Workshop 6: Rede MOIFOPA-PIV Brasil 06_SBTE_MOIFOPA.P65 4/8/2010, 17:27

Transcription

Workshop 6:
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Rede MOIFOPA-PIV Brasil
4/8/2010, 17:27
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Acta Scientiae Veterinariae . 38(Supl 2): s417-s447, 2010.
ISSN 1678-0345 (Print)
ISSN 1679-9216 (Online)
Advances in Artificial Ovary in Goats
José Ricardo de Figueiredo1, Juliana Jales de Hollanda Celestino1, Luciana Rocha Faustino1
& Ana Paula Ribeiro Rodrigues1
ABSTRACT
Background: In livestock including goats, the modern assisted reproductive technologies are being used for the
improvement and preservation of livestock genetics and the enhancement of reproductive efficiency. It is well known
that mammalian ovaries contain many thousands of immature oocytes enclosed predominantly in preantral follicles
(PF). However, more than 99.9% of follicles will not ovulate but instead they will be eliminated by a physiological process
called atresia. The rescue of PF from the ovaries follow by the development of in vitro culture systems (artificial ovary)
which allow the in vitro growth and maturation (IVGM) of their enclosed immature oocytes up to maturation stage by
preventing follicular atresia could have a major impact on the in vitro embryo production in the future. In vitro studies
have demonstrated satisfactory results with laboratory animals. The birth of a mouse from primordial follicles grown,
matured and fertilized in vitro was obtained. Despite the good results in mice, in other animals, including livestock, at
most a few embryos have been produced from IVGM oocytes from in vitro grown oocytes.
Review: The in vitro culture of preantral follicle, also called artificial ovary, is an important step of the Manipulation of
Oocytes Enclosed in Preantral Follicles (MOEPF). It aims to create in vitro an appropriated culture system which allows
the survival, grow, maturation and further fertilization of small oocytes from preantral follicles, by preventing the follicular
atresia that occurs abundantly in the ovaries. This article emphasizes the different steps of the MOEPF, its importance
and finally, the main results obtained by our research team related to the in vitro culture of caprine preantral follicles. Our
group has developed research with PF including isolation, conservation (cooling and cryopreservation) and in vitro
culture of PF, focus on caprine species. In our laboratory, it was established efficient protocols for the isolation and
conservation of PF, being the in vitro culture of PF up to maturation stages the major limitation at present. The types of
in vitro culture systems available for preantral follicles include the culture of whole ovary, ovarian cortical slices and
mechanically and/or enzimatically isolated follicles. We have been tested the effects of several substances including
coconut water solution, antioxidants, serum, different types of hormones and growth factors on in vitro culture of
preantral follicles enclosed in ovarian fragments. The early work of our group with culture of isolated secondary follicles
were conducted involving the basic conditions of culture, i.e., ideal oxygen tension, regime of medium change, and how
to culture the isolated follicles, individual or group, with or without FSH, in the presence or absence of antral follicles. The
basic culture system for the in vitro culture of caprine PF which maintains follicular survival is well established. Primordial
follicle activation and their further growth up to secondary stage in vitro were achieved. Isolated primary follicles can
growth up to antral stage although this rate is still low. Antrum formation and fully grown oocytes were obtained from in
vitro culture of large secondary follicles even yielding a few mature oocytes and embryos.
Conclusion: In vitro embryo production from caprine preantral follicles grown in vitro was successfully achieved by
our research group. At present, the key challenge for researchers in this important field of reproduction is to increase
the rates of maturation and in vitro production of embryos from oocytes enclosed in preantral follicles at early stages,
in order to produce, in the future, large number of offsprings.
Keywords: MOIFOPA, in vitro culture, preantral follicles, caprine
1
Laboratório de Manipulação de Oócitos e Folículos Ovarianos Pré-antrais (LAMOFOPA), Faculdade de Veterinária, Universidade
Estadual do Ceará (UECE), Fortaleza, Av. Paranjana, n 1700, Campus do Itaperi, Bairro Itaperi, CEP 60700-000 Fortaleza,CE, Brasil.
CORRESPONDENCE:: J.R. Figueiredo [jrfig@pesquisador.cnpq.br - Fax: + 55 (51) 3101-9860].
1
Estadual do Ceará (UECE), Fortaleza, Av. Paranjana, n 1700, Campus do Itaperi, Bairro Itaperi, CEP 60700-000 Fortaleza,CE, Brasil.
CORRESPONDENCE:: J.R. Figueiredo [jrfig@pesquisador.cnpq.br - Fax: + 55 (51) 3101-9860].
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Acta Scientiae Veterinariae. 38 (Supl 2): s417-s447
I. INTRODUCTION
II. STEPS OF MOEPF
III. IMPORTANCE OF MOEPF
IV. RESULTS OBTAINED BY OUR RESEARCH TEAM RELATED TO IN VITRO
CULTURE OF GOAT PREANTRAL FOLLICLES
IN VITRO CULTURE OF CAPRINE PREANTRAL FOLLICLES ENCLOSED IN OVARIAN CORTICAL
SLICESIN VITRO CULTURE OF ISOLATED CAPRINE PREANTRAL FOLLICLES
V. CONCLUSION
I. INTRODUCTION
Goats are present on all continents and are commercially viewed as highly attractive livestock animals,
since they have been used for many purposes such as milk, meat and skin production. Extraordinary developments
have been achieved in the field of assisted reproductive technology both in human and animals in the last two
decades. In livestock including goats, the modern assisted reproductive technologies are being used for the improvement
and preservation of livestock genetics and the enhancement of reproductive efficiency [28].
It is well known that mammalian ovaries contain many thousands of immature oocytes enclosed
predominantly in preantral follicles (PF). However, more than 99.9% of follicles will not ovulate but instead they will be
eliminated by a physiological process called atresia. The rescue of PF from the ovaries follow by the development of
in vitro culture systems (artificial ovary) which allow the in vitro growth and maturation (IVGM) of their enclosed
immature oocytes up to maturation stage by preventing follicular atresia could have a major impact on the in vitro
embryo production in the future. Because this biotechnology intends to mimic what happens with a few oocytes able
to escape from atresia during their growth and maturation inside the ovary we call this technique “artificial ovary”. This
term is easily understood to ordinary people facilitating interaction between scientists and society.
In vitro studies have demonstrated satisfactory results with laboratory animals. Eppig & O’ Brien [12]
obtained the birth of a mouse from primordial follicles grown, matured and fertilized in vitro. Some years later, the
same group improves their previously protocol and related the embryo production and further birth of 59 healthy mice
from PF cultured and matured in vitro [27]. Despite these good results in mice, in other animals, including livestock,
at most a few embryos have been produced from IVGM oocytes (rat [10]; pig [37]; buffalo [16]; goat – Saraiva et al.,
unpublished data and Magalhães et al., unpublished data).
This article emphasizes the different steps of the Manipulation of Oocytes Enclosed in Preantral Follicles
(MOEPF), its importance and finally, the main results obtained by our research team related to the in vitro culture of
caprine preantral follicles (artificial ovary)..
II. STEPS OF MOEPF
The MOEPF consists: 1) Isolation or rescue of PF from the ovaries, 2) PF conservation -aiming the storage
for a short (cooling) or a long (cryopreservation) period and 3) In vitro follicular culture with the purpose to promote the
growth, maturation and in vitro fertilization of oocytes from PF, providing the embryo production and offspring after
embryo transfer. The steps of isolation and conservation of PF are well established [14], being the lack of an appropriated
in vitro culture system the limit step for the development of the biotechnique of MOEPF. The different steps of
MOEPF are illustrated in figure 1.
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Figure 1. Different steps of MOEPF. IVM: in vitro maturation; IVF: in vitro fertilization; IVC: in vitro culture of embryos; ET: embryos transfer.
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III. IMPORTANCE OF MOEPF
1- Fundamental or basic research- Providing the study of the in vitro effect of different substances on the PF, being an
essential tool in understanding underlying mechanisms of oocyte and follicle growth and differentiation;
2- Pharmaceutical industry- In vitro testing the action of the drugs on the follicles before its application in the
experiments with human or animals;
3- Formation of genetic banks (germoplasm) - Through the conservation of the oocytes enclosed in PF from animals
of zootecnic interest or in extinction;
4- Assisted human reproduction- Preserving the female fertility in woman which will be submitted to radio and
quimioterapy treatments and which need to have their ovaries removed and cryopreserved for further autotransplant
or in vitro culture;
5- Multiplication of animals - It will allow to the in vitro embryo production in large scale from the oocytes enclosed in
PF, retrieved from the whole ovaries or ovarian fragments (biopsy);
6- Animal welfare - MOEPF will contribute to the reduction of the stress in animals, representing an alternative to the
procedures, such as superovulation, embryo collection and ovum pick-up by ultrassonography, as well as the use of
life animals in experiments.
IV. RESULTS OBTAINED BY OUR RESEARCH TEAM RELATED TO IN VITRO
CULTURE OF GOAT PREANTRAL FOLLICLES
Our group has developed research with PF including isolation, conservation (cooling and cryopreservation) and in
vitro culture of PF, focus on caprine species. In our laboratory, it was established efficient protocols for the isolation and
conservation of PF [14], being the in vitro culture of PF up to maturation stages the major limitation at present.
The types of in vitro culture systems available for preantral follicles include the culture of whole ovary [12], ovarian
cortical slices [31,32] and mechanically and/or enzimatically isolated follicles [35,36]. The performance of in vitro culture of
preantral may be affected by many factors including animal species, type of culture system (bi or tridimensional), pH,
oxygen tension and medium composition which must supply appropriated amounts of electrolytes, antioxidants, amino
acids, energetic substrates, vitamins, hormones and growth factors [13,3,15]. Due to the importance of medium composition,
our team has focused on the development of culture media which ensure follicular survival and development.
