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Endocrinology, doi:10.1210/en.2006-1668
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Endocrinology Vol. 148, No. 8 3711-3721
Copyright © 2007 by The Endocrine Society

Fibroblast Growth Factor-9, a Local Regulator of Ovarian Function

Ann E. Drummond, Marianne Tellbach, Mitzi Dyson and Jock K. Findlay

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. Ann Drummond, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: ann.drummond{at}princehenrys.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fibroblast growth factor 9 (FGF9) is widely expressed in embryos and fetuses and has been shown to be involved in male sex determination, testicular cord formation, and Sertoli cell differentiation. Given its male gender bias, the ovary has not been reported to express FGF9, nor has a role in ovarian function been explored. We report here that FGF9 mRNA and protein are present in the rat ovary and provide evidence that supports a role for FGF9 in ovarian progesterone production. FGF9 mRNA levels as determined by real-time PCR were high in 4-d-old rat ovaries, thereafter declining and stabilizing at levels approximately 30% of d 4 levels at d 12–25. Levels of FGF9 mRNA in the ovary were significantly higher than that present in adult testis, at all ages studied. The FGF9 receptors FGFR2 and FGFR3 mRNAs were present in postnatal and immature rat ovary and appeared to be constitutively expressed. FGF9 protein was localized to theca, stromal cells, and corpora lutea and FGFR2 and FGFR3 proteins to granulosa cells, theca cells, oocytes, and corpora lutea, by immunohistochemistry. Follicular differentiation induced by gonadotropin treatment reduced the expression of FGF9 mRNA by immature rat ovaries, whereas the estrogen-stimulated development of large preantral follicles had no significant effect. In vitro, FGF9 stimulated progesterone production by granulosa cells beyond that elicited by a maximally stimulating dose of FSH. When the granulosa cells were pretreated with FSH to induce LH receptors, FGF9 was found not to be as potent as LH in stimulating progesterone production, nor did it enhance LH-stimulated production. The combined treatments of FSH/FGF9 and FSH/LH, however, were most effective at stimulating progesterone production by these differentiated granulosa cells. Analyses of steroidogenic regulatory proteins indicate that steroidogenic acute regulatory protein and P450 side chain cleavage mRNA levels were enhanced by FGF9, providing a mechanism of action for the increased progesterone synthesis. In summary, the data are consistent with a paracrine role for FGF9 in the ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE VERTEBRATE FIBROBLAST growth factor (FGF) family consists of 23 members classified on the basis of conserved gene structure and amino acid sequence (1, 2). These heparin-binding polypeptides play roles in development, proliferation, differentiation, cellular migration, tissue repair and injury response, angiogenesis, and cancer metastasis.

FGF superfamily members signal via transmembrane tyrosine kinase receptors, of which there are four types, designated FGFR1–FGFR4. Except for FGFR4, two to three isoforms exist for each receptor as a result of alternate splicing (3, 4, 5). The transmembrane localization of these receptors does not preclude their nuclear localization, with FGF ligand and receptor complexes previously identified in the nucleus (6, 7, 8) and roles in cellular differentiation and proliferation demonstrated (6, 8). These "nuclear" FGFRs are thought to act at the level of gene transcription. In addition, low-affinity binding sites contributed by heparin and heparin sulfate proteoglycans facilitate signaling by FGF family members (9, 10, 11, 12). In the rat ovary, FGFR1 and FGFR2 have been localized to granulosa cells and theca cells (13), with FGFR1 also present in luteinized ovaries (14). Ovine corpora lutea localize FGFR1 and FGFR2 (15). In the bovine, FGFR1–FGFR3 have been localized to theca and granulosa cells (16, 17), with FGFR4 expression confined to theca cells (18). All four FGFR mRNA transcripts have been detected in human ovary, with protein for FGFR2–FGFR4 localized to oocytes, granulosa cells, and surface epithelium (19, 20).

Some members of the FGF superfamily have been localized to the ovary. FGF1 (also known as acidic FGF) and FGF2 (also known as basic FGF) have been found in oocytes of small follicles, granulosa cells, theca cells, and corpora lutea (16, 21, 22, 23, 24, 25). Both FGF1 and FGF2 have been shown to stimulate granulosa cell, germinal epithelial cell, theca cell, and luteal cell proliferation (26, 27, 28, 29, 30). FGF2 has also been shown to exert a variety of effects on granulosa cell function, notably, steroidogenesis (31, 32) and apoptosis (33, 34, 35). Roles in angiogenesis of developing corpora lutea (36) and primordial follicle development have also been proposed for FGF2 (25). FGF7 (also known as keratinocyte growth factor) has been localized to bovine theca cells (37) and luteal cells (38) and shown to stimulate bovine granulosa cell proliferation and the survival, growth, and differentiation of rat preantral follicles in vitro (39). FGF8 [also known as AIGF (androgen-induced growth factor)] has been localized to maturing mouse oocytes (40) and bovine oocytes, granulosa cells, and theca cells (18). FGF4 [also known as kaposi FGF and hst 1 (human stomach tumor-1)] has been localized to ovulated mouse oocytes (41). To date, no other family members have been identified in the ovary.

