturation medium comprising TCM199
supplemented with 1 mg/mL BSA, 5 g/mL luteinizing hormone (LH) and 0.5 g/mL
rFSH®, 10 ng/mL epidermal growth factor (EGF), 50 ng/mL insulin-like growth factor1 (IGF-1), 0.911 mMol/L pyruvate, 100 µMol/L cysteamine, and 1 µg/mL estradiol.
After washing, the oocytes were transferred to 100 µL drops of maturation medium
under mineral oil and incubated for 40 h at 39ºC with 5% CO2 in air. At the end of the
maturation period, oocytes were labeled with 10 µM Hoechst 33342 (483 nm) for the
assessment of chromatin configuration. The maturation rate was calculated as the
number of oocytes that resumed meiosis relative to the total oocytes retrieved for IVM.
2.8. Viability assessment of oocytes cultured in vitro by fluorescence microscopy
To further evaluate oocyte integrity, after IVM, the caprine oocytes from in vitro
cultured secondary follicles were denuded, and live/dead fluorescent staining was
performed in 100-µL droplets of TCM-HEPES containing 4 μM calcein-AM and 2 μM
ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany). CalceinAM was used to detect the intracellular esterase activity of the viable cells, and
ethidium homodimer-1 was used to label the nucleic acids of non-viable cells showing
plasma membrane disruptions. The oocytes were incubated at 39°C for 15 min and
subsequently examined using a fluorescence microscope (400X; Nikon, Eclipse 80i,
Tokyo, Japan). The fluorescent signals from calcein-AM and ethidium homodimer-1
were measured at 488 and 568 nm, respectively. The oocytes were considered live when
the cytoplasm was positive for calcein-AM (green) fluorescence and dead when the
chromatin was labeled with ethidium homodimer-1 (red).
2.9. Caprine follicle mRNA extraction and quantitative PCR (qPCR) for bmpr1A,
bmpr2, smad1, smad4, smad5, smad6, smad7 and smad8 genes
Considering that BMP-6 at 1 ng/mL without FSH presented a higher antrum
formation rate than the cultured control (MEM) treatment, we examined the levels of
mRNA for BMP receptor/Smad signaling in vivo (non-cultured control) and in vitro
(MEM and BMP-6 at 1 ng/mL without FSH). For mRNA isolation, 3 pools of 10
follicles from each treatment were collected and stored in microcentrifuge tubes (1.5
mL), frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total mRNA
190
was isolated using a TRIzol Plus Purification Kit (Invitrogen, São Paulo, Brazil). The
RNA preparations were subjected to DNase I digestion and treated with the RNeasy
Micro Kit (Invitrogen Life Technologies). Complementary DNA (cDNA) was
synthesized from the mRNA (0.15 μg from each sample) using Superscript II RNase HReverse Transcriptase (Invitrogen Life Technologies).
The qPCR reaction was performed in a final volume of 20 µl containing 1 µl of
cDNA, 1X Power SYBR® Green PCR Master Mix (10 μl), 7.4 μl of ultra-pure water
and 0.4 μM of both sense and antisense primers. The gene-specific primers for the
amplification of different transcripts are shown in Table 1. Briefly, the gene sequences
were searched on NCBI data base and primers were designed using the Primer3
platform (http://bioinfo.ut.ee/primer3-0.4.0/) and tested using idtDNA data base
(http://www.idtdna.com). Glyceraldehyde-3-phosphate-dehydrogenase (GADPH) was
selected as an endogenous control to assess the expression stability and normalization of
gene expression in all samples and it was chosen from previous results (Chaves et al.,
2012).
The cycle profile for the first PCR step included initial denaturation and
polymerase activation for 15 minutes at 94ºC, followed by 40 cycles of 15 seconds at
94ºC, 30 seconds at 60ºC and 45 seconds at 72ºC. The final extension was performed
for 10 minutes at 72ºC. The specificity for each primer set was tested using a melting
curve, performed between 60 and 95°C for all genes. All amplifications were performed
in triplicate using an iQ5 Real-Time PCR system (Bio-Rad iQ5, CA, USA). The deltadelta-Ct method (Livak and Schmittgen 2001) was used to transform the Ct values into
normalized relative expression levels.
191
Table 1. Oligonucleotide primers used for the real-time polymerase chain reaction
analysis of caprine follicles before (Day 0) and after in vitro culture (Day 18).
Target gene
bmp-r1a
bmp-r2
smad1
smad5
smad8
smad4
smad6
smad7
gapdh
Primer sequence (5´→ 3´)
Sense
TGGATTGCCCTTACTGGTTC
Forward
CGCATTAGCGCAGTTTGATA
Reverse
AACAATTCAGTGGGCCAGAC Forward
GATCTGAGCAGGTGGGACAT Reverse
TGCCTCACGTCATCTACTGC
Forward
ATTCGCTGTGTCTTGGAACC
Reverse
CAGCTCCCAGCTGATACTCC
Forward
CCAATGTTTGGGCTCTTCAT
Reverse
TCCTATGACATCCGTGGACA
Forward
CTTCATCTCCCTGCTTCCAG
Reverse
GCCACTGAAGGACATTCGAT
Forward
GCCCTGAAGCTATCTGCAAC
Reverse
GAGACAGAGTTGGCCTTTCG
Forward
GCTGAACTCCCCAAATGTGT
Reverse
CAGCTCAATTCGGACAACAA Forward
GGCTGTACGCCTTCTCGTAG
Reverse
ATGCCTCCTGCACCACCA
Forward
AGTCCCTCCACGATGCCAA
Reverse
Genbank reference
NM_001076800.1
XM_002685492.1
AY035385.1
AF508027.1
AY145520.1
AY185301.1
NM_001206145.1
AF436855.1
NM_001190390.1
2.10. Statistical analysis
Goats provide different numbers of follicles, which were collected as a pool for
use in subsequent experimental procedures. The isolated follicles were considered to be
the experimental unit, according to the methods of Araújo et al. (2011). The data for
follicular survival, oocyte development, antrum formation and meiotic resumption after
in vitro culture in each treatment were compared using the Chi-square test, with the
results expressed as percentages. The data for the follicular diameters were subjected to
analysis using the Kolmogorov-Smirnov and Bartlett tests to confirm normal
distribution and homoscedasticity, respectively. ANOVA was subsequently performed,
and the treatments were compared using the Student-Newman-Keuls (SNK) test.
192
Because of the heterogeneity of variances, the days of culture were compared using the
Kruskal-Wallis non-parametric test.
For real-time RT-PCR treatment, the fresh control samples were randomly
assigned in blocks, and the relative expression values (2-ΔΔCt) were subjected to the
Shapiro-Wilk normality test using the univariate procedure of the SAS 9.0 software
package. The data for the mRNA expression in preantral follicles cultured in vitro were
analyzed using the Tukey test. The statistical significance of the differences among the
non-cultured control and the treatments (MEM and BMP-6 at 1 ng/mL without FSH)
was assessed using SAS 9.0. The results were expressed as the means ± standard error
of means (SEM), and significant differences were considered at P<0.05.
3. Results
3.1. Morphological evaluation of caprine secondary follicles cultured in vitro
A total of 264 secondary isolated follicles were evaluated and distributed
through several treatments (MEM, FSH, BMP-6 at 1, and 10 ng/mL in the absence or
presence of FSH), with at least 40 follicles cultured per treatment. At the beginning of
the culture period, all follicles were morphologically normal (Fig. 1A), i.e., with intact
basement membranes and oocytes with homogeneous cytoplasm surrounded by
granulosa cells. From day 0 to day 18, there was a reduction (P<0.05) in the percentage
of morphological normal follicles in all treatments (Table 2). Overall, the percentage of
morphologically normal follicles was similar (P>0.05) among the treatments, regardless
of the day of culture.
Figure 1. (A) Morphologically normal preantral (day 0) and (B) antral follicles (day 6)
using BMP-6 at 1 ng/mL withou rFSH®.
193
Table 2. Percentage of morphological normal follicles, means ± SEM of follicular
diameter (µm) and overall growth rate (µm/day) of caprine follicles after long-term
culture (18 days) in αMEM+ or medium supplemented with BMP-6 at 1 or 10 ng/mL in
the absence or presence of rFSH®.
Treatments
Survival (%)
n
Day 0
Follicular diameter (µm)
Day 18
(µm/day)
BMP-6
FSH
-
-
42
42 (100) a
13 (31) b
220.0 ± 6.6 b 366.6 ± 46.5 a
12.6 ± 1.4
-
+
40
40 (100) a
12 (30) b
217.4 ± 7.3 b 491.9 ± 49.7 a
15.4 ± 1.6
a
b
230.4 ± 7.4
Day 18
b
428.1 ± 35.0
a
13.7 ± 1.5
1 ng
-
47
47 (100)
1 ng
+
48
48 (100) a
13 (27.1) b
221.0 ± 6.6 b 490.6 ± 50.7 a
15.7 ± 1.5
10 ng
-
43
43 (100) a
12 (27.9) b
242.4 ± 7.7 b 471.1 ± 72.3 a
13.8 ± 1.8
10 ng
+
44
44 (100) a
15 (34.1) b
235.3 ± 7.8 b 397.3 ± 36.8 a
13.5 ± 1.6
a,b
22 (46.8)
Day 0
Growth rate
Different letters denote significant differences between the culture periods within the
same medium (P<0.05).
