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生精小管中持续进行的。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，通过在小鼠身上产生导致男性不育的突变，哺乳动物精子发生的研究得到了加强。其中一个突变，蛋白磷酸酶1cg(PP1cg)基因敲除小鼠是我们实验室制造的，也是这次拨款申请的重点。我们之前的研究揭示了PP1cg基因表型的各个方面，表明PP1cg基因可能具有多种功能(早产生殖细胞萎缩；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一致，该酶由PP1cg等催化亚基和大约50个不同调节亚基中的一个组成。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，但不能取代PP1cg的丢失。这导致我们假设PP1cg必须与一个或多个无法与PP1ca或PP1cb结合的睾丸特异性调节亚基结合。酵母双杂交筛选出5个克隆，它们可能是PP1cg的睾丸特异结合伙伴，产生与睾丸特异功能相关的全酶，即睾丸特异的去磷酸化靶标。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蛋白质组分析，使用尖端技术比较突变和正常睾丸中的磷酸化蛋白质。另一种方法将使用我们实验室中确定的调节亚单位作为筛选相互作用蛋白质的诱饵。这些实验将帮助我们了解PP1cg如何通过识别在共同途径中发挥作用的其他蛋白质来影响精子发生。我们的研究将丰富对哺乳动物精子发生的一般理解，特别是当它出错的时候。

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.