Insulin-Like and Fibroblast Growth Factors and Their Receptors Are

Insulin-Like and Fibroblast Growth Factors and Their Receptors Are

BIOLOGY OF REPRODUCTION 58, 1451-1457 (1998)
Insulin-Like and Fibroblast Growth Factors and Their Receptors Are Differentially
Expressed in the Oviducts of the Common Marmoset Monkey (Callithrix jacchus)
during the Ovulatory Cycle'
Christoph Gabler,3 Annette Plath-Gabler, 3 Almuth Einspanier, 4 and Ralf Einspanier 2 ,3
Institute of Physiology,3 Technical University Miinchen-Weihenstephan, Freising, Germany
Department of Reproductive Biology,4 German Primate Center, Gottingen, Germany
The mammalian oviduct is the site of fertilization and
is the site at which essential early embryonic events occur,
including the first cell divisions and the onset of mRNA
transcription. The oviduct has a ciliated and secretory epithelium, cells increasing their height and activity (ciliation and secretion) under the influence of estrogen [16,
17], possibly acting via local growth factors. Several reports indicate that the oviduct could be an essential source
of growth factors: in human fallopian tubes, the presence
of mRNA and specific immunoreactivity for IGF-1, IGF2, and IGFR-1 [18, 19] as well as epidermal growth factor
(EGF), transforming growth factor a (TGFa), and EGF
receptor (EGFR) [20-23] have been reported. Recently
FGF-1, FGF-2, and FGFR-2 were identified in bovine oviducts [24, 25], and IGF-1 and -2 and the corresponding
receptor IGFR-1 were identified in the ovine oviduct [26].
Both FGF bind to the same receptor types, though FGF-1
may be 30- to 100-fold less potent than FGF-2 [27, 28].
However, it has been shown [29] that the mitogenity of
FGF-1 is greatly enhanced by heparin; thus the two
growth factors are assigned equal potency in cell activation. To our knowledge, no such published data about the
FGF system in the primate oviduct are available. IGF-1 is
known to play a major role in the cross-talk between gonadotropins and sex steroids within the reproductive tract,
acting mainly via IGFR-1 [30].
This study was done to analyze the expression at the
mRNA and protein levels of FGF-1, FGF-2, IGF-1, and
IGF-2 as well as of their corresponding receptors in marmoset oviducts during the ovulatory cycle. Results were
compared with plasma estradiol and progesterone concentrations.
ABSTRACT
It issuggested that growth factors support the process of maturation and differentiation in the mammalian oviduct. Fibroblast
growth factors (FGF) and insulin-like growth factors (IGF) are
possible influences on these processes. The present study describes for the first time the expression of FGF-1 and -2 and
their receptors as well as IGF-1 and -2 and the corresponding
IGF receptor type 1 in the oviduct of the New World monkey
Callithrix jacchus. Because of the limited RNA yields from oviducts, reverse transcription-polymerase chain reaction (RT-PCR)
was performed to estimate expression levels. Expression patterns
were found to be similar for all examined growth factors and
receptors: the highest mRNA contents were obtained at the late
proliferative and early to midsecretory phases, compared with
lower levels during the early proliferative phase. Elevated
amounts of these RNAs were correlated with high serum estradiol but not with progesterone concentrations. Each PCR product showed a high degree of homology (> 92%) to the known
human sequences.
Immunohistochemical analysis indicated greater specific
staining for FGF-1 and -2 and IGF-1 before ovulation on the
luminal epithelial surface of marmoset oviducts in comparison
to the other cycle phases. Differences in staining intensity were
not observed between the ampulla and isthmus. In summary, the
marmoset oviduct expresses all components of functional FGF
and IGF systems, thus suggesting auto-/paracrine effects of these
growth factors within the primate oviduct, possibly under the
control of estrogenic hormones.
