Molecular analysis of male infertility

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Gamete production is central to species survival. In mammals, production of sperm takes place in the seminiferous tubules of the testis in a continuous fashion. This requires complex cell-cell interactions between the supporting somatic Sertoli cells and the germ cells (sperm precursors). The study of spermatogenesis in mammals has been augmented in recent years by the generation of mutations in mice that cause male infertility. One of these mutations, the Protein phosphatase 1cg (PP1cg) knockout mouse was made in our lab, and is the focus of this grant application. Our previous studies have revealed various aspects of the phenotype that suggest the PP1cg gene may have more than one function (premature germ cell sloughing; abnormal sperm development; loss of germline stem cells). These observations are consistent with the multifunctionality of Type 1 serine threonine phosphatases, enzymes composed of catalytic subunits like PP1cg and one of approximately 50 different regulatory subunits. In our initial analysis of the mutants, we observed that two other almost identical Type 1 catalytic subunits, PP1ca and PP1cb, are expressed in the testis, yet are unable to substitute for loss of PP1cg. This led us to hypothesize that PP1cg must bind to one or more testis specific regulatory subunits that are unable to bind to PP1ca or PP1cb. A yeast two hybrid screen for such isoform specific interacting proteins revealed five clones which may be the testis specific binding partners of PP1cg that produce unique holoenzymes with testis specific functions, ie testis specific dephosphorylation targets. Our research plan involves searching for these targets using several approaches. One approach is whole tissue phosphoproteome analysis, using cutting edge technology to compare the phosphorylated proteins in mutant and normal testes. Another approach will involve the use of the regulatory subunits identified in our lab as bait in a screen for interacting proteins. These experiments will help us understand how PP1cg affects spermatogenesis by identifying other proteins that act in a common pathway. Our studies will enrich the general understanding of mammalian spermatogenesis, especially when it goes awry.