The first studies performed by our group and majority of the results obtained up to now involved the in vitro
culture of caprine preantral enclosed in ovarian cortical slices.
In vitro culture of caprine preantral follicles enclosed in ovarian cortical slices
Using this culture system, the effects of several substances including coconut water solution, antioxidants,
serum, different types of hormones and growth factors on in vitro culture of PF have been evaluated.
In our first study, we evaluated the effect of coconut water solution (CWS) on goat primordial follicle growth,
viability and granulosa cell proliferation compared with those of Minimum Essential Medium (MEM) and mixtures of
MEM and CWS. The results demonstrated that after 5 days of culture addition of CWS (25, 50, 75 and 100%) to MEM
maintained the proportion of follicle activation similar to that observed in pure MEM. However, when pure CWS was
used the follicular degeneration is enhanced [31].
In another study we tested the effects of indole-3-acetic acid (IAA), an important ingredient of coconut, on
survival, activation, and growth of caprine PF using histological and ultrastructural criteria. The results showed a
greater percentage of histologically-normal follicles in MEM alone or MEM supplemented with IAA (20 ng/ml) than
other treatments. Indole-3 acetic acid added at a concentration of 20 or 40 ng/ml increased the proportion of primordial
follicles that entered the growth phase after 5 days. Ultrastructural studies, however, did not confirm maintenance of
the morphological integrity of caprine follicles cultured for 1 or 5 days in MEM supplemented with IAA [23].
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The importance of some antioxidants such as á-tocoferol, ternatin and ascorbic acid was also investigated.
The addition of á-tocoferol, ternatin did not maintain the ultrastructural integrity of caprine PF cultured in vitro neither
showed additional effects on follicular activation and growth [17]. However, ascorbic acid showed to be very important
for the performance of long-term (14 days) in vitro culture of caprine follicles. The combination of 50 µg/mL of ascorbic
acid and FSH (50 ng/mL) maintained ultrastructural integrity and promoted follicular activation and growth [29].
Due to the fact that serum may be rich in nutrients, hormones and growth we investigated the importance
of different sources of serum (fetal calf serum, estrus and diestrus goat serum) and concentrations (0, 10 or 20%) on
in vitro culture of caprine PF. As described for á-tocoferol and ternatin, regardless the source or concentration, the
serum did not maintain the ultrastructural integrity of caprine PF cultured in vitro neither showed additional effects on
follicular activation and growth [4].
Because of the importance of hormones and ovarian growth factors for the regulation of folliculogenesis we
investigated their effects on in vitro culture of PF in many experiments.
FSH is one of the most studied substances by our group. We have investigated the effect of different
sources and concentration of FSH on caprine PF development. In a first study we investigated the effects of pFSH
(Stimufol®) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural
studies.Three FSH concentrations were tested (10, 50 or 100 ng/mL) and it was demonstrated that FSH at concentration
of 50 ng/ml not only maintained the morphological integrity of 7 days cultured caprine PF, but also stimulated the
activation of primordial follicles and the growth of activated follicles [24].
Later on, we study the effects of different pFSH (Stimufol® and Folltropin®) on the in vitro survival and
growth of caprine PF after 7 days of culture. The results showed that FSH preparations affect differently the performance
of in vitro culture of caprine PF. Stimufol® was better to preserve follicular ultrastructure while Folltropin® was more
efficient to promote follicular growth [19]. Next, we compared different sources of FSH (pituitary and recombinant) in
different concentrations. Recombinant FSH (rFSH) was superior to pFSH because 50 ng/ml of rFSH maintained the
ultrastructural integrity of caprine PF and promoted primordial follicles activation and further growth of follicles cultured
for seven days [20].
Another gonadotrophin tested in vitro was Luteining Hormone. Saraiva et al. [30] showed that the addition of
low concentrations of LH (1 ng/ml) combined with or without FSH maintained the goat follicular ultrastructural integrity,
but LH in doses higher than 5 ng/ml induced atresia of in vitro goat PF.
Estradiol in dose-dependent manner presented beneficial effects on caprine preantral culture. Ultrastructural
studies confirmed follicular integrity after 7 days of culture in the presence of 1 pg/ml estradiol plus FSH [18].
Besides hormones, the influence of intra-ovarian factors on caprine PF culture was also investigated by our
group. Many of the extracellular signaling molecules implicated in regulation of early folliculogenesis belong to the
transforming growth factor (TGF)-â superfamily. Of these molecules we studied the activin, Nervous Growth Factors
(NGF) and Growth and Differentiation Factor 9 (GDF 9).
Silva et al. [33] investigated the effects of activin-A and follistatin on in vitro primordial and primary follicle
development in goats. It was found that goat primordial follicles were activated to develop into more advanced stages,
i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of
primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal
follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by
follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin,
KL, GDF-9 and BMP-15.
Nervous growth factor (NGF) in low concentration (1ng/ml) showed positive effects on follicular survival
after culture but has no effect on follicular actitvation and growth. [9].
When BMP -6 and -7 were tested in different concentrations (0, 1, 10, 50 or 100 ng/mL) on caprine preantral
follicles in vitro culture, BMP-6 negatively affects the follicular survival [1], whereas BMP-7 in low concentrations
improves follicular survival (1 ng/mL) and growth (10 ng/mL) [2].
In general, several studies with farm animals and primates have successfully shown the activation and
transition of primordial follicles to primary stages during in vitro culture of ovarian cortical slices. However, using these
mammalian models primary follicles do not grow to secondary stages. Despite that, we succeeded to overcome this
problem using an appropriate concentration of growth and differentiation factor-9 (GDF-9). Indeed, Martins et al. [22]
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demonstrated that GDF-9 at a concentration of 200 ng/ml maintained the survival of PF and promoted activation of
primordial follicles. Furthermore, GDF-9 stimulated the transition from primary to secondary follicles, maintaining
ultrastructural integrity of the follicles.
In other studies we investigated the effects of some mitogenic substances such as, Fibroblast Growth
Factor 2, (FGF-2) and Epidermal Growth Factor (EGF), as well as other substances like Kit Ligant (KL), Vascular
Endotelial Growth Factor (VEGF) and Vasoactive Intestinal Pepitide (VIP).
Matos et al. [25] demonstrated that at a concentration of 50 ng/ml of FGF-2 not only maintained the
morphological integrity of caprine PF cultured for 5 days, but also stimulated the activation of primordial follicles and
the growth of activated follicles. A positive interaction between FSH and FGF-2 to promote the initiation of primordial
follicles growth and oocyte growth was also observed [26]. In contrast to that observed to FGF-2, previous work using
a single tested concentration of EGF (100 ng/mL) associated with pFSH (100 ng/mL) showed no interaction between
these two substances [32].
Celestino et al. [7] investigated the effects of different concentrations of EGF (0, 1, 10, 50, 100, or 200 ng/
mL) on the survival and growth of caprine preantral. They demonstrated that the low concentrations of EGF (1 or 10
ng/mL) maintained caprine follicular viability and promote the transition from primordial to primary follicles after 7 days
of culture. Similar results were observed using KL (50 ng/mL) testing the same concentrations reported for EGF [8].
Angiogenic factors, like VEGF, also played an important role on follicular development in vitro. Bruno et al.
[5] investigated the effect VEGF on the survival and growth of goat PF after in vitro culture and verified the expression
of VEGF receptor (VEGFR)-2 in goat ovaries. Immunohistochemical studies demonstrated the expression of VEGFR2 in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial follicles. The best
concentrations of VEGF to promote follicular growth and to maintain follicular survival after 7 days of culture were 10
ng/mL and 200 ng/mL, respectively [5]. VIP at a concentration of 10 ng/mL showed similar effects [6].
In vitro culture of isolated caprine preantral follicles
In addition to several studies using in vitro culture of goat PF enclosed in ovarian tissue, described above, in
the last three years, our group has been worked intensively with the in vitro culture of isolated caprine PF. Although the
number of results, so far, with this system is still small, the results are of great importance, with rates of antrum
formation and follicular survival above 70%, obtaining in vitro grown oocytes (e” 110 mm) capable of resuming meiosis,
including some of them reaching nuclear maturation (metaphase II stage with the extrusion of first polar body).
The early work of our group with culture of isolated secondary follicles were conducted involving the basic
conditions of culture, i.e., ideal oxygen tension, regime of medium change, and how to culture the isolated follicles,
individual or group, with or without FSH, in the presence or absence of antral follicles.
By testing two different oxygen tensions (5 or 20%), it was found that the concentration of 20% O2 was
more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when
grown in vitro after 30 days of culture [34].
In other study, we evaluated the effects of different medium replacement intervals (2 or 6 days) on the
viability, antral cavity formation, growth and in vitro maturation of oocytes from caprine and ovine isolated preantral
follicles cultured of 24 days. Goat and sheep preantral follicles behaved completely different. To improve the efficiency
of the culture system, the medium must be replaced every two and six days for goat and sheep preantral follicles,
respectively [21].
We also tested different forms of follicular culture, ie, individually or group , with or without FSH, and in the
presence or absence of antral follicles. The duration of culture period was 24 days. In this study, in the absence of
FSH, the culture of preantral in group showed greater rates of follicular survival and growth than individual follicles. On
the other hand, in the presence of FSH these parameters were not affect, but larger number of grown oocytes and
rates of meiosis resumption were observed when follicles were cultured in group. Finally, the co-culture of antral
follicle with individual preantral follicle affected negatively follicular survival and growth while opposite results were
observed with follicles in group [11].
Last, but not least, the results even more relevant, though not yet published, were achieved in which
embryos were produced from isolated goat PF grown, matured and fertilized in vitro (Saraiva et al., unpublished data
and Magalhães et al., unpublished data). These results were submitted for publication and the papers are under
revision.
