Insulin-Like Growth Factor-I Regulation of Luteinizing Hormone (LH) Receptor Messenger Ribonucleic Acid Expression and LH-Stimulated Signal Transduction in Rat Ovarian Theca-Interstitial Cells 1

Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) secreted by small preantral follicles may be involved in stimulating the initial differentiation of the theca interna and, in particular, expression of the LH receptor in pre-theca cells. To test this hypothesis, we examined the effects of IGF-I on LH receptor mRNA expression in theca-interstitial cells (TIC) isolated from the ovaries of hypophysectomized immature rats by percoll gradient centrifugation. TIC (3.5 x 104 viable cells/well) were cultured up to 6 days with and without LH (0-10 ng/ml) and IGF-I (0-100 ng/ml). Androsterone in the medium was measured by RIA, and LH receptor mRNA was measured by specific reverse transcriptase-polymerase chain reaction assay. LH receptor mRNA was low in control (untreated) TIC. IGF-I stimulated a dose-related increase (2-fold) in LH receptor mRNA at 2 days (ED5 = 9.0 + 1.9 ng/ml) that remained constant at 4 days and then declined to basal levels at 6 days. LH stimulated a dose-related (ED 50 = 17.6 ± 1.0 pg/ml) increase in LH receptor mRNA that reached a maximum of 4-fold at 2 days. At 4 days, LH down-regulated LH receptor mRNA below basal levels, and it had no effect at 6 days. Addition of IGF-I (30 ng/ml) to LH-treated TIC abolished the stimulatory effect of LH throughout the culture period. LH receptor mRNA was highly sensitive to LH since the ED, was approximately 2.5-fold lower than for stimulation of androsterone production (39.8 ± 3.8 pg/ml). To understand the molecular mechanism of the synergistic stimulation of androgen production by IGF-I and LH, the effects of IGF-I on the cAMP/protein kinase A (PKA) signaling pathway were examined. When freshly isolated TIC were challenged with IGF-I alone (30 ng/ml), there was no effect on cAMP production or PKA activity, but IGF-I augmented LH stimulation of cAMP production slightly at high concentrations of LH and blocked stimulation of PKA activity by a saturating concentration of LH (3 ng/ml), suggesting that IGF-I increased LH down-regulation of PKA. We next examined the effects of IGF-I on LH receptor number. When TIC were placed into culture, LH/hCG binding sites decreased to approximately 35% of the initial number at 24 h and 25% at 2 days. This decrease was accompanied by a similar loss of cholera toxin- and hCG-stimulated cAMP production. IGF-I treatment did not prevent the loss of binding sites at 24 h but by 48 h increased the number of binding sites and hCG-stimulated cAMP production to initial values. These responses were specific to the LH/hCG receptor since prostaglandin E 2 -stimulated cAMP production did not change in the presence or absence of IGF-I. The results of our studies demonstrate that IGF-I alone can stimulate the expression of LH receptor mRNA and increase the number of LH/hCG binding sites functionally coupled to cAMP production in TIC. These data support the hypothesis that IGF-I may be involved in stimulating the initial expression of LH receptors during the early stages of thecal differentiation.