FGF9 is widely expressed in embryos and fetuses (42, 43) and has been shown to be involved in male sex determination (44). Furthermore, FGF9 can regulate multiple SRY (sex-determining region on the Y gene)-dependent processes, such as mesenchymal proliferation and mesonephric migration into the gonad and Sertoli cell differentiation (45). FGF9 has been shown to signal preferentially via FGFR2–FGFR4 (46, 47, 48, 49). The gonads of XY FGF9 knockouts are sex reversed, presenting with oviducts and fused uteri (45). FGF9 knockout mice die at birth as a result of lung hypoplasia (50), so although normal reproductive structures (uteri and ovaries) are present in female knockout mice (45), it has not been possible to investigate ovarian follicular formation and development after birth. To date, there have been no reports on the localization or action of FGF9 in the postnatal or adult ovary.

Our studies in the aromatase knockout mouse, in which sex-reversed cells (Sertoli and Leydig cells) were identified in the estrogen-deficient ovary (51), led us to believe that FGF9 could be a positive marker for the aromatase knockout mouse ovary and thus a negative control for normal ovary. The studies reported herein arose because of the fact that FGF9 was found to be present in adult rat ovary. Therefore, the aims of these studies were as follows: investigate the expression of FGF9 mRNA during postnatal ovarian development in the rat; localize FGF9 protein to the ovary; and investigate the regulation of FGF9 mRNA and protein in ovaries from rats treated with steroids and gonadotropic hormones to induce defined stages of follicle growth and differentiation. Having identified FGF9 mRNA in the ovary and localized FGF9 protein to ovarian cells, we hypothesized that FGF9 regulates the function of ovarian cells in growing follicles. We therefore examined the expression of FGFR2 and FGFR3 by the ovary and investigated the effect of FGF9 on steroid production using progesterone production as an endpoint. To elucidate a mechanism of action for the apparent stimulatory effect of FGF9 on progesterone production, the expression of steroidogenic enzymes/proteins by cultured rat granulosa cells was investigated. The findings of these studies are consistent with a local regulatory role for FGF9 on steroidogenesis in the ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley rats were obtained from Central Animal Services, Monash University (Melbourne, Victoria, Australia). Ovaries were collected from untreated rats at 4, 8, 12 (postnatal), 22, 23, 25 (immature), and 52 and 70 (adult) days of age. Some immature animals at 21 d received a single sc injection of pregnant mare serum gonadotropin (PMSG) (10 IU for 48 h) or human chorionic gonadotropin (hCG) (10 IU for 8 h), a combination treatment regimen of PMSG and hCG, or a diethylstilboestrol (DES) implant for 24 or 96 h (52), before ovary collection. Ovaries were used for either RNA extraction or the preparation of formalin-fixed, paraffin-embedded tissues. Testes were collected from adult rats 16 wk of age for RNA extraction. Animals were maintained under standard conditions of lighting and temperature and received laboratory feed pellets and water ad libitum. The project was approved by the Institutional Animal Experimentation and Ethics Committee as conforming to the guidelines of the National Health and Medical Research Council of Australia.

RNA extraction
Ovaries, dissected free of fat and adhering tissue or isolated granulosa cells, were homogenized in 1 ml Ultraspec RNA reagent (Biotecx; Fisher Biotec, Melbourne, Victoria, Australia). After 5 min on ice, 0.2 ml chloroform per milliliter of Ultraspec RNA reagent was added to the samples, which were then shaken vigorously and stored at 4 C for 5 min before centrifuging for 15 min at 12,000 x g. RNA was precipitated from the aqueous phase with 1 vol of isopropanol, after which the pellet was washed twice with ethanol, air dried, and resuspended in sterile water. To ensure that the RNA was completely dissolved, the samples were incubated for 10 min at 60 C. The samples were then treated with deoxyribonuclease (DNA-free; Ambion, Austin, TX) to remove any contaminating DNA. At least three independent pools of RNA were prepared for each age/treatment group. The number of ovaries per pool ranged from 24 to 40 for postnatal animals and two per pool for immature and DES-treated animals. Individual testes were used for RNA extraction.

