Protease regulation of ovarian recrudescence

描述（由申请人提供）：卵巢功能，包括卵泡发育、排卵和黄体形成/降解，取决于组织重塑事件，其中许多与Zn+依赖性内肽酶家族（基质金属蛋白酶（MMP））相关。在光周期物种中观察到卵巢组织的极端重塑，其中日照长度的季节性变化可以抑制或刺激下丘脑/垂体分泌GnRH/促性腺激素，导致卵巢功能萎缩或恢复。因此，光反应个体是基本卵巢功能的极好模型，因为日照长度的改变自然诱导并随后逆转卵巢萎缩。在光周期西伯利亚仓鼠，卵巢重塑提示白天长度的变化与基质金属蛋白酶的差异表达。事实上，在体内施用广谱MMP抑制剂GM 6001后，光刺激的卵巢功能恢复（复发）可被阻碍。虽然这表明这些蛋白酶的重要作用，但MMPs在复发期间的作用以及静止卵巢如何在萎缩数周后恢复循环的过程尚不清楚。该提议假设1）响应于GM 6001的复发抑制发生，因为正常介导卵巢功能恢复的MMP底物的切割被阻碍，2）GM 6001处理的卵巢不能恢复功能，因为关键的卵巢过程如血管生成、颗粒细胞增殖和类固醇生成依赖于MMP活性，和3）由于明胶酶（MMP-2/-9）是卵巢周期性的关键参与者，并且明胶酶活性在GM 6001施用后显著下调，因此大部分重塑可归因于明胶酶。最初的实验将使用假设驱动的蛋白质组学方法，通过比较GM 6001处理卵巢与对照卵巢的蛋白质组来鉴定复发期间MMP切割的底物。检查mRNA和蛋白质两者的血管生成的关键标志物（例如，CD 34、VEGF-R1）、颗粒细胞增殖（PCNA）和类固醇生成（例如，Cyp 19，32 HSD）将揭示复发期间MMP作用的机制，并定义MMP在这些过程中的作用，因为GM 6001处理的组织与对照相比。最后，明胶酶特异性抑制剂（SB-3CT）将在光刺激复发期间体内给药，以提供明胶酶在恢复卵巢中的作用和功能的直接证据。总之，这些研究将为卵巢功能复发的细胞和分子调控提供新的见解。此外，这些数据将提供第一个证据的目标MMP的行动作为卵巢周期的回报。通过使用光周期模型了解MMPs在哺乳动物卵巢功能中的作用有助于阐明如何用非激素机制关闭（避孕）和重新启动（辅助生殖，过早绝经）卵巢活动的关键临床问题。

DESCRIPTION (provided by applicant): Ovarian function, including follicle development, ovulation, and corpus luteum formation/degradation, is dependent upon tissue remodeling events, many of which are associated with a family of Zn+dependent endopeptidases, the matrix metalloproteinases (MMPs). Extreme remodeling of ovarian tissue is observed in photoperiodic species where seasonal changes in day length can either inhibit or stimulate hypothalamic/ pituitary secretion of GnRH/gonadotropins, leading to atrophy or resumption of ovarian function. Photo- responsive individuals are therefore excellent models for basic ovarian function, because modifications of day length naturally induce and then reverse ovarian atrophy. In photoperiodic Siberian hamsters, ovarian remodeling prompted by changes in day length is associated with differential expression of MMPs. Indeed, photostimulated return to ovarian function (recrudescence) can be impeded following in vivo administration of a broad-spectrum MMP inhibitor, GM6001. While this suggests an important role for these proteases, the action of MMPs during recrudescence and the process of how the quiescent ovary can resume cycling following weeks of atrophy is unknown. This proposal hypothesizes that 1) inhibition of recrudescence in response to GM6001 occurs because cleavage of MMP substrates that normally mediate return to ovarian function is impeded, 2) that the GM6001-treated ovary fails to return to function because key ovarian processes such as angiogenesis, granulosa cell proliferation, and steroidogenesis are dependent on MMP activity, and 3) that because gelatinases (MMPs-2/-9) are key players in ovarian cyclicity, and gelatinase activity is significantly down regulated following GM6001 administration, much of the remodeling can be attributed to gelatinases. Initial experiments will use a hypothesis-driven proteomics approach to identify substrates cleaved by MMPs during recrudescence by comparing the proteome of GM6001-treated vs. control ovaries. Examination of both mRNA and protein for key markers of angiogenesis (e.g., CD34, VEGF-R1), proliferation of granulosa cells (PCNA), and steroidogenesis (e.g., Cyp19, 32HSD) will reveal a mechanism of MMP action during recrudescence, and define a role for MMPs in these processes as GM6001 treated tissue is compared to controls. Finally, a gelatinase-specific inhibitor (SB-3CT) will be administered in vivo during photostimulated recrudescence to provide direct evidence of gelatinase action and function in the recovering ovary. Together, these studies should provide novel insight into the cellular and molecular regulation of recrudescence of ovarian function. In addition, these data will provide the first evidence of the targets of MMP action as ovarian cyclicity returns. Understanding the role that MMPs play in mammalian ovarian function by using a photoperiodic model helps to elucidate critical clinical questions of how to shut down (contraception) and restart (assisted reproduction, premature menopause) ovarian activity with non-hormonal mechanisms.