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V. CONCLUSION
The basic culture system for the in vitro culture of caprine preantral follicles which maintains follicular
survival is well established. Primordial follicle activation and their further growth up to secondary stage in vitro were
achieved. Antrum formation and fully grown oocytes were obtained from in vitro culture of large secondary follicles
even yielding a few mature oocytes and embryos. At present, the key challenge for researchers in this important field
of reproduction is to increase the rates of maturation and in vitro production of embryos from oocytes enclosed in
preantral follicles at early stages, in order to produce, in the future, large number of offsprings.
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Figueiredo J.R. 2010. Effect of the medium replacement interval on the viability, growth and in vitro maturation of isolated caprine and
ovine pre-antral follicles. Reproduction in Domestic Animals. [in press].
23 Martins F.S., Celestino J.J.H., Saraiva M.V.A., Matos M.H.T., Bruno J.B., Rocha-Junior C.M.C., Lima-Verde I.B., Lucci C.M., Báo
S.N. & Figueiredo J. R. 2008. Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their
progression to secondary follicles. Reproduction Fertility and Development. 20: 916-924.
24 Matos M.H.T., van den Hurk R., Martins F.S., Santos R.R., Luque M.C.A., Silva J.R.V., Celestino J.J.H., Báo S.N. & Figueiredo J.R.
2006. Histological and ultrastructural features of caprine preantral follicles after in vitro culture in the presence or absence of indole-3acetic acid. Animal Reproduction. 3(4): 415-422.
25 Matos M.H.T., Lima-Verde I.B., Luque M.C.A. Maia Jr. J.E., Silva J.R.V., Celestino J.J.H., Martins F.S., Báo S.N., Lucci C.M. &
Figueiredo J.R. 2007. Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro.
Zygote. 15: 173-182.
26 Matos M.H.T., van den Hurk R., Lima-Verde I.B., Luque M.C.A., Santos K.D.B., Martins F.S., Báo S.N., Lucci C.M. & Figueiredo
J.R. 2007. Effects of Fibroblast Growth Factor-2 on the in vitro Culture of Caprine Preantral Follicles. Cells Tissues Organs. 186: 112120.
27 Matos M.H.T., Lima-Verde I. B., Bruno J. B., Lopes C. A. P., Martins F. S., Santos K.D.B., Rocha R.M.P., Silva J.R.V., Báo S.N. &
Figueiredo J.R. 2007. Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development
in vitro. Reproduction Fertility and Development. 19: 677-684.
28 O´Brien M.J., Pendola J.K. & Eppig J.J. 2003. A revised protocol for in vitro development of mouse oocyte from primordial follicles
dramatically improves their development competence. Biology of Reproduction. 68: 1682-1686.
29 Rahman A.N.M.A., Abdullah R.B. & Wan Khadijah W.E. 2008. A review of reproductive biotechnologies and their applications in goat.
Biotechnology. 7: 371-384.
30 Rossetto R., Lima-Verde I.B., Matos M.H.T., Saraiva M.V.A., Martins F.S., Faustino L.R., Araújo V.R., Silva C.M.G., Name K.P.O.,
Báo S.N., Campello C.C., Figueiredo J.R. & Blume H. 2009. Interaction between ascorbic acid and follicle-stimulating hormone
maintains follicular viability after long-term in vitro culture of caprine preantral follicles. Domestic Animal Endocrinology. 37: 112-123.
31 Saraiva M.V.A., Celestino J.J.H., Chaves R.N., Martins F.S., Bruno J.B., Lima Verde I.B., Matos M.H.T., Silva G.M., Porfirio E.P.,
Báo S.N., Campello C.C., Silva J.R.V. & Figueiredo J.R. 2008. Influence of different concentrations of LH and FSH on in vitro caprine
primordial ovarian follicle development. Small Ruminant Research. 78: 87-95.
32 Silva J.R.V., van den Hurk R., Costa S.H.F., Andrade E.R., Nunes A.P.A., Ferreira F.V.A., Lôbo R.N.B. & Figueiredo J.R. 2004.
Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water. Animal
Reproduction Science. 81: 273-286.
33 Silva J.R.V., van den Hurk R., Matos M.H.T., Santos R.R., Pessoa C., Moraes M. O. & Figueiredo J.R. 2004. Influences of FSH and
EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue. Theriogenology. 61: 1691-1704.
34 Silva J.R.V., Tharasanit T., Taverne., M.A.M., van der Weijden G.C., Santos R.R., Figueiredo J.R. & van den Hurk R. 2006. The
activin-follistatin system and in vitro early follicle development in goats. Journal of Endocrinology. 189: 113-125.
35 Silva C.M.G., Matos M.H.T., Rodrigues G.Q., Faustino L.R., Pinto L.C., Chaves R.N., Araújo V.R., Campello C.C. & Figueiredo
J.R. 2010. In vitro survival and development of goat preantral follicles in two different oxygen tensions. Animal Reproduction Science.
117: 83-89.
36 Telfer E.E., McLaughlin M., Ding C. & Thong KJ. 2008. A two-step serum-free culture system supports development of human
oocytes from primordial follicles in the presence of activin. Human Reproduction. 23: 1151-1158.
37 West E.R., Xu M., Woodruff T.K. & Shea L.D. 2007. Physical properties of alginate hydrogels and their effects on in vitro follicle
development. Biomaterials. 28: 4439-4448.
38 Wu J. & Tian Q. 2007. Role of follicle stimulating hormone and epidermal growth factor in the development of porcine preantral follicle in
vitro. Zygote. 15: 233-240.
Supl 1
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Progress in Caprine and Ovine Preantral Follicles Cryopreservation
Ana Paula Ribeiro Rodrigues1, Luciana Rocha Faustino1, Juliana Jales de Hollanda
Celestino1 & José Ricardo de Figueiredo1
ABSTRACT
The use of cryobiology has been of extreme importance for the preservation of genetic material from males and
females for subsequent use in assisted reproduction programs. Especially to cryopreservation of ovarian tissue the
goal is to ensure the follicular viability, notably, preantral follicles, which represent 95% of the shares present in
follicular ovarian cortex. In addition, cryopreservation procedures must maintain the integrity of tissue compartments
and the cell-to-cell contacts. Researching for an ideal protocol for cryopreservation of ovarian cortex has been widely
performed in animals such as mice and some production species such as goat and sheep. Studies carried out in
those species are of great importance for the preservation and enjoyment of the maximum reproductive potential of
goats and sheep of valuable genetic, economic and social development for the Northeast of Brazil. Moreover, the
similarities observed between ovarian tissue and folliculogenesis between small ruminants and humans shows that
the application of cryopreservation of ovarian fragments in sheep and goats can be potentially applied to humans.
Therefore, the use of cryopreservation of oocytes enclosed in ovarian preantral follicles has been shown as a
complementary tool for reproductive biotechnology aimed at maximizing the reproductive potential of animals of high
genetic value even after the death or programmed disposal. Preantral follicles can be cryopreserved in situ or isolated,
i.e. included or free of the ovarian cortex, respectively. Although results of ovarian tissue cryopreservation have been
promising, this technique has disadvantages: difficulties in assessment of the quality or quantity of the follicular
population in the tissue, the possibility of transmission of disease or infection present in the tissue to the graft
recipient, and the possible presence of chromosomal defects or autoimmune conditions that affect the whole ovary.
Furthermore, cryopreservation of isolated preantral follicles is less practical, the chances of losing them during the
different stages of cryopreservation are larger. Moreover, there is not established a culture system that ensures the
fully growth and maturation of these follicles in vitro, ensuring thus the production of viable embryos. Regardless, one
way or another, the application of cryopreservation of preantral follicles, isolated or enclosed in ovarian tissue, will
allow the establishment of animal/human germplasm banks in order to preserve the genetic heritage and hence the
preservation and maintenance of female fertility. Several studies in different species, especially humans, have
demonstrated the resumption of reproductive function including reporting the birth of healthy offspring after
transplantation of cryopreserved ovarian tissue. However, in small ruminants, especially goats, advances are still
limited. In this review, will present the main results obtained with the cryopreservation of preantral follicles, emphasizing
the efforts that have been performed by our team in the Laboratory of Manipulation of Oocytes and Ovarian Preantral
Follicles (LAMOFOPA) for the ovarian tissue banking from goats and sheep. Our findings have allowed us to conclude
that further studies are required in order to define a protocol (slow frozen or vitrification) to ensure the viability and
ovarian function and follicular normal after thawing/warming.
Keywords: cryopreservation, preantral follicles, ovary, transplantation, in vitro culture.
1
Estadual do Ceará (UECE), Fortaleza, Av. Paranjana,n 1700, Campus do Itaperi, Bairro Itaperi, CEP 60700-000 Fortaleza,CE, Brasil.
CORRESPONDÊNCIA: A.P.R Rodrigues [aprrodrigues@pq.cnpq.br - Fax: + 55 (51) 3101-9860].
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I. INTRODUCTION
II. GENERALITIES OF CRYOPRESERVATION
III. STEPS OF CRYOPRESERVATION
IV. APPLICATIONS OF CRYOPRESERVATION OF PREANTRAL FOLLICLES
V. CURRENT STATUS OF CRYOPRESERVATION OF PREANTRAL FOLLICLES IN
MAMMALS
IV. MAIN RESULTS OBTAINED BY LAMOFOPA ABOUT CRYOPRESERVATION OF
CAPRINE AND OVINE PREANTRAL FOLLICLES
VII. CONCLUSION
I. INTRODUCTION
In last decades, the researches on animal assisted reproduction have provided a major revolution in the
multiplication of animals, especially those with high economic potential, such as caprine and ovine. Reproductive
biotechnics such as artificial insemination, in vitro fertilization and embryo transfer are the most known and used [23],
however, other biotechnics have also been the target of intense study. An example is the biotechnic Manipulation of
oocytes enclosed in ovarian preantral follicles (MOIFOPA), which has gained considerable importance in basic research
by the major advances achieved as demonstrated by the establishment of in vitro culture systems that ensure the
formation of the antrum [64] and even the production of embryos in an advanced stage of development from preantral
follicles (secondary) (Magalhães et al., unpublished data). Beyond of a deeply involved study of the initial folliculogenesis
process (preantral phase), the MOIFOPA is also dedicated to the preservation of preantral follicles (oocytes, follicular
cells and other structures), whose process can be accomplished by short (cooling) or long periods (cryopreservation).