INTRODUCTION
Growth factors are expressed in a wide variety of tissues, including those of the female reproductive tract, and
are considered to play an important role in cellular proliferation and differentiation. Fibroblast growth factor
(FGF)-1 and -2 and their receptors (FGFR), as well as
insulin-like growth factor (IGF)-1, IGF-2, and IGF receptor (IGFR)-1 and -2, have been detected in the ovary [17]; and it is suggested that they are involved in the development of the ovarian follicle and corpus luteum,
where they may influence angiogenesis and granulosa cell
proliferation in the developing follicle [8-10] as well as
increase ovarian androgen production [6]. Additionally,
these growth factors and their related receptors are reported to be expressed in uterine tissue [11-14]. FGF-1
and -2, IGF-1, and IGFR-1 immunoreactivity was observed predominantly in glandular epithelial cells [11, 15],
and it is proposed that they are involved in the cyclic
growth of the endometrium.
Accepted January 26, 1998.
Received November 11, 1997.
'This research was supported by the DFG (Ei 296/4-2).
2
Correspondence: R. Einspanier, FML-lnstitute of Physiology, V6ttingerstr. 45, 85350 Freising, Germany. FAX: 49-8161-714204; e-mail:
einspani@pollux.edv.agrar.tu-muenchen.de
MATERIALS AND METHODS
Animals and Sample Collection
Oviducts from 13 normally cycling female common
marmoset monkeys (Callithrixjacchus), as well as from
an early-pregnant monkey and from an ovarian-dysfunctional (follicle cysts) marmoset monkey, were used for this
study (15 monkeys total). The animals were housed in
pairs as described previously [31]. Animals were bled
twice weekly to determine the plasma progesterone concentration and to monitor ovarian cyclicity. Ovarian cycles
were regulated by injection of prostaglandin (PG) F 2, (0.8
pIg Estrumate; Pitman-Moore, GmbH, Burgwedel, Germany) on Day 12 of the luteal phase to induce luteolysis
and the onset of preovulatory follicle development [32].
Ovulation in the marmoset monkey occurs, on average,
10.7 days after PGF 2 ,-induced luteolysis [32]. The day of
ovulation was defined as the day preceding a rise in plasma progesterone concentration above 10 ng/ml [33]. The
proliferative phase has a mean length of 10 days, and the
1451
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GABLER ET AL.
FIG. 1. Specific RT-PCR products for
FGF-1 (317 bp), FGF-2 (288 bp), FGFR
(471 bp), and ubiquitin (189 + 417 bp)
separated by agarose gel electrophoresis.
(1) Early and (2) late proliferative phase;
(3) early and (4) mid secretory phase; (5)
animal with ovarian dysfunction; (6)pregnant animal.
interrupted secretory phase has a mean length of 12 days.
Normal cycle length for marmoset monkeys in this colony
is on average 28 days.
For mRNA analysis, oviducts were collected under halothane anesthesia: 1) prior to the LH rise on Day 7 of the
proliferative phase (n = 3); 2) on Day 8 of the proliferative
phase (22 h after exogenous hCG [75 IU; Vemie Veterinir
Chemie GmbH, Kempen, Germany]) administration as described previously [34-35]) but prior to ovulation (n = 4);
3) during the secretory phase on Day 5 (early secretory
phase) (n = 3); and 4) during the secretory phase on Day
9 (midsecretory phase) (n = 3).
Additionally, one monkey showing cystic ovaries and
permanently elevated progesterone and a further animal at
Day 44 of pregnancy were included in the study. The excised oviducts were immediately transferred to liquid nitrogen and subsequently stored at -80°C until examined.
Tissue samples to be used for immunohistochemistry
from each cycle stage (2 animals, respectively) were
mounted on Tissue Tek (Sakura Finetek Europe BN, Zoeter
Wounde, Netherlands) and then transferred to liquid nitrogen and stored at -80°C until analysis.
Enzyme Immunoassay (EIA) for Progesterone and
Estradiol
For estradiol measurements, 50 pxl plasma was extracted with 1 ml tert.-butylmethyl-ether (Riedel-de-Haen,
Seelze, Germany), and the aqueous phase was frozen out
at -60°C. The organic supernatant was recovered and
evaporated, and the residue was dissolved in 150 1 assay
buffer (40 mM NaH 2PO 4 [Merck, Darmstadt, Germany],
0.15 mM NaCl [Merck; pH 7.2], 0.1% BSA [A-7888; Sigma Chemical Co., St. Louis, MO]). Then 50 L1 was ana-
lyzed by EIA using a 6-keto-17[-estradiol-6-carboxymethyloxime-BSA antibody as described previously [36].
The intra- and interassay variations were 6.3% and 9.5%,
respectively.
