Ovarian follicular development is dependent on hormone (estradiol and FSH)-induced changes in granulosa cell function which lead to the amplification of cAMP production and estradiol biosynthesis. Estradiol and cAMP act, in turn, to regulate the intracellular content of mRNA for specific prudes in granulosa cells of preovulatory follicles. Despite the importance of cAMP in mediating he actions of FSH in granulosa cells and the importance of estradiol in modulating these responses, the precise molecular processes that are being regulated by these signals remain largely unknown, Therefore to determine the molecular mechanisms by which FSH and estradiol regulate granulosa cell differentiation, we propose to study two genes known to be regulated by these hormones during follicular development. These are RIIB, the regulatory subunit of cAMP- dependent protein kinase type II and aromatase cytochrome P450 (P450arom). RIIB transcription is increased by FSH and estradiol but is turned off as a consequence of elevated intracellular cAMP associated with the LH surge and the initiation of luteinization. RIIB is not expressed in luteinized cells. In contrast, P450arom is induced in granulosa cells of preovulatory follicles, is maintained in luteal cells and is increased markedly during pregnancy by placental-derived hormones, primarily rate placental lactogen. Based on these considerations the specific aims of the proposed research are to: 1) Analyze the hormonal regulation of aromatase gene expression using in vitro systems; 2) Identify the 5' ends of the RIIB and aromatase gene transcription in granulosa and luteal cells. Accordingly, we have: 1) begun isolating and sequencing genomic DNA clones for aromatase and RIIB; 2) set up and validated transient transfection assays using a granulosa cell culture system; 3) established S2 nuclease protection assays; and begun constructing RIIB - CAT fusion genes for analysis of 5' flanking regulatory regions. Using these approaches we hope to determine the molecular events that mediate cAMP and estradiol action during granulosa cell differentiation.
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