Expression of Fibroblast Growth Factors 17 and 18 (Fgf-17 And Fgf-18) in Bovine Antral Follicles.

Abstract

Fibroblast growth factors (FGF) have been recognised as important local regulators of follicle development. FGF-17 and FGF-18 belong to the FGF-8 subfamily and exhibit similar amino acid sequences and receptor affinity. They induce cell proliferation and differentiation through activation of FGFR-3c and FGFR-4, recently detected (mRNA) in granulosa (GC) and theca cells (TC) of bovine antral follicles. The first aim of this study was to determine the spacial and temporal patterns of FGF-17 and FGF-18 expression in bovine antral follicles. As FGF-17 mRNA was found to be altered by follicle health status, a granulosa cell culture system was used to test the effect of FSH on GC FGF-17 expression. Antral follicles greater than 5mm in diameter were dissected from abattoir ovaries, GC and TC separated and total RNA extracted. Estradiol (E) and progesterone (P) concentrations of the follicular fluid were measured by RIA and follicles were grouped according to E:P ratios of <0.01 (highly atretic), 0.01-1 (transitional) and >1 (healthy). Cumulus oocyte complexes were aspirated from antral follicles, cumulus cells were removed and total RNA extracted from pools of 50 oocytes. To investigate regulation of FGF-17 mRNA by FSH, GC from small follicles (2-5 mm) were placed into serum-free medium supplemented with insulin and bovine FSH (0, 0.1, 1, 10 or 100 ng/ml). Expression of FGF-17 and FGF-18 mRNA was investigated by real time RT-PCR using bovine-specific primers (TaqMan Assay by Design, ABI) and GAPDH as the endogenous control. Relative expression was determined by the Pfaffl equation. FGF-17 mRNA was detected in pools of oocytes, and in TC and GC, where expression was higher in highly atretic follicles compared with transitional and healthy follicles. FGF-17 protein was localized to oocytes, GC and TC by immunohistochemistry. FSH inhibited FGF-17 mRNA expression in cultured GC in a dose-dependent manner. FGF-18 mRNA was detected in GC and TC, where it did not change with follicle status, but was absent in the oocyte. In conclusion, the present data suggest a role for FGF-17 in the control of folliculogenesis. FGF-17 expression is low in growing follicles and is inhibited by FSH, which indicates a role in the control of follicle atresia. Supported by FAPESP.