"To Survive or Die" - Adamts9 in Folliculogenesis and Germ Cell Loss

卵泡的发育和维持，因此功能性卵巢是一个显着的组织 需要各种蛋白酶参与的重塑过程。然而，我们的知识 负责的酶和潜在的机制是不完整的。我们的长期目标 是确定影响女性生育能力的分子和途径，从而提高对女性生育能力的认识。 雌性动物生殖过程和生育力的调节剂。该补助金的具体目的 应用是阐明功能，过程，相互作用的分子和途径，和目标， 被忽视的金属蛋白酶，即，Adamts9（一种具有血小板反应蛋白1的去整合素和金属蛋白酶 基序，成员9）在卵泡的发育和维持中。我们已公布或初步 结果有力地表明Adamts 9在卵泡的存活和程序性死亡中具有关键作用。 我们假设卵母细胞来源的Adamts9酶对正常卵泡发育至关重要， 因此，通过改变细胞外基质（ECM）蛋白的周转， 滤泡内信号传导。我们将通过全面检查卵巢癌的状态来检验这一假设。 毛囊，即，野生型或各种敲除、靶向分子和途径中的健康卵泡与垂死卵泡 使用传统方法，包括但不限于共聚焦显微镜成像，细胞凋亡测定， 增殖和蛋白质印迹，RNA或蛋白质染色。我们还将应用先进的工具， 荧光报告物或分子生物传感器以及利用无偏的方法，例如，单细胞 RNA测序（scRNAseq）和质谱。为了阐明细胞或分子特异性事件，我们 已经建立或获得了用于靶细胞或信号传导的各种敲除、报告或生物传感器系 分子，使我们能够表征生殖细胞，性腺体细胞或靶向细胞的详细变化， 高分辨率的分子。利用这些体内系统和荧光报告子的优点， 我们将描述和阐明Adamts9依赖的ECM周转，基因表达，信号分子， 卵泡发育和维持中的变化、途径和过程。第一 时间，功能和机制的亚当斯9，特别是在卵泡发生，将全面 其特征在于脊椎动物模型，这将填补知识空白，即，生殖细胞如何产生酶 如Adatmts9，调节其ECM环境，从而决定了细胞的存活或程序性死亡。 卵泡获得的新知识将有助于理解或设计新的治疗方法 用于生殖系统疾病，如不孕症、性腺发育不全、绝经早期、原发性卵巢癌、 卵巢功能不全（POI）和多囊卵巢综合征（PCOS）。该地区赠款还将提供关键的 在一所主要为农村地区服务的大学为不同的本科生提供研究机会和培训， 弱势群体，并解决国家卫生研究院的生物医学劳动力增强使命。

Development and maintenance of an ovarian follicle, and thus a functional ovary is a remarkable tissue remodeling processes that requires involvements of various proteases. However, our knowledge concerning the enzymes that are responsible for, and underlying mechanisms are incomplete. Our long-term objective is to identify molecules and pathways that affect female fertility, and therefore to advance knowledge on the regulators for reproductive processes and fertility in female animals. The specific aims of this grant application are to elucidate functions, processes, interacting molecules and pathways, and targets of an overlooked metalloprotease, i.e., Adamts9 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 9) in the development and maintenance of an ovarian follicle. Our published or preliminary results strongly indicate Adamts9 has a critical role in survival and programmed death of an ovarian follicle. We hypothesize that oocyte originated Adamts9 enzyme is vital for normal follicle development and survival by varying turnover of extracellular matrix (ECM) proteins therefore increase or decrease of intra-follicular signaling. We will test this hypothesis via a comprehensive examination of status of ovarian follicles, i.e., healthy vs. dying follicles in wildtype or various knockouts, targeted molecules and pathways using tradition approaches including but not limited to confocal microscope imaging, assays of apoptosis, proliferation & Western blots, staining of RNA or proteins. We will also apply advanced tools using in vivo fluorescence reporters or molecular biosensors as well as utilizing unbiased approaches, e.g., single cell RNA sequencing (scRNAseq) and mass spectrometry. To elucidate cell- or molecule-specific events, we have established or obtained various knockouts, reporter or biosensor lines for targeted cells or signaling molecules, which allow us to characterize detailed changes of germ cells, gonad somatic cells, or targeted molecules at a high resolution. Using the advantages of these in vivo systems and fluorescence reporters, we will characterize and elucidate Adamts9 dependent ECM turnover, gene expression, signaling molecule changes, pathways, and processes in development and maintenance of an ovarian follicle. For the first time, functions, and mechanisms of Adamts9, particularly in folliculogenesis, will be comprehensively characterized in a vertebrate model, which will fill a knowledge gap, i.e., how a germ cell originated enzyme such as Adatmts9, regulates its ECM environments, thus determines survival or programmed death of an ovarian follicle. New knowledge gained will be appreciated for understanding or designing new treatments for reproductive disorders such as infertility, gonadal dysgenesis, early menopause, primary ovarian insufficiency (POI) and polycystic ovarian syndrome (PCOS). This AREA grant will also provide critical research opportunities and training for diverse undergraduates at a university mainly serving rural areas and disadvantage populations, and also address NIH mission for biomedical workforce enhancement.