Specific insulin-like growth factor (IGF) I- and II-binding sites on rat granulosa cells: relation to IGF action.

Although insulin-like growth factor-I (IGF-I) and insulin have been shown to augment rat granulosa cell differentiation, their mechanism(s) of action has not yet been elucidated. In the present study, we have examined granulosa cells obtained from immature hypophysectomized estrogen-treated rats for specific IGF-binding sites that might mediate the effects of the insulin-like peptides. Using synthetic [125I]iodo-IGF-I, we have found specific high affinity, low capacity (Kd = 1.36 +/- 0.131 nM; 3250 +/- 662 sites/cell) IGF-I-binding sites that have lower affinities for the related peptides IGF-II and insulin (potency ratio, 1:9:700 for IGF-I, IGF-II, and insulin). We have also found specific binding sites for [125I]iodo-IGF-II, a newly available synthetic peptide. The IGF-II-preferring sites were of a single class (Kd = 1.54 +/- 0.32 nM; 4728 sites/cell) and exhibited a rank competition order of IGF-II greater than IGF-I much greater than insulin. To study the functional correlates of these binding activities, granulosa cells were cultured for 2 days in serum-free medium in the presence of FSH, with or without increasing concentrations of IGF-I, IGF-II, or insulin. Medium steroids were then determined by specific RIA, and cellular LH/hCG receptors were measured by specific [125I]iodo-hCG binding. Treatment with FSH increased estrogen and progestin production and induced the formation of LH/hCG receptors. Concomitant treatment with the three peptides dose-dependently enhanced both FSH-stimulated steroidogenesis and LH/hCG receptor induction, with a rank order of potency of IGF-I greater than IGF-II greater than insulin (potency ratio, 1:8:36). This rank order of potency of the insulin-like peptides was more closely correlated with their ability to compete for IGF-I binding rather than IGF-II binding, suggesting the preferential involvement of IGF-I receptors in the ovarian actions of the IGFs, although the involvement of IGF-II and insulin receptors cannot be dismissed. Our results demonstrate, for the first time, a biological action of synthetic IGF-II in granulosa cells and further show a novel insulin effect, enhancement of LH/hCG receptor induction. These findings also indicate that rat granulosa cells possess specific IGF-I and IGF-II-binding sites that may mediate the gonadotropin-enhancing actions of the insulin-like peptides. Since IGF-I appears to be the most biologically potent peptide, it is likely to be the most important insulin-like peptide involved in granulosa cell differentiation in vivo.