Effect of insulin-like growth factor-I on cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid expression in ovarian theca-interstitial cells stimulated to differentiate in vitro

Abstract

Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating ovarian theca-interstitial cell (TIC) differentiation in preantral follicles. The purpose of the present studies was to examine the potential role of IGF-I in TIC differentiation by determining the effects of IGF-I on cholesterol side-chain cleavage cytochrome P450 (P450 SCC) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I up to 6 days. At various times cytoplasmic RNA was extracted from the TIC and P450 SCC mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Increasing concentrations of LH (0–1 μg/ml) stimulated a dose-related increase in P450 SCC mRNA (ED 50 = 36.2 ± 5.5 ng/ml) which reached maximal levels at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) caused a small increase in P450 SCC mRNA over TIC treated with LH alone but did not alter the ED 50 for LH stimulation. IGF-I alone also stimulated an increase in P450 SCC mRNA which reached approximately 3-fold over unstimulated levels at 100 ng/ml. In the presence of LH, IGF-I stimulated a dose-related increase in P450 SCC mRNA (ED 50 = 1.2 ± 0.05 ng/ml). Time-course studies revealed that expression of P450 SCC mRNA was greatest at 2 days in TIC treated with IGF-I alone, LH alone or LH plus IGF-I and then declined at 4 and 6 days. Detailed studies demonstrated that LH stimulated a rapid increase in progesterone production after an approximately 2 h lag. Progesterone levels reached maximal levels at 10 h and were maintained until approximately 20 h after which they began to decline. Although IGF-I alone did not alter progesterone production, concomitant treatment with LH and IGF-I caused progesterone to increase in the culture medium until 18 h when the levels were approximately 2-fold higher than in TIC treated with LH alone. After 20 h progesterone levels began to decrease. P450 SCC mRNA expression followed a pattern very similar to the pattern of progesterone production with two exceptions. First, IGF-I alone stimulated an approximately 3-fold increase in P450 SCC mRNA at 24 h of treatment which was sustained throughout the culture period. Second, maximal levels of P450 SCC mRNA in TIC treated with LH plus IGF-I were maintained until 30 h before a decline was observed. The results of our studies demonstrate that IGF-I stimulates the expression of P450 SCC mRNA in ovarian TIC and support the hypothesis that IGF-I may play a role in stimulating TIC differentiation in developing follicles.