Defining Roles of PP1 Phosphatases in Sperm Meiosis

精子减数分裂染色体分离错误对人类有严重的后果：男性不育， 胚胎致死和发育异常许多这样的错误是由于特殊的 精子减数分裂染色体分离的生物学。我们的目标是确定精子特异性分子 紧凑和分离父系DNA的机制，适当的繁殖成功。在这 应用，我们的目标是确定四个PP 1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起作用。优雅在我们 这些PP 1磷酸酶的初步研究是通过与精子相关的蛋白质组学分析鉴定的 在雄性育性和染色体分离中具有保守的功能。 然而，每个PP 1家族成员如何控制一般的减数分裂机制， 实现精子减数分裂的不同方面是未知的。为了确定这些蛋白质在精子中的功能 减数分裂，我们将追求三个具体目标。第一，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，以区分精子特异性 从GSP-1和GSP-2的卵母细胞特异性功能。我们还将发现， GSP-1和GSP-2与GSP-3和GSP-4（仅在精子染色质上发现）的差异 减数分裂第二，我们将描述PP 1磷酸酶与已知作用于 染色体分离，像动粒的组成部分。动粒蛋白在纺锤体中的功能 我们在初步研究中发现，有些DNA在精子中的定位差异很大， 相对于卵母细胞减数分裂。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成分的相互作用。第三、 我们将确定信号通路的组成部分，物理上与PP 1磷酸酶， 通过免疫共沉淀和质谱分析精子发生，然后进行喂食RNAi分析 以确定与GSP蛋白在生育中具有相似作用的候选蛋白。通过这种方法，我们不仅可以识别 信号通路成员，而且是控制精子减数分裂所需的核心分离机制 分裂 与公共卫生的相关性：染色体上PP 1磷酸酶的精子特异性功能 分离已经被描绘，并且这些保守的磷酸酶的分子靶标或调节剂 一旦发现，我们将发现以前未知的男性不育和生殖失败的原因。 因为这些蛋白质从蠕虫到人类都是保守的，我们发现了它们的组成和机制， 对了解人类不孕症很重要

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.