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程序的最后一步，释放可受精的卵母细胞。尽管在过去的四十年里进行了深入的研究，但仍然缺乏对卵泡破裂分子机制的全面了解，部分原因是哺乳动物模型系统利用遗传筛选的限制。我们最近开发了一种新的果蝇系统，允许快速应用遗传方法来揭示卵泡破裂分子事件的精确细节。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是高度保守的;例如，这两种系统都需要基质金属蛋白酶（Mmp）活性才能使卵泡破裂。利用丰富的遗传工具和我们的离体排卵测定，该项目将系统地询问精确调节Mmp活性和卵泡破裂所需的保守因子。我们的初步数据表明，细胞内游离Ca 2+的增加是Mmp激活所需的，但不是表达。相反，滤泡NADPH氧化酶（Nox），产生活性氧（ROS），和氧化应激诱导的c-Jun N-末端激酶（JNK）途径调节时空Mmp蛋白的表达。因此，我们建议：1）阐明Mmp激活所需的保守的钙依赖性信号转导途径，2）研究卵泡ROS和JNK信号在Mmp表达中的作用，3）使用遗传筛选鉴定用于Mmp调节和排卵的新卵泡因子。这项工作将提供一个全面的了解卵巢信号网络，精确地调节Mmp活性和卵泡破裂，这将是很难在哺乳动物模型系统。这些信号通路的保守性将使从本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，这项工作将最终揭示有希望的新的药物靶点，用于缓解无排卵性不孕症或避孕药的开发，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.