Multihormone regulation of steroidogenesis in cultured porcine granulosa cells: studies in serum-free medium.

FSH, LH, and estradiol are known to modulate ovarian follicular differentiation. However, the cellular site of action and relative importance of the three hormones have remained uncertain. The recent development of a serum-free system for the culture of immature porcine granulosa cells has enabled us to reinvestigate these issues with better control of pituitary peptides and gonadal steroids. Progesterone production in response to FSH was higher in cells cultured in serum-free complete medium than in those grown in the presence of 10% fetal calf serum [10-fold vs. 1.5-2 fold (control)]. Ovine LH alone was also able to stimulate progesterone production in serum-free free complete medium (6-fold); this effect could not be accounted for by FSH contamination. The LH stimulation, however, was enhanced by FSH. Insulin was required for both FSH and LH stimulation of progesterone production. Estradiol stimulated progesterone production per se (2- to 3-fold) and also enhanced FSH and LH actions. The estimated ED50 for estradiol in FSH-treated cells was 20 ng/ml. Maximal levels of progesterone after 6 days were observed when the combination of FSH, LH, and estradiol was present from the onset of the culture. Incubations carried out in the presence of 5-cholesten-3 beta-25-diol indicated that the hormonal interactions take place, at least in part, at the level of the side-chain cleavage enzyme. These results indicate that FSH is the most important hormonal stimulus for progesterone synthesis in immature granulosa cells. However, LH, estradiol, and insulin (or insulin-like growth factors) exert direct actions on the granulosa cell that may be required for the development of optimal steroidogenic potential.