Specifically in the case of cryopreservation, this technique has a greater emphasis, for the oocyte and
follicular survival outside the animal body for an indefinite period, allowing the establishment of animal germplasm
banks for preservation of threatened species and/or animals of high genetic an zootechnical value. In this context,
several studies have been conducted in the field of cryopreservation (slow freezing or vitrification) of preantral follicles,
in order to develop efficient protocols that allow the maintenance of follicular viability after freezing or vitrification
procedure similar to that observed in fresh follicle or not cryopreserved.
Given the importance of this technique for animal assisted reproduction, this review aims to 1) provide a brief
description of the cryopreservation process and 2) show the main results obtained with slow freezing or vitrification of
preantral follicles, isolated or enclosed in ovarian tissue (in situ) in different species, highlighting the results achieved by
our team (laboratory of manipulation of oocytes and ovarian preantral follicles - LAMOFOPA) in caprine and ovine.
II. GENERALITIES OF CRYOPRESERVATION
The cryobiology is the branch of science that studies the effects of low temperatures, usually lowers the
freezing point of water, in biological systems, and the cryopreservation of cells and biological tissues an important
medical application of this science [19]. Cryopreservation can be defined as the long-term maintenance of biological
material under ultra low temperatures (£-80°C) [8], and the liquid nitrogen (-196°C) temperature is the usually adopted.
At this temperature, diffusion is considered insignificant, depending on the scale of storage time, the molecular
kinetic energy is very low and the metabolic reactions, driven by thermal energy, will occur very slowly or be completely
paralyzed [30]. In this state, theoretically, the cryopreserved material can be stored indefinitely [7].
The cryopreservation methods widely employed are the slow freezing and vitrification. The first consists in
cellular dehydration with a gradual reduction of temperature in the presence of low concentrations of cryoprotectants
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[46]. In this method, the material is cooled slowly under the control of a programmable freezer, which freezing curve
begins, in general, at room temperature and ends around -30 to -140°C when cellular dehydration necessary to
prevent or reduce the formation of intracellular ice crystals (FCGI) is reached sufficiently [65].
Despite the slow freezing is more widespread as the conventional method of cryopreservation, with important
applications in medically assisted reproduction programs, the FCGI and osmotic shock are two lethal factors that can
lead to cell death during this procedure [65]. Thus, in order to circumvent the obstacles of slow freezing, vitrification
has been considered as an alternative method.
Vitrification is considered a relatively inexpensive method, since it does not require the use of sophisticated
equipment and high cost (programmable freezer) and uses an extremely fast cooling rate, where the cryoprotectant
solutions pass from the aqueous phase for vitreous state, without exposure to the crystalline state [48], hence
without the FCGI. However, to obtain the vitreous state is necessary to use high concentrations of cryoprotectants
(CPA), which give greater viscosity to the cryopreservation solution [53], but on the other hand, increase the toxicity
of these substances on the glazed biological material.
III. STEPS OF CRYOPRESERVATION
Generally, the cryopreservation protocols comprise the steps of exposure to the CPA, cooling, storage,
thawing or heating and removal of the CPA. Exposure to the CPA, also known as equilibrium period, is the time
required for of CPA penetration or infusion into cells and/or tissues [20]. This is a very important stage and is strongly
influenced by CPA concentration, time and temperature which is realized the exposure [19]. In slow freezing, the
cooling stage is carried out gradually so that the dehydration occurs progressively [65], while the vitrification the
cooling rate is extremely rapid, ranging around 20000-40000°C/min [33], and as a result, the water goes from liquid to
a vitreous state without the FCGI [48,66]. Finished cooling, the sample is stored, usually in liquid nitrogen (-196 °C)
until the immediate moment from the thawing and heating. Thawing and heating should be immediately followed by
removal of the CPA, using one or more washings of cryopreserved material [57].
IV. APPLICATIONS OF CRYOPRESERVATION OF PREANTRAL FOLLICLES
Given the features of mature oocytes that threaten their survival after cryopreservation and ethical drawbacks
of embryo cryopreservation, cryopreservation of preantral follicles, isolated or enclosed in ovarian tissue (in situ), has
become increasingly prominent in preserving females genetic material, especially the primordial follicles that represent
a large follicular population [23] and housed inside immature oocytes. Besides being smaller than the mature oocytes,
these oocytes are less differentiated, possess fewer organelles, lack pellucida zone and cortical granules, besides
being less metabolically active [12]. All these features are potentially beneficial to cryopreservation [12,43] and thus
preantral follicles are regarded as an important target of cryopreservation programs. Furthermore, recovery of this
class follicular, hormonal treatment is not necessary for the production of embryos, and thus can be collected at any
time in the life of female (reproductive or not), and after the death of it.
In human medicine, cryopreservation of preantral follicles has been used as an important tool for preserving
fertility in women of reproductive age with cancer, before gonadotoxic treatment [6,42] Specifically in veterinary medicine,
this technique can be applied to the preservation of preantral follicles from ovaries of animals of major economic interest;
from females who are at risk of extinction or even for preservation of genetic material from pets and transgenic animals,
aiming to implement animal germplasm banks for subsequent transplantation or in vitro culture, as illustrated in Figure 1.
It should be noted also that the cryopreservation of preantral follicles is an essential part of the MOIFOPA
biotechnic, which aims to isolate and culture in vitro a large number of preantral follicles until complete maturation.
However, due to the limitations still found in culture in vitro systems, with respect to capacity to ensure full development
and maturation of these follicles, cryopreservation could allow the preservation of preantral follicles until such efficient
culture protocols are fully established [22].
Given the importance of cryopreservation of preantral follicles, several authors have studied and developed
protocols for freezing or vitrification gametes to preserve and restore the fertility of females of different species, as
will be discussed in subsequent topics.
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Figure 1. Forms of use of preantral follicles after cryopreservation and storage in animal/human germplasm banks.
V. CURRENT STATUS OF CRYOPRESERVATION OF PREANTRAL FOLLICLES IN
MAMMALS
The first attempts at cryopreservation of preantral follicles began in the 50s, with freezing of ovarian
fragments of rodents [15,44,45]. Since then, several studies have been performed to establish a protocol that maintains
the ideal follicular viability similar to that obtained before cryopreservation or results in minor possible damage after
cryopreservation of isolated preantral follicles or in situ.
In mice, the results have been promising. Several authors have reported the resumption of ovarian function
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[25,35]; the birth of healthy offspring obtained from previously frozen ovary transplanted, followed by in vitro culture
[31,34] or mating [35]. Studies involving vitrification have also shown satisfactory results in this species. Chen et al.
[14], for instance, noted that the cryopreservation of ovarian tissue resulted in a high percentage of preantral follicles
with normal morphology, and a pregnancy rate (87%) and a number of offspring (n=64) after transplantation similar to
non-vitrified tissue (93%; n=66). Offspring from preantral follicles that grew, matured and were fertilized, only using in
vitro culture after cryopreservation, has also been reported in the literature [16,26].
In domestic animals, the results are not significant, except for sheep, whose specie has been reported
birth of lambs after ovarian tissue cryopreservation followed by transplantation. The first result of great impact was
reported by Gosden et al. [24], who obtained the resumption of cyclic activity and pregnancy, giving birth a healthy
offspring after orthotopic autotransplantation of frozen/thawed ovarian cortex fragments. Almost one decade later,
other teams have also reported the birth of lambs after freezing and transplantation of ovarian tissue [27,54,55]. Other
healthy lambs were also born from females that received half ovary orthotopic autografts [9] or ovarian fragments [36]
previously vitrified.
In caprine, although there is still no reported case of birth of animals after cryopreservation of ovarian
tissue, has been shown that preantral follicles can be successfully frozen [38,49,50] or vitrified [63], with reports
including the recovery of gonadal function after transplantation [58].
In bovine and swine, few studies have been documented about cryopreservation of ovarian tissue. However,
some researchers have obtained satisfactory results showing that bovine preantral follicles may show high survival
rates after slow freezing: 77.0% [13] and 88.4% [37]. Recently it was observed that after incubation (38.5°C, 5% CO2)
for a short period (2 h) of swine ovarian tissue after thawing, the percentage of morphologically normal follicles was
similar to that observed in non-frozen or fresh tissue under the same conditions of incubation. Furthermore, the ultrastructure of preantral follicles was also well preserved [10]. After xenotransplantation of swine vitrified ovarian tissue
was observed a satisfactory revascularization and follicular development until the antral stage [40].
In pets, like cats, has also been developed protocols for cryopreservation of ovarian tissue [32]. Encouraging
results were obtained after freezing of the ovarian cortex of feline followed by xenotransplantation under renal capsule
of immunodeficient mice, been observed development of follicles in advanced stages [11]. Already after vitrification
of ovarian tissue of bitch were observed damage to the follicle structure and reduction in the number of primordial and
primary follicles [29].
Regarding the cryopreservation of human preantral follicles, this practice began when Newton et al. [41]
demonstrated that ovarian tissue remained viable after freezing. The vitrification of human ovarian tissue has also
been shown to be feasible, since the primordial follicles developed and showed increased levels of estradiol and
progesterone after in vitro culture [28]. Probably due to the great interest in preserving fertility in women, major efforts
have been made to establish this practice, with some reports of births of healthy children after transplantation of
cryopreserved ovarian tissue [5,17,18,39,56]. Recently, one of these women conceived a child again and therefore
the first to have two children from different pregnancies, born as a result of transplantation of frozen/thawed ovarian
tissue [21].