3.2. Follicular diameter, growth rate, antrum formation and oocyte maturation
Irrespective of treatment, the follicular diameter significantly increased from day
0 to day 18 of culture (data not shown). However, the mean daily follicular increase was
similar (P>0.05) among the treatments after 18 days of culture (Table 2). Antral follicles
(Fig. 1B) were observed as early as day 6 of culture in all treatments. Importantly, from
day 12, compared with the MEM treatment, the percentage of antral formation in the
BMP-6 at 1 ng/mL treatment was significantly higher (P<0.05) (Fig. 2). At the end of
the culture period (day 18) and after in vitro maturation, the percentage of viable
oocytes ranged from 87.5 to 100% (Table 3). The addition of FSH and/or BMP6 to the
culture medium had no effect on either the recovery rate of oocytes ≥110 µm or the
percentage of meiotic resumption. However, metaphase II (Fig. 3) oocytes were only
observed in the groups treated with FSH and/or BMP-6 (Table 3).
194
Figure 2. Antrum formation rate (%) in follicles cultured for 18 days in αMEM+ or
medium supplemented with BMP-6 (1 or 10 ng/mL) in the absence or presence of
rFSH®. A,B Different letters denote significant differences among treatments in the same
period (P<0.05).
195
Table 3. Oocyte viability (%) and diameter (µm), recovery rate of oocytes cultured in vitro (%), and meiotic stages (%) of caprine oocytes from
preantral follicles after long-term culture (18 days) in αMEM+ or medium supplemented with BMP-6 (1 or 10 ng/mL) in the absence or presence
of rFSH®.
Treatments
Number of viable
Average of
Number of
Number of
Number of
Number of cells
oocytes/number
marked oocytea
oocytesb/number
oocytes with
oocytes showing
in metaphase
of marked ocytesa
diameter (µm)
of follicles (%)
germinal
germinal vesicle
II/number of
vesicles/number
breakdown/numb
oocytesb (%)
of oocytesb (%)
er of oocytesb (%)
(%)
BMP-6
FSH
-
-
27/27 (100)
114.4 ± 2.2
13/42 (31) AB
9/13 (69.2)
4/13 (30.8)
0/13 (0)
-
+
21/24 (87.5)
112.3 ± 2.0
8/40 (20) B
4/8 (50)
4/8 (50)
1/8 (12.5)
1 ng
-
24/24 (100)
116.9 ± 2.9
12/47 (25.5) B
6/12 (50)
6/12 (50)
1/12 (8.3)
1 ng
+
35/36 (97.2)
115.9 ± 3.0
10/48 (20.8) B
3/10 (30)
7/10 (70)
1/10 (10)
A
11/21 (52.4)
10/21 (47.6)
1/21 (4.8)
9/16 (56.3)
7/16 (43.7)
1/16 (6.3)
10 ng
-
29/31 (93.6)
117.4 ± 2.7
21/43 (48.8)
10 ng
+
27/27 (100)
111.7 ± 1.5
16/44 (36.4) AB
a
Total oocytes recovered from cultured follicles
b
Only oocytes (≥110 μm) were selected for the in vitro maturation procedure
A,B
Different letters denote significant differences among treatments (P<0.05).
196
Figure 3. The oocytes from follicles grown in vitro in αMEM+ medium (A-C) or under
treatment with BMP-6 at 1 ng/mL without FSH® (D-F) after 18 days. Note the presence
of the intact germinal vesicle (GV; white arrow) in the MEM treatment and the
metaphase II (MII; black arrow) stage indicated in blue after Hoechst 33342 staining in
BMP-6 treatment.
3.3. Expression of mRNA for bmpr1A, bmpr2, smad1, smad4, smad5, smad6, smad7 and
smad8 in caprine secondary follicles before and after in vitro culture
The effects of BMP-6 were evaluated for the mRNA expression levels of BMP
receptors (bmpr1A and bmpr2) and Smads (smad1, smad4, smad5, smad6, smad7 and
smad8) (Fig. 4). The mRNA expression for bmpr2 (Fig. 4A), smad1 (Fig. 4B), smad5
(Fig. 4C) and smad6 (Fig. 4E) was detected in all treatments. The mRNA expression of
smad8 (Fig. 4D) and smad7 (Fig. 4F) was detected only in the MEM and BMP-6 at 1
ng/mL without FSH treatments. The mRNA expression for smad5 was higher (P<0.05)
after in vitro culture in both the MEM and BMP-6 (1 ng/mL) treatments compared with
the non-cultured control. However, at day 18, the MEM and BMP-6 (1 ng/mL)
treatments presented lower (P<0.05) mRNA expression for the bmpr2 and smad1 genes
compared with the non-cultured control. The mRNA expression for smad6 was similar
(P>0.05) among the treatments. The mRNA expression for Smad4 was detected only in
197
the BMP-6 (1 ng/mL) treatment, whereas the mRNA expression for bmpr1A was not
detected at all.
Figure 4. Relative expression of mRNA (means ± SD) of (A) bmpr2; (B) smad1; (C)
smad5; (D) smad8; (E) smad6; and (F) smad7 in the non-cultured control (D0) and after
18 days of culture in αMEM+ medium or BMP-6 at 1 ng/mL without rFSH®.
A,B
Different letters denote significant differences among treatments (P<0.05).
4. Discussion
There is little knowledge regarding the effects of BMP-6 on folliculogenesis in
goats. This study is the first to demonstrate the effects of BMP-6 during the in vitro
development of isolated caprine secondary follicles and its influence on BMP
receptors/Smad signaling. In a recent study, we showed the deleterious effect of BMP-6
on the in vitro culture of preantral follicles enclosed in caprine ovarian cortex (in situ
culture system; Araújo et al., 2010). Histological and ultrastructural analyses revealed
that BMP-6, even at low concentrations (1 ng/mL), induced atresia in primordial
follicles (Araújo et al., 2010). However, Frota et al. (2011) verified that addition of
BMP-6 to the culture medium enhances the growth of cultured caprine secondary
198
follicles mainly by antrum formation after 6 days of in vitro culture. Therefore,
considering that follicular categories (primordial, primary, secondary and tertiary
follicles) have different medium requirements and that the type of follicle culture (in
situ versus isolated form) might affect the in vitro culture performance, we used isolated
secondary follicles in the present study. The secondary follicle morphology is
characterized by at least two complete layers of granulosa cells in the proliferative and
undifferentiated phase and oocytes with higher transcription and protein synthesis rates.
The isolated follicle culture system facilitated enhanced and rapid in vitro growth, as the
substances from the culture medium are directly infused into follicular cells. In addition,
the isolated follicle culture system might explain the follicular requirement during the
preantral and antral phases in in vitro culture (Araújo et al., 2011).
Although the addition of BMP-6 to the culture medium did not influence
follicular growth, oocyte maturation or the percentage of morphologically normal
follicles, these results showed that the addition of 1 ng/mL of BMP-6 without FSH
significantly increased the antral formation rate compared with the cultured control
(MEM) and BMP-6 at 10 ng/mL (without FSH) treatments. Thus, these results suggest
that low concentrations of BMP-6 (1 ng/mL) might positively affect folliculogenesis.
The acquisition of the antral cavity was considered a positive aspect of follicular
quality, as the oocyte needs an adequate environment for growth. Higher antrum
formation rates were observed in the BMP-6 at 1 ng/mL without FSH treatment.
Similarly, Frota et al. (2011) verified that BMP-6 enhanced antrum formation in
cultured caprine secondary follicle. Additionally in our experiment, compared with the
cultured control (MEM), after in vitro culture, only the BMP-6 (1 ng/mL without FSH)
treatment showed the entire BMP receptor/Smad signaling, including smad4 gene
expression. Because smad4 knockout impairs the development of antral follicles
(Pangas et al., 2006), the presence of this molecule after the in vitro culture of caprine
secondary follicles might reflect the high antrum formation rates induced through BMP6. In mammals, smad4 is the only co-mediating Smad; it translocates to the nucleus
with phosphorylated R-Smads and subsequently modulates the transcription of BMP
target genes (Wang et al., 2010). Smad complexes regulate many biological processes,
including
cell
proliferation,
differentiation
and
apoptosis
during
embryonic
development as well as adult tissue homeostasis (Costello et al., 2009). Interrupted
BMP/Smad signaling through smad4 significantly inhibited the growth of granulosa
cells (Wang et al., 2010). Moreover, during the development of early secondary stage
199
preantral follicles, smad4 knockout might inhibit communication between oocytegranulosa cells and increase the numbers of atretic follicles in mice (Pangas et al.,
2006). Therefore, the maintenance of the entire BMP/Smad signaling, including smad4
expression, after in vitro culture of caprine secondary follicles is crucial for the BMP
response.
The results obtained in the present study showed that while the mRNA
expression for bmpr1A was not detectable, there was a reduction of the mRNA
expression for bmpr2 after in vitro culture. Chen et al. (2009) and Costa et al. (2012)
observed similar results, showing the down-regulation of bmpr1A and bmpr2 expression
after the in vitro culture of ovine granulosa cells and caprine secondary follicles,
respectively. Previous studies demonstrated that BMP-6 plays a role in follicular
development through the proliferation and differentiation of granulosa cells (Juengel et
al., 2006; Krysko et al., 2008; Frota et al., 2011). The results of a recent study showed
that the mRNA expression for bmp6 increases with in vivo follicular growth (Costa et
al., 2012), indicating the importance of BMP-6 during the follicular growth in goats.
Moreover, the protein and mRNA expression of bmp6 was demonstrated in the caprine
oocytes of follicles during all stages of development, particularly in those from antral
follicles (Frota et al., 2011).
