Ovulation is essential for mammalian reproduction. It is a complex inflammation-like process that occurs only when mature follicles are exposed to the luteinizing hormone (LH) surge and is dependent on LH induction of specific genes. Those identified are the progesterone receptor (PR), the secreted metalloproteinase ADAMTS-4 and the hyaluronon binding, matrix associated proteoglycan versican. Ovulation is also dependent on several PR-regulated genes since mice null for both PR subtypes (PRKO) fail to ovulate (are infertile) even when supplemented with exogenous hormones. PR target genes include other secreted metalloproteinases ADAMTS-1 and ADAM8, as well as the G-protein coupled receptor Frizzled-1 (FZ-1), a member of the Wnt/Frizzled signaling cascade. Like PRKO mice ADAMTS-1 null mice are infertile indicating this metalloproteinase mediates some effects of LH and PR in ovulation. Since the molecular mechanisms by which LH induces PR and how PR impacts expression of ADAMTS-1 appear to involve critical Spl/Sp3 binding sites in the proximal promoters of these two genes, we propose to determine the critical regulatory factors that interact with Spl/Sp3 to control transcription. We propose further to determine the mechanisms by which the structurally and functionally related metalloproteinases ADAMTS-1 and ADAMTS-4 (as well as ADAM8) impact ovulation. We hypothesize that these proteinases impact the formation and function of the extracellular matrix (ECM) that is made during cumulus oocyte expansion, which is essential for extrusion of the ooctye from the ovulation pore and its uptake by the oviduct. This hypothesis is based on the evidence that versican, ADAMTS-1 and ADAMTS-4 are made by cumulus cells, co-localize in the matrix and that versican is a preferred substrate for these enzymes. Lastly, we propose that ovulation is dependent on the induction of Fz-1, a G-protein coupled receptor that mediates beta-catenin signaling and gene expression. Using new molecular approaches for analyzing factors that regulate gene activation (ChIP, DAPA and siRNA), as well as mutant mouse models that permit conditional disruption of target genes in granulosa cells, we propose to: I) Determine the molecular mechanisms by which LH induces expression of ovulation-related genes, PR and ADAMTS- 1; II) Determine the functional roles of LH and PR induced genes ADAMTS-1, ADAMTS-4 and Fz-1 in ovulation; III) Identify novel LH and PR regulated genes that impact ovulation. These studies should provide novel information about the hormonal regulation of PR and its target genes in the ovulation process and lead to new insights into causes of infertility in women as well as to new methods of contraception.
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