VI. MAIN RESULTS OBTAINED BY LAMOFOPA ABOUT CRYOPRESERVATION OF
CAPRINE AND OVINE PREANTRAL FOLLICLES
Aiming to preserve female gametes of important races of caprine and ovine, and thus to maintain the
reproductive capacity of these animals after death or removal of female reproductive activity for several reasons, the
LAMOFOPA started its studies in 2000 about cryopreservation of preantral follicles, isolated or enclosed in ovarian
tissue in both species. In Figure 2 can be seen a summary of key results.
Amorim et al. [1,3,4] evaluated different cryoprotectants concentrations (0, 0.5, 1.0, 1.5, 2.0 and 2.5 M) of
ethylene glycol (EG), dimethylsulfoxide (DMSO), propanediol (PROH) and glycerol (GLY), after a period of equilibrium
and freezing of primordial follicles isolated from ovine ovarian tissue. In the treatment with EG, the number of viable
follicles after cryopreservation using concentrations of 1.0, 1.5 and 2.0 M (724±246,69; 824±291,90 and 844±296,29
follicles, respectively) was similar to that observed for fresh follicles or control (3764±795,21 follicles). With DMSO
concentrations of 1.0 and 1.5 M showed the highest numbers of viable follicles after freezing (636±161,82 and
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628181,28 follicles, respectively) being, however, lower than the control (800±203,86 follicles). When PROH or GLY
were used, the percentage of viable follicles decreased significantly compared to control, the treatments did not differ
among themselves after freezing. Working with different CPA for cryopreservation of caprine preantral follicles, Rodrigues
et al. [49,50] evaluated the morphological and ultra-structural features of these follicles after freezing of ovarian tissue
with 1.5 or 3.0 M PROH and DMSO [50] or GLY and EG [51]. In the first study, results showed that the percentage of
frozen preantral follicles morphologically normal for both cryoprotectants was significantly less than control (77%).
However, the percentage of normal follicles after freezing and thawing with 1.5 M DMSO (26%) was significantly
higher than other treatments, this result was confirmed by electron microscopy. In the second study, the treatments
were also significantly less than the fresh control (89%). After freezing/thawing, the percentage of preantral follicles
with normal morphology did not differ between treatments, except 3.0 M EG that was higher than 1.5 M (47% and
31% respectively). Electron microscopy revealed that preantral follicles present in cryopreserved ovarian tissue with
1.5 M EG, considered normal after histological analysis, were highly vacuolated, while in other treatments, the ultrastructure was preserved.
In caprine, isolated primordial follicles, freezing with DMSO or PROH at concentrations of 1.5 or 3.0 M
showed that the percentage of viable follicles did not differ from control [59]. In the same work, for the first time, in
vitro culture for 24 h was applied in this specie, after freezing with 1.5 M DMSO or PROH. Before culture, the viability
of primordial follicles (65-77%) immediately after thawing was similar to fresh control (93%). Moreover, the percentage
of viable follicles cryopreserved with DMSO (63%) or PROH (63%) and subsequently culture was similar between
both, but significantly lower than primordial follicles cultured without cryopreservation (81%).
Establish an optimal method for cryopreservation of ovine enclosed preantral follicles in ovarian tissue, an
in situ study of morphology and ultra-structure of preantral follicles after freezing of ovarian tissue using different
cryoprotectants such as DMSO, PROH , GLY and EG at concentrations of 1.5 or 3.0 M [52]. The authors found that
all cryoprotectants at both concentrations, showed percentages of preantral follicles with normal morphology significantly
lower than the fresh control (77.6%). The highest percentage of normal follicles was observed after freezing with EG
(1.5 M - 59.6% and 3.0 M - 64.5%) or DMSO (1.5 M - 67.6%), while analysis by transmission electron microscopy
only confirmed the ultra-structural integrity of follicles frozen in 1.5 M of DMSO.
In an attempt to better evaluate the effects of freezing of caprine preantral follicles, isolated or enclosed in
ovarian tissue, using DMSO (1.5 M) and EG (1.5 and 3.0 M for isolated and in situ follicles, respectively), was
performed after cryopreservation a follicular culture for up to five days [52]. The in situ viability of the cryopreserved
and cultured follicles (DMSO - 60% and EG - 45%) was similar to cultured in situ follicles without previous freezing
(68%). For the isolated follicles, the percentage of viability was only similar to follicles cultured without before freezing
(81%) after freezing with DMSO (77%), which did not differ in the percentage of frozen follicles with EG (63%).
In order to optimize the protocols for cryopreservation of preantral follicles, our team froze caprine enclosed
preantral follicles in ovarian tissue, in the absence or presence of an extracellular cryoprotectant (0.5 M sucrose) with
or without 1.0 M DMSO and/or EG. Histological analysis and follicular viability analysis showed beneficial effects of
the addition of sugar in the freezing solution. Cryopreserved follicles in EG; EG added sucrose or sucrose only were
further in vitro cultured for 24 h. The results showed that treatment containing EG and sucrose (59%) showed percentage
of viable follicles similar to cultured without freezing (57%) [61].
Evaluating isolated ovine preantral follicles after freezing with 1.5 or 3.0 M DMSO, EG, PROH or GLY,
Santos et al. [60] showed that all treatments resulted in a reduction in the percentage of viable follicles compared to
control or fresh follicles (83.1%). The highest percentage of viable preantral follicles after cryopreservation was
observed when EG or DMSO were used, in both concentrations. Amorim et al. [2] also showed that isolated ovine
primordial follicles survived after cryopreservation when DMSO, EG, PROH and GLY were used at a concentration of
1.0 M. In this study the authors showed that DMSO (87%) or EG (78%) had the highest follicular survival rates.
However, based on the results of survival after in vitro culture, the authors recommended only the EG (42%) for
conservation of ovine primordial follicles, which did not differ from control cultured (55%). Later, isolated or in situ
ovine primordial follicles were frozen with 1.5 M DMSO or EG, followed or not by in vitro cultivation. After cryopreservation
and in vitro culture of isolated or in situ follicles, there was no increase in diameter or follicular development. However,
follicular development was best observed when follicles were cultured and cryopreserved included in ovarian tissue
[63]. In 2007 it was realized for the first time vitrification of preantral follicles in caprine ovarian tissue, comparing two
different techniques of vitrification, i.e., conventional vitrification and solid surface vitrification. In this study, the
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DMSO = dimethylsulfoxide; EG = ethylene glycol; GLI = glycerol; PROH = propanediol; SUC = sucrose; CH = classical histology; TEM = transmission electron microscopy; TB = trypan blue; FL =
florescent lable; HPLC = high performance liquid chromatography.
Figure 2. Main results obtained with the cryopreservation of caprine and ovine preantral follicles.
.
.
authors concluded that to maintain the viability of vitrified and cultured caprine preantral follicles, shall be used solid
surface vitrification in solution containing sucrose and EG, which showed follicular viability (58%) after 24 h of
cultivation, similar to the cultivated control (65%) [64].
In more recent studies, fragments of ovaries of both species were exposed to DMSO at different
concentrations (1.0, 1.5 and 2.0 M) and exposure times (05-40 minutes), with the objective of determining the
perfusion rates (amount in mg) of CPA in ovarian tissue, using High Performance Liquid Chromatography (HPLC). In
ovine specie [38], it was shown that, in general, the exposure time had no influence on tissue levels of DMSO.
However, after exposure for 30 minutes, the ovarian fragments in 2.0 M solution retained significantly higher levels of
DMSO than the solution of 1.0 M. After freezing, follicular viability was more efficient when the ovarian tissue was
exposed to 1.5 and 2.0 M DMSO for 5 minutes or 1.0 M for 10 minutes. Regarding the caprine [47], the infusion of
DMSO was similar to the ovine, in which the exposure time and concentration did not influence the tissue levels of
DMSO, except after 30 and 40 minutes of exposure to 2.0 and 1.5 M DMSO, respectively. After freezing, the
percentage of viable preantral follicles was similar to control in fragments previously exposed to 1.0 and 1.5 M DMSO
for 10 minutes.
VII. CONCLUSION
As evidenced in this review, the application of cryopreservation of preantral follicles, isolated or enclosed in
ovarian tissue, will allow the establishment of animal/human germplasm banks in order to preserve the genetic
heritage and hence the preservation and maintenance of female fertility. Moreover, cryopreservation of immature
oocytes derived from preantral follicles, which are more resistant to cryopreservation procedures, presents itself as a
valuable support to the MOIFOPA biotechnic, until efficient protocols for complete follicle development are not yet
routine applied. With regard to the various studies of cryopreservation of preantral follicles in different species,
scientific advances appear to be significant, however, the procedure of cryopreservation in reality, is still carried out
empirically. Accordingly, intensive studies have yet to be performed so that cryopreservation becomes an option
clinically consistent, promising, for (animal or human) assisted reproduction to preserve the reproductive function.
Specifically, in the species studied in LAMOFOPA, intense efforts are still required, especially studies with the
vitrification of preantral follicles.
Unpublished data: Magalhães et al (dmmvet@hotmail.com)
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freezing. Fertility & Sterility. 83(1): 1248-1254.
