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

0013-7227/07/$15.00/0 Printed in U.S.A. Endocrinology 148(8):3711–3721 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1668 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 T HE 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 (3–5). The transmembrane localization of these receptors does not preclude their nuclear localization, with FGF ligand and receptor complexes previously identified in the nucleus (6 – 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 –12). In the rat ovary, FGFR1 and FGFR2 have been localized to granulosa cells and theca cells (13), with FGFR1 also First Published Online May 10, 2007 Abbreviations: DES, Diethylstilboestrol; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; 3␤HSD, 3␤-hydroxysteroid dehydrogenase; PMSG, pregnant mare serum gonadotropin; SCC, P450 side chain cleavage; StAR, steroidogenic acute regulatory protein. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. nadotropin 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. (Endocrinology 148: 3711–3721, 2007) 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, 21–25). Both FGF1 and FGF2 have been shown to stimulate granulosa cell, germinal epithelial cell, theca cell, and luteal cell proliferation (26 –30). FGF2 has also been shown to exert a variety of effects on granulosa cell function, notably, steroidogenesis (31, 32) and apoptosis (33–35). 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 (androgeninduced 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) 3711 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 go- 3712 Endocrinology, August 2007, 148(8):3711–3721 Materials and Methods 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 E (...truncated)