After in vitro culture, the down- and up-regulation of R-Smads (smad1 and
smad5, respectively) was observed, while there was no difference in the I-Smads
(smad6 and smad7) expression. R-Smads act as transcriptional factors in the nucleus;
therefore, the nuclear translocation of R-Smads upon BMP-6 stimulation is a critical
event for signal transduction (Ebisawa et al., 1999). The caprine preantral and antral
follicles expressed mRNA for smad1, smad5 and smad8 before and after in vitro culture
(Costa et al., 2012). In cattle, antral follicles expressed smad1 protein, and its
expression was activated through BMP-6 in granulosa cells (Glister et al., 2004). In
addition, BMP-6 induced the phosphorylation of smad1 and smad5, but not smad8
(Ebisawa et al., 1999; Aoki et al., 2001). Similar to the findings of Ebisawa et al.
(1999), in the present study, the reduced expression of smad1 and the increased
expression of smad5 after the in vitro culture of secondary caprine follicles
demonstrated that smad5 is the principal R-Smad for BMP-6 and that smad1 might also
act downstream of BMP-6 signaling. Furthermore, in the present study, smad8
expression was not stimulated after in vitro culture; similarly, Ebisawa et al. (1999)
200
observed that smad8 was phosphorylated in both the presence and absence of BMP-6.
Thus, smad8 might act in other signaling pathways.
In conclusion, the low BMP-6 concentration (1 ng/mL) positively influenced
antrum formation after the in vitro culture of caprine preantral follicles. Moreover,
complete mRNA expression for BMP receptors and their intracellular signaling proteins
(Smads) were obtained only in treatment containing BMP-6. Therefore, further studies
on the need to use FSH on the culture medium for advanced caprine follicles are
needed.
Acknowledgements
The authors would like to thank Dr. Isabel C.C. Santos for providing the
samples of recombinant FSH (rFSH®) used in this study. This work was supported
through funding from the National Council for Scientific and Technological
Development (CNPq, Brazil, grant number: 554.812/2006-1-RENORBIO). Valdevane
R. Araújo is the recipient of a grant from the Coordination for the Improvement of
Higher Education Personnel (CAPES-Brazil).
Conflict of interest
The authors declare no conflicts of interest that would prejudice the impartiality of the
research reported.
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12 CAPÍTULO 7
Fator de crescimento do endotélio vascular-A165 (VEGF-A165) estimula o
desenvolvimento in vitro e a competência oocitária de folículos pré-antrais caprinos
“Vascular endothelial growth factor-A165 (VEGF-A165) stimulates the in vitro
development and oocyte competence of goat preantral follicles”
Periódico: Cell and Tissue Research, v. 346, p. 273-281, 2011.
206
RESUMO
O objetivo desse estudo foi avaliar o efeito do fator de crescimento do endotélio
vascular-A165 (VEGF-A165) sobre o desenvovimento in vitro de folículos secundários
caprinos. Folículos pré-antrais (≥150 μm de diâmetro) foram isolados de ovários de
cabras adultas sem padrão racial definido e cultivados por 18 dias em αMEM na
ausência (controle) ou na presença de VEGF-A165 nas concentrações de 10 (VEGF10) e
100 ng/ml (VEGF100). Análises da sobrevivência follicular, diâmetro, formação de
antro e taxa de crescimento diário foram realizadas a cada 6 dias. No final do período de
cultivo, oócitos morfologicamente normais (≥110 μm de diâmetro) foram destinados à
maturação in vitro (MIV). Os resultados demonstraram que todos os folículos
apresentaram oócitos e células da granulosa morfologicamente normais e após a
marcação com calcein-AM, altas taxas de oócitos viáveis foram observadas em todos os
tratamentos. O diâmetro follicular e a taxa de crescimento observados no tratamento
VEGF10 foram maiores que ao observado no controle. Ambos os tratamentos com
adição de VEGF-A165 resultaram em altas taxas de oócitos recuperados para MIV
quando comparado ao controle. Além disso, apenas a adição de VEGF-A165 permitiu
que oócitos crescidos in vitro atingissem metáfase II. Desta forma, pode-se concluir que
a adição de VEGF-A165 ao meio de cultivo melhorou o desenvolvimento in vitro de
folículos pré-antrais caprinos permitindo a produção de oócitos maturos.
Palavras-chave: Crescimento in vitro. Maturação. VEGF-A165. Folículos ovarianos.
Fatores de crescimento. Cabra.
207
Vascular endothelial growth factor-A165 (VEGF-A165) stimulates the in vitro
development and oocyte competence of goat preantral follicles
Abridged title: VEGF-A165 produces mature oocytes of preantral follicles
V.R. Araújo*, G.M. Silva, A.B.G. Duarte; D.M. Magalhães, A.P. Almeida, R.F.B.
Gonçalves, J.B. Bruno, T.F.P. Silva, C.C. Campello, A.P.R. Rodrigues and J.R.
Figueiredo
Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), Faculty
of Veterinary Medicine, State University of Ceara, Fortaleza, CE, Brazil
*Corresponding address:
Programa de Pós-Graduação em Ciências Veterinárias (PPGCV)
Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais (LAMOFOPA)
Universidade Estadual do Ceará (UECE)
Av. Paranjana, 1700, Campus do Itaperi.
Fortaleza – CE – Brasil. CEP: 60740-000
Tel.: +55.85. 3101.9852; Fax: +55.85.3101.9840
E-mail address: val_exclusiva@yahoo.com.br (Valdevane R. Araújo)
208
ABSTRACT
The aim of this study was to evaluate the effect of vascular endothelial growth factorA165 (VEGF-A165) on the in vitro development of goat secondary preantral follicles.
Preantral follicles (≥150 μm in diameter) were isolated from the ovaries of adult mixedbreed goats and individually cultured for 18 days in αMEM in the absence (control) or
presence of VEGF-A165 at concentrations of 10 ng/ml (VEGF10) and 100 ng/ml
(VEGF100). Analyses of follicular survival, diameter, antrum formation and rate of
daily growth were performed every 6 days. At the end of the culture period,
morphologically normal oocytes (≥110 μm in diameter) were taken for in vitro
maturation (IVM). The results demonstrated that all follicles presented oocytes and
granulosa cells that were morphologically normal and after labeling with calcein-AM,
high rates of oocyte viability were observed in all treatments. The follicular diameter
and the growth rate achieved in the presence of VEGF10 were higher than those of the
control. Both treatments with VEGF-A165 showed higher rates of oocyte recovery for
IVM when compared with the control. Moreover, only the addition of VEGF-A165
permitted oocytes grown in vitro to reach metaphase II. Thus, the addition of VEGFA165 to the culture medium improves the development of goat preantral follicles
cultured in vitro, allowing the production of mature oocytes.
Keywords: In vitro growth, Maturation, VEGF-A165, Ovarian follicles, Growth factors,
Goat
209
Introduction
In mammals, the majority of oocytes are stored in preantral follicles. As the
follicles grow, they need a higher nutrient intake and become dependent on
gonadotropins. Some in vitro studies of preantral secondary follicles suggest that these
follicles are able to generate competent oocytes that are able to undergo subsequent
embryo development in vitro (Wu et al. 2001; Gupta et al. 2008; Magalhães et al. 2011;
Arunakumari et al. 2010). However, the mechanisms that control the regulation of the in
vitro growth of preantral follicules are not completely understood, particularly those
that control the development of large secondary follicles. Evidence has been
accumulated over recent decades suggesting that follicular development is a continuous
process regulated by various endocrine and paracrine factors, among which we highlight
vascular endothelial growth factor (VEGF).
VEGF protein and its receptors are present in many cell types including ovary
cells. The VEGF receptors comprise a family of tyrosine-kinase-type receptors, e.g., Flt1 (VEGFR-1), KDR/Flk-1 (VEGFR-2) and Flt-4 (VEGFR- 3) and have been detected in
mammalian ovaries (Neufeld et al. 1999). VEGF is a factor that acts by stimulating the
mitosis of endothelial cells and by increasing vascular permeability (Redmer and
Reynolds 1996). In the ovary, both granulosa and theca cells of bovine secondary
follicles express mRNA for VEGF-A165 and its receptors (Yang and Fortune 2007). In
rats, the expression of VEGF-A188 also occurs in granulosa and theca cells of secondary
follicles and can be intensified in response to the gonadotropins follicle stimulating
hormone (FSH), luteinizing hormone and human chorionic gonadotropin (Yang et al.
2008). During follicular growth, the antral cavity becomes filled with follicular fluid,
which in turn is rich in VEGF (Ferrari et al. 2006). VEGF production is stagedependent, increasing as the follicle grows, together with the amount of VEGF-A
present in follicular fluid (Barboni et al. 2000). Furthermore, VEGF in primates is also
produced by preovulatory follicle cells and luteal cells (Taylor et al. 2004).
Previous studies have evaluated the effect of VEGF-A165 on follicular
development. In vivo, Danforth et al. (2003) have found an increase in the number of
initial primary and secondary follicles after the injection of VEGF into rat ovaries.
