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，其中许多与锌依赖性内肽酶家族、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，其中日长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，光敏感个体是基本卵巢功能的优秀模型，因为改变日长自然会诱导并逆转卵巢萎缩。在光周期西伯利亚仓鼠中，日长变化引起的卵巢重塑与 MMP 的差异表达相关。事实上，在体内施用广谱 MMP 抑制剂 GM6001 后，光刺激卵巢功能的恢复（复发）可能会受到阻碍。虽然这表明这些蛋白酶发挥着重要作用，但 MMP 在复发过程中的作用以及静止卵巢在萎缩数周后如何恢复循环的过程尚不清楚。该提议假设 1) GM6001 抑制复发的原因是通常介导卵巢功能恢复的 MMP 底物的裂解受到阻碍，2) GM6001 处理的卵巢无法恢复功能，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，3) 因为明胶酶(MMPs-2/-9) 是卵巢周期的关键参与者，施用 GM6001 后明胶酶活性显着下调，大部分重塑可归因于明胶酶。初步实验将使用假设驱动的蛋白质组学方法，通过比较 GM6001 处理与对照卵巢的蛋白质组来识别复发期间 MMP 裂解的底物。检查 mRNA 和蛋白质中血管生成（例如 CD34、VEGF-R1）、颗粒细胞增殖 (PCNA) 和类固醇生成（例如 Cyp19、32HSD）的关键标志物将揭示复发期间 MMP 的作用机制，并在将 GM6001 处理的组织与对照组织进行比较时确定 MMP 在这些过程中的作用。最后，在光刺激复发期间体内施用明胶酶特异性抑制剂（SB-3CT），以提供恢复卵巢中明胶酶作用和功能的直接证据。总之，这些研究应该为卵巢功能复发的细胞和分子调控提供新的见解。此外，随着卵巢周期的恢复，这些数据将提供 MMP 作用目标的第一个证据。通过使用光周期模型了解 MMP 在哺乳动物卵巢功能中的作用有助于阐明如何通过非激素机制关闭（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.