The hypothesis that GH may affect gonadal function by increasing local levels of the GH-dependent somatomedin C/insulin-like growth factor I (IGF I) was tested. Ovine GH (200 micrograms) was injected into immature, hypophysectomized, estrogen-treated female rats; animals were sacrificed 8 or 12 h later. Renal and ovarian homogenates were acid extracted and chromatographed over Sephadex G-50. Eight h after GH injection, 3.6 to 6.4-fold increases in immunoreactive IGF I (IR-IGF I) levels were observed in either ovarian or renal extracts subjected to acid chromatography. Twelve h after GH treatment, IR-IGF I levels remained elevated, but were lower than after 8 h. In neither case could IR-IGF I levels be accounted for by serum contamination. IR-IGF I eluted with an apparent mol wt near that of synthetic human IGF I in both kidney and ovary. Thus, GH can directly increase ovarian and renal tissue IR-IGF I levels in vivo. Taken with previous observations showing a direct gonadotropin-enhancing effect of IGF I on rat granulosa cells in vitro, our results support the hypothesis that GH may affect ovarian differentiation by inducing the local production or accumulation of IGF I, providing evidence for a novel intraovarian paracrine control mechanism.

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.

Growth hormone (GH) deficiency in rats is associated with decreased ovarian steroidal responsiveness to gonadotropins, possibly through a reduction in the production of the GH-dependent Somatomedin C/insulinlike growth factor I (SM C/IGF I). We have investigated the direct effects of synthetic SM C/IGF I on gonadotropin-stimulated ovarian steroidogenesis in vitro. Granulosa cells were cultured in a serum-free medium for 48 h in the presence of follicle-stimulating hormone (FSH), with or without SM C/IGF I. FSH dose-dependently increased both estrogen and progestin production. Concomitant treatment with SM C/IGF I led to a dose-dependent augmentation of progestin secretion over the full range of FSH doses tested, by a maximum of 2.3- to 2.6-fold. FSH-stimulated estrogen was enhanced by up to 2.4-fold but only at low doses of FSH. SM C/IGF I-enhanced progestin production was associated with increased pregnenolone production and 3 beta-hydroxysteroid dehydrogenase activity, whereas augmented estrogen production appeared to be due to enhanced aromatase activity. The actions of SM C/IGF I, at physiologically relevant concentrations were correlated with increased extracellular cAMP accumulation and cellular protein content but were independent of any change in cell number or viability. In contrast to SM C/IGF I, the closely related peptide multiplication-stimulating activity decreased estrogen production while increasing progestin metabolite accumulation. The present results indicate that the GH-dependent peptide SM C/IGF I may play a role in ovarian development by enhancing gonadotropin-stimulated granulosa cell steroidogenesis.

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