Our long-term research goal is to further our understanding of the cAMP response mechanisms in ovarian follicular and luteal cells: their regulation at the molecular level by luteinizing hormone (LH), follicle stimulating hormone (FSH) and steroid hormones like estrogen; their physiological significance in the ovarian follicle and corpus luteum. At the level of the adenylyl cyclase enzyme, we propose to evaluate the molecular mechanism by which both Mg2+ and LH induce desensitization of the adenylyl cyclase to LH in a cell-free membrane preparation derived from preovulatory porcine follicles. At the level of the cAMP-dependent protein kinase enzyme, we propose to determine the biochemical properties of the regulatory and catalytic protein kinase subunits; the regulation of the regulatory and catalytic subunit activities and regulatory subunit levels by LH, FSH and estrogen; and the hormonal regulation of protein kinase subunit associations to form the holoenzymes. These protein kinases parameters will be followed in ovarian follicles and corpora lutea at specified times during the reproductive phases of rat and rabbits. Methods include standard assays for adenylyl cyclase, phosphotransferase cAMP binding activities; protesterone and cAMP radioimmunoassays; standard protein purification techniques; and recombinant DNA technology. It is through such studies on the molecular regulation of these key enzyme systems by the gonadohormones that we can understand the mechanism of action and therefore the functions of LH, FSH and estrogen in ovarian follicles and corpora lutea.
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