Cellular Signals in Ovulation and Luteinization

描述（申请人提供）：排卵对哺乳动物的生殖至关重要。这是一个复杂的炎症样过程，仅在成熟卵泡暴露于促黄体激素（LH）激增时发生，并依赖于特定基因的LH诱导。鉴定的是孕酮受体（PR）、分泌的金属蛋白酶ADAMTS-4和透明质酸结合的基质相关蛋白聚糖多功能蛋白聚糖。排卵也依赖于几种PR调节基因，因为两种PR亚型无效的小鼠（PRKO）即使补充外源性激素也不能排卵（不育）。PR靶基因包括其他分泌型金属蛋白酶ADAMTS-1和ADAM 8，以及G蛋白偶联受体Frizzled-1（FZ-1），Wnt/Frizzled信号级联的成员。与PRKO小鼠一样，ADAMTS-1基因敲除小鼠是不育的，表明这种金属蛋白酶介导LH和PR在排卵中的一些作用。由于LH诱导PR和PR如何影响ADAMTS-1表达的分子机制似乎涉及这两个基因的近端启动子中的关键Spl/Sp3结合位点，我们建议确定与Spl/Sp3相互作用以控制转录的关键调控因子。我们建议进一步确定结构和功能相关的金属蛋白酶ADAMTS-1和ADAMTS-4（以及ADAM 8）影响排卵的机制。我们假设这些蛋白酶影响 卵丘卵母细胞扩增过程中产生的细胞外基质（ECM）的形成和功能，这对于卵母细胞从排卵孔中挤出并被输卵管吸收至关重要。该假说基于多功能蛋白聚糖、ADAMTS-1和ADAMTS-4由卵丘细胞产生、共定位于基质中并且多功能蛋白聚糖是这些酶的优选底物的证据。最后，我们提出排卵依赖于Fz-1的诱导，Fz-1是一种G蛋白偶联受体，介导β-连环蛋白信号传导和基因表达。使用新的分子方法分析调节基因激活的因素（ChIP、达帕和SiRNA），以及允许有条件破坏颗粒细胞中靶基因的突变小鼠模型，我们建议：I）确定LH诱导排卵相关基因PR和ADAMTS- 1表达的分子机制; II）确定LH和PR诱导基因ADAMTS-1、ADAMTS-4和Fz-1在排卵中的功能作用; III）鉴定影响排卵的新的LH和PR调节基因。这些研究将为PR及其靶基因在排卵过程中的激素调控提供新的信息 研究和引导对妇女不孕原因的新认识以及新的避孕方法。

Description (provided by applicant): 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.