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（应用程序提供）：蛋白质磷酸酶1的四种同工型（pp1¿，pp1¿，pp1¿和pp1？2），源自三个基因，实际上是相同的，除了其极端的C-末端。同工型PP1？1和哺乳动物特异性的PP1？2源自单个基因（PPP1CC）的剪接变体转录本。尽管所有四种PP1同工型均在睾丸中表达，但pp1？2在雄性生殖细胞中占主导地位，并且哺乳动物精子中是否存在唯一的PP1同工型。 PP1？2同工型具有独特的22个氨基酸C末端尾巴。 PPP1CC的靶向缺失导致 - / - 雄性的无菌性，由于精子的形态学出现严重缺陷和精子错误。我们测试了由睾丸特异性PGK2启动子驱动的PP1？2或PP1？1的转基因表达，还是PPP1CC内源性启动子可以恢复PPP1CC - / - 雄性（救援小鼠）的生育能力。我们的初步研究表明，正常的精子形态发生和生育能够恢复，只有在分化的男性生殖细胞中表达足够高的PP1？2时。即使PP1？1同工型完全不存在，这种生育能力也会发生。 PP1？2的水平低导致运动精子较差，几种形态异常。 PP1？1救援小鼠的精子数也比PPP1CC - / - 小鼠更大。但是，精子严重畸形，完全不受影响。该提案的重点是确定对PP1的足够水平的绝对和同工型特异性要求，从而导致哺乳动物精子的正常结构和功能。我们将确定PP1的同工型特异性功能是否由其C端确定。最后，我们将确定限制PP1的水平？2如何改变精子蛋白组，从而导致结构畸形和精子功能受损。这些研究将继续揭示涉及哺乳动物精子形态发生和功能的新型机制，从而根据精子中的PP1？2的水平进行简单的人类和其他哺乳动物的诊断测试。公共卫生相关性：陈述替代剪接蛋白质磷酸酶PP1？2 PP1的同工型？基因在精子发生和哺乳动物繁殖中起着至关重要的作用。然而，尚不清楚负责其在睾丸和精子中调节其调节和作用的分子机制尚不清楚。我们将确定PP1的方式？基因及其蛋白质产物PP1？基因及其蛋白质产物PP1？ 2在睾丸和精子中是负责精子的形成和功能。