RT
RNA (400 ng) was reverse transcribed with 50 U Moloney murine leukemia virus (Expand) reverse transcriptase (Roche, Sydney, New South Wales, Australia) and final concentrations of 1x cDNA synthesis buffer (supplied with enzyme), 1 mM NTPs (Roche, Sydney, New South Wales, Australia), 20 U RNasin (Promega, Sydney, New South Wales, Australia), 10 mM dithiothreitol, and 25 pmol oligo-dT15 (Roche, Sydney, New South Wales, Australia), as described previously (52).

Real-time PCR
mRNA expression was analyzed using the Roche LightCycler (Roche, Mannheim, Germany) as described previously (53). Briefly, two standards were used for PCR: a purified FGF9 cDNA pool diluted between 1000 and 0.001 fg/µl for FGF9 analyses and an ovarian cDNA pool diluted 1:2 to 1:2000 for FGFR2, FGFR3, steroidogenic acute regulatory protein (StAR), P450 side chain cleavage (SCC), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2 microglobulin analyses. Sample cDNAs diluted 1:2 to 1:10 in sterile water were added to individual capillaries. Taq enzyme, dNTPs, reaction buffer, and SYBR GREEN I dye were supplied in the FastStart DNA Master SYBR Green I kit (Roche, Mannheim, Germany), of which 2 µl/capillary was added. Primer concentrations of 10 pmol and 3 mM Mg were added to each capillary. Primer sequences are given in Table 1Go. The capillary volume was made up to 20 µl with sterile water. Forty cycles of PCR were programmed to ensure that the threshold crossing point (cycle number) was attained. Fluorescence emission was monitored continuously during cycling. At the completion of cycling, melting curve analysis was performed to establish the specificity of the amplicons produced. In addition, each amplicon was sequenced to verify the identity of the amplified product (data not shown). The level of expression of each mRNA and their estimated crossing points in each sample were determined relative to the standard preparation using the LightCycler computer software. A ratio of specific mRNA/housekeeper gene amplification was then calculated. In most instances, individual pools for each age group or treatment with each primer set, were performed in a single PCR experiment. The intraassay variation was never more than 5% (n = 7) regardless of the primer set. The nature of LightCycler PCR diminishes issues such as assay sensitivity, and, at the concentrations of standard and sample used in these studies, the sensitivity threshold was never approached.


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  		TABLE 1. Oligonucleotide primer sequences used to cDNAs

 
Immunohistochemistry
Antibodies directed against FGF9 and FGFR3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody directed at FGFR2 was obtained from Abcam (Cambridge, UK). The FGF9 antibody was a goat polyclonal raised against a peptide mapping to the carboxy terminus of human FGF9 (identical sequence to rat). The FGFR3 antibody was a rabbit polyclonal raised against a peptide mapping to the carboxy terminus of human FGFR3 (cross-reactive with rat). The FGFR2 antibody was a rabbit polyclonal raised against a synthetic peptide corresponding to amino acids 362–374 of human FGFR2 (cross-reactive with rat). The antisera were validated by the manufacturers in a number of formats including Western blots. The manufacturer has determined that the antibody for FGFR3 does not cross-react with FGFR1, FGFR2, or FGFR4, that the antibody for FGFR2 does not cross-react with FGFR1 and FGFR3, and that the FGF9 antibody is also highly specific. FGF9 and FGFR3 antibodies were used to localize their respective proteins in 5 µm sections of formalin-fixed paraffin-embedded rat ovary and Bouins-fixed paraffin-embedded testis (provided by Drs. Sarah Meachem and Pavel Sluka, Prince Henry’s Institute of Medical Research) using standard immunohistochemical protocols. Briefly, sections were dewaxed in histosol (Australian Biostain, Traralgon, Victorian, Australia), dehydrated in ethanol, and washed in water. The sections were equilibrated in 0.1 M PBS (pH 7.4). Endogenous peroxidase activity was blocked by incubating the sections for 30 min in 0.3% hydrogen peroxide followed by three washes in distilled water. Enzymatic (pepsin) antigen retrieval was then performed. The sections were incubated with Digest-All (Zymed, South San Francisco, CA) for 5 min at 37 C and then washed in PBS. The sections were blocked for 30 min in 1% blocking reagent (Roche, Mannheim, Germany), after which the primary antibody diluted 1:200 was added and the sections were incubated for 18 h at 4 C. After extensive washing in PBS, the biotinylated second antibody diluted 1:200 (DakoCytomation, Carpinteria, CA) was added to the sections for 60 min at room temperature. After washing in PBS, the sections were incubated with a peroxidase-conjugated avidin-biotin complex (Vector Elite; Vector Laboratories, Burlingame, CA) for 60 min at room temperature after which the reaction product was developed using 3,3' diaminobenzidine tetrahydrochloride (DakoCytomation) and hydrogen peroxide in PBS. The sections were counterstained with hematoxylin, dehydrated in ethanol, and cleared in histosol, and coverslips were mounted using DPX (BDH, Poole, UK). Control sections received buffer, IgG (goat) diluted appropriately, or antibody that had been preabsorbed with peptide against which the antisera had been raised, in place of primary antibody. The ovaries of 6–10 rats, two sections per ovary, were examined for each antibody in three separate experiments. Follicles were classified as described previously (54).

