INSULIN-LIKE GROWTH FACTOR-I STIMULATES THE EXPRESSION OF 3-BETA-HYDROXYSTEROID DEHYDROGENASE MESSENGER-RIBONUCLEIC-ACID IN OVARIAN THECA-INTERSTITIAL CELLS

Evidence accumulating in the literature supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating differentiation of the ovarian theca-interstitial cells (TIC) during early follicular development. IGF-I has been shown to synergistically enhance the stimulation of androgen biosynthesis and to increase LH binding in TIC. The purpose of the present studies was to examine the role of IGF-I in TIC differentiation by determining the effects of IGF-I on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) 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 for up to 6 days. At various times cytoplasmic RNA was extracted from the TIC, and 3beta-HSD mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Amplification of 3beta-HSD mRNA using primers designed to distinguish between the type I and type II 3beta-HSD gene products revealed that the TIC expressed primarily the type I gene. Increasing concentrations of LH (0-1 mug/ml) stimulated a dose-related increase in 3beta-HSD mRNA that was approximately 3-fold at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) increased 3beta-HSD mRNA approximately 2-fold over TIC treated with LH alone. IGF-I alone also stimulated a dose-related increase in 3beta-HSD mRNA that reached a level approximately 3-fold greater than unstimulated levels. Addition of LH (100 ng/ml) increased 3beta-HSD mRNA at each concentration of IGF-I. Time-course studies revealed that expression of 3beta-HSD 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. More detailed studies demonstrated that LH stimulated a rapid increase in progesterone production by the TIC after an approximately 2-h lag. Progesterone levels began to decline after approximately 20 h in culture. Although IGF-I alone did not alter progesterone production, concomitant treatment with LH and IGF-I increased progesterone to levels approximately 2-fold higher than in TIC treated with LH alone without altering the temporal pattern of progesterone production. When expression of 3beta-HSD mRNA was examined, LH stimulated an initial increase between 2 and 4 h and a secondary increase at approximately 30 h. During the first 30 h of culture, concomitant treatment with LH and IGF-I caused an approximately 3-fold increase in 3beta-HSD mRNA levels above the levels in TIC treated with LH alone, but did not change the LH-stimulated levels after 30 h. Interestingly, the time course of 3beta-HSD expression was much slower in response to IGF-I alone, rising after an approximately 20-h lag to maximum levels at 30 h. The results of our studies demonstrate that IGF-I stimulates the expression of 3beta-HSD mRNA in ovarian TIC, supporting the hypothesis that IGF-I may play a role in stimulating TIC differentiation in developing follicles.