Mattioli et al. (2001) have observed that VEGF-A increases the blood supply and
activates the change of primordial follicles into primary follicles. In addition, during the
in vitro culture of preantral follicles enclosed in ovarian fragments, i.e., in situ, Yang
210
and Fortune (2007) have found a role for VEGF-A165 in the transition from primary to
secondary follicles and in the increase in follicular diameter after the in vitro culture of
ovarian tissue from bovine fetuses. Recently, our team has obtained similar results in
goats, in which we have shown follicle survivability and an increase in follicular and
oocyte diameter (Bruno et al. 2009). In situ culture favors the interaction between
follicles at different stages of development and stroma cells. Moreover, this culture
system can be successfully used to study the earliest stages of follicular development,
specifically the activation of primordial follicles. The isolated follicle culture system
allows for better individual monitoring, promotes nutrition and is an excellent
alternative to the in vitro production of embryos from oocytes grown, matured and
fertilized in vitro. However, no reports are available concerning the effect of VEGFA165 on the in vitro culture of isolated large secondary follicles. Thus, this study has
aimed to evaluate the influence of VEGF-A165 on the in vitro development of caprine
isolated secondary follicles.
Materials and methods
Chemicals and media
Unless mentioned otherwise, the culture media, ascorbic acid and other
chemicals used were purchased from Sigma (St. Louis, Mo., USA).
Source of ovaries
Ovaries (n=32) from 16 adult mixed-breed goats (1–3 years old) were collected
at a local slaughterhouse. The surrounding fat tissue and ligaments were removed and
the ovaries were washed in 70% alcohol, followed by two washes in minimum essential
medium (MEM). The ovaries were placed into tubes containing 15 ml MEM plus
HEPES (MEM HEPES), supplemented with 100 μg/ml penicillin and 100 μg/ml
streptomycin and then transported to the laboratory at 4°C within 1 h.
211
Isolation and selection of caprine preantral follicles
In the laboratory, the surrounding fat tissue and ligaments were stripped from the
ovaries. Ovarian cortical slices (1 mm thick) were cut from the ovarian surface by using
a surgical blade under sterile conditions. Then, the ovarian cortex tissues were placed
and washed in fragmentation medium, which consisted of MEM HEPES. Preantral
follicles (≥150 μm in diameter) were visualized under a stereomicroscope (SMZ 645
Nikon, Tokyo, Japan), manually dissected from the strips of ovarian cortex by using
27.5-gauge (27.5-G) needles and transferred to the culture medium for further
evaluation of follicular quality. Follicles with a visible central oocyte, surrounded by
two or more granulosa cell layers and with an intact basement membrane and no antral
cavity, were selected for culture.
Culture of caprine preantral follicles
After selection, follicles were individually cultured in 100-μl drops of culture
medium in Petri dishes (60×15 mm; Corning, USA). The medium used was αMEM (pH
7.2-7.4), supplemented with 3 mg/ml bovine serum albumin (BSA), ITS (10 μg/ml
insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium), 2 mM glutamine, 2 mM hypoxanthine,
50 μg/ml ascorbic acid and increasing concentrations of recombinant follicle stimulating
hormone (rFSH: 100 ng/ml until day 6; 500 ng/ml until day 12; 1000 ng/ml until day
18), which constituted the control medium. For the experimental conditions, two
different concentrations of human recombinant VEGF-A165 expressed in Escherichia
coli (10 and 100 ng/ml) were tested. Incubation was carried out at 39°C in 5% CO2 in
air for 18 days. Fresh media were prepared immediately before use and incubated for 1
h prior to use, with 60 μl medium being changed in each drop every 2 days. The
concentrations of rFSH (Nanocore, Brazil; Saraiva et al. 2011), ascorbic acid (G.M.
Silva et al., unpublished), and VEGF (Bruno et al. 2009) were chosen based on previous
studies performed in our laboratory. The cultures were replicated four times and at least
60 follicles were used per treatment.
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Morphological evaluation of follicle development
Follicular features were evaluated during culture including the integrity of the
basement membrane, the morphological aspects of the oocyte and surrounding
granulosa cells and the morphological signs of degeneration, such as darkness or any
abnormality of oocytes and the surrounding granulosa cells. The percentage of normal
follicular morphology was calculated by excluding the follicles in which the rupture of
the basement membrane occurred. The follicular diameter was measured only in normal
follicles every 6 days with the aid of an ocular micrometer attached to a
stereomicroscope (SMZ 645 Nikon, Tokyo, Japan; 100x magnification). Two
perpendicular diameters were recorded for each follicle and the average of these two
values was reported as the follicular diameter. With regard to the follicular growth, the
mean increase in follicular diameter was calculated as follows: the diameter of viable
follicles at day 18 minus the diameter of viable follicles at day 0, divided by the total
number of viable follicles at day 18. Antral cavity formation was defined as a visible
translucent cavity within the granulosa cell layers.
Viability assessment of oocytes cultured in vitro
For a better evaluation of follicular integrity, after 18 days of culture, live/dead
fluorescent staining was performed on caprine oocytes from in-vitro-cultured preantral
follicles in 100-μl droplets of MEM HEPES mounted in glass slides with 4 μM calceinAM and 2 μM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe,
Germany), followed by an incubation at 37°C for 15 min. Finally, the follicles were
examined by using a fluorescence microscope (Nikon, Eclipse 80i, Tokyo, Japan). The
emitted fluorescent signals of calceinAM and ethidium homodimer were collected at
488 and 568 nm, respectively. Whereas the first probe detected the intracellular esterase
activity of viable cells, the later labeled the nucleic acids of non-viable cells after
plasma membrane disruption. The oocytes were considered live if the cytoplasm was
stained positively with calcein-AM (green) and if chromatin was not labeled with
ethidium homodimer (red).
213
In vitro maturation of caprine oocytes from in-vitro-cultured preantral follicles
At the end of the 18-day culture period, all the healthy follicles were carefully
and mechanically opened with 27.5-G needles under a stereomicroscope for oocyte
recovery. Only oocytes (≥110 μm) with homogeneous cytoplasm and surrounded by at
least one compact layer of cumulus cells were selected for in vitro maturation (IVM).
The recovery rate was calculated by dividing the number of oocytes (≥110 μm) by the
number of viable follicles at day 18 of culture and multiplying this value by 100. The
selected cumulus oocyte complexes were washed three times in maturation medium
composed of TCM199 supplemented with 1% BSA, 5 μg/ml luteinizing hormone and
0.5 μg/ml rFSH, 10 ng/ml epidermal growth factor, 50 ng/ml insulinlike growth factor,
0.911 mMol/l pyruvate and 1 μg/ml estradiol. After being washed, the oocytes were
transferred to 100-μl drops of maturation medium under mineral oil and then incubated
for 40 h at 39°C with 5% CO2 in air. At the end of the maturation period, oocytes were
stained with 10 μM Hoechst 33342 (483 nm) for the assessment of chromatin
configuration. In parallel, to establish the efficiency of the maturation medium, cumulus
oocyte complexes were aspirated from antral follicles (more than 2 mm in diameter) and
cultured under the same conditions as described above. Thus, the rate of maturation of
oocytes grown in vivo could be determined.
Statistical analysis
Goats yielded various numbers of follicles, which were then taken as a pool for
experimental procedures. Follicles were subsequently considered as the experimental
unit, following the same approach of Araújo et al. (2011). Data concerning follicular
survival, oocyte development, antrum formation and meiotic resumption after in vitro
culture in each treatment were compared by using the Chi-square test, with the results
being expressed as percentages. Data concerning follicular diameters were submitted to
Kolmogorov-Smirnov and Bartlett tests to confirm the normal distribution and
homoscedasticity, respectively. An analysis of variance was then carried out and
treatments were compared by using the Student-Newman-Keuls test. Because of the
heterogeneity of variances, days of culture were compared by using the Kruskal-Wallis
non-parametric test. Results were expressed as mean ± standard deviation (SD) and
differences were considered to be significant when P<0.05.
214
Results
Follicular morphology and viability of oocytes from caprine preantral follicles cultured
in vitro
A total of 185 preantral follicles were isolated from goat ovaries, selected and
cultured in various concentrations of VEGF-A165 and at least 60 follicles were used per
treatment. During the culture period, on days 0, 6, 12 and 18, the follicles were
evaluated for oocytes, surrounding granulosa cell morphology and basement membrane
integrity. At the end of the culture period, even these follicles extruded presented
morphologically normal oocytes and granulosa cells. The granulosa cells were shiny,
cohesive and arranged in several layers (Fig. 1a-c) and the oocytes had cytoplasm with
regular contours and homogeneous staining. On day 12, an increased rate of extrusion
was noted when compared with that on day 0 (Fig. 2), with no significant increases in
this parameter until day 18. Moreover, independent of the culture period, no significant
differences were seen between treatments for this parameter. Viability analysis with
fluorescent markers demonstrated that the oocyte viability was not correlated to the rate
of extrusion as both oocytes in intact follicles and oocytes extruded from follicles were
viable at the end of the culture period. Viable (Fig. 1d-f) and degenerated (Fig. 1j-l)
oocytes were obtained at the end of the culture period regardless of treatment, i.e., those
oocytes marked in green by calcein-AM or in red by ethidium homodimer, respectively.
The percentages of viable oocyte were 95.7%, 95.7% and 98.1% in the control,
VEGF10 (10 ng/ml VEGF) and VEGF100 (100 ng/ml VEGF) groups, respectively
(Table 1, P>0.05).
