Molecular analysis of male infertility

配子的产生是物种生存的核心。在哺乳动物中，精子的产生是在睾丸的精管中以连续的方式进行的。这需要支持体细胞支持细胞和生殖细胞（精子前体）之间复杂的细胞-细胞相互作用。近年来，对哺乳动物精子发生的研究得到了加强，因为老鼠体内产生了导致雄性不育的突变。其中一个突变，蛋白磷酸酶1cg （PP1cg）敲除小鼠是在我们的实验室制造的，也是本次资助申请的重点。我们之前的研究已经揭示了表型的各个方面，表明PP1cg基因可能具有不止一种功能（生殖细胞过早脱落、精子发育异常、生殖系干细胞丢失）。这些观察结果与1型丝氨酸苏氨酸磷酸酶的多功能性相一致，这种酶由催化亚基如PP1cg和大约50种不同的调节亚基之一组成。在我们对突变体的初步分析中，我们观察到另外两个几乎相同的1型催化亚基PP1ca和PP1cb在睾丸中表达，但无法替代PP1cg的缺失。这导致我们假设PP1cg必须结合一个或多个不能结合PP1ca或PP1cb的睾丸特异性调节亚基。对这类异构体特异性相互作用蛋白进行酵母双杂交筛选，发现了5个可能是PP1cg睾丸特异性结合伙伴的克隆，它们产生具有睾丸特异性功能的独特全酶，即睾丸特异性去磷酸化靶点。我们的研究计划包括使用几种方法寻找这些目标。一种方法是全组织磷酸化蛋白组分析，使用尖端技术比较突变和正常睾丸中的磷酸化蛋白。另一种方法将涉及使用我们实验室鉴定的调节亚基作为筛选相互作用蛋白质的诱饵。这些实验将通过识别在共同途径中起作用的其他蛋白质，帮助我们了解PP1cg如何影响精子发生。我们的研究将丰富对哺乳动物精子发生的一般理解，特别是当它出错时。

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.