The surge of luteinizing hormone (LH) initiates the unique biological process of ovulation by inducing the expression of a specific set of genes in a highly regulated manner. One gene encodes prostaglandin endoperoxide synthase-2 (PGS-2) that controls the rate-limiting step in the conversion of arachadonic acid to prostaglandins and related eicosanoids. Our laboratory was instrumental in identifying, by biochemical and immunological approaches, that PGS-2 is a novel and inducible isoform of the enzyme. The rapidly elevated, but transient induction of PGS-2 mRNA by LH requires surge concentrations of hormone and occurs exclusively in granulosa cells of preovulatory follicles. The molecular mechanisms by which LH regulates the expression of the PGS-2 gene in granulosa cells are complex and not yet fully understood. Using deletional and mutational analyses, we have determined that the rat PGS-2 promoter contains an E-box consensus sequence that is essential for transcription of PGS-2 promoter-reporter transgenes in granulosa cells and binds Upstream Stimulatory Factor (USF). USF also binds to E-box regions in promoters of other genes expressed in ovarian granulosa cells: the FSH receptor, the regulatory subunit type II (RII beta) of cyclic AMP dependent protein kinase, and Steroidogenic Factor-1 (SF-1). The rat PGS-2 promoter also contains at least one CAAT enhancer binding (C/EBP) site that binds C/EBP alpha and C/EBP beta present in granulosa cell extracts. However, sites in addition to the E-box and CAAT box are essential for expression of the PGS-2 gene in granulosa cells. A critical role for C/EBPs in the ovary is underscored by the observations that C/EBP beta, like PGS-2, is rapidly but transiently induced exclusively in granulosa cells of preovulatory follicles in response to the LH surge. Targeted deletion of the PGS-2 and C/EBP beta genes in mice has been shown to block ovulation reinforcing the importance of prostaglandins the critical functional role of C/EBP beta in regulating molecular event(s) (including the expression of PGS-2?) that control the ovulation process. The availability of mice deficient in either PGS-2 or C/EBP beta provides us with the unique opportunity to extend our studies on the hormonal regulation of PGS-2, as well as other genes in granulosa cells, and to determine their functional roles in the physiology of ovulation. Therefore, one specific aim of this proposal is to identify the factor(s) and mechanisms by which LH mediates expression of the PGS-2 gene in granulosa cells. A related and longer term goal of the research is to identify and characterize the genes regulated by LH, prostaglandins (PGs), and C/EBPs that control key steps in the ovulatory process.
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