Granulosa cell cultures
Granulosa cells were released from 25-d-old, DES-treated rat ovaries by repeated puncture with fine gauge needles as described previously (55). Granulosa cells were plated in serum-free McCoys 5C (2 mM containing glutamine, 100 µg/ml transferrin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone). Three culture conditions were established. In the first, the granulosa cells (2 x 105 per well) were incubated for 48 h at 37 C in the presence or absence of FSH (100 ng/ml) (rFSH-I8; obtained from the National Hormone and Pituitary Distribution Program and the National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health, Baltimore, MD) or human recombinant FGF9 (0.1–50 ng/ml) (Sigma, St. Louis, MO). In the second, the granulosa cells (2 x 105 per well) were pretreated with FSH (100 ng/ml) for 24 h at 37 C to induce LH receptors, the media was then removed and replaced with fresh McCoys 5C and treatments, including FSH (100 ng/ml), FGF9 (25 ng/ml), and LH (100 ng/ml) (rLH-I9; obtained from the National Hormone and Pituitary Distribution Program and the National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health) either alone or in combination. The incubation was stopped 48 h after the addition of treatments. The conditioned media from each culture was collected and stored at –20 C until assayed for progesterone. In the third, culture wells were pretreated with 0.5% fetal calf serum for 1 h at 37 C to aid plating of the cells. The media containing fetal calf serum was removed, and fresh serum-free media containing 5 x 105 cells per well was added. The next day, the media was changed, and treatments (100 ng/ml FSH with or without 0.1 or 50 ng/ml FGF9) were added for 16 h. At the end of the incubation period, the media were removed, and 1 ml of Ultraspec solution was added to lyse the cells. The RNA was then extracted from the cells and reverse transcribed, and PCR was performed. The doses selected for each treatment were based on either previous studies undertaken in our laboratory, as was the case for FSH, in which 100 ng/ml was shown to be maximally stimulating (our unpublished data), or the recommendations of the manufacturers and published work (56), in the case of FGF9. The dose of LH (100 ng/ml) was matched to that of FSH.

Progesterone RIA
Progesterone concentrations in granulosa cell conditioned media were measured directly, without extraction, by an RIA using a World Health Organization polyclonal sheep antisera, as described previously (57). The interassay and intraassay variations were 6.0 and 3.8%, respectively, and the sensitivity of the assay was 0.27 ng/ml.

Statistics
The data were analyzed using GBstat and GraphPad (San Diego, CA) Prism 4. Statistical significance was determined by ANOVA in conjunction with a post hoc multiple comparison test (either Fisher’s least significant difference or Tukey-Kramer’s test). Each experiment was repeated at least three times, with n equal to a minimum of three per age group or treatment, in each experiment. The data are presented as the mean ± SD. P values of <0.05 compared with the appropriate control were regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FGF9 mRNA expression by rat ovaries and testes
FGF9 mRNA expression by the ovary declined between postnatal d 4 to 12, with levels on d 25 similar to those on d 12 (Fig. 1Go). FGF9 mRNA was present in adult testes but was least abundant in this tissue relative to the mRNA present in ovaries at all stages analyzed (Fig. 1Go).


Figure 1
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  		FIG. 1. Levels of expression of FGF9 mRNA in postnatal (d 4, 8, and 12) and immature (d 25) rat ovaries and adult testes, normalized for GAPDH mRNA expression. The number of pools (n) analyzed separately at each age is indicated below each histogram. The data are mean ± SD, with different letters denoting statistical significance (P < 0.05).

 
FGFR2 and FGFR3 mRNA expression by rat ovaries
FGFR2 and FGFR3 mRNAs were detectable in postnatal d 4, 8, and 12 ovaries and immature d 25 rat ovaries (Fig. 2Go). There were no statistically significant differences in the expression of these receptors during this period of development.


Figure 2
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  		FIG. 2. Levels of expression of FGFR2 and FGFR3 mRNA in postnatal (d 4, 8, and 12) and immature (d 25) rat ovaries, normalized for GAPDH mRNA expression. The number of pools (n) analyzed separately at each age is indicated below each histogram. The data are mean ± SD. There were no statistically significant differences between age groups.

