Mechanisms subserving insulin's differentiating actions on progestin biosynthesis by ovarian cells: studies with cultured swine granulosa cells.

To characterize the nature of insulin action on ovarian cells, an in vitro system of swine granulosa cells was developed in which 3- to 50-fold stimulation of progesterone production was observed in response to insulin under serum-free or sparsely (1%) serum-supplemented conditions. These studies demonstrate that optimal cell density is critical for the full expression of insulin action, and that the dose-dependent responses of granulosa cells to insulin are also significantly influenced by the maturational status of the parent follicle. The striking (greater than 30-fold) responses of medium-sized and healthy preovulatory follicles to insulin were not attributable to the presence or absence of atresia or to a selective inhibition of progesterone's catabolism to 20 alpha-dihydroprogesterone, since 20 alpha-dihydroprogesterone production was also markedly increased in response to insulin. The mechanisms subserving insulin action were explored further by testing the capacity of insulin to 1) increase progesterone accumulation in response to exogenously provided pregnenolone, and 2) stimulate pregnenolone biosynthesis in the presence of trilostane, an inhibitor of pregnenolone metabolism. The effects of insulin were to increase the biosynthesis of progesterone from available pregnenolone and increase the production of pregnenolone from endogenous sterol substrate. The physiological relevance of these differentiative actions of insulin is suggested by insulin's ability to significantly enhance the stimulatory effects of FSH, LH, epinephrine, prostaglandin E2, and cAMP effectors, cholera toxin and 8-bromo-cAMP. In summary, the present studies delineate culture conditions in which swine granulosa cells exhibit a high degree of responsiveness to the stimulatory actions of insulin on progestin biosynthesis. The effects of insulin are critically influenced by cell density, stage of follicle maturation, and the presence or absence of classical ovarian effector hormones with which insulin can interact synergistically. Moreover, our studies indicate that insulin exerts significant actions at several levels of progestin biosynthesis, including the production of pregnenolone, progesterone, and 20 alpha-dihydroprogesterone by granulosa cells. In view of the high concentrations of insulin-like growth factors and somatomedins attained in porcine Graafian follicles, we suggest that trophic actions of insulin or insulin-like peptides and the synergism of insulin with classical ovarian effector hormones are likely to be of physiological importance to the differentiation of granulosa cells in the developing ovarian follicle.