215
Fig. 1 Oocytes from goat follicles, grown in vitro, at the end of the culture period (after
18 days) with various treatments: control (a, d, g, j), with 10 ng/ml VEGF (b, e, h k), or
with 100 ng/ml VEGF (c, f, i, l). Oocytes are marked in green by Calceina-AM in d-f
and in red by ethidium homodimer in j-l for all treatments. Bars 50 μm
216
Fig. 2 Percentages of goat preantral follicles with normal morphology (healthy follicles)
cultured for 18 days (D0, D6, D12, D18) in αMEM+ (Control) and αMEM+
supplemented with 10 ng/ml VEGF (VEGF10) or 100 ng/ml VEGF (VEGF100).
Different lowercase letters denote significant differences among culture periods within
the same medium (P<0.05)
Follicular diameter and growth rate of goat follicles cultured in vitro
With the progression of the culture period, a significant increase occurred in
follicular diameter in all treatments (Fig. 3, P<0.05). When treatments were compared
with each other from day 12 onward, VEGF10 was the only treatment that showed a
follicular diameter significantly higher than that in the control group. Moreover, the
VEGF10 treatment exhibited significantly higher growth rates when compared with the
control (20.85±10.19 μm versus 16.72±8.94 μm, respectively, P<0.05). When the
VEGF10 treatment was compared with VEGF100, no significant difference was
observed (20.85±10.19 μm versus 17.88±7.25 μm, respectively, P>0.05)
217
Fig. 3 Diameter of goat follicles cultured for 18 days (D0, D6, D12, D18) in αMEM+
(Control) and αMEM+ supplemented with 10 ng/ml VEGF (VEGF10) or 100 ng/ml
VEGF (VEGF100). Different lowercase letters denote significant differences among
culture periods within the same medium (P<0.05). Different uppercase letters denote
significant differences among treatments in the same period (P<0.05)
Antrum formation
In all treatments, a significant increase occurred in antrum formation rate from
day 0 to day 6 and, later, from day 6 to day 18 (Fig. 4). Moreover, when treatments
were compared within each period of culture, the VEGF100 treatment showed an
antrum formation rate that was significantly higher than that of the control (75.4%
versus 58.3%, respectively, P<0.05) at day 6.
218
Fig. 4 Antrum formation in goat follicles cultured for 18 days (D0, D6, D12, D18) in
αMEM+ (Control) and αMEM+ supplemented with 10 ng/ml VEGF (VEGF10) or 100
ng/ml VEGF (VEGF100). Different lowercase letters denote significant differences
among culture periods within the same medium (P<0.05). Different uppercase letters
denote significant differences among treatments in the same period (P<0.05)
Recovery rate and chromatin configuration of oocytes from in-vitro-grown caprine
preantral follicles
The recovery rate of oocytes with diameters ≥110 μm for IVM (Table 1) was
significantly higher in the VEGF100 group than in the control group (P<0.05). With
respect to the chromatin configuration of oocytes competent to resume meiosis,
numbers were significantly higher only in the VEGF100 group. Notably, only oocytes
from follicles grown in vitro in the presence of VEGF-A165 (VEGF10: 9.1%; VEGF100:
29.4%) reached the stage of metaphase II (MII). Figure 5 shows oocytes at MII from
follicles grown in vivo, i.e., obtained from antral follicles (Fig. 5a-c), grown in vitro
(Fig. 5d–f) and grown in vitro but after the addition of 10 ng/ml VEGF (Fig. 5g-i) or
100 ng/ml VEGF (Fig. 5j-l). The maturation rate of oocytes grown in vivo was 54.05%
and all oocytes resumed meiosis.
219
Table 1 Recovery rate of oocytes (≥110 μm) grown in vitro and meiotic stages of goat oocytes from preantral follicles cultured for 18 days in
αMEM+ (Control) and αMEM+ supplemented with 10 ng/ml VEGF (VEGF10) or 100 ng/ml VEGF (VEGF100). Significant differences between
treatments in the same column are indicated by uppercase letters (P<0.05)
Treatments
Number of viable
Number of
Number of oocytes with
Number of oocytes showing
Number of oocytes in
oocytes/number of
oocytesb/number
germinal vesicles/number
germinal vesicle breakdown/
metaphase II/number
marked oocytesa (%)
of follicles (%)
of oocytesb (%)
number of oocytesb (%)
of oocytesb (%)
αMEM+
44/46 (95.7)A
7/60 (11.7)B
5/7 (71.4)A
2/7 (28.6)B
0/2 (0.0)
VEGF10
44/46 (95.7)A
11/64 (17.2)AB
7/11 (63.6)A
4/11 (36.4)B
1/11 (9.1)A
VEGF100
52/53 (98.1)A
17/61 (27.9)A
4/17 (23.5)B
13/17 (76.5)A
5/17 (29.4)A
a
Total oocytes recovered from cultured follicles
b
Only oocytes ≥110 μm were selected for the in vitro maturation procedure
A,B
Different letters denote significant differences among treatments (P<0.05).
220
Fig. 5 Oocytes from goat follicles grown in vivo (a-c) and in vitro under control
conditions (d-f) and after treatment with 10 ng/ml VEGF (g-i) or 100 ng/ml VEGF (j-l).
b, e, h, k Viable oocytes marked in green by Calcein-AM for all the treatments. Note the
presence of the germinal vesicle in the controls (f) and metaphase II in oocytes in vivo
(c) and after treatment with 10 ng/ml VEGF (i) or 100 ng/ml VEGF (l), marked in blue
by Hoechst 33342. Bars 50 μm
221
Discussion
This study demonstrated the importance of VEGF-A165 as a component of the
medium for the in vitro culture of isolated caprine preantral follicles. We found high
rates of healthy follicules and of oocyte viability at the end of the culture period in all
treatments. However, VEGF-A165 was able to increase follicular diameter and the
growth rate of cultured follicles at both concentrations of VEGF used. A study in
primates has shown that, in preantral follicles, a positive correlation is present between
the increase in follicular diameter and the production of VEGF-A165 during the culture
period (Fisher et al. 2009). The ability of VEGFA165 to stimulate the growth of preantral
follicles might be attributable to this factor, which is present in granulosa and thecal
cells. These follicular compartments are the sites of the expression of mRNA for VEGF
and its receptors and of VEGF protein (Yang and Fortune 2007). Furthermore, VEGFA165 might act indirectly by increasing cell permeability, allowing a greater supply of
growth factors, gonadotropins, steroids and oxygen important to the growth of follicles.
This condition has been verified in vivo by Danforth et al. (2003) who have
demonstrated that the direct injection of VEGF-A into the ovarian bursa of rats
improves the neovascularization and vascular permeability close to the developing
follicles.
All treatments tested in this study showed a progressive increase in the antrum
formation rate; however, no significant difference was observed between treatments
after 18 days of culture. This can be explained by the presence of FSH in the culture
medium (control). Although the antral cavity is formed spontaneously in advanced
preantral follicles cultured in vitro, the presence of FSH significantly improves the rates
of antrum formation (Saraiva et al. 2011). However, the addition of 100 ng/ml VEGFA165 stimulates early antrum formation, as observed at day 6 of culture. This is the first
report of the role of VEGF-A165 in the antrum formation of goat preantral follicles.
During follicular development, the production of follicular fluid is known to be
enhanced by the increased follicular vascularization and permeability of blood vessels
(van den Hurk and Zhao 2005). Although no blood supplementation is available in the
in vitro environment, the culture medium may exert similar actions. Furthermore, the
ability of VEGF-A165 to increase the permeability of the cells might have favored the
emergence and subsequent development of antral follicles.
222
In this study, VEGF-A165 also significantly increased the rate of suitable oocytes
(≥110 μm) for IVM and was able to stimulate meiotic resumption, especially during
VEGF100 treatment. Notably, only in treatments with VEGF-A165 did the oocytes reach
MII. In goats, the rates of MII oocytes grown in vitro is still low compared with those of
in-vivo grown oocytes (60-70% of MII) obtained from antral follicles (Chauan and
Anand 1991); the production of mature oocytes from preantral follicles grown in vitro
in domestic animals has only been reported in sheep (Tamilmani et al. 2005;
Arunakumari et al. 2007, 2010), pigs (Wu et al. 2001) and buffalo (Gupta et al. 2008).
To date, in goat preantral follicles, a low MII rate has been reported (Duarte et al. 2010;
Magalhães et al. 2011). However, we report, for the first time, that the addition of
VEGF-A165 enhances the progression of meiosis to the MII stage, this rate being 29.4%
in preantral follicles grown in vitro. In sheep oocytes grown in vivo, Cao et al. (2009)
have observed that VEGF-A165 significantly increases the percentage of oocytes at MII
and promotes a normal distribution of α-tubulin and chromosomes in the spindle. This
suggests that the exposure of oocytes to VEGF-A165 improves the organization of the
cytoskeleton and that this cellular modification is beneficial to the progression of
meiosis. In bovine oocytes, supplemention of the medium with VEGF-A results in the
increased extrusion of the first polar body and in the improved development potential of
oocytes (Einspanier et al. 2002; Luo et al. 2002). These results might be attributable to
the direct action of VEGF-A165 via VEGFR-2 (a VEGF receptor), which is expressed in
oocytes of follicles from all developmental stages and in the cumulus cells of antral
follicles (Bruno et al. 2009). The presence of this receptor on both oocytes and cumulus
cells is strong evidence for the role of VEGF in the acquisition of oocyte competence.
Furthermore, the presence of VEGF-A receptors, especially on granulosa cells, suggests
that this factor might be involved in proliferation events and have an effect on the onset
of primordial follicle development in humans (Abir et al. 2010). During the transition of
primordial follicles to the primary stage, an increase in VEGF and its mRNA takes
place in rats (Kezele et al. 2005). Moreover, as mentioned earlier, the indirect action of
VEGF-A165 on cell permeability might have promoted the increased availability of
nutrients and substances important for the growth of oocytes and the acquisition of
meiotic competence.