 
Localization of FGF9 and FGFR proteins to rat ovaries
FGF9 protein was localized to the cytoplasm of stromal cells and basement membranes surrounding granulosa cells in d 4, 8, and 12 ovaries (Fig. 3Go). In d 12 ovaries, theca cell precursors were also positively stained for FGF9 protein (Fig. 3Go). In the immature 23- and 25-d-old (see Fig. 7Go) and adult rat ovaries (Fig. 3Go, D70), FGF9 protein was localized to the cytoplasm of stromal cells and theca cells and to basement membranes of follicles. Some corpora lutea also expressed FGF9 protein (Fig. 3Go, D70). FGF9 protein was not detected in granulosa cells or oocytes of any of the ovaries examined (Fig. 3Go) (see Fig. 7Go). Sections incubated with preabsorbed FGF9 antisera, or in the absence of primary antibody, did not localize FGF9 protein (Fig. 3Go) (see Fig. 7Go). Testes, used as a positive control, localized FGF9 protein to interstitial Leydig cells and the basement membrane of seminiferous tubules (Fig. 3Go).


Figure 3
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  		FIG. 3. Immunohistochemical localization of FGF9 protein to days (D) 4, 8, and 12 (postnatal) and 70 (adult) rat ovaries and adult testis. Control sections in which primary antibody (FGF9 antibody) was omitted are included for each age group. GC, Granulosa cell; P, primary follicle; p, primordial follicle; CL, corpus luteum; T, theca; TP, theca cell precursor; B, basement membrane; O, oocyte; ST, seminiferous tubules; LC, Leydig cells. Scale bars are indicated on each image.

 

Figure 7
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  		FIG. 7. Immunohistochemical (IHC) localization of FGF9 protein to ovaries of DES-, PMSG-, hCG-, and PMSG/hCG-treated rats. No primary antibody controls (b) and antisera preabsorbed against the immunizing peptide (a) for FGF9 are also included. GC, Granulosa cell; p, primordial follicle; T, theca; O, oocyte; B, basement membrane. Scale bars are indicated on each image, with low- and high-power images shown.

 
FGFR3 protein was localized to the nucleus of granulosa cells and some oocytes and theca cells of immature rat ovaries (Fig. 4Go). The staining pattern of FGFR3 in adult ovaries was similar to that of immature ovaries (Fig. 4Go, E and F). In addition, the luteal cells of some corpora lutea showed nuclear staining for FGFR3 (Fig. 4Go). Sections incubated with preabsorbed FGFR3 antisera (data not shown) or in the absence of primary antibody did not localize protein (Fig. 4Go). In contrast to FGFR3, FGFR2 protein was localized to the cytoplasm of ovarian cells. Granulosa cells, theca cells, and oocytes of immature and adult ovaries localized FGFR2 protein (Fig. 4Go). Copora lutea of adult ovaries also stained positively for FGFR2. Sections incubated in the absence of primary antibody did not localize protein (Fig. 4Go).


Figure 4
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  		FIG. 4. Immunohistochemical localization of FGFR3 and FGFR2 proteins to immature (d 22) and adult (d 70 and 52) rat ovaries. No primary antibody controls are shown for each antisera. D, Day; GC, granulosa cell; PF, preantral follicle; AF, antral follicle; O, oocyte; CL, corpus luteum; T, theca; P, primary follicle; p, primordial follicle. Scale bars are indicated on each image.

 
Effect of hormone treatment on FGF9 mRNA expression by rat ovaries
There was no significant effect of DES treatment on FGF9 mRNA expression by immature rat ovaries after 24 or 96 h (Fig. 5Go). In our first analyses of the PMSG with or without hCG-treated ovaries, it was noted that GAPDH mRNA appeared to be enhanced by PMSG treatment (data not shown). Hence, the data were reanalyzed using ß2 microglobulin as the housekeeping gene. Gonadotropin treatment, whether it be PMSG or hCG, reduced the levels of FGF9 mRNA by approximately 30% in the rat (Fig. 6Go), whereas combined PMSG/hCG treatment reduced FGF9 mRNA expression by approximately 60% (Fig. 6Go).


Figure 5
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  		FIG. 5. Levels of expression of FGF9 mRNA in immature rat ovaries with or without (control) DES treatment for 24 or 96 h, normalized for GAPDH mRNA expression. The number of pools (n) analyzed separately at each age is indicated below each histogram. The data are mean ± SD. There were no statistically significant differences between the groups.

 

Figure 6
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  		FIG. 6. Levels of expression of FGF9 mRNA in immature rat ovaries after PMSG (48 h), hCG (8 h), or combined PMSG/hCG (P/h) (48 plus 8 h) treatment, normalized for ß2 microglobulin mRNA expression. The number of pools (n) analyzed separately at each age is indicated below each histogram. The data are mean ± SD, with different letters denoting statistical significance (P < 0.05).

