INSULIN-LIKE GROWTH FACTOR-I SELECTIVELY STIMULATES CHOLESTEROL SIDE-CHAIN CLEAVAGE EXPRESSION IN OVARIAN THECA-INTERSTITIAL CELLS

Evidence accumulating in the literature supports the concept that insulin-like growth factor I (IGF-I) may be an important local regulator of ovarian function. Recent studies have demonstrated that IGF-I synergistically augments LH stimulation of theca-interstitial cell (TIC) androgen biosynthesis. The purpose of the present studies was to begin to elucidate the molecular mechanisms of the interaction between IGF-I and LH. TIC were purified from ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When isolated TIC (5 x 106viable cells per dish) were cultured (4 days) in serum-free medium, low amounts (<10 ng/ml) of androsterone were produced. Basal androsterone production was not changed by incubation with IGF-I (30 ng/ml). Treatment with LH (50 ng/ml) caused an 85-fold stimulation of androsterone synthesis that was further increased 2.1-fold by concomitant treatment with IGF-I. Immunoblot analysis demonstrated that untreated TIC contained low levels of 17α-hydroxylase/C17-20 lyase enzyme (P45017α) that were unchanged by incubation with IGF-I alone. LH treatment increased P45017α content 5.5-fold and coincubation with LH plus IGF-I increased P45017α content 16-fold above control levels. Cholesterol side chain cleavage enzyme (P450scc) was readily detected in immunoblots from untreated TIC. P450scc content was increased 2.6-fold by LH treatment and 4.2-fold by LH plus IGF-I. Interestingly, IGF-I alone induced a 2-fold increase in P450scc. To determine if the increases in P450scc content were associated with increased enzyme activity, progesterone production was measured. Progesterone synthesis was increased 6-fold above basal levels by LH and 7.5-fold by LH plus IGF-I. Surprisingly, IGF-I treatment did not stimulate progesterone production. To determine if the P450scc induced by IGF-I was catalytically active, TIC were pretreated (2 days) with IGF-I to induce P450scc and then challenged (4 h) with LH, 8-bromo- cAMP, or 25-hydroxycholesterol. In each case P450sccactivity was significantly greater in TIC pretreated with IGF-I compared with unprimed controls. There was no increase in TIC P45017α activity after any of the challenges with or without IGF-I pretreatment. Northern blot analysis showed that control TIC contained detectable amounts of P450sccmRNA which were increased 4-fold by incubation with IGF-I alone. LH caused a 50% decrease in P450scc mRNA while IGF-I plus LH increased mRNA 2-fold above control levels. P45017α mRNA was also detectable in untreated TIC. The P45017α mRNA was coordinately regulated with P450scc mRNA. These experiments demonstrated that IGF-I alone causes a selective increase in functional P450sccactivity by stimulating increases in P450scc enzyme content and P450scc mRNA levels in the TIC, and that IGF-I augments the ability of LH to increase the content of P450scc and P45017α content in the TIC. © 1990 by The Endocrine Society.