Progesterone in plasma was determined by a direct,
nonextraction EIA using an antiserum raised in sheep
against progesterone-ll-a-hemisuccinate-BSA and alkaline phosphatase linked to progesterone- 11-glucuronide as
enzyme conjugate. This assay has been previously described for the marmoset monkey [37].
RNA Isolation
Total RNA was extracted from whole monkey oviducts
according to the method of Chomczynski and Sacchi [38]
using Trizol reagent (Gibco BRL, Gaithersburg, MD). The
yield of total RNA was spectroscopically determined at 260
nm. Quality and quantity of oviductal RNA was verified
after denaturing electrophoresis on a 1% (w:v) formaldehyde-containing agarose gel followed by ethidium bromide
staining.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)
Four micrograms total RNA was reverse transcribed to
obtain cDNA using Superscript II reverse transcriptase
(Gibco BRL) at 45°C for 30 min, followed by 90°C for 2
min. Reaction mixtures contained 50 mM Tris/HCl (pH
8.3), 75 mM KC1, 3 mM MgCl 2, 10 mM dithiothreitol,
0.588 mM dNTPs, 2.5 mM random hexamers, and 140 U
of reverse transcriptase in a final volume of 60 ul.
The following commercially synthesized primers (Pharmacia, Freiburg, Germany) were used to amplify specific
monkey transcripts:
GROWTH FACTORS AND THEIR RECEPTORS IN MARMOSET OVIDUCTS
1453
FIG. 2. Specific RT-PCR products for IGF1 (210 bp), IGF-2 (215 bp), IGFR-1 (314
bp), and ubiquitin (189 + 417 bp) separated by agarose gel electrophoresis. (1) Early
and (2) late proliferative phase; (3) early
and (4) mid secretory phase; (5) animal
with ovarian dysfunction; (6) pregnant animal.
FGF-1
(318
FGF-2
(289
[24]
bp)
[24]
bp)
FGFR [39]
(471 bp)
Ubiquitin [24]
(189 bp)
IGF-1 [40]
(210 bp)
forward 5'
reverse 5'
forward 5'
reverse 5'
GCT GAA GGA GAA ACC ACG AC 3'
GTT TTC CTC CAA CCT TTC CA 3'
GAA CGG GGG CTT CTT CCT 3'
CCC AGT TCG TTT CAG TGC C 3'
forward 5'
reverse 5'
forward 5'
reverse 5'
forward 5'
reverse 5'
GAR
CCC
ATG
CTT
GGC
GGA
ATG
RAA
CAG
CTG
CGA
CCC
GAG
RGA
ATC
GAT
CTT
GAG
RTG
CCA
TTT
GTT
GGC
ACC
ATG
SAC
GTG
GTA
GGG
CTC
AAG MTG ATY GG 3'
RTC ACT CTG 3'
AAG AC 3'
TC 3'
CTT GA 3'
TGC GGG 3'
IGF-2 (corresponding to bases 619-833 of the human sequence:
EMBL no.: J03242)
(215 bp)
forward 5' TAT GCT GCT TAC CGC CCC AG 3'
reverse 5' ACA TCC CTC TCG GAC TTG GC 3'
IGFR-1 (corresponding to bases 2593-2906 of the human sequence:
EMBL no.: X04434)
(314 bp)
forward 5' TTA AAA TGC CCA GAA CCT GAG 3'
reverse 5' ATT ATA ACC AAG CCT CCC AC 3'
The predicted size of each RT-PCR product is shown in
parentheses. The PCR reactions were performed as described previously [24]. Individual amplification programs
were applied for FGF-1 and FGFR (35 cycles at 94°C and
60°C, 1 min each), FGF-2 and IGF-1 (30 cycles at 94°C
and 60°C, 1 min each), and ubiquitin (22 cycles at 94°C,
55°C and 72°C, 45 sec each). Five microliters of each PCR
product was run on 1.5% agarose gels containing 1 xg/ml
ethidium bromide. As a negative control, water instead of
RNA was used for the RT-PCR, and all reactions were performed three times for each RNA preparation. Specificity
of RT-PCR products was checked by subcloning into the
pCR-Script SK(+) cloning vector (Stratagene, La Jolla,
CA), followed by double-stranded DNA sequencing (Sequiserve, Vaterstetten, Germany).