 
Impact of hormone treatment on FGF9 protein localization
FGF9 protein localization was investigated in ovaries containing follicles at defined stages of follicle growth and differentiation. The administration of DES to immature rats stimulates the proliferation of undifferentiated granulosa cells and the formation of large preantral follicles in the ovary. The ovaries of DES-treated rats had more intense staining for FGF9 protein in the theca, stromal cells, and basement membranes relative to the untreated 25-d-old control ovaries (Fig. 7Go). FGF9 protein localized to theca interna and externa after DES treatment, whereas untreated ovary only localized protein to theca externa. The administration of PMSG to immature rats stimulates follicle growth and the development of preovulatory follicles in the ovary. When followed by an injection of hCG, ovulation and luteinization are induced. PMSG treatment reduced the intensity of FGF9 protein staining in the ovary with only the theca externa showing immunolocalization. There was virtually no basement membrane localization of FGF9 in PMSG-treated ovaries compared with the untreated control ovaries (Fig. 7Go). The FGF9 staining pattern was similar in hCG- and PMSG/hCG treated-ovaries with theca cells, stromal cells, and basement membrane localizing FGF9 protein (Fig. 7Go). Sections incubated with preabsorbed antisera or in the absence of primary antibody did not localize protein (Fig. 7Go, A and B).

Effect of FGF9 on progesterone production by isolated granulosa cells
FGF9 dose dependently enhanced progesterone production beyond that elicited by FSH (100 ng/ml) alone (Fig. 8Go), with significance achieved at doses of FGF9 of at least 10 ng/ml. In the presence of FSH (100 ng/ml), FGF9 dose dependently and synergistically increased progesterone production by granulosa cells in culture, beyond that elicited by FGF9 alone (Fig. 8Go). To evaluate the impact that LH may have on FGF9-stimulated progesterone production, granulosa cells were pretreated with FSH to induce LH receptors. LH stimulated progesterone production to a significantly greater degree than FSH or FGF9 alone (Fig. 9Go). In fact, FSH pretreatment reduced the capacity of the cells to respond to FGF9 stimulation with 25 ng/ml FGF9, eliciting 25 ng/ml progesterone in the absence of pretreatment and only 7 ng/ml after FSH pretreatment (Figs. 8Go and 9Go). The addition of FGF9 to FSH-stimulated granulosa cells significantly enhanced progesterone production beyond that with either FGF9 or FSH alone, whereas there was no effect of FGF9 on LH-stimulated progesterone production beyond that elicited by LH alone. The addition of FSH and FGF9 to LH-stimulated granulosa cells enhanced progesterone production beyond that elicited by LH alone but did not exceed the levels stimulated by either LH/FSH or FSH/FGF9 (Fig. 9Go).


Figure 8
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  		FIG. 8. Progesterone production by rat granulosa cells cultured with FGF9, FSH, and combined FGF9 plus FSH for 48 h. Each treatment was performed in triplicate. The results are representative of at least three separate experiments. The data are mean ± SD, with different letters denoting statistical significance (P < 0.05). C, Untreated control group.

 

Figure 9
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  		FIG. 9. Progesterone production by rat granulosa cells cultured with FGF9 (25 ng/ml), FSH (100 ng/ml), LH (100 ng/ml), or the combined treatments of FGF9 plus FSH, FGF9 plus LH, or FGF9 plus FSH and LH for 48 h. The cells were pretreated for 24 h with FSH (100 ng/ml) before the addition of treatments to induce LH receptors. Each treatment was performed in quadruplicate. The results are representative of at least three separate experiments. The data are mean ± SD, with different letters denoting statistical significance (P < 0.05).

 
Effect of FGF9 on the expression of StAR, SCC, and 3ß-HSD mRNAs by cultured granulosa cells
There are three proteins in the steroidogenic pathway leading to progesterone production that maybe regulated. The first in the pathway is StAR, which facilitates the transport of cholesterol to the inner mitochondrial membrane. Next is SCC, which converts cholesterol to pregnenolone and finally 3ß-HSD, which converts pregnenolone to progesterone. We investigated the mRNA expression for all three genes encoding these proteins in granulosa cells in response to FGF9. FSH enhanced the expression of StAR, SCC, and 3ß-HSD mRNAs by cultured granulosa cells (Fig. 10Go). FGF9 at 0.1 ng/ml had no significant effect on expression, but, at 50 ng/ml, FGF9 stimulated StAR mRNA expression by granulosa cells 3-fold beyond that elicited by FSH alone. In the presence of both FGF9 and FSH, StAR mRNA expression by granulosa cells was significantly enhanced. FGF9 (50 ng/ml) stimulated SCC mRNA expression to the same degree as FSH, and, when FSH and FGF9 were added together, the expression of SCC by granulosa cells was enhanced (Fig. 10Go). FGF9 at 0.1 and 50 ng/ml had no effect on 3ß-HSD mRNA expression by granulosa cells. In the presence of FSH, FGF9 at its highest dose (50 ng/ml) significantly inhibited 3ß-HSD mRNA expression by granulosa cells (Fig. 10Go).


