Ovulation and luteinization are two processes which are induced in preovulatory ovarian follicles as a consequence of the LH surge. Ovulation is preceded by a pronounced but transient increase in prostaglandin endoperoxide H synthase (PGS) the rate-limiting enzyme for synthesis of prostaglandins. We have recently purified this novel LH- induced enzyme (M(r)=72,000; PGS72; PGSi) and shown that it is immunologically and biochemically distinct (based on N-terminal amino acid sequence, enzymatic activity and tryptic digests) from the other isoform of the enzyme (M(r)=69,000; PGS69) which is present in many tissues, such as uterus, kidney and heart. Luteinization, on the other hand, is associated with a rapid but sustained increase in cholesterol side-chain cleavage cytochrome P450 (P450scc). LH-induction of luteinization is established within 7h of exposure of preovulatory follicles to LH/FSH in vivo or in vitro. Following this exposure to hormones, luteinization can proceed and be maintained in vitro in the absence of hormones. Thus, in association with luteinization regulation of the P450scc gene appears to shift from cAMP-dependent to cAMP- independent mechanisms. The cellular signalling pathways which determine the responses of granulosa cells to ovulatory levels of gonadotropins have yet to be clearly defined but appear to involve distinct pathways. For example, we have recently shown that ovulatory but not basal concentrations of LH and FSH induce both PGSi and P450scc. In contrast, gonadotropin releasing hormone (GnRH) can mimic LH-induction of PGSi but not P450scc. Tyrphostins (inhibitors of tyrosine kinases) block LH and GnRH induction of PGSi and prevent LH stimulation of luteinization and constitutive expression of P450scc. Although the cAMP-A-kinase pathway appears to play a primary role in mediating LH action, tyrosine kinases may also be involved. To characterize the hormonal regulation of PGSi and P450scc expression, we have isolated both genes, sequenced 5' flanking regions containing putative promoter elements and have begun characterizing the functional activity of these promoters. Therefore, the specific aims of the proposed research are: (1) to determine the molecular mechanisms involved in LH-mediated transient induction of the novel, distinct isoform of prostaglandin endoperoxide synthase (PGSi); (2) to determine the molecular mechanisms by which LH induces P450scc and its sustained expression in luteal cells and (3) to determine the specific cellular signalling pathways by which LH mediates induction of PGSi (ovulation) and P450scc (luteinization). Understanding the regulation of these genes should provide greater insight into the events controlling ovulation and luteinization, and perhaps the broader areas of inflammation and terminal cell differentiation, respectively.
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