As a control for quantification, sense cRNA transcripts
were synthesized from the cloned inserts using the appro-
priate T3 or T7 RNA polymerase (Stratagene) according to
the manufacturer's instructions. Such generated cRNAs and
a mass ladder (Gibco BRL) were subjected to a 1% agarose
gel electrophoresis followed by ethidium bromide staining.
Resultant band intensities were scanned by a video documentation system (Pharmacia) and analyzed with the Image
Master 1D program (Pharmacia). To estimate cRNA concentrations, signal intensities were compared with the
known concentrations of the marker. Calibrated amounts of
the specific sense cRNAs were then used in each RT-PCR
performed with monkey total RNA.
Immunohistochemistry
Frozen cryostat sections (7 lim) from the marmoset
monkey isthmus and ampulla were mounted together on
gelatine-coated slides. The sections were treated as described previously [24] and then analyzed for the presence
of FGF-1 and FGF-2 (both polyclonal antibodies, 1:5000),
IGF-1 (polyclonal; 1:1000), and heparan sulfate (Clone
7E12, 1:500; Boehringer, Mannheim, Germany). The crossreactivities of the antibodies to other growth factors and of
FGF-1 versus FGF-2 and vice versa were below 0.1%. Immunoreactivity was visualized using the alkaline phosphatase technique (APAAP complex; Dianova, Hamburg, Germany) and the conjugated avidin-biotin method (LSAB kit;
Dakopatts, Hamburg, Germany). To show specificity of the
staining reactions, antibodies that had been preabsorbed
with excess antigen were used. As further controls for specificity, in some sections the primary antibody was omitted
or replaced by preimmune IgG. Sections were counterstained with hematoxylin, mounted in glycerin jelly, and examined using a Zeiss (Carl Zeiss, Thornwood, NY) microscope. For morphological investigations, sections were
stained with hematoxylin.
1454
GABLER ET AL.
TABLE 1. Homology relative to the human of the partial PCR-derived
cDNA sequences for various growth factors and receptors.
Factors
(EMBL No.)a
Ubiquitin (Z49056)
FGF-1 (Z49053)
FGF-2 (Z49054)
FGFR-2 (Z68149)
IGF-1 (Z49055)
IGF-2 (AJ001297)
IGFR-1 (AJ001298)
Homology (%) to human cDNA
(EMBL No.)b
84.6
91.5
96.5
96.8
92.4
94.9
94.6
(M26880)
(X65778)
(M27968)
(M37715)
(X57025)
(J03242)
(X04434)
a The EMBL access numbers of the Callithrix jacchus cDNA sequences
are in parentheses.
b Percentage homology between the marmoset monkey cDNAs and the
known genes (EMBL access number in parentheses).
RESULTS
Estradiol and Progesterone Blood Concentrations and
Quality of the RNA
Plasma concentrations of estradiol and progesterone
were measured in six marmoset monkeys during three successive prostaglandin-primed cycles. These six animals
showed the same hormone profiles through this controlled
cycle: as expected, PGF 2. rapidly terminated the luteal
phase as indicated by a decline of progesterone levels and
ovulation within the next 11 days (data not shown). For
each animal, plasma concentrations of these hormones were
determined at the time of halothane anesthesia and allocated to the appropriate cycle stage. Physiological hormone
levels were within the previously described ranges [33, 41]:
1) progesterone exceeded 10 ng/ml during the secretory
phase (up to a maximum of 160 ng/ml) and 2) estradiol
concentrations were found to be below 300 pg/ml during
the secretory phase after a short increase before ovulation.
RT-PCR Data
As a control for the quantity and quality of the RNA,
total RNA was first subjected to denaturing agarose gel
electrophoresis. Because of the limited RNA yields (< 50
[ag), an additional estimation of RNA quantity was obtained
through estimation of expression of the mRNA for the
housekeeping gene ubiquitin: two ubiquitin-specific RTPCR products (189 + 417 base pairs [bp]) were found to
be stably expressed during the ovulatory cycle in the Callithrix jacchus oviduct (Figs. 1 and 2). These ubiquitin
products were used to control for the efficiency of the RTPCR between samples. In a separate analysis, ubiquitin
transcript intensity was shown to be equivalent for all RNA
samples by Northern blot hybridization analysis (data not
shown).