Figure 10
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  		FIG. 10. Levels of expression of StAR, SCC, and 3ß-HSD mRNAs in granulosa cells cultured with FSH (100 ng/ml), FGF9 (0.1 or 50 ng/ml), and combined FSH/FGF9 treatments for 16 h. Each treatment was performed in quadruplicate. The results are representative of at least three separate experiments. The data are mean ± SD, with different letters denoting statistical significance (P < 0.05). C, Untreated control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes, for the first time, the expression of FGF9 mRNA and localization of protein by postnatal, immature, and adult rat ovaries and provides evidence for a functional role of FGF9 in ovarian progesterone production. The localization of FGF9 protein to theca, stroma, and basement membranes surrounding granulosa and theca cells and FGFR3 protein to granulosa cells, theca cells, and oocytes, is consistent with a paracrine regulatory role of FGF9 within the ovarian follicle.

The levels of FGF9 mRNA changed during prepubertal follicular development, being highest 4 d after birth, when the only defined follicular structures in the ovary are primordial and a few primary follicles and stabilizing at d 12, when a range of follicle types, including antral follicles, are present (57). This decline in FGF9 mRNA may be attributed to real changes in FGF9 mRNA in a given cell type over time, or, alternatively, it may reflect a dilution of the mRNA as a result of developmental changes in the ovarian cell composition and mass. Protein localization data suggest that theca/stromal cell precursors express FGF9 in d 4 ovaries. Between d 8 and 12, theca cells differentiate from the interstitial stromal cells and are recruited to preantral follicles in which they express FGF9 protein. During this time, granulosa cells proliferate, antral follicles form, and the ovary increases in size, which would be consistent with a decline in the FGF9 mRNA attributable to dilution. In adult rats, some but not all corpora lutea of adult ovaries localized FGF9 protein. Both theca and granulosa cells differentiate into luteal cells during formation of the corpus luteum, consistent with luteinized theca cells producing FGF9.

The selective expression of FGF9 protein by corpora lutea may be explained by the heterogeneity of corpora lutea present in the ovary at any time. Both newly formed and preexisting corpora lutea from earlier cycles reside in the ovary simultaneously. At any time, four to five different cycles worth of corpora lutea may be present in the ovary (58), many at varying stages of luteolysis. The responsiveness of these corpora lutea has been shown to vary with the stage of the estrous cycle. For example, studies on the luteolytic action of prolactin indicate that regressing corpora lutea were responsive to the proapoptotic action of prolactin during the transition between estrus and proestrus and were refractory at all other times (59). FGF9 produced by luteal cells might activate autocrine production of progesterone by corpora lutea during regression. This would be consistent with the demonstrated antiapoptotic action of progesterone on large granulosa cells (23, 60).

FGF9 was significantly more potent than FSH in stimulating progesterone production, with 10 ng/ml FGF9 eliciting 7-fold greater levels of progesterone than a maximally stimulating dose of FSH. Another member of the FGF family, FGF2, has also been shown to stimulate granulosa cell progesterone production (61, 62). The studies reported herein indicate that FGF9 stimulates progesterone production by enhancing the expression of StAR and SCC. StAR facilitates the transport of cholesterol to the inner mitochondrial membrane in which it is converted to pregnenolone by SCC. Thus, all of the substrates required for progesterone production are effectively increased by FGF9. Our studies with granulosa cells differentiated in vitro to express LH receptor indicated that FGF9 was not as effective at stimulating progesterone production as LH, nor did it enhance LH-stimulated production in this system. Nevertheless, the combined treatment of FSH/FGF9 was as effective at stimulating progesterone production as FSH/LH, supporting a role for FGF9 in luteinization.

It was of interest to note that FSH pretreatment of granulosa cells led to a reduced capacity of FGF9 to stimulate progesterone production. There are two possible explanations for this observation. First, FSH might down-regulate FGFR expression, thereby reducing the effectiveness of FGF9. FSH has been reported to influence the expression of FGFR by which FGF2 transmits signals in rat granulosa cells (63). Second, the functional status of the granulosa cells at the time they receive FGF9 treatment is different in the two models. In vitro, FSH induces functional luteinization of the granulosa cell as evidenced by their ability to produce progesterone. Thus, with an FSH pretreatment period, the granulosa cells are "luteinized" before they see FGF9, and this may impact their ability to respond to FGF9 stimulation.