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Xenotransplantation of Isolated Preantral Bovine Ovarian Follicles
Under the Kidney Capsule of Nude Mice
P.E.J. Bols1*, J.M.J. Aerts1, A. Langbeen1, E. Jorssen1, I.G.F. Goovaerts1 & J.L.M.R. Leroy1
ABSTRACT
Background: Because of limited availability of in vitro culture systems for preantral bovine follicles, additional insights
might be generated by grafting ovarian tissue into immunodeficient mice to study preantral follicular dynamics. While
a large amount of data has already been gathered using laboratory animals and through clinical use in human
assisted reproductive technologies, the bovine model offers exciting additional prospects, not at least because of the
many similarities between the bovine and the human species with regard to ovarian physiology. Different grafting
strategies are available and their follicular survival rates largely depends on the grafting site and the physiological
status of the recipient animal. A very important key factor in this respect is the re-establishment of blood and nutrient
supply following transplantation. This abstract reports on recent developments related to preantral follicle
xenotransplantation of bovine ovarian tissue strips, more specifically isolated preantral follicles transferred underneath
the kidney capsule of nude mice.
Review: Bovine ovaries from fetuses and calves were enzymatically disaggregated and isolated preantral follicles
were suspended in PBS. Granulosa and stroma cells originating from the ovarian digest served as embedded matrix.
This suspension was subsequently injected under the kidney capsule of adult immunodeficient mice. Two weeks
following transplantation, preantral follicular survival and proliferation was assessed by histology and immunostaining
with proliferating cell nuclear antigen (PCNA). Results were compared with ungrafted control tissue. The proportion of
primordial follicles decreased considerably from 58.2% in control tissue to 17.1% in transplants in the fetal group and
from 76.0% to 17.2% in the calf group. Correspondingly, the proportion of primary follicles increased from 13.4% to
62.2% in the fetal group and from 5.4% to 63.5% in the calf group. Follicular growth and proliferation, as measured by
PCNA immunostaining, showed an increase from 40.6% growing follicles to 81.9% in the fetal group and from 21.0%
to 80.7% in the calf group (Aerts et al., 2009).
Conclusion: Although prolonged in vitro follicular culture of bovine preantral follicles remains difficult under the
current circumstances, the massive follicular activation following xenotransplantation indicated that isolated preantral
follicles are able to survive and proliferate 14 days after being transferred to a subcapsular renal site. This offers
promising possibilities for additional research using this bovine-mouse model.
Keywords: xenotransplantation, preantral, mice.
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Gene Expression During Early Follicular Development and
Mechanisms of Activation and Growth of Primordial Follicles in
Mammals
José R.V. Silva1, Cintia C.F. Leitão1, Ticiana F.P. Silva2, Fabrício S. Martins2, Claudio A.P.
Lopes2 & Robert van den Hurk3
ABSTRACT
Background: For decades, the mechanisms maintaining the dormancy, survival and growth of mammalian primordial
follicles as well as their growth up to an early antral stage have been not well understood. In recent years, data
obtained from studies on the expression and quantification of mRNA for several growth factors and from studies with
genetically modified mouse models have revealed a number of molecules, whose functions are indispensable for (i)
the maintenance of follicular quiescence, (ii) primordial follicle survival and (iii) activation, and/or (iv) the growth of
primary follicles up to an early antral stage.
Review: This review focuses on expression of mRNA and protein for growth factors, cytokines and their respective
receptors in early follicles, as well as on the intrafollicular signaling cascades that lead to the in-vitro activation of
primordial follicles. Furthermore, fundamental information on the levels of mRNA for growth factors at different stages
of follicular development and the in-vitro effects of several locally expressed factors on ovarian follicular development
will be discussed. During the transition into the primary stage or from the primary into the secondary follicle stage of
goats there is a significant increase in the mRNA expression of different factors (BMP-6, BMP-15, KL, EGF, VIP,
GDF-9). The detection of these different factors depends on the species. In rat ovaries, GHR is detected in oocytes,
granulosa and theca cells. The presence of the mRNA for GHR, but not that for GH has been detected in rat pre-antral
follicles. GHR mRNA has only been found in granulosa cells, while positive immunostaining for GHR has been
observed in both oocytes and granulosa cells. Also in vitro studies with goat primordial follicles have demonstrated
that different factors, estradiol and progesterone promote primordial follicle activation and/or oocyte growth. Recently,
several studies have been performed with mouse primordial follicles to understand how intrafollicular cytokines and
growth factors may control the fate of primordial follicles in the ovary. In rodents, anti-Mullerian hormone has been
shown to inhibit mouse primordial follicle growth and to downregulate c-kit expression in rats, suggesting that, at least
in rodents, the kit system plays a key role in initiation of follicular growth. In relation to control of primary and
secondary follicles, several in vitro studies have demonstrated that FSH, activin-A, EGF, GH, IGF-I and IGF-II
stimulate oocyte growth and follicular development in different species.
Conclusion: This review updates the information on expression of mRNA and proteins for growth factors and their
role in the regulation of development from primordial to early antral follicles. The growing knowledge of the molecules
that control the dormancy, survival, activation and growth of primordial follicles will not only contribute for a better
understanding of the physiology of the mammalian ovary, but will also enable researchers to develop more promising
methods for promoting growth of oocytes from primordial follicles, the richest follicular resource in the mammalian
ovary.
Keywords: primordial follicles, gene expression, growth factors, in vitro culture.
1
Biotechnology Nucleus of Sobral - NUBIS, Federal University of Ceara, 62041-040, Sobral, CE, Brazil. 2Faculty of Veterinary Medicine,
LAMOFOPA, State University of Ceara, Fortaleza, CE, Brazil. 3Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands. CORRESPONDENCE: J.R.V. Silva [jrvsilva@ufc.br - TEL: +55 (88) 36132603].
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I. INTRODUCTION
II. GENE EXPRESSION IN MAMMALIAN EARLY-STAGED FOLLICLES
III. LOCAL FACTORS AND SIGNALING MECHANISMS IN OOCYTES WHICH
CONTROL PRIMORDIAL FOLLICLE ACTIVATION AND SURVIVAL
IV. LOCAL FACTORS THAT CONTROL PRIMARY FOLLICLE DEVELOPMENT UP TO
THE ANTRAL STAGE
V. CONCLUSIONS
I. INTRODUCTION
In most mammalian species, the pool of ovarian primordial follicles is formed during fetal development,
thousands of primordial follicles being present in the ovaries of newborn females. By degeneration of the oocyte,
many of these relatively quiescent follicles will gradually be lost [70], which dictates the process of reproductive aging
[8]. In order to ensure a proper length of reproductive life, an adequate number of such follicles survive in the ovary for
decades, without further growth and differentiation. Only limited numbers of primordial follicles are continuously
recruited into the growing follicle pool [25]. The molecular mechanisms controlling the balance between the survival
and loss of primordial follicles are, however, poorly defined. In an attempt to understand these mechanisms for
primordial and later-staged pre-antral follicles, expression and quantification of mRNA for local growth factors and
cytokines, and their receptors have received increasing attention in recent years. In this respect, we performed
different studies with goats, sheep, cows and rats as animal models (for reviews, see [78, 79, 71]. Most of our mRNA
quantification studies, however, were carried out with caprine ovarian follicles.
The current review updates the knowledge on expression and level of mRNA for several growth factors and
cytokines in primordial, primary and secondary follicles, and discusses intracellular signaling pathways that control
activation and growth of primordial follicles. It furthermore includes data from in-vitro studies and knockout experiments
that demonstrated the importance of growth factors for early follicle development.
II. GENE EXPRESSION IN MAMMALIAN EARLY-STAGED FOLLICLES
Caprine primordial follicles show a significant increase in the levels of bone morphogenetic protein-6 (BMP6) mRNA during their transition into the primary stage, BMP-6 protein being expressed in the oocyte of these follicles
[22]. During the transition from the primary into the secondary follicle stage, a significant increase (P<0.05) in the
levels of mRNA for kit ligand (KL) [14], epidermal growth factor (EGF) [73], bone morphogenetic protein-15 (BMP-15)
[74], growth and differentiation factor-9 (GDF-9) [72] and vasoactive intestinal peptide (VIP) [10] has been observed,
while the levels in primordial follicles did not vary from those of primary follicles. Previously, the presence of proteins
for KL [72], EGF [73], BMP-15 and GDF-9 [74] have been demonstrated in goat early-staged follicles. The mRNAs for
IGF-1 [41], FGF-2 and FSH-R [38] were demonstrated at primordial, primary and secondary follicle stages, but no
significant change in their levels were observed during growth of these pre-antral follicles. The mRNA for the receptors
of KL (c-kit) [72], EGF (EGF-R) [73], BMPs (BMP-RIA, BMP-RIB, BMP-RII) [74] and activins (Act-RIA, Act-RIB, ActRIIA, Act-RIIB) are also expressed in goat primordial primary and secondary follicles, indicating that these growth
factor-receptor complexes may have an important role in the control of early follicular development in goats. In
contrast, both mRNA and protein for BMP-7 [22] and GH-R [41] were not expressed in caprine pre-antral follicles.
In rat ovaries, growth hormone receptor (GHR) is detected in oocytes, granulosa and theca cells [13]. The
presence of the mRNA for GHR, but not that for GH has been detected in rat pre-antral follicles [84]. GHR mRNA has
only been found in granulosa cells, while positive immunostaining for GHR has been observed in both oocytes and
granulosa cells. The gene expression of GHR in pre-antral follicles suggests that the observed actions of GH are
mediated by these receptors. Immunolocalization of GHR in theca cells indicates that GH directly affects the theca cells
[84]. Immunostaining for GH in absence of their mRNA expression could be due to existing receptor bound GH. The
findings are supported by those of Abir et al. [1], who detected proteins and mRNA transcripts for GHR in oocytes and
granulosa cells from early-staged human follicles, but contrast, those of Sharara & Nieman [67] and Martins [41], who
could not detect GHR mRNA in pre-antral follicles from human and goat ovaries, respectively. Thus far, Abir et al. [1] are
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the only ones who demonstrated mRNA transcripts for GH in the somatic compartments of early-staged follicles. The
presence of GH mRNA in follicles is remarkable since, to exert its stimulatory effect on follicular development, it is more
likely that systemically produced GH binds to follicular GHR than locally produced GH. The observed positive expression
for GH mRNA in human follicles could be due to the used in-situ hybridization method, which may have given a false
reaction. On the other hand, one cannot rule out, that methods used for GH mRNA expression by other researchers were
inadequate to detect the messenger. Future studies have to confirm whether follicles are able or unable to form GH.
