Trophic actions of human somatomedin c/insulin-like growth factor i on ovarian cells: In vitro studies with swine granulosa cells

We have investigated the responsiveness of swine granulosa cells to somatomedin C/insulin-like growth factor I (IGF I) under serum-free conditions in vitro. Somatomedin C/IGFI (<80% pure) exerted dose-dependent stimulatory effects on the production of progesterone and its reduced metabolite, 20α-hydroxypregn-4-en-3-one. A 50-fold stimulation of progesterone production could be observed after 48-96 h of hormone treatment. These effects were not mimicked by platelet-xderived growth factor, epidermal growth factor, fibroblast growth factor, desoctapeptide insulin, or porcine relaxin. The stimulatory action of somatomedin C/IGF I was not attributable to changes in cell protein or DNA content or cell number and reflected enhanced steroidogenesis per se rather than simply release of stored progesterone. In addition, augmented progesterone production was accompanied by increased biosynthesis of its precursor, pregnenolone, measured in the presence of trilostane to block 3/3-hydroxysteroid dehydrogenase activity. Moreover, somatomedin C/IGF I enhanced progesterone biosynthesis in response to a maximally effective concentration of the soluble sterol substrate for side-chain cleavage, 25-hydroxycholesterol, suggesting that somatomedin C increases the effective activity of the mitochondrial cholesterol side-chain cleavage system. The stimulatory effects of somatomedin C/IGF I were inhibited by cycloheximide and actinomycin D, indicating that protein and RNA synthesis are required for the full expression of somatomedin C′s differentiative effects. The physiological importance of such trophic actions was suggested by the capacity of nanomolar concentrations of somatomedin C/IGF I to augment progesterone production further in the presence of maximally stimulating concentrations of β-bromo-cAMP or estradiol. In addition, equilibrium competition curves and saturation analysis of [¹²⁵I]somatomedin C/IGF I binding revealed high affinity [dissociation constant (K_d) - 0.69 nM], low capacity (0.57 pmol somatomedin C bound/mg DNA) receptor sites on swine granulosa cells. [¹²⁵I]Somatomedin C/IGF I binding was highly specific in that multiplication stimulating activity, porcine insulin, desoctapeptide insulin, and insulin-receptor antisera competed only sparingly for these binding sites. In summary, human somatomedin C/IGF I exerts potent and specific differentiative effects on swine granulosa cells cultured under serum-free conditions in vitro. Somatomedin C/IGF I increases the biosynthesis of progesterone and pregnenolone, probably by stimulating the cholesterol side-chain cleavage reaction, rather than by impeding progesterone′s catabolism. Moreover, somatomedin C/IGF I enhances the stimulatory effects of β-bromo-cAMP and estradiol. The stimulatory actions of this peptide are dependent upon protein and RNA synthesis and are presumably mediated via high affinity, specific, low capacity binding sites for somatomedin CI/GF I demonstrable on swine granulosa cells. We conclude that ovarian cells must be considered targets for trophic actions of somatomedin C/IGF I.