We investigated how follicular differentiation might impact on FGF9 mRNA expression and protein localization in the ovary using hormone-treated rat models. DES treatment to induce large preantral follicles had no significant impact on FGF9 mRNA expression. However, protein localization studies suggested that more protein was present in the theca, particularly in the theca interna, after DES treatment. Estradiol has been shown to stimulate FGF9 production by endometrial stromal cells (56). Because estrogen receptor {alpha} is the predominant estrogen receptor subtype in the uterus (64, 65, 66) and theca cells (67), it is reasonable to suggest that the stimulatory action of estradiol on FGF9 production might be mediated via this receptor. This hypothesis is consistent with the data we collected for FGF9 protein. The absence of a change in FGF9 mRNA may simply be attributable to the manner in which the expression studies were undertaken. Whole ovary from DES-treated rats was used. DES stimulates granulosa cell proliferation (52), thereby increasing ovarian size, so that the contribution of theca to the total cellular content of the ovary is reduced. Thus, changes in mRNAs contributed by the theca may be diluted in this model. Collection of theca by laser capture microdissection and subsequent RT-PCR studies will resolve this issue. Gonadotropin-induced follicular differentiation led to a reduction in FGF9 mRNA expression by immature rat ovaries, which was exacerbated when the early events associated with ovulation and luteinization were triggered by the combination of PMSG/hCG. This indicates that mRNA produced by theca/stromal cells is under gonadotropic hormonal regulation, and, given that PMSG equates primarily with FSH, whose receptors are present on granulosa cells, it would appear that both direct and indirect regulation are involved.

FGFR2–FGFR4 are thought to be capable of transducing FGF9 signals (46, 47, 48, 49). In these studies, we have shown that FGFR2 and FGFR3 mRNAs are expressed by rat ovary as early as 4 d of age and that the ovarian levels do not change significantly during the period in which folliculogenesis is established. Previously, mRNA for FGFR3 has not been detected in rat ovary by either in situ hybridization or Northern blot analyses (13, 41), suggesting that levels of the receptor must be low in the ovary, if present at all. Despite these findings, we were also able to localize FGFR3 protein to granulosa and theca cells and oocytes of the rat ovary by immunohistochemistry. The nuclear localization of the FGFR3 staining pattern, although surprising for a membrane-bound receptor, is not without precedent. FGF9 has been shown to induce nuclear localization of FGFR2 in differentiating Sertoli cells (6). Furthermore, FGF ligand-receptor complexes have been shown to be internalized and subsequently localized to intracellular and nuclear compartments of various cells (7, 8, 68, 69, 70, 71), in which they are thought to mediate biological responses. The localization of FGFR3 to granulosa cells is consistent with a paracrine regulatory role of FGF9, in this case mediating progesterone production by these cells. FGFR2 mRNA has been shown to be expressed by granulosa cells and theca/interstitial cells of the rat ovary (13), and we now report the localization of FGFR2 protein to the cytoplasm of granulosa cells, theca cells, oocytes, and luteal cells. The proximity of FGFR2 to sites of FGF9 protein localization may result in activation of this receptor pathway. To date, FGFR4 mRNA and protein have been identified only in human and bovine ovary (19, 20). The localization of FGF9 protein to ovarian basement membranes is consistent with the affinity of FGF family members for heparin and heparin sulfate proteoglycans (low-affinity FGFR) (9, 10, 11, 12) found on these surfaces and in the associated extracellular matrix.

In summary, we have shown that mRNA and protein for FGF9 and the receptors FGFR2 and FGFR3 relevant to the transduction of signals by FGF9 are present in the rat ovary. The data support a role for FGF9 in the regulation of ovarian function and in particular in steroid production and confirm that FGF9 has roles to play outside of male sex determination and differentiation.


   Acknowledgments
 
We thank Drs. Sarah Meachem and Pavel Sluka for providing testis sections, Dr. Morag Young for whole testes, and Ileana Kuyznierewicz for her assistance with granulosa cells cultures and progesterone assays. Sue Panckridge is thanked for her assistance in the preparation of the figures.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia (Regkeys 241000 and 198705).

Disclosure Statement: A.E.D., M.T., M.D., and J.K.F. have nothing to declare.

First Published Online May 10, 2007

Abbreviations: DES, Diethylstilboestrol; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; PMSG, pregnant mare serum gonadotropin; SCC, P450 side chain cleavage; StAR, steroidogenic acute regulatory protein.

Received December 12, 2006.

Accepted for publication May 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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