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 specialbiology of sperm meiotic chromosome segregation. Our goal is to define sperm-specific molecularmechanisms that compact and segregate paternal DMA properly for reproductive success. In thisapplication, our objective is to determine how four PP1 phosphatase proteins (GSP-1, GSP-2, GSP-3 andGSP-4) function in chromosome segregation during sperm formation in the nematode C. elegans. In ourpreliminary studies these PP1 phosphatases were identified by proteomic analysis associated with spermmeiotic 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 andimplements distinct aspects of sperm meiosis is not known. To define how these proteins function in spermmeiosis, we will pursue three specific aims. First, because GSP-1 and GSP-2 are associated with bothsperm and oocyte chromatin, we will analyze the loss-of-function of gsp-7 and gsp-2 to differentiate spermspecificfrom oocyte-specific functions of GSP-1 and GSP-2. We will also find differences in function ofGSP-1 and GSP-2 from those of GSP-3 and GSP-4 (which are found only on sperm chromatin) in spermmeiosis. Second, we will characterize PP1 phosphatase interactions with proteins that are known to act inchromosome segregation, like components of the kinetochore. Kinetochore proteins function in spindleattachment to DMA and we have found in preliminary studies that some are localized differentially in spermversus oocyte meiosis. We will therefore conduct colocalization and coimmunopreciptiation experiments tofind 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 duringspermatogenesis through coimmunoprecipitation and mass spectrometry, followed by feeding RNAi analysisto pinpoint candidates with roles in fertility similar to GSP proteins. By this approach we will identify not onlysignaling pathway members, but also core segregation machinery required to control sperm meioticdivisions.Relevance to Public Health: Once sperm-specific functions of PP1 phosphatases in chromosomesegregation have been delineated and molecular targets or regulators of these conserved phosphatasesfound, 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 uncoverwill be important to understanding human infertility.
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