RT-PCR analysis followed by sequence determination of
the products verified that FGF-1, FGF-2, IGF-1, and IGF2 and their related receptors, FGFR and IGFR-1, were specifically expressed in the oviduct of the marmoset monkey
(Figs. 1 and 2). Each cloned cDNA shared a high homology
with the known human gene (> 92%) (Table 1). Only the
sequence homology between the marmoset ubiquitin cDNA
and the human gene was lower (> 85%) owing to an unusual codon usage.
The relative signal intensities for PCR products specific
for all growth factors and receptors and for all animals were
assessed after correction based on the ubiquitin mRNA signals. Individual mRNAs were then additionally evaluated
FIG. 3. RT-PCR signals for FGF-1 and -2, FGFR, IGF-1, IGF-2, and IGFR1 during the late proliferative (A) and the early secretory phases (B) in
both oviducts from a single individual.
for their approximate content with the use of known
amounts of sense cRNA.
For all examined growth factors and receptors, the expression levels during the early proliferative phase (numbered 1) appeared lower when compared with those of the
late proliferative samples (2) and early secretory-stage samples (3 and 4) (Figs. 1 and 2).
On the basis of the sense cRNA standards, the following
mean mRNA levels were measured after ovulation in the
monkey oviduct: 50 pg ubiquitin mRNA, 30 fg FGF-1
mRNA, 300 fg FGF-2 mRNA, 25 pg FGFR mRNA, 30 pg
IGF-1 mRNA, 25 pg IGF-2 mRNA, and 30 pg mRNA encoding IGFR-1 per microgram total RNA, respectively.
Additionally, the expression pattern of both oviducts
from one individual was examined separately during the
late proliferative and early secretory phases of the cycle (n
= 2). Oviducts of the late proliferative stage with two ipsilateral follicles showed an increased mRNA expression
for FGF-1, FGF-2, IGF-1, and IGFR-1 in comparison to
the opposite oviduct with only one follicle (Fig. 3). In contrast, during the early secretory phase, no obvious difference in signal strength was visible, although one ovary contained three corpora lutea and the opposite ovary none.
Oviducts obtained from one early-pregnant animal (sample
6), as well as one marmoset with ovarian dysfunction (sample 5), showed expression of all growth factor system components examined (Figs. 1 and 2).
Immunohistochemistry
Immunohistochemical observations indicated that marmoset oviductal cells contained immunoreactive FGF-1,
FGF-2, and IGF-1. Staining for FGF-1 and -2 as well as
for IGF-1 was detected specifically on the epithelial cell
GROWTH FACTORS AND THEIR RECEPTORS IN MARMOSET OVIDUCTS
1455
FIG. 4. Immunohistochemical localization of IGF-1 (B) and (C), heparan sulfate (D), FGF-1 (E), and FGF-2 (F) in the marmoset oviduct (Day 8 of the
proliferative phase, 22 h after exogenous hCG administration). Positive staining is shown by a black chromogen. Negative control (A) with rabbit
immunoglobulin. The arrow shows blood capillaries. Original magnification: A, B, D-F) x375; C) x625 (reproduced at 72%).
layer (Fig. 4). Additionally, some positive immunostaining
for these antigens was found in the nuclei of oviduct epithelial cells. For FGF-2 only, there was also strong immunostaining in the basal lamina and in capillary endothelial cells. The intensity of immunostaining differed between
the proliferative and secretory phases, with highest signal
intensities before ovulation (after hCG) in comparison with
the other ovulatory cycle stages. No such differences in
immunostaining intensity were found between the ampulla
and isthmus, or between oviducts from the same animal.
Heparan sulfate was localized to the epithelial cell surface
at the oviductal lumen, as well as in capillary endothelial cells,
and showed no obvious cycle dependency (Fig. 4).
