Insulin-Like Growth Factor I and Insulin Potentiate Luteinizing Hormone-Induced Androgen Synthesis by Rat Ovarian Thecal-Interstitial Cells*

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Abstract
We tested the hypothesis that insulin-like growth factor I (IGF-I) and insulin play a role in androgen production by rat ovarian thecal-interstitial cells. Collagenase/DNase-dispersed rat ovarian thecal-interstitial cells obtained from immature hypophysectomized Sprague-Dawley rats were cultured at a concentration of 106 cells/ml in serum-free medium in the presence of increasing concentrations of LH IGF-I, or insulin. The medium was replaced every 48 h, and the androsterone concentration in the cultures supernatants was used as an index of androgen production. In the absence of added hormones (control) androsterone levels were consistently less than 0.1 ng/ml. Increasing concentrations of LH stimulated androsterone synthesis in a dose-dependent manner. IGF-I, in the absence of LH, did not significantly increase androsterone levels above control values. However, when combined with 10 ng/ml LH, IGF-I increased androsterone synthesis above levels seen with LH alone in a dose-related fashion: for example, the peak androsterone levels seen with LH and 100 ng/ml (13 nM) IGF-I at 96 h of culture were significantly greater than the peak level seen with 10 ng/ml LH alone (302 .+-. 71 vs. 17 .+-. 7 ng/ml; P < 0.0125). Similarly, while insulin alone did not increase androsterone synthesis above control values, androsterone concentrations were increased by insulin in combination with 10 ng/ml LH; a peak value of 240 .+-. 67.7 ng/ml was observed at 96 h of culture with 100 ng/ml (18 nM) insulin (P < 0.025 vs. LH alone) Androsterone levels were slightly less with insulin than with IGF-I, but this difference was not significant. The combination of IGF-I and insulin did not increase levels of androsterone synthesis above those observed with each hormone alone. IGF-I bound to a high affinity binding site on ovarian cell monolayer cultures with an apparent binding affinity of 1.3 .times. 10-9 M. Insulin also competed for binding with radiolabeled IGF-I in a dose-dependent manner, but the affinity of insulin was approximately 500-fold less; half-maximal inhibition [125I]IGF-I binding occurred with an insulin concentration of approximately 300 nM (or .apprx. 1700 ng/ml). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thecal-interstitial cell monolayers affinity labeled with radiolabeled IGF-I in the absence and presence of unlabeled hormone revealed proteins with characteristics of type I IGF receptors. Affinity labeling to a protein of a relative molecular mass of approximately 45,000 was also noted, probably representing IGF carrier proteins synthesized by thecal-interstitial cell monolayers. Unlabeled IGF-I and insulin inhibited the affinity labeling of the type I IGF receptor, but the doses of insulin required were several-fold greater than those of IGF-I, in keeping with the relative affinities of these peptides for the type I IGF receptor. We conclude that IGF-I an insulin synergize with LH to stimulate androgen production by normal ovarian thecal-interstitial cells. These finding suggests a role for IGF-I and insulin in the pathophysiology of ovarian dysfunction syndromes involving thecal-interstitial cell hyperplasia and hyperandrogenism.