GROWTH FACTOR CONTROL OF OVARIAN ANDROGEN BIOSYNTHESIS

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Ovarian theca-interstitial cells (TIC) are the obligatory source of androgen substrate for follicle estrogen biosynthesis. Evidence in the literature clearly shows that the timely progression of TIC differentiation is essential for normal ovarian function, however the mechanisms by which hormones regulate TIC differentiation are poorly understood. In order to study the molecular mechanisms of TIC differentiation in healthy and diseased states, we have developed a unique method for isolating a population of highly purified TIC which are free from granulosa cell contamination. Significantly, the purified TIC are responsive to hormone regulation in serum-free medium and provide the best available model to study hormone regulation of gene expression during TIC differentiation. We have shown that LH is an important hormone regulating TIC differentiation, but LH alone cannot account for the pattern of gene expression observed in TIC from developing follicles. During the first stages of differentiation, TIC express LH receptors and cholesterol side chain cleavage (P450scc) but not 17alpha-hydroxylase/C17-20 lyase (P45017alpha) even though the mRNA for both P450scc and P45017alpha are transcribed. We have shown that insulin-like growth factor I (IGF-I) alone stimulates both P450scc and P45017alpha mRNA and the selective expression of P450scc protein in TIC. The implication of this finding is that IGF-I may play an important physiological role in regulating the early stages of TIC differentiation. The overall goals of this application are to understand the physiological role of IGF-I in regulating TIC differentiation and to elucidate the molecular mechanisms by which IGF-I acts alone and in concert with LH to regulate the expression of genes in TIC. In situ hybridization will be used together with autoradiography, histochemistry and immunofluorescence to examine gene expression during follicle development and atresia. The expression of IGF-I in the granulosa cells will be correlated with the expression in TIC of LH and IGF-I receptors, cAMP-dependent protein kinase regulatory and catalytic subunits, and P450scc, 3beta-hydroxysteroid dehydrogenase and P45017alpha. In addition we attempt to determine the sequence of gene expression that occurs during the first stages of thecal differentiation when TIC differentiate from fibroblast-like precursor cells into characteristic theca interna cells. Studies will be performed to compare and contrast the transcriptional and translational effects of IGF-I with LH using nuclear runoff assays, RT-PCR and immunoprecipitation techniques. Finally, studies will be performed to determine the details of the second messenger system interactions responsible for the synergistic interactions between IGF-I and LH. From these studies we expect to learn fundamental new information regarding the physiology of early thecal differentiation, follicle development and atresia and how these processes are regulated by locally produced hormones. These results will help us to understand the mechanism of hyperandrogenism in diseases such as hyperinsulinemia and polycystic ovarian disease.