DISCUSSION
In this study the marmoset oviduct was identified as an
important source of growth factors. Using RT-PCR, transcripts for FGF- 1, FGF-2, IGF- 1, and IGF-2 were identified,
local biosynthesis of these factors being supported by specific immunostaining with an epithelial localization, suggesting a secretion of the mature proteins. These results thus
are in agreement with reports describing mRNAs encoding
IGF-1, IGF-2, and IGFR-1 in human fallopian tubes [19] or
IGF-1 transcripts in oviducts of the rat, mouse, and cow [40,
42, 43]. In the cow there was increased expression of IGF1 after ovulation, and in the rat oviduct, highest IGF-1 transcript levels were present during proestrus, similar to what
we observe in the marmoset oviduct. Previously, we showed
[24] that in bovine oviducts, only FGF-1 expression indicated cycle-dependant variation, with highest mRNA contents during the postovulatory stage; contents of mRNA for
FGF-2 and FGFR remained unchanged. These results differ
from those described here for the marmoset, where the highest
mRNA contents of both FGFs and their receptors could be
detected during the late proliferative and early secretory
stages. This is similar to the expression pattern of another
growth factor system, the EGF/TGFo family [20-23]. Increase in the amounts of EGF, EGF receptor, and IGF-1
occurred in the oviducts in association with a rise in estradiol,
but not in progesterone levels, that has been described for
humans and rats in vivo and in vitro in oviductal cells [21,
22, 43, 44]. Since serum estradiol and progesterone profiles
during the human and marmoset cycles appear similar, expression of the growth factors might also be regulated in a
comparable fashion in the marmoset. The observations made
in the present study on the pregnant animal and on the animal
with ovarian dysfunction would support this view. In the latter,
expression of the growth factors appeared to be similar in
1456
GABLER ET AL.
content to that at ovulation, during which the estradiol concentration in plasma is maximal-whereas in the pregnant
animal, the expression data for the investigated growth factor
systems were similar to those from the late secretory phase.
One would thus expect there to be a distinctly different
expression pattern between the two oviducts within one animal just before ovulation, possibly corresponding with the
individual ovarian follicle status.
The successful immunostaining for FGF-1 and -2 and
IGF-1 in the marmoset oviducts indicates that these growth
factors are located at the luminal surface of the epithelium,
suggesting that the proteins may be secreted into the lumen
around ovulation. This agrees with a recent report that immunoreactive FGF-1 and FGF-2 proteins were measured in
bovine oviduct flushings around ovulation [24]. FGF-2 was
additionally found on the basal membrane and in endothelial cells. Thus, the possibility of additional import of FGF2 from the circulation cannot be excluded.
Within the human oviduct, staining for EGF, TGFoa, and
EGFR [23], as well as for IGF-1 and IGFR-1 [18], is cycle
dependent, with higher signals before ovulation. This
agrees with the results obtained in the present study showing higher immunostaining signal corresponding with higher expression levels before ovulation.
The appearance of heparan-like structures on the epithelial surface indicates that the heparin-binding FGF-1 and
-2 could be easily fixed on the oviductal surface. This matrix is known to serve as a reservoir for these growth factors
and thus may serve as a modulator of their action, possibly
mediated by the FGFR receptors (four human receptor
types are known). The present observations suggest that
functional growth factor systems for IGF and FGF could
influence early embryonic development within the marmoset oviduct. Secondly, an auto-/paracrine mechanism between the oviduct cells themselves might additionally be
involved in epithelial physiology linked to support of the
embryo. In this regard, recent studies have demonstrated
that FGF-2 can promote the in vitro formation of mesoderm
in rabbits [45] as well as the transition of bovine morulae
to blastocysts [46]. Such results would support the concept
that growth factor systems like FGF and IGF could serve
as important auto-/paracrine mediators of the primate oviduct and be driven by estrogenic hormones. Important cycle-specific changes within the primate oviduct have been
shown previously for the estrogen receptor and for epithelial morphology (reciliation and deciliation) [47, 48].
Given that the patterns of hormone and growth factor
expression appear to be similar between the marmoset and
the human, and given the high degree of homology (>
92%) at the DNA sequence level, it is suggested that the
marmoset monkey could serve as a convenient primate
model for human reproductive research.
3.
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6.
7.
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9.
10.
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20.
ACKNOWLEDGMENTS
21.
We
FGFzinski,
for the
22.
would like to thank D. Schams for providing the estradiol assay,
and -2, and IGF-1 antibodies; A. Zuber, G. Schwentker, A. Jurdand K. Fuhrmann for their motivated assistance; and Prof. Hodges
animal facilities.
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