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Protein Phosphatase Action in Mammalian Spermatogenesis and Sperm Function

蛋白磷酸酶在哺乳动物精子发生和精子功能中的作用

基本信息

批准号：
8289861
负责人：
SRINIVASAN VIJAYARAGHAVAN
金额：
$ 42.21万
依托单位：
KENT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-16 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8289861
关键词：
Amino Acid Sequence Homology Amino Acids Binding Biochemical C-terminal DNA-Binding Proteins Defect Deformity Development Diagnostic tests Diamond Enzymes Epididymis Fertility Fluorescence Genes Germ Cells Human In Vitro Infertility Knockout Mice Lead Male Sterility Mammals Messenger RNA Molecular Morphogenesis Mus Phenotype Phosphorylation Play Protamines Protein Isoforms Protein phosphatase Proteins Proteome RNA Splicing Regulation Reproduction Role Serine Signal Transduction Somatic Cell Sperm Count Procedure Sperm Motility Spermatids Spermatogenesis Staining method Stains Structural Protein Structure Tail Testing Testis Threonine Transcript Transgenic Mice Transgenic Organisms Translating Variant Wild Type Mouse base cell motility fascinate gel electrophoresis in vivo male mammalian genome man novel promoter protein profiling restoration sertoli cell sperm cell sperm function sperm morphology

项目摘要

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.

描述（申请人提供）：蛋白磷酸酶1的四种亚型（PP 1 <$、PP 1 <$、PP 1？1、PP1？2），来自三个基因，实际上是相同的，除了在其极端的C-末端。异构体PP 1？1和特定的PP 1？2来源于单个基因的剪接变体转录本（Ppp 1cc）。虽然所有四个PP 1亚型表达在睾丸，PP 1？2在雄性生殖细胞中占主导地位，是哺乳动物精子中唯一的PP 1亚型。PP1？2同种型具有独特的22个氨基酸的C-末端尾。Ppp 1cc的靶向缺失由于精子细胞的形态发育严重缺陷和精子形成缺陷而导致-/-雄性不育。我们测试是否转基因表达PP 1？2还是PP 1？由睾丸特异性Pgk 2启动子或Ppp 1cc内源性启动子驱动的1可以恢复Ppp 1cc-/-雄性（拯救小鼠）的生育力。我们的初步研究表明，正常的精子形态发生和生育能力恢复时，只有足够高水平的PP 1？2在分化的雄性生殖细胞中表达。这种恢复生育能力发生时，即使PP 1？1同种型完全不存在。低水平的PP 1？2导致精子运动不良，伴有几种形态异常。PP1？1救援小鼠产生的精子数量也高于Ppp 1cc-/-小鼠。然而，精子严重畸形，完全不动。本提案的重点是确定PP 1适当水平的绝对和亚型特定要求？2导致哺乳动物精子的正常结构和功能。我们将确定是否亚型特异性功能的PP 1？2由其C-末端决定。最后，我们将确定如何限制PP 1的水平？2改变精子蛋白质组，从而导致结构畸形和精子功能受损。这些研究将继续解开新的机制，参与哺乳动物精子的形态和功能，从而导致简单的诊断测试的基础上的水平，在人类和其他哺乳动物的生育能力？精子中2个。公共卫生相关性：声明选择性剪接蛋白磷酸酶PP 1？2亚型PP 1？基因在精子发生和哺乳动物生殖中起着重要作用。然而，其在睾丸和精子中的调节和作用的分子机制尚不清楚。我们将如何识别PP 1？基因及其蛋白产物PP 1？2在睾丸和精子中的表达与精子的形成和功能有关。