Defining Roles of PP1 Phosphatases in Sperm Meiosis

PP1 磷酸酶在精子减数分裂中的作用

基本信息

批准号：
8015300
负责人：
Diana S. Chu
金额：
$ 18.99万
依托单位：
SAN FRANCISCO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
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/8015300
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 Signal Pathway Signal Transduction Specificity Spermatogenesis Techniques Testing Time 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

项目摘要

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分离错误对人类产生严重后果：男性不育、胚胎致死率和发育异常。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正确压缩和分离父本 DMA 以实现繁殖成功的机制。在这个应用中，我们的目标是确定四种 PP1 磷酸酶蛋白（GSP-1、GSP-2、GSP-3 和 GSP-4）在线虫精子形成过程中的染色体分离中发挥作用。秀丽隐杆线虫。在我们的初步研究通过与精子相关的蛋白质组学分析鉴定了这些 PP1 磷酸酶减数分裂染色质，并在雄性生育力和染色体分离方面显示出保守的功能。线虫到哺乳动物；然而，每个 PP1 家族成员如何控制一般减数分裂机器以及精子减数分裂的不同方面的实现尚不清楚。定义这些蛋白质如何在精子中发挥作用减数分裂，我们将追求三个具体目标。首先，因为 GSP-1 和 GSP-2 都与两者相关精子和卵母细胞染色质，我们将分析 gsp-7 和 gsp-2 的功能丧失来区分精子特异性来自 GSP-1 和 GSP-2 的卵母细胞特异性功能。我们还会发现功能上的差异精子中的 GSP-1 和 GSP-2 与 GSP-3 和 GSP-4（仅在精子染色质上发现）的区别减数分裂。其次，我们将表征 PP1 磷酸酶与已知作用于的蛋白质的相互作用。染色体分离，如着丝粒的组成部分。着丝粒蛋白在纺锤体中发挥作用 DMA 的附着，我们在初步研究中发现，有些在精子中存在差异与卵母细胞减数分裂。因此，我们将进行共定位和共免疫沉淀实验发现与动粒和其他核心分离机制的特定组件的相互作用。第三，我们将鉴定与 PP1 磷酸酶物理相关的信号通路成分通过免疫共沉淀和质谱法进行精子发生，然后进行 RNAi 分析以确定与 GSP 蛋白类似的生育作用的候选者。通过这种方法，我们不仅可以识别信号通路成员，也是控制精子减数分裂所需的核心分离机制部门。与公共健康的相关性：染色体中 PP1 磷酸酶的精子特异性功能分离已经被描绘出来，并且这些保守磷酸酶的分子靶点或调节剂如果发现的话，我们将发现以前未知的男性不育和生殖障碍的原因。由于这些蛋白质从蠕虫到人类都是保守的，因此我们发现了其成分和机制对于了解人类不孕症非常重要。