In conclusion, the addition of VEGF-A165 to the culture medium improves the
development of caprine preantral follicles cultured in vitro, allowing the production of
mature oocytes. However, more studies are needed for the development of an efficient
223
medium that encourages the production of a large number of mature oocytes from goat
preantral follicles grown in vitro.
Acknowledgements We thank Dr. Isabel C.C. Santos for providing samples of the
rFSH used in these experiments.
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13 CAPÍTULO 8
Desenvolvimento in vitro de folículos secundários bovinos em sistemas bi e
tridimensional utilizando fator de crescimento do endotélio vascular (VEGF), fator
de crescimento semelhante à insulina-1 (IGF-1) e hormônio do crescimento (GH)
“In vitro Development of Bovine Secondary Follicles in Two- and Three-Dimensional
Culture System Using Vascular Endothelial Growth Factor (VEGF), Insulin-Like
Growth Factor-1 (IGF-1) and Growth Hormone (GH)”
Periódico: Animal Reproduction Science (Submetido em: 1 de junho de 2013).
227
RESUMO
O objetivo deste estudo foi avaliar o desenvolvimento folicular e a produção de
estradiol de folículos secundários bovinos isolados em sistemas de cultivo bi (2D:
Experimento 1) ou tridimensional (3D utilizando alginato: Experimento 2) na ausência
(Controle, apenas α-MEM+) ou presença do fator de crescimento do endotélio vascular
(VEGF), fator de crescimento semelhante à insulina-1 (IGF-1) ou hormônio do
crescimento (GH) sozinhos ou em combinação por longos períodos. Um total de 363
folículos secundários foram isolados e individualmente cultivados por 32 dias a 38,5oC
e 5% de CO2 com adição (5 µl) de meio a cada dois dias. No Experimento 1, o
crescimento folicular e a formação de antro foram superiores (P<0.05) no tratamento
VEGF quando comparado aos demais tratamentos. No Experimento 2, apenas as
concentrações de estradiol foram superiores (P<0.05) no tratamento GH quando
comparado ao controle, enquanto que os outros parâmetros foram similares (P>0.05).
Em conclusão, este estudo demonstrou que os efeitos benéficos de usar uma
determinada suplementação de meio de cultivo depende do sistema de cultivo utilizado
(2D vs 3D). O VEGF e o GH foram os suplementos mais efetivos para o cultivo in vitro
de folículos secundário bovinos se os sistemas de cultivo 2D e 3D forem utilizados,
respectivamente.
Palavras-chave: Vaca. Sistemas de cultivo. Estradiol. Fatores de crescimento.
Folículos pré-antrais.
228
In vitro Development of Bovine Secondary Follicles in Two- and
Three-Dimensional Culture Systems Using Vascular Endothelial
Growth Factor (VEGF), Insulin-Like Growth Factor-1 (IGF-1), and
Growth Hormone (GH)
V.R. Araújoa,b, M.O. Gastala, A. Wischrala, J.R. Figueiredob, E.L. Gastala,*
a
Department of Animal Science, Food and Nutrition, Southern Illinois University,
1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
b
Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), Faculty
of Veterinary Medicine, State University of Ceará, Av. Paranjana 1700, Campus do
Itaperi, Fortaleza, 60.740-903, CE, Brazil.
*Corresponding author: Eduardo Gastal, Department of Animal Science, Food and
Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL,
62901, USA. FAX: (618) 453-5231; e-mail: egastal@siu.edu
229
Abstract
The aim of this study was to evaluate the development and estradiol production of
isolated bovine secondary follicles in two- (2D: Experiment 1) and three-dimensional
(3D using alginate: Experiment 2) long-term culture systems in the absence (Control
group; only α-MEM+) or presence of vascular endothelial growth factor (VEGF),
insulin-like growth factor-1 (IGF-1), or growth hormone (GH) alone, or a combination
of all. A total of 363 isolated secondary follicles were cultured individually for 32 days
at 38.5°C in 5% CO2 in a humidified incubator with addition of medium (5 µl) every
other day. In Experiment 1, follicular growth and antrum formation rates were higher
(P<0.05) in VEGF treatment when compared to the other treatments. In Experiment 2,
only estradiol concentration was greater (P<0.05) in the GH than the Control group,
whereas the other end points were similar (P>0.05). In summary, this study
demonstrated that the benefits of using a certain type of medium supplement depended
on the culture system (2D vs. 3D). VEGF and GH were effective supplements for the in
vitro culture of bovine secondary follicles when 2D and 3D culture systems were used,
respectively.
Keywords: Cattle, Culture systems, Estradiol, Growth factors, Preantral follicles
230
1. Introduction
Improvements in ovarian culture systems have increased the growth and
preserved the survival of bovine preantral follicles after long-term culture period.
Preantral follicles have been cultured in vitro enclosed in ovarian tissue (in situ; Yang
and Fortune, 2007; Tang et al., 2012) or in the isolated form (McLaughlin et al., 2010;
Rossetto et al., 2012). Isolated follicles have been cultured using a two-dimensional
(2D: Figueiredo et al., 1995; McLaughlin et al., 2010; McLaughlin and Telfer, 2010;
Rossetto et al., 2012, 2013; Araújo et al., 2012) or three-dimensional system (3D:
Figueiredo et al., 1995; Xu et al., 2010, 2011) in the absence (Wycherley et al., 2004) or
presence of extracellular matrix (ECM) such as collagen type 1 (Figueiredo et al., 1995;
Itoh et al., 2002), laminin, fibronectin, matrigel (Figueiredo et al., 1995), and alginate
(Xu et al., 2010; 2011).
A variety of hormones and growth factors have been used for the in vitro culture
of preantral follicles in many species (for review see Thomas et al., 2003). In small
ruminant species, culture medium containing EGF and LH (goat; Saraiva et al., 2010);
GH (goat; Magalhães et al., 2011); tyroxin, FSH, IGF-1, and GH (sheep; Arunakumari
et al., 2010); and LIF (sheep; Luz et al., 2012) resulted in in vitro embryo production
after culture of isolated secondary follicles. However, in cattle the results have been
limited to the follicular activation of small (primordial) preantral follicles (Wandji et al.,
1996; Fortune et al., 1998; Yang and Fortune, 2007, 2008; Tang et al., 2012) and early
antral follicles from large (secondary) preantral follicles (Gutierrez et al., 2000; Itoh et
al., 2002; McLaughlin et al., 2010; McLaughlin and Telfer, 2010; Rossetto et al., 2012,
2013; Araújo et al., 2012).
Currently, the culture systems for bovine preantral follicles do not produce good
results; however, researchers have been trying to improve these systems through the
addition of growth factors and/or hormones to the culture media. The addition of
vascular endothelial growth factor (VEGF) to the culture medium increased the
percentage of primordial follicle activation and formation of secondary follicles, and
increased follicle and oocyte diameters after in situ culture of bovine preantral follicles
(Yang and Fortune, 2007). Additionally, follicle-stimulating hormone (FSH) and
insulin-like growth factor (IGF-1) seemed to interact positively to enhance the growth
rate of isolated bovine secondary follicles (Gutierrez et al., 2000). Despite the potential
importance of these substances for bovine preantral follicle culture, to our knowledge,
231
there are no reports on the effects of VEGF and GH on the in vitro culture of isolated
secondary bovine follicles nor on the interaction of these factors with FSH and IGF-1
using 2D and 3D culture systems. The aim of this study was to evaluate the
development and estradiol production from isolated bovine secondary follicles in two(2D: Experiment 1) and three-dimensional (3D using alginate: Experiment 2) long-term
culture systems in the absence (Control group; only α-MEM+) or presence of VEGF,
IGF-I, or GH alone, or a combination of all.
2. Materials and methods
2.1. Chemicals and media
Unless otherwise stated, the culture media and chemicals used in the present
study were purchased from Sigma Chemical Co. (St. Louis, MO).
2.2. Source of ovaries
Ovaries (n = 188) from 94 adult Black Angus cows were collected at a local
slaughterhouse. The surrounding fat tissue and ligaments were removed, and the ovaries
were washed in 70% alcohol, followed by two washes in minimum essential medium
alpha (α-MEM). The ovaries were placed into tubes containing 20 ml of α-MEM plus
HEPES (α-MEM-HEPES), supplemented with 100 µg/ml penicillin and 100 µg/ml
streptomycin, and transported to the laboratory at 4°C within 1.5 h.
2.3. Isolation and selection of secondary follicles
In the laboratory, the surrounding fat tissue and ligaments were stripped off from
the ovaries. Ovarian cortical slices (1 mm thick) were cut from the ovarian surface using
a surgical blade under sterile conditions. Ovarian cortices were placed and washed in
fragmentation medium, which consisted of α-MEM-HEPES. Preantral follicles with
190.0 ± 6.6 m (mean ± SEM) in diameter were selected under a stereomicroscope
(SMZ 645 Nikon, Tokyo, Japan), manually dissected from the strips of ovarian cortex
using 25 gauge needles, and transferred to the culture medium for further evaluation of
the follicular quality. Preantral follicles with a visible central oocyte, surrounded by two
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or more granulosa cell layers, an intact basement membrane, and no antral cavity were
considered normal secondary follicles (Gutierrez et al., 2000) and selected for in vitro
culture.
