Genetic interrogation of conserved follicular factors for matrix metalloproteinase regulation and ovulation

 描述(申请人提供)：卵泡破裂是复杂排卵计划的最后一步，释放可受精的卵母细胞。尽管在过去的40年里进行了大量的研究，但对卵泡破裂的分子机制仍然缺乏全面的了解，部分原因是哺乳动物模型系统使用遗传筛选的限制。我们最近开发了一种新的果蝇系统，它允许快速应用遗传方法来揭示关于卵泡破裂的分子事件的准确细节。此外，我们实验室最近的研究表明，从苍蝇到人类，排卵的基本细胞和分子机制都是高度保守的；例如，这两个系统都需要基质金属蛋白酶(MMPs)活性才能使卵泡破裂。利用丰富的遗传工具和我们的体外排卵试验，该项目将系统地询问精确调节基质金属蛋白酶活性和卵泡破裂所需的保守因子。初步数据显示，细胞内游离钙离子的增加是激活基质金属蛋白酶所必需的，但不是表达所必需的。相反，产生活性氧(ROS)的卵泡NADPH氧化酶(NOX)和氧化应激诱导的c-Jun氨基末端激酶(JNK)通路调节时空MMP蛋白的表达。因此，我们建议1)阐明基质金属蛋白酶激活所需的保守的钙依赖信号转导途径，2)研究卵泡ROS和JNK信号在基质金属蛋白酶表达中的作用，3)利用基因筛选寻找新的卵泡因子来调节基质金属蛋白酶和排卵。这项工作将提供对卵巢信号网络的全面理解，这些信号网络精确地调节基质金属蛋白酶的活性和卵泡破裂，这在哺乳动物模型系统中是困难的。这些信号通路的保守性将使从这项研究中获得的知识在哺乳动物和人类排卵中得到进一步验证。因此，这项工作最终将揭示缓解无排卵性不孕症或避孕开发的有前景的新药靶点，这两者都与人类健康高度相关。

 DESCRIPTION (provided by applicant): Follicle rupture is the final step of the complex ovulation program, which releases fertilizable oocytes. Despite intensive study in the past four decades, a comprehensive understanding of the molecular mechanisms of follicle rupture is still lacking, in part due to the limitation of mammalian model systems to utilize genetic screens. We recently developed a novel Drosophila system that allows rapid application of genetic approaches to reveal precise details regarding the molecular events of follicular rupture. Moreover, recent studies from our lab have shown that the basic cellular and molecular mechanisms of ovulation are highly conserved from flies to humans; for instance, both systems require matrix metalloproteinase (Mmp) activity for follicle rupture. Leveraging the wealth of genetic tools and our ex vivo ovulation assay, this project will systematically interrogate conserved factors that are required for the precise regulation of Mmp activity and follicle rupture Our preliminary data reveals that an increase in intracellular free Ca2+ is required for Mmp activation but not expression. Conversely, follicular NADPH oxidase (Nox), which generates reactive oxygen species (ROS), and the oxidative stress-induced c-Jun N-terminal kinase (JNK) pathway regulate spatiotemporal Mmp protein expression. Therefore, we propose to 1) elucidate the conserved calcium-dependent signal transduction pathways that are required for Mmp activation, 2) investigate the role of follicular ROS and JNK signaling in Mmp expression, and 3) identify novel follicular factors for Mmp regulation and ovulation using genetic screens. This work will provide a comprehensive understanding of the ovarian signaling networks that precisely regulate Mmp activity and follicle rupture, which would be difficult in mammalian model systems. The conserved nature of these signaling pathways will allow the knowledge gained from this study to be further validated in mammalian and human ovulation. Therefore, this work will ultimately reveal promising new drug targets for the alleviation of anovulatory infertility orfor contraceptive development, both of which are highly relevant to human health.