Defining Roles of PP1 Phosphatases in Sperm Meiosis

PP1 磷酸酶在精子减数分裂中的作用

• 批准号: 7751320
• 负责人: Diana S. Chu
• 金额: $ 18.85万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7751320
• 关键词: Antibodies; Biochemistry; Biology; Caenorhabditis elegans; Chromatin; Chromosome Condensation; Chromosome Segregation; Chromosomes; Cytology; DNA; Data; Defect; Development; Diagnosis; Diagnostic tests; Embryo; Failure; Family member; Fertility; Genes; Goals; Human; Infertility; Kinetochores; Laboratories; Male Infertility; Mammals; Mass Spectrum Analysis; Mechanics; Meiosis; Methods; Mitosis; Molecular; Molecular Abnormality; Molecular Target; Nematoda; Oocytes; Outcome; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Protein Dephosphorylation; Protein phosphatase; Proteins; Proteomics; Public Health; Publishing; RNA Interference; Research; Resources; Role; San Francisco; Signal Pathway; Signal Transduction; Specificity; Spermatogenesis; Techniques; Testing; Time; Universities; Work; base; cohesion; feeding; genetic analysis; genetic regulatory protein; human male; loss of function; male; member; mutant; protein function; reproductive; reproductive success; research study; segregation; sperm cell; sperm function; tool

Sperm meiotic chromosome segregation errors have serious consequences in humans: male infertility, embryonic lethality, and developmental abnormalities. Many such errors are a consequence of the special biology of sperm meiotic chromosome segregation. Our goal is to define sperm-specific molecular mechanisms that compact and segregate paternal DMA properly for reproductive success. In this application, our objective is to determine how four PP1 phosphatase proteins (GSP-1, GSP-2, GSP-3 and GSP-4) function in chromosome segregation during sperm formation in the nematode C. elegans. In our preliminary studies these PP1 phosphatases were identified by proteomic analysis associated with sperm meiotic chromatin and show conserved function in male fertility and chromosome segregation from C. elegans to mammals; however, how each PP1 family member controls general meiotic machinery and implements distinct aspects of sperm meiosis is not known. To define how these proteins function in sperm meiosis, we will pursue three specific aims. First, because GSP-1 and GSP-2 are associated with both sperm and oocyte chromatin, we will analyze the loss-of-function of gsp-7 and gsp-2 to differentiate spermspecific from oocyte-specific functions of GSP-1 and GSP-2. We will also find differences in function of GSP-1 and GSP-2 from those of GSP-3 and GSP-4 (which are found only on sperm chromatin) in sperm meiosis. Second, we will characterize PP1 phosphatase interactions with proteins that are known to act in chromosome segregation, like components of the kinetochore. Kinetochore proteins function in spindle attachment to DMA and we have found in preliminary studies that some are localized differentially in sperm versus oocyte meiosis. We will therefore conduct colocalization and coimmunopreciptiation experiments to find interactions with specific components of the kinetochore and other core segregation machinery. Third, we will identify signaling pathway components that physically associate with PP1 phosphatases during spermatogenesis through coimmunoprecipitation and mass spectrometry, followed by feeding RNAi analysis to pinpoint candidates with roles in fertility similar to GSP proteins. By this approach we will identify not only signaling pathway members, but also core segregation machinery required to control sperm meiotic divisions. Relevance to Public Health: Once sperm-specific functions of PP1 phosphatases in chromosome segregation have been delineated and molecular targets or regulators of these conserved phosphatases found, we will have uncovered previously unknown causes of male infertility and reproductive failure. Because these proteins are conserved from worms to humans, components and mechanisms we uncover will be important to understanding human infertility.

精子减数分裂染色体隔离错误对人类有严重的后果：男性不育， 胚胎致死性和发育异常。许多这样的错误是特殊的结果 精子减数分裂染色体分离的生物学。我们的目标是定义精子特异性分子 适当地紧凑和隔离父亲DMA的机制，以获得生殖成功。在这个 应用，我们的目标是确定四种PP1磷酸酶蛋白如何（GSP-1，GSP-2，GSP-3和 GSP-4）在线虫秀丽隐杆线虫中精子形成期间染色体分离中的功能。在我们的 初步研究通过与精子相关的蛋白质组学分析来鉴定这些PP1磷酸酶 减数分裂染色质和在男性生育能力和染色体隔离中表现出保守的功能。 秀丽隐杆线到哺乳动物；但是，每个PP1家庭成员如何控制一般的减数分裂机械和 实现精子减数分裂的不同方面尚不清楚。定义这些蛋白质如何在精子中起作用 减数分裂，我们将追求三个具体目标。首先，因为GSP-1和GSP-2都与 精子和卵母细胞染色质，我们将分析GSP-7和GSP-2的功能丧失，以区分精子特定 来自GSP-1和GSP-2的卵母细胞特异性功能。我们还会发现 GSP-3和GSP-4的GSP-1和GSP-2（仅在精子染色质上发现） 减数分裂。其次，我们将表征PP1磷酸酶与已知作用在内的蛋白质的相互作用 染色体隔离，例如动力学的成分。动力学蛋白在纺锤体中的功能 与DMA的依恋，我们在初步研究中发现，有些在精子中差异化 与卵母细胞减数分裂。因此，我们将进行共定位和共免疫实验 找到与动力学和其他核心分离机制的特定组件的相互作用。第三， 我们将确定在物理与PP1磷酸酶物理相关的信号通路成分 通过共免疫沉淀和质谱法进行了精子发生，然后进行RNAi分析 指出具有类似于GSP蛋白的生育能力的候选者。通过这种方法，我们不仅会确定 信号途径成员，但也需要控制精子减数分裂所需的核心隔离机制 部门。 与公共卫生的相关性：染色体中PP1磷酸酶的精子特异性功能 已经划分了分离和这些保守磷酸酶的分子靶标或调节剂 发现，我们将发现以前未知的男性不育症和生殖失败的原因。 由于这些蛋白质是从蠕虫到人的保守的，所以我们发现的组成部分和机制 对于理解人类不育将很重要。