2.4. Culture of secondary follicles
Follicles (n=363) were cultured for 32 days at 38.5oC in a humidified
atmosphere with 5% CO2 in air. Fresh medium was prepared immediately before use
and incubated for at least 1 h and a small supplementation medium replacement method
was used in which was performed the addition of 5 µl of fresh medium to an initial
small drop (50 µl) every other day, resulting in a final volume of 125 µl on the last day
of culture (Araújo et al., 2012). The control medium, named α-MEM+ (Araújo et al.,
2012) was composed of α-MEM (pH 7.2-7.4) supplemented with 3 mg/ml bovine serum
albumin (BSA), ITS (insulin 10 µg/ml, transferrin 5.5 µg/ml, and sodium selenite 6.7
ng/ml; CellGro Mediatech, Inc., Manassas, VA), 2 mM glutamine, 2 mM hypoxanthine,
50 μg/ml ascorbic acid, and 100 ng/ml recombinant follicle stimulating hormone (rFSH,
BioVision, Inc., Milpitas, CA).
2.5. Experimental design
In each experiment, selected follicles were individually distributed in single 50
µl culture medium drops under mineral oil in Petri dishes (60 x 15 mm, Corning, USA)
as previously described (Araújo et al., 2012). Follicles were randomly assigned to five
different treatments: α-MEM+ (Control group) or α-MEM+ supplemented with 100
ng/ml VEGF (VEGF group), 50 ng/ml IGF-1 (IGF-1 group), or 50 ng/ml GH (GH
group) alone or a combination of all (VEGF+IGF+GH group). Two different culture
systems, two- (2D using plastic Petri dishes: Experiment 1) and three-dimensional (3D
using alginate: Experiment 2), were used to compare all treatments. Each of the five
treatments during each experiment was repeated six times with 32-43 secondary
follicles cultured per treatment.
The concentrations of VEGF, IGF-1 (PeproTech Inc., Rocky Hill, NJ), and GH
(MPBio Biomedicals, LLC., Solon, OH) were chosen based on previous studies (Araújo
et al., 2011a; Thomas et al., 2007; Magalhães et al., 2011, respectively). In Experiment
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2, 0.25% of a matrix alginate-based (Xu et al., 2011) was used for the three-dimensional
culture system.
2.6. Alginate hydrogel preparation and follicle encapsulation
For the three-dimensional culture system in Experiment 2, sodium alginate (5565% guluronic acid) was provided by the Institute for Women's Health Research
(Chicago, IL, USA). Alginate aliquots were reconstituted by mixing on a racking
platform at room temperature overnight with sterile PBS (137 mM NaCl, 10 mM
phosphate, and 2.7 mM KCl; Invitrogen) to a concentration of 0.25% (w/v).
After isolation and selection, the follicles were encapsulated individually in
alginate beads. Single follicles were transferred to droplets of alginate (5 µl), and the
droplets were immersed in a cross-linking solution (50 mM CaCl2 and 140 mM NaCl).
Following cross-linking for 2-3 min, the alginate beads were removed and rinsed in
culture medium (Xu et al., 2011).
2.7. Morphological evaluation of follicle development
Follicular features were evaluated during culture including the integrity of the
basement membrane, the morphological aspects of the oocyte, and surrounding
granulosa cells. Morphological signs of degeneration, such as darkness or abnormality
of oocytes and surrounding granulosa cells, were considered as previously reported
(Rossetto et al., 2012). Follicular diameter was measured every 8 days in
morphologically normal follicles with the aid of an ocular micrometer inserted into a
stereomicroscope (SMZ 645 Nikon, Tokyo, Japan) (75X magnification). Two
perpendicular measures were recorded for each follicle and the average of the two
values was reported as follicular diameter (µm). Regarding follicular growth rate, the
mean increase in follicular diameter was calculated as follows: the diameter of
morphologically normal follicles at day 32 minus the diameter of normal follicles at day
0, divided by 32. Antral cavity formation was defined as a visible translucent cavity
within the layers of granulosa cells.
2.8. Viability assessment of follicles cultured in vitro
234
For a more accurate evaluation of follicular integrity after 32 days of culture,
live/dead fluorescent labeling (Rossetto et al., 2012) was performed. Follicles were
placed in droplets of α-MEM-HEPES with 4 μM calcein-AM and 2 μM ethidium
homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany), followed by
incubation at 38.5°C for 15 min. Finally, the follicles were examined using a
fluorescence microscope (Zeiss, Axiovert 10, NY, USA). The emitted fluorescent
signals of calcein-AM and ethidium homodimer were collected at 450-490 nm. The
probe detected the intracellular esterase activity of viable cells first, and then the nucleic
acids of non-viable cells by plasma membrane disruption. The follicles were considered
live if the cytoplasm was labeled positively with calcein-AM (green) or dead if cellular
chromatin was labeled with ethidium homodimer (red).
2.9. Estradiol concentration measured by enzyme immunoassay
To evaluate follicular steroidogenesis in vitro, concentrations of estradiol were
measured in reserved culture media against standard dilutions using an estradiol
Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (Neogen, Lexington KY, USA).
Media were removed from all the treatments in both experiments on days 0 and 32 of in
vitro culture. Briefly, the reserved media were diluted with EIA buffer (1:10), placed in
microplate wells coated with polyclonal (rabbit) antibody raised against the estradiol
antigenic site, mixed with estradiol enzyme conjugate, and incubated for 60 min. After
incubation, the unbound conjugate was washed three times with diluted EIA buffer, and
a substrate solution of tetramethylbenzidine (TMB) was added to allow development of
color. After 30 min, the absorbance of the plate was read at 650 nm using a microplate
reader (Synergy 2 Multi-Mode Microplate Reader, Winooski, VT). Results were
obtained using the 4 parameter logistic (4PL) curve with the Readerfit © program
(Hitachi Solutions America, Ltd., 2012). The intra-assay coefficient of variation and
sensitivity of the assay were 3.2 and 0.02 ng/ml, respectively.
2.10. Statistical analyses
Follicle and estradiol data were challenged for extreme values with the Dixon
outlier test (Zar, 1984). Data for end points that were not normally distributed,
according to the Shapiro-Wilk test, were transformed to logarithms or ranks. Single-
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point data were analyzed by one-way ANOVA. If a main effect of group was
significant, the differences between groups were examined by Duncan’s multiple range
tests. Frequency data were analyzed by the chi-square test. A probability of P<0.05
indicated that a difference was significant. Data are given as the mean ± SEM unless
otherwise stated.
3. Results
For Experiments 1 (2D) and 2 (3D), a total of 207 (2.3/ovary) and 156
(1.6/ovary) bovine secondary follicles, with two or more layers of granulosa cells and
usually surrounded by theca cells (Fig. 1A), were isolated and used for the in vitro
culture, respectively. No difference was observed for follicular morphology, viability,
and diameter among treatments in each experiment and time point (i.e., days 0, 8, 16, 24
and 32 of culture). The overall mean (± SEM) of morphologically normal follicles (%),
follicular viability (%), and diameter (µm) for 2D and 3D systems were: 86.2 ± 17.2%
and 68.8 ± 13.8%; 71.0 ± 22.4% and 86.8 ± 27.5%; 226.7 ± 7.1 µm and 287.3 ± 12.3
µm, respectively. Overall, when follicles were cultured in the 2D system (Experiment
1), the addition of VEGF to the culture medium increased (P<0.05) the growth rate and
the percentage of antrum formation when compared to the other treatments (Table 1).
However, such effects were not observed when the 3D culture system (Experiment 2)
was used. Furthermore, the addition of GH to the culture medium resulted in the highest
(P<0.05) estradiol concentration when compared to all other treatments. Antral follicles
after in vitro culture (day 32) using only α-MEM+, α-MEM+ plus VEGF, or α-MEM+
plus GH in 2D (Fig. 1B-C) or 3D (Fig. 1D) culture systems are shown (Fig. 1),
respectively.
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Fig. 1. Bovine follicles before (A; day 0) and after in vitro culture (B-D; day 32) in
medium containing only α-MEM+ (B), or α-MEM+ plus VEGF (C), or α-MEM+ plus
GH (D). Normal in vitro grown preantral (A) and antral follicles (B-D) using 2D (B-C)
or 3D (D) culture systems, respectively. o: oocyte; gc: granulosa cells; tc: theca cells; a:
antral cavity formation. Scale bars = 50 µm. Images were captured at 32X (A) and 10X
(B-D).
237
Table 1. Morphologically normal follicles (%), antrum formation (%), growth rate (µm/day), and estradiol concentration (ng/ml) of bovine
follicles after 32 days of in vitro culture in two- (2D: Experiment 1) and three-dimensional (3D using alginate: Experiment 2) culture systems in
the absence (Control group: only α-MEM+) or presence of VEGF, IGF-I, or GH alone, or a combination of all (VEGF+IGF+GH).
