Insulin as an Amplifier of Gonadotropin Action on Steroid Production: Mechanisms and Sites of Action in Goldfish Prematurational Full-Grown Ovarian Follicles

The present study examines the effects of mammalian insulin and insulin-like growth factor-I (IGF-I) on steroid production by goldfish vitellogenic and prematurational full-grown ovarian follicles in vitro. Insulin (0.4-40 micrograms/ml) had no effect on its own, but potentiated human chorionic gonadotropin (hCG) and 25-hydroxycholesterol-stimulated testosterone production by prematurational full-grown follicles in both a dose- and time-dependent manner. By comparison, in vitellogenic follicles insulin (4 micrograms/ml) had no effect on hCG- or 25-hydroxycholesterol-stimulated testosterone and 17 beta-estradiol production. IGF-I (11-100 ng/ml) neither affected basal nor hCG- and 25-hydroxycholesterol-stimulated testosterone production by vitellogenic or prematurational full-grown follicles. Studies to determine the site(s) of action of insulin showed that insulin had no effect on basal cyclic adenosine 3',5'-monophosphate (cAMP) generation, but it enhanced hCG-stimulated cAMP and testosterone production by prematurational full-grown follicles. Insulin also enhanced conversion of 25-hydroxycholesterol to testosterone, but had no effect on the metabolism of pregnenolone, suggesting that insulin either enhances mobilization of cholesterol to the mitochondria or increases the activity of the cytochrome P450 side-chain cleavage enzyme. The ability of insulin to enhance hCG and/or 25-hydroxycholesterol-stimulated testosterone production was blocked by the addition of the protein kinase A (PKA) inhibitor H89, which confirmed that these actions of insulin are exerted, at least in part, by the cAMP/PKA pathway. These findings suggest that insulin, but not IGF-I, is capable of participating in the regulation of ovarian steroid biosynthesis through effects at multiple steps within the steroid cascade and that ovarian responsiveness to insulin changes during the course of follicular development in the goldfish. Together, these results provide new insights into the biological action of insulin in the fish ovary.