Several components of the insulin like growth factor (IGF) system are expressed in early-staged follicles of
various mammalian species. Armstrong et al. [5] showed that IGF receptor type I, IGFBP-2 and IGFBP-3 are expressed
in granulosa cells and in the oocyte of bovine pre-antral follicles, whereas IGF-I and IGF-II are absent. Expression of
IGF type I receptor has been reported to increase during the development of a bovine primary follicle to the large
antral stage [81]. In rats, mRNA transcripts for IGF-I and type 1 IGF receptor have been detected in somatic cells and
oocytes of secondary follicles [83]. The expression of IGF type I receptor increases during the development of a
primary follicle to an early antral stage, i.e., to a follicle of 1 mm in diameter [59]. In non-human primates, IGF-I gene
expression has also been detected in oocytes from primary follicles and more advanced follicle stages, while IGFRI is detected in all oocytes, i.e., inclusive those from primordial follicles [80]. In rats, immunoreactive IGF-I and IGFR
has been visualized in pre-antral follicles, particularly in their oocytes [83].
III. LOCAL FACTORS AND SIGNALING MECHANISMS IN OOCYTES WHICH
CONTROL PRIMORDIAL FOLLICLE ACTIVATION AND SURVIVAL
In-vitro studies with goat primordial follicles have demonstrated that KL [14], FSH [74], BMP-7 [4], FGF-2
[43], GDF-9 [40], IGF-1 [41], EGF [74], activin-A [72], VEGF [9], VIP [10] estradiol and progesterone [35] promote
primordial follicle activation and/or oocyte growth. Like KL, the cytokine leukemia inhibitory factor (LIF) promotes
transition of primordial into primary follicle in rat ovaries [51]. Besides, these authors showed that LIF increases
expression of KL mRNA in cultured rat granulosa cells. They conclude from their findings that LIF may interact with
KL to promote primordial follicle development.
In rodents, anti-Mullerian hormone (AMH) has been shown to inhibit primordial follicle growth (mouse) [17]
and to downregulate c-kit expression (rat) [50], suggesting that, at least in rodents, the kit system plays a key role in
initiation of follicular growth. Indeed, mutations in mice, that prevent the production of the soluble form of the cytokine
kit ligand, lead to failure of follicular growth beyond the primary stage [27, 6]. Additionally, KL is necessary and
sufficient to induce primordial follicle development and thus to initiate folliculogenesis in the rat [57].
Recently, several studies have been performed with mouse primordial follicles to understand how intrafollicular
cytokines and growth factors may control the fate of primordial follicles in the ovary [63]. Cytokines, like KL, activate the
phosphatidylinositol 3 kinase (PI3K) pathway in oocytes.The PI3K pathway includes components like serine/threonine kinase
(Akt), forkhead transcription factor 3 (FOXO3), glycogen synthase kinase 3A (GSK3A), GSK3B and phosphatase and tensin
homologue deleted on chromosome 10 (PTEN) [36]. FOXO3a is a downstream effector of the PTEN/PI3K/AKT pathway [77].
In mouse ovaries, FOXO3a causes suppression of follicular activation, preserving the follicular reserve pool [16].
A considerable proportion of the signaling mediated by PI3Ks converges at 3-phosphoinositide-dependent
protein kinase-1 (PDK1). The PI3K–PDK1 cascade in oocytes regulates ovarian aging by regulating the survival of
primordial follicles [63]. Tensin homologue deleted on chromosome 10 (PTEN) may act as a phosphoinositide-3
(PIP3)-phosphatase that antagonizes the activity of PI3K by dephosphorylating PIP3 to PIP2 [37]. In mouse, oocytespecific deletion of PTEN causes premature activation of the primordial follicle pool, suggesting that the mammalian
oocyte is the initiator of follicle activation and that the oocyte PTEN-PI3K pathway governs follicle activation through
control of initiation of oocyte growth [63]. Using oocyte-specific loss of PTEN to induce PI3K activation of Akt
activation resulted in FOXO3 hyperphosphorylation, and FOXO3 nuclear export. This was concomitant with primordial
follicle activation, thus indicating that PI3K pathway and FOXO3 actually control primordial follicle activation [32].
Mammalian target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase that regulates cell
growth and proliferation in response to growth factors and nutrients. It functions by modulating processes such as
protein synthesis, ribosome biogenesis and autophagy [23, 66, 82]. Specific inhibition of the mammalian serine/
threonine kinase mammalian target of rapamycin (mTOR) indicated a role for mTOR in parallel to P13K in primordial
follicle activation [63]. Adhikari et al. [3] recently provided genetic evidence to show that, in mutant mice, the tumor
suppressor tuberous sclerosis complex 1 (TSC1), which negatively regulates mTORC1, functions in oocytes to
maintain the quiescence of primordial follicles. They further showed that maintenance of the quiescence of primordial
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follicles requires synergistic, collaborative functioning of both TSC and PTEN and that these two molecules suppress
follicular activation through distinct ways. Adhikari et al. [3] suggested that TSC/mTORC1 signaling and PTEN/PI3K
signaling act in parallel, in a synergistical and collaborative way. Synergistically, they regulate the dormancy and
activation of primordial follicles; together, they ensure the proper length of female reproductive life. Similarly, the tumor
suppressor Tsc2 plays an essential physiological role in oocytes to preserve the female reproductive lifespan by
suppressing activation of primordial follicles [2].
Recent studies on mutant mouse models have demonstrated that the quiescence of primordial follicles is
not only maintained by molecules like PTEN, Tsc1-Tsc2 complex and Foxo3a, but also by cyclin-dependent kinase
(Cdk) inhibitor (p27), AMH, and forkhead box L2 (Foxl2) and possibly by other unidentified molecules [63]. At the
same time, the survival of primordial follicles is maintained by PDK1 signaling, and rpS6. In the absence of any of the
primordial follicle suppressors such as PTEN, Tsc1, Tsc2, Foxo3a, or p27, rapid global activation of primordial
follicles occurs, indicating that the combined functions of these molecules are required to maintain the quiescence of
the primordial follicle pool, and so to equip animals with fertilizable oocytes at later reproductive life. In the absence
of the maintainer molecules that safeguard the survival of primordial follicles, including PDK1 and ribosomal protein
S6 (rpS6), all primordial follicles are lost prematurely, straight from their quiescent state.
IV. LOCAL FACTORS THAT CONTROL PRIMARY FOLLICLE DEVELOPMENT UP TO
THE ANTRAL STAGE
Regarding the factors that control primary and secondary follicles, several in- vitro studies have demonstrated
that FSH (cow: [28]; mouse: [15, 20,76]; rat: [45]; goat: [39]) activin-A (rat: [83]; goat: [72]; cow: [29;46], EGF (human: [60]), GH
(rat: [83]), IGF-I (rat: [83]), and IGF-II (goat: [61]) stimulate oocyte growth and follicular development. Other regulators of
follicle growth are fibroblastic growth factor-2 (FGF-2: [68]); members of the transforming growth factor-â (TGF-â) superfamily
members [34], including somatically derived anti-Mullerian hormone (AMH) and oocyte-derived growth differentiation factor9 (GDF-9 (goat: [40]; rat: [24], and the bone morphogenetic proteins (BMPs), in particular BMP4, BMP7 and BMP15 [69, 55].
V. CONCLUSIONS
This review updates the information on expression of mRNA and proteins for growth factors and their role in
the regulation of development from primordial to early antral follicles. The growing knowledge of the molecules that
control the dormancy, survival, activation and growth of primordial follicles will not only contribute for a better understanding
of the physiology of the mammalian ovary, but will also enable researchers to develop more promising methods for
promoting growth of oocytes from primordial follicles, the richest follicular resource in the mammalian ovary.
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82 Wullschleger S., Loewith R. & Hall M.N. 2006. TOR signaling in growth and metabolism. Cell. 124: 471-484.
83 Zhao J., Taverne M.A., Van der Weijden G.C., Bevers M.M. & Van den Hurk R. 2001. Insulin-like growth factor-I (IGF-I) stimulates the
development of cultured rat pre-antral follicles. Molecular Reproduction and Development. 58: 287-296.
84 Zhao J., Taverne M.A., Van der Weijden G.C., Bevers M.M. & van den Hurk R. 2002. Immunohistochemical localisation of growth
hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I) and type I IGF-I receptor, and gene expression of GH and GHR
in rat pre-antral follicles. Zygote. 10: 85-94.
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The Resumption of Meiosis Mediated by Angiotensin II is Dependent
on Progesterone, Oxytocin and Prostaglandin
Paulo Bayard Gonçalves1, Marcos Henrique Barreta1, João Francisco Oliveira1, Rogério
Ferreira1, Bernardo Garziera Gasperin1 & Lucas Carvalho Siqueira1
ABSTRACT
Background: Oocytes are arrested in the first meiotic prophase during follicular development until near ovulation. In
vitro, oocytes resume meiosis spontaneously, germinal vesicle breakdown (GVBD) occurs and oocytes progress to
the MII stage when they are removed from their follicles and cultured under suitable conditions. On the other hand,
meiotic maturation of bovine oocytes occurs within the follicle, raising the question regarding possible involvement of
positive signal(s) to induce meiotic resumption in vivo. Meiotic resumption is dependent on the preovulatory surge of
LH, but the bovine cumulus–oocyte complexes (COCs) do not have LH receptors, so the signal must occur through
theca and mural granulosa cells. The LH surge stimulated the ovarian renin-angiotensin system (RAS), including an
increase in the renin activity and angiotensin II (AngII) concentration in bovine follicular fluid. Also, the interactions
among LH, AngII increased follicular production of prostaglandins (PGs) and modulated steroidogenesis in the bovine
preovulatory follicle. AngII has also been associated with follicular growth and ovulation in cows. Furthermore, AngII
stimulated the resumption of meiosis in co-culture systems of cumulus enclosed bovine oocytes and follicular cells.
