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。许多这样的错误是特殊的 精子减数分裂染色体分离生物学。我们的目标是定义精子特异性分子 适当地压缩和分离父亲的DNA以获得生殖成功的机制。在这 应用，我们的目标是确定四个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起作用。在我们的 通过与精子相关的蛋白质组学分析，初步鉴定了这些PP1磷酸酶 减数分裂染色质在雄性育性和染色体分离中显示出保守功能。 然而，每个PP1家族成员如何控制一般的减数分裂机制和 实施精子减数分裂的不同方面尚不清楚。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。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，以区分精子特异性 GSP-1和GSP-2的卵母细胞特异性功能。我们还会发现在功能上的差异 精子中GSP-1和GSP-2来自GSP-3和GSP-4(仅在精子染色质中发现) 减数分裂。其次，我们将描述PP1磷酸酶与已知作用于 染色体分离，类似于着丝粒的成分。动粒蛋白在纺锤体中的功能 附着在DMA上，我们在初步研究中发现，一些在精子中存在差异 与卵母细胞减数分裂的对比。因此，我们将进行共定位和免疫共沉淀实验，以 找出与动粒和其他核心分离机制的特定组件之间的相互作用。第三, 我们将确定与PP1磷酸酶物理上相关的信号通路组件 通过免疫共沉淀和质谱分析精子发生，随后进行喂养RNAi分析 以确定与GSP蛋白类似的在生育中起作用的候选基因。通过这种方法，我们不仅将识别 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 组织。 与公共健康相关：染色体上的PP1磷酸酶曾经具有精子特有的功能 已经描述了这些保守的磷酸酶的分离和分子靶点或调节因子 一旦发现，我们就会揭开以前不为人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，我们发现了这些蛋白质的成分和机制 对于了解人类不孕不育将是非常重要的。

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.