End point
α-MEM+
VEGF
IGF-1
GH
VEGF+IGF+GH
Experiment 1: 2D culture system
Morphologically normal follicles (%)
30/41 (73.2)a
31/41 (75.6)a
29/43 (67.4)a
31/43 (72.1)a
27/39 (69.2)a
Antrum formation (%)
17/41 (41.5)b
29/41 (70.7)a
18/43 (41.9)b
21/43 (48.8)b
17/39 (43.6)b
Growth rate (µm/day)
3.3 ± 0.4b
5.0 ± 0.5a
3.0 ± 0.5b
2.8 ± 0.4b
2.3 ± 0.3b
Estradiol production (ng/ml)
2.4 ± 0.7a
3.1 ± 1.0a
0.8 ± 0.3a
2.7 ± 1.0a
0.8 ± 0.3a
Morphologically normal follicles (%)
18/32 (56.3)a
18/32 (56.3)a
16/32 (50)a
14/30 (46.7)a
10/30 (33.3)a
Antrum formation (%)
10/32 (31.3)ab
6/32 (18.8)b
13/32 (40.6)a
7/30 (23.3)b
4/30 (13.3)b
Experiment 2: 3D culture system
Growth rate (µm/day)
6.7 ± 1.1a
7.5 ± 1.2a
6.1 ± 1.0a
6.4 ± 1.1a
5.0 ± 1.3a
Estradiol production (ng/ml)
1.3 ± 0.7b
0.3 ± 0.3b
1.6 ± 0.6b
22.1 ± 5.3a
0.9 ± 0.4b
a-b
No common superscripts within same end point means that treatments were different (P<0.05).
238
4. Discussion
This study demonstrated for the first time that the addition of VEGF affected
positively the in vitro culture of bovine isolated secondary follicles using a twodimensional culture system. It was observed that the addition of VEGF to the culture
medium improved the percentage of antrum cavity formation, as well as follicular
growth rate. Based on the fact that VEGF is a cellular mitogenic factor, the presence of
exogenous VEGF in the culture medium might have increased the number of theca and
granulosa cells, resulting in the development of larger follicles. VEGF has been shown
to activate the PI3K/AKT pathway in different cell types, such as endothelial cells
(Gerber et al., 1998; Fujio et al., 1999), neurons (Jin et al., 2000), and smooth muscle
cells (Banerjee et al., 2008), promoting cell survival and proliferation (Datta et al.,
1999). Moreover, the mechanism involved in the proliferative role of VEGF seems to be
distinct of its classic angiogenic action, the PI3K/AKT intracellular pathway
(Abramovich et al., 2010). Increased follicular growth rate has been correlated with
increased VEGF production during antrum formation of primate preantral follicles (Xu
et al. 2010). Recently, we observed that the addition of VEGF to the culture medium of
caprine preantral follicles allowed the increase of follicular diameter and growth rate
(Araújo et al., 2011a). These results might be associated with the presence of mRNA for
VEGF receptors and ligand (bovine: Yang and Fortune, 2007), as well as the
immunoreactivity for VEGF protein (caprine: Sharma and Sudan, 2010; Bruno et al.,
2009), which have been detected in preantral follicles. Additionally, the inhibition of
VEGF decreased the expression of the proliferating cell nuclear antigen (PCNA) marker
in the theca and granulosa cells from rat ovaries (Abramovich et al., 2010).
This study demonstrated a positive effect of VEGF in the antrum cavity
formation in bovine secondary follicles after long-term two-dimensional in vitro culture.
Recent study from our team demonstrated the ability of VEGF to increase the
permeability of the cells and subsequent development of antral follicles in goats (Araújo
et al., 2011a). As mentioned before, VEGF is a cellular mitogenic factor that could act
indirectly on the accumulation of intrafollicular fluid and antral cavity formation,
consequently increasing the follicular diameter. In vivo, ovarian cell permeabilization
via VEGF occurs by reorganization and neoformation of ovarian vessels (Danforth et
al., 2003; Quintana et al., 2004), which improves follicle growth by the increase of the
239
availability of growth factors, gonadotropins, steroids, and oxygen, essential for follicle
development (for review see Araújo et al., 2011b). However, in vitro, there is no
blood/plasma to release these needed substances to the cells for continuous
development. In this case, the culture medium plays a role similar to that of
blood/plasma during the in vitro culture, acting as nutritional support to the cells in
culture (Araújo et al., 2011c). Therefore, VEGF might influence the accumulation of
antral fluid through additional mechanisms, such as an osmotic potential through the
sodium pump or cleavage of glycosaminoglycan, osmotically active molecules of the
follicular fluid (e.g., hialuronon; Rodgers and Irving-Rodgers, 2010). From all enzymes
involved in antral fluid formation, it has been reported that bovine preantral follicles and
granulosa cells in culture express hyaluronan synthase 1 (HAS1; Vasconcelos et al.,
2013) and 2 (HAS2; Schoenfelder and Einspanier, 2003); these two enzymes lead to
follicle growth and cumulus-oocyte complex expansion during follicular development
and final oocyte maturation, respectively. Therefore, the increase of cell permeability
attributed to VEGF in our results might have been associated with the increase of
synthesis of some of those molecules.
In Experiment 2 (alginate-based 3D system), the addition of GH to the culture
medium resulted in higher estradiol concentrations. Kobayashi et al. (2000)
demonstrated that murine preantral follicles stimulated by GH formed complete theca
layers abundant in mitochondria and rough endoplasmic reticulum and many lipid
droplets, indicating that GH causes proliferative effects on theca cells. Moreover, these
authors also observed that there was no estradiol production in preantral follicles
without theca cells, suggesting that theca cells are mandatory for estradiol secretion of
preantral follicles. Thus, we could speculate that the higher estradiol concentrations may
have been attributed to increased theca cell presence in the follicles cultured with GH.
Higher concentrations of estradiol in the 3D culture system containing GH could have
also been associated with the fact that this system might have better preserved the
follicular architecture and consequently the follicle function as previously documented
when using the 3D culture system for other species (Wycherley et al., 2004; Xu et al.,
2010; 2011). It is worth mentioning that this is the first study with encapsulation of
bovine secondary follicles using alginate-based matrix. Recently, Xu et al. (2011)
suggested that granulosa cells from monkey preantral follicles may utilize
androstenedione and progesterone efficiently to synthesize high levels of estradiol after
240
appropriate proliferation using alginate hydrogel. However, in the present study, the
higher estradiol concentration observed in the GH group using a 3D alginate-based
culture system was not associated with any other improvement of follicle development.
In conclusion, this study demonstrated that the benefits of using a certain type of
medium supplement depended on the culture system (2D vs. 3D). VEGF and GH were
effective supplements for the in vitro culture of bovine secondary follicles when 2D and
3D culture systems were used, respectively.
Acknowledgments
The authors thank Dr. Teresa K. Woodruff from Northwestern University,
Chicago, IL, USA for generously donating the alginate used in the 3D culture system.
We are also grateful to Mr. Alvin Kasten for providing the ovaries at the
slaughterhouse. This work was supported by a start-up package (Gastal EL) from SIU.
Araújo VR is the recipient of a PhD scholarship from CNPq, Brazil.
Conflict of interest
There is no conflict of interest that could be perceived as prejudicing the
impartiality of the research reported.
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CONCLUSÕES
Diante dos resultados apresentados concluiu-se que:
a) A adição periódica de meio melhorou o desenvolvimento de oócitos oriundos de
folículos secundários caprinos crescidos in vitro;
b) O tipo de meio de base (αMEM e TCM-199) e o regime de troca de meio afetaram
diferentemente o desenvolvimento folicular, a expressão gênica e a produção de
estradiol após cultivo in vitro de folículos secundários bovinos isolados; sendo
recomendada nesta espécie, a utilização do αMEM em regime de adição periódica de
meio;
c) A BMP-6, mesmo em baixas concentrações (1 ng/mL), promoveu atresia e alterações
ultraestruturais em folículos primordiais durante o cultivo in vitro de tecido cortical
ovariano caprino;
d) A BMP-6 (1 ng/mL), na ausência de FSH, estimulou a formação de antro, bem como
a expressão do RNAm para os receptores de BMP (BMPR-2) e seus sinalizadores
intracelulares (Smads) após cultivo in vitro de folículos secundários caprinos
isolados;
e) O VEGF promoveu o desenvolvimento de oócitos oriundos de folículos secundários
caprinos crescidos in vitro, permitindo a produção de oócitos maturos (metáfase II);
f) A adição de VEGF, utilizando sistema de cultivo 2D, melhorou o desenvolvimento
(formação de antro e taxa de crescimento) in vitro de folículos secundários bovinos.
Além disso, o GH aumentou os níveis de estradiol produzidos por folículos
secundários bovinos cultivados in vitro, utilizando o sistema de cultivo 3D.
246
PERSPECTIVAS
Os resultados da presente tese monstraram que a adição periódica de meio
melhorou o desenvolvimento de folículos pré-antrais caprinos e bovinos isolados. Vale
salientar que os sistemas de cultivo desenvolvidos garantiram taxas aceitáveis de
sobrevivência e crescimento folicular mesmo em cultivo de longa duração. Um
importante achado do presente estudo foi a determinação de uma concentração de
VEGF (100 ng/mL) que aumentou, de forma significativa, as taxas de maturação in
vitro de oócitos oriundos de folículos pré-antrais caprinos crescidos in vitro, fato inédito
na literatura.
Neste contexto, estudos complementares que explorem a utilização das
substâncias testadas no cultivo in vitro de folículos pré-antrais na presente tese,
empregando diferentes estágios de desenvolvimento folicular, inclusive em cultivos
com meios sequenciais, ou ainda, em diferentes sistemas de cultivo, poderão ser
realizados para melhor entender o processo de desenvolvimento folicular e oocitário.
Desta forma, será factível a elaboração de meios de cultivo capazes de proporcionar
condições ótimas para um completo crescimento e maturação folicular e oocitária,
contribuindo de forma significativa para a melhoria da eficiência reprodutiva dos
rebanhos.
247
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