Effects of fibroblast growth factor 9 on steroidogenesis and control of FGFR2IIIc mRNA in porcine granulosa cells.

摘要

The objectives of this study were to investigate the effects of fibroblast growth factor 9 (FGF9) on hormone-stimulated porcine granulosa cell proliferation and steroid production and to further elucidate the hormonal and developmental control of FGFR2IIIc gene expression in granulosa cells. Porcine ovaries were collected from a local slaughterhouse and granulosa cells were collected from small to medium (1 to 5 mm) follicles for 5 in vitro studies that were conducted. Cells were cultured for 48 h in 5% fetal calf serum plus 5% porcine serum and then treated with various combinations of FSH, IGF-I, FGF9, Sonic hedgehog (SHH), cortisol, PGE₂, and/or wingless-type mouse mammary tumor virus integration site family member 5A (WNT5A) in serum-free medium for an additional 24 or 48 h. Medium was collected for analysis of steroid concentration via RIA, or RNA was collected for gene expression analysis of FGFR2IIIc via quantitative reverse transcription PCR. Fibroblast growth factor 9 stimulated (P<0.05) IGF-I-induced estradiol production in the presence of FSH and testosterone. However, FGF9 had inconsistent effects on progesterone production, stimulating progesterone production in the presence of FSH and testosterone but inhibiting progesterone production in the presence of IGF-I, FSH, and testosterone. Cell numbers were increased (P<0.05) by FGF9 in the presence of IGF-I and FSH but not in the presence of FSH and absence of IGF-I. For FGFR2IIIc mRNA studies, granulosa cells were treated with FSH, IGF-I, FGF9, SHH, cortisol, PGE₂, or WNT5A. Follicle-stimulating hormone alone had no effect (P>0.10) whereas IGF-I increased (P<0.05) FGFR2IIIc mRNA abundance. Cortisol, PGE₂, SHH, and WNT5A had no effect (P>0.10) on FGFR2IIIc gene expression whereas FGF9 in the presence of FSH and IGF-I inhibited (P<0.05) FGFR2IIIc gene expression. In an in vivo study, granulosa cells from large (7 to 14 mm) follicles had greater (P<0.05) abundance of FGFR2IIIc mRNA than small (1 to 3 mm) or medium (4 to 6 mm) follicles. In conclusion, IGF-I-induced FGFR2IIIc mRNA may be a mechanism for increased responses to FGF9 in FSH plus IGF-I-treated granulosa cells. Fibroblast growth factor 9 and IGF-I may work together as amplifiers of follicular growth and granulosa cell differentiation by stimulating estradiol production and concomitantly stimulating granulosa cell growth in pigs.