Effect of a partially purified 30.1 kDa ovine follicular fluid protein on ovine follicle and ovarian somatic cell growth, and oocyte maturation in vitro

Aim: Regulation of folliculogenesis and oocyte-somatic cell interactions in the ovarian follicles is under the control of gonadotrophins and various local factors. In the present study, an attempt was made to isolate and examine the biological activities of ovarian follicular fluid protein(s) in sheep in vitro. Methods: Follicular fluids aspirated from ovarian follicles of slaughterhouse-derived ovaries were made cell free by centrifugation (5000 g for 30 min) and steroid free by charcoal treatment. The follicular fluid was then subjected to ammonium sulphate precipitation and gel filtration chromatography using G-75 Sephadex. Protein detection was performed using a UV spectrophotometer at 280 nm. The 35-50% fraction yielded a detectable peak and a protein of 30.1 kDa as examined by SDS-PAGE. The effect of increasing doses of the 30.1 kDa ovine follicular fluid protein (oFFP) was tested at different doses on pre-antral and antral follicle growth; cumulus cell expansion; oocyte maturation; changes in protein, calcium and phosphorus levels of oocytes after culture in media containing different levels of isolated protein; mural granulosa cell, polar granulosa cell (cumulus cell), oviductal epithelial cell monolayer formation and granulosa cell proliferation in vitro. Results: The oFFP significantly inhibited antral follicle growth, cumulus expansion, oocyte maturation and somatic cell growth in vitro in a dose-dependent manner. The oFFP did not have a significant effect on the pre-antral follicle growth in vitro. The protein, calcium and phosphorus contents of oocytes were found to decrease in oocytes cultured in maturation medium containing the oFFP. Conclusion: The present study demonstrates a follicular fluid factor regulating folliculogenesis and oocyte maturation in sheep.