Review: The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II (Ang II)
and Ang II receptors in the ovary is suggestive of a functional ovarian RAS. In the last few years, our research group
has focused on studying the contribution of RAS in antral follicle development, ovulation and oocyte maturation. A
new concept of mechanism and factors involved in oocyte meiotic resumption has been established using cattle as
a model. Transvaginal ultrasound has been used for intrafollicular injection to investigate factors that play a part in the
resumption of meiosis. With this in vivo and an in vitro model, culturing oocytes in the presence of theca and
granulosa cells, we have demonstrated that AngII via the AT2 receptor subtype plays a pivotal role in the antral follicle
development, early mechanism of ovulation and oocyte meiotic resumption in cattle. When bovine cumulus-oocyte
complexes (COCs) are cultured with follicular hemisections, oocyte maturation is inhibited; however, follicular cells or
conditioned medium were unable to inhibit nuclear maturation in the presence of AngII. In vivo, intrafollicular injection
of saralasin (a competitive AngII antagonist) completely inhibited the oocyte meiotic resumption in follicles (larger
than 12 mm) challenged with an GnRH agonist. Moreover, oocytes co-cultured with follicular hemisections progressed
nuclear maturation when prostaglandin E2 (PGE2), PGF2a, progesterone, oxitocin or AngII was present in the coculture system and indomethacin inhibited AngII-induced meiotic resumption.
Conclusion: our results provide strong evidence that AngII mediates the oocyte meiotic resumption induced by an
LH surge in cattle and that this event is dependent on progesterone, oxytocin and prostaglandin.
Keywords: oocyte maturation, resumption of meiosis, angiotensin II, progesterone, oxytocin, prostaglandin.
1
Laboratoìrio de Biotecnologia e ReproducaÞo Animal, Universidade Federal de Santa Maria, Prédio do Hospital Veterinário, Sala 417,
Campus Universitário, Camobi, CEP 97105-900 Santa Maria, RS, Brasil. CORRESPONDÊNCIA: P.B. Gonçalves [bayard@biorep.ufsm.br;
pbayard@terra.com.br - FAX: +(55) 55 3220-8484].
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I. INTRODUCTION
II. ANGIOTENSIN II ON FOLLICLE DEVELOPMENT AND OVULATION
III. ANGIOTENSIN II ON RESUMPTION OF MEIOSIS
IV. ANGIOTENSIN II DEPENDS ON PROGESTERONE, OXYTOCIN AND
PROSTAGLANDIN TO INDUCE MEIOTIC RESUMPTION.
V. FINAL CONSIDERATIONS
I. INTRODUCTION
The oocyte meiotic resumption is dependent of signals provided by LH peak around ovulation in cattle.
However, the absence of LH receptors in bovine oocyte [6,16] suggests that gonadotropin does not act directly on the
female gamete, but rather stimulates intrafollicular mediators. The concentration of angiotensin II (AngII) increases in
follicular fluid after the LH surge [2] and this peptide has been linked to steroidogenesis [1,21], follicular development
[8,14,18] and ovulation [2,7]. Furthermore, our group demonstrated that AngII mediates the resumption of meiosis
induced by an LH surge and that this event is dependent on progesterone, oxytocin and prostaglandin [3,4,11,19]. The
aim of this review was to compile the most relevant data of our studies about the role of AngII on the oocyte
maturation and to propose a new model for the resumption of meiosis in cattle.
II. ANGIOTENSIN II ON FOLLICLE DEVELOPMENT AND OVULATION
Paracrine regulations between the oocyte and follicular cells are essential for follicular development, oocyte
maturation and ovulation. Considering this interaction, our group has used a system of co-culture of cumulus oocytecomplexes (COCs) and follicle hemisections to study the role of the renin-angiotensin system in the resumption of
meiosis of bovine oocyte. Firstly, we showed that AT1 and AT2 receptors are expressed in both granulosa and theca
cells. The abundance of AT2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas
both receptors in theca cells and AT1 in granulosa cells did not change [18]. Granulosa cells cultured with hormones
that stimulate estradiol secretion increased AT2 mRNA and protein levels, whereas fibroblast growth factors (FGF-7
and 10) inhibited estradiol secretion and AT2 protein levels [18]. We also found that the concentration of AngII
increases in dominant follicle at time expected for follicular deviation [9]. Transvaginal ultrasound has been used for
intrafollicular injection to understand the regulation of follicular wave and ovulation. With this in vivo model, we have
demonstrated that AngII-receptor blocker inhibits follicular growth and decreases estradiol concentration in follicular
fluid and downregulates mRNA expression of follicular growth-related genes, such as aromatase (CYP19), 3âhydroxysteroid dehydrogenase (HSD3b), LH receptor, SerpinE2 and cyclinD2 in granulosa cells. Moreover, intrafollicular
injection of AngII or AT2-specific agonist prevented the expected atresia of the second largest follicle, which continued
to grow at a rate similar to the dominant follicle for 24h [8]. These findings have provided evidence that AngII signaling
promotes follicle growth and dominance in cattle. AngII acts through promoting differentiation (LHr, aromatase, 3âHSD)
and proliferation (cyclinD2) of granulosa cells. In regarding to ovulation, we demonstrated that AngII antagonists block
ovulation in cattle when intrafollicularly injected at 0 and 6 h after GnRH-challenge in vivo. Ovulation was also inhibited
by AT2- but not by AT1-AngII receptor antagonist [7]. Furthermore, AngII stimulates an enhancement in mRNA
abundance of disintegrin metalloproteinase domain 17 (ADAM17), EGF-like ligands amphiregulin (AREG), epiregulin
(EREG) and prostaglandin synthase 2 (PTGS2). In addition, AngII stimulates genes involved in extracellular remodeling
and follicular wall rupture. The inhibition of sheddase activity abolished the stimulatory effect of AngII on AREG,
EREG and PTGS2 mRNA [17].
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III. ANGIOTENSIN II ON RESUMPTION OF MEIOSIS
Oocytes are arrested in the first meiotic prophase during follicular development. Meiotic resumption is
dependent on the preovulatory surge of LH, but the bovine cumulus-oocyte complexes (COCs) do not have LH
receptors, so the effect must occur through theca and mural granulosa cells [15,20]. The LH surge stimulates the
ovarian renin-angiotensin system (RAS), including an increase in the renin activity [14] and AngII concentration in
bovine follicular fluid [2]. Using the in vitro model, we cultured COCs with or without follicular cells and AngII or
saralasin (a competitive AngII antagonist). In the absence of follicular cells, AngII did not affect the percentage of
oocytes reaching the germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stage after 7-h,
12-h and 18-h of culture, respectively. Similarly, saralasin did not affect the percentage of oocytes reaching MII stage.
Theca cells or medium conditioned with follicular cells that inhibited oocyte maturation were not able to inhibit nuclear
maturation when Ang II was present in the culture system [11].
Using our in vivo model, the role of AngII in LH-induced meiotic resumption was assessed. Cows were
superovulated and when follicles reached diameters larger than 12 mm, transvaginal ultrasound intrafollicular injections
were performed with saralasin or saline and GnRH agonist was intramuscularly injected to induce an LH surge. Fifteen
hours after GnRH agonist, the oocytes from follicles that received saline underwent resumption of meiosis (100%)
and most of them were in metaphase I (69.2%) stage while those that received saralasin were in the GV stage
(100%). Our results supported the hypothesis that AngII mediates the resumption of meiosis induced by an LH surge
in bovine oocytes [3].
IV. ANGIOTENSIN II DEPENDS ON PROGESTERONE, OXYTOCIN AND
PROSTAGLANDIN TO INDUCE MEIOTIC RESUMPTION.
The interactions among LH, AngII, endothelin-1, and atrial natriuretic peptide increased follicular production
of prostaglandins (PGs) and modulated steroidogenesis in the bovine preovulatory follicle [1]. AngII stimulates PGendoperoxide synthase 2 (PTGS2) and PG synthesis in the vascular endothelium [10], renal tissue [12], and human
monocytes [13]. In rabbit ovaries perfused in vitro, AngII was found to stimulate PGE2 and PGF2á production in the
absence of gonadotropins [21]. Recently, our group demonstrated that indomethacin (nonselective prostaglandin
inhibitor) inhibited AngII-induced meiotic resumption and PGE2, PGF2á or AngII induced GVBD in oocytes cocultured with follicular cells [3].
There are strong evidences that progesterone mediates the LH action to increase cyclooxygenase-2 mRNA,
PGE, and PGF2a in the ovulatory cascade [5]. We observed that the competitive inhibitor of progesterone receptors
mifepristone (MIFE) inhibited the stimulatory action of AngII in the resumption of meiosis. Similarly to the action of
AngII, progesterone was able to stimulate the resumption of meiosis when oocytes were cultured with follicle
hemisections. However, atosiban (an oxytocin inhibitor), but not saralasin, was able to maintain oocyte in germinal
vesicle stage when progesterone was present in the culture system. Also, oxitocin induced the oocyte meiotic
resumpetion and indomethacin, but not MIFE, blocked this oxitocin effect (data not published).
V. FINAL CONSIDERATIONS
Our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge
in bovine oocytes and stimulates a cascade of events involving the interaction of progesterone, oxytocin and
prostaglandin.
REFERENCES
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