Induction and maintenance of oestradiol and immunoreactive inhibin production with FSH by ovine granulosa cells cultured in serum-free media

Abstract

A serum-free ovine granulosa cell culture system is described that allows the induction of FSH-responsive oestradiol production by undifferentiated cells from small (< 3.5 mm) follicles (P < 0.001) and the maintenance of oestradiol production by differentiated cells from large (≥ 3.5 mm) follicles. Physiological doses of FSH stimulated (P < 0.01) proliferation of cultured granulosa cells from both small and large follicles. The synthesis of immunoreactive inhibin and progesterone by granulosa cells from small and large follicles increased (P < 0.01) with time of culture, and was not dependent on FSH. Inhibin secretion expressed on a per cell basis was not FSH responsive. Insulin and insulin-like growth factor I (IGF-I), in the presence of FSH, stimulated (P < 0.001) cell proliferation and oestradiol and inhibin production by granulosa cells from small and large follicles. There was a significant (P < 0.001) interaction between insulin and IGF-I in the stimulation of granulosa cell proliferation and differentiation. Both epidermal growth factor (EGF) and transforming growth factor α (TGF-α) in the presence of FSH stimulated cellular proliferation (P < 0.001) in a dose-responsive manner and concomitantly inhibited (P < 0.001) oestradiol and inhibin secretion. The development of this granulosa cell culture system will make it possible to study, in vitro, the cascade of events that controls granulosa cell differentiation and ultimately follicle selection in sheep.