Defining Roles of PP1 Phosphatases in Sperm Meiosis

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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.