A knock-in mouse model for male fertility: basis for the mammal-specific protein phosphatase isoform PP1y2 in sperm

雄性生育能力的敲入小鼠模型：精子中哺乳动物特异性蛋白磷酸酶亚型 PP1y2 的基础

• 批准号: 10675027
• 负责人: SRINIVASAN VIJAYARAGHAVAN
• 金额: $ 7.6万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10675027
• 关键词: Affect; Alternative Splicing; Amino Acid Sequence; Animals; Binding; Biochemical; Brain; CRISPR/Cas technology; Calcineurin; Calcium; Cells; Characteristics; Clinical; Complex; Consensus; Couples; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Energy Metabolism; Engineering; Enzymes; Epididymis; Exons; Female; Fertility; Gene Expression; Genes; Genetic; Genome; Germ Cells; Glycogen Synthase Kinase 3; Impairment; In Vitro; Infertility; Introns; Invertebrates; Knock-in Mouse; Knock-out; Knockout Mice; Male Infertility; Mammals; Messenger RNA; Metabolism; Morphogenesis; Mus; Nucleotides; Penetration; Phenotype; Phosphoproteins; Physiological Processes; Process; Protein Isoforms; Protein phosphatase; Proteins; RNA Splicing; Role; Second Messenger Systems; Seminiferous tubule structure; Signal Transduction; Signaling Protein; Site; Somatic Cell; Sperm Motility; Spermatogenesis; Spermatogenic Cell; Testis; Tissues; Transcript; Vertebrates; Wild Type Mouse; cell motility; egg; genetic manipulation; inorganic phosphate; insight; knockout gene; male; male fertility; man; mouse model; paralogous gene; placental mammal; promoter; reproductive tract; sperm cell; sperm function; sperm protein; transgene expression; zygote

Spermatogenesis and the development of fertility competent sperm involve complex processes in the testis, epididymis and female reproductive tract. These physiological processes of sperm formation and development are difficult if not impossible to be recapitulated and studied in vitro. Mouse models amenable to genetic manipulation are essential for understanding the basis for male gamete function. Considerable progress has been made in identifying signaling proteins essential for sperm function which include components of cyclic AMP metabolism, proteins controlling sperm intracellular pH and calcium levels of sperm in the epididymis and in the female reproductive tract. Numerous gene knock out approaches targeting the signaling proteins involved in metabolism and in the action of second messengers result in male infertility. Yet the mechanistic basis for normal and disrupted sperm function remains largely unknown. We discovered that a protein phosphatase PP12, which is one of two paralogs from one gene Ppp1cc, is highly expressed in testis and present in sperm. The other paralog, PP11, is expressed in brain and several somatic cells and tissues and cells. Two other genes encode the PP1 and PP1 isoforms. These four PP1 isoforms are highly conserved between themselves and across species. The isoform PP12 is present only in placental mammals suggesting an essential role for it in the unique features of mammalian sperm function. It is notable that sperm from other species contain one of the protein phosphate isoforms PP11, PP1 or PP1. The targeted knock out of Ppp1cc results in male infertility. Transgenic expression of PP12, but not PP11, driven by a testis specific promoter restores fertility in the Ppp1cc null mice, highlighting the essential requirement for PP12. In this proposal we will examine a mouse line we have generated where the Ppp1cc gene is edited by Crispr/Cas9 to express PP11 alone in testis. Determination of the impaired functions in PP11 bearing mice and sperm should lead to the identification of key proteins essential for normal sperm function. Male infertility is responsible for about 10% of infertile couples. Identification of the causes and treatments for male infertility are limited. A number of factors can affect male infertility: major factors are likely environmental or genetic. This mouse model bearing the non-mammalian PP1 isoform in sperm should also be valuable in not only understanding the significance of the presence of PP12 in all mammals but also in understanding the biochemical basis for fertility and infertility in man.

精子发生与具有生育能力的精子发育关系复杂 睾丸、附睾部和女性生殖道的突起。这些生理过程 即使不是不可能，也很难概括和研究。 在试管中。能够进行基因操作的小鼠模型对于理解这一基础是必不可少的 用于雄配子功能。 在识别信号转导蛋白方面已经取得了相当大的进展 精子功能包括环磷酸腺苷代谢成分、控制精子的蛋白质 附睾和女性生殖道内精子的细胞内pH和钙水平。 大量针对信号蛋白的基因敲除方法涉及新陈代谢和 在第二信使的作用下会导致男性不育。然而，正常的机械论基础 而精子功能受损在很大程度上仍是未知的。 我们发现了一种蛋白磷酸酶Pp12，它是来自 Ppp1cc基因在睾丸中高度表达，在精子中存在。另一个Paralog，pp11， 在大脑和几个体细胞以及组织和细胞中表达。另外两个基因编码 Pp1和pp1亚型。这四种PP1亚型之间高度保守 跨物种。Pp12的亚型只存在于胎盘哺乳动物中，这表明 对于它在哺乳动物精子功能中的独特功能。值得注意的是，来自其他地方的精子 物种含有蛋白质磷酸异构体pp11、pp1或pp1中的一种。定向敲打 Ppp1cc的缺失会导致男性不育。PP12的转基因表达，而不是PP11的转基因表达 睾丸特异性启动子可恢复pp1cc基因缺失小鼠的生育能力，突出了 对于pp12的要求。在本提案中，我们将检查我们在以下位置生成的鼠标行 Pp1cc基因由Crispr/Cas9编辑，在睾丸中单独表达pp11。确定了 携带Pp11的小鼠和精子的功能受损应导致关键蛋白的鉴定 对于正常的精子功能来说是必不可少的。 男性不育约占不育夫妇总数的10%。身份识别 男性不育的原因和治疗是有限的。影响男性不育的因素有很多： 主要因素可能是环境或遗传因素。这是一种非哺乳动物的小鼠模型 精子中的PP1亚型也应该不仅在理解PP1的意义上有价值 Pp12在所有哺乳动物中的存在，也有助于理解生育和 男性不育症。