Protein Phosphatase Action in Mammalian Spermatogenesis and Sperm Function

描述（由申请人提供）：蛋白磷酸酶1的四种亚型(PP1¿，PP1¿，PP1？1和PP1？2)，由三个基因衍生而来，除了极端的c端外，几乎是相同的。同工异构体PP1？和哺乳动物特有的PP1？2个来源于单个基因的剪接变异转录本（Ppp1cc）。尽管所有四种PP1亚型都在睾丸中表达，但PP1？到目前为止，PP1 2在男性生殖细胞中占主导地位，是哺乳动物精子中唯一的PP1亚型。PP1吗?2异构体具有独特的22个氨基酸的c端尾部。Ppp1cc的靶向缺失导致-/-雄性不育，这是由于精子形态发育的严重缺陷和精子的错误。我们测试了转基因PP1？2还是PP1？1由睾丸特异性Pgk2启动子或Ppp1cc内源性启动子驱动，可以恢复Ppp1cc -/-雄性（拯救小鼠）的生育能力。我们的初步研究表明，只有当足够高的PP1？2在分化的雄性生殖细胞中表达。这种生育能力的恢复即使在PP1？1异构体完全不存在。低水平的PP1？2导致精子运动能力差，并伴有一些形态异常。PP1吗?1救援小鼠也比Ppp1cc -/-小鼠产生更多的精子数量。然而，精子严重畸形，完全不动。本建议的重点是确定适当水平PP1的绝对和同种形式的具体要求。2导致哺乳动物精子的正常结构和功能。我们将确定PP1的异构体是否具有特异性功能？2是由c端决定的。最后，我们将确定PP1的极限水平如何？2改变精子蛋白质组，从而导致结构畸形和精子功能受损。这些研究将继续揭示哺乳动物精子形态发生和功能的新机制，从而基于PP1？2个在精子中。

DESCRIPTION (provided by applicant): Four isoforms of protein phosphatase 1 (PP1¿, PP1¿, PP1? 1, and PP1?2), derived from three genes, are virtually identical, except at their extreme C-termini. The isoforms PP1?1 and mammalian-specific PP1?2 derive from splice-variant transcripts of a single gene (Ppp1cc). Although all four PP1 isoforms are expressed in testis, PP1?2 is by far predominant in male germ cells, and is the sole PP1 isoform present in mammalian spermatozoa. The PP1?2 isoform has a unique 22 amino acid C-terminal tail. Targeted deletion of Ppp1cc leads to sterility of -/- males due to grossly defective morphological development of spermatids and faulty spermiation. We tested whether transgenic expression of PP1?2 or PP1?1 driven by the testis- specific Pgk2 promoter or the Ppp1cc endogenous promoter could restore fertility of Ppp1cc -/- males (rescue mice). Our preliminary studies show that normal sperm morphogenesis and fertility are restored only when adequately high levels of PP1?2 are expressed in differentiating male germ cells. This restoration of fertility occurs even when the PP1?1 isoform is completely absent. Low levels of PP1?2 result in poorly motile sperm with several morphological abnormalities. PP1?1 rescue mice also produce greater sperm numbers than Ppp1cc -/- mice. However, sperm are grossly malformed and completely immotile. The focus of this proposal is to identify the absolute and isoform specific requirement for adequate levels of PP1?2 leading to normal structure and function of mammalian spermatozoa. We will determine whether the isoform specific function of PP1?2 is determined by its C-terminus. Finally, we will ascertain how limiting levels of PP1?2 alter the sperm proteome, thus resulting in structural deformities and impaired sperm function. These studies will continue to unravel novel mechanisms involved in mammalian sperm morphogenesis and function, and thus lead to simple diagnostic tests for fertility in man and other mammals based on the level of PP1?2 in spermatozoa. PUBLIC HEALTH RELEVANCE: Statement Alternatively spliced protein phosphatase PP1?2 isoform of PP1 ? gene plays an essential role in spermatogenesis and mammalian reproduction. However, molecular mechanisms responsible for its regulation and action in testis and spermatozoa are unclear. We will identify how PP1? gene and its protein product PP1?2 in testis and spermatozoa are responsible for sperm formation and function.