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: 10527437
• 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/10527437
• 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; Knock-in Mouse; Knock-out; Knockout Mice; Lead; Male Infertility; Mammals; Messenger RNA; Metabolism; Morphogenesis; Mus; Nucleotides; 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; Transgenic Organisms; Vertebrates; Wild Type Mouse; cell motility; egg; genetic manipulation; inorganic phosphate; insight; male; male fertility; man; mouse model; paralogous gene; placental mammal; promoter; reproductive tract; sperm cell; sperm function; sperm protein; 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.

精子发生和生育能力精子的发展涉及复合物 睾丸，附睾和雌性生殖道的过程。这些物理过程 即使不是不可能，很难概括和研究精子的形成和发育 体外。适合遗传操作的小鼠模型对于理解基础至关重要 用于男配子功能。 在识别信号蛋白中已经取得了很大进展 精子功能，包括环状AMP代谢的组成部分，控制精子的蛋白质 附睾和雌性复制道中的细胞内pH和精子水平。 许多基因淘汰了针对与代谢有关的信号蛋白的方法 在第二使者的行动中，导致男性不育症。但是正常的机械基础 而被破坏的精子功能仍然在很大程度上未知。 我们发现蛋白质磷酸酶PP12是来自 一个基因Ppp1cc，在睾丸中高度表达，并存在于精子中。另一个旁系同源物pp11是 在大脑以及几个体细胞，组织和细胞中表达。其他两个基因编码 PP1和PP1同工型。这四个PP1同工型在他们之间高度保守， 跨物种。同工型PP12仅存在于斑点哺乳动物中，这表明必不可少 它在哺乳动物精子功能的独特特征中的作用。值得注意的是其他精子 物种含有磷酸蛋白质同工型PP11，PP1或PP1。有针对性的敲门 从PPP1CC中导致男性不育症。 pp12的转基因表达，而不是pp11，由pp11驱动 睾丸特异性启动子恢复了PPP1CC无效小鼠的生育能力，突出了必不可少的 PP12的要求。在此提案中，我们将检查我们已经生成的鼠标线 PPP1CC基因由CRISPR/CAS9编辑以在睾丸中单独表达PP11。确定 PP11轴承小鼠和精子中功能受损应导致关键蛋白的鉴定 正常精子功能必不可少的。 男性不育症约有10％的不育夫妇。识别 男性不育的原因和治疗量受到限制。许多因素会影响男性不育症： 主要因素可能是环境或遗传因素。这个带有非哺乳动物的鼠标模型 精子中的PP1同工型也不仅在理解的意义方面都很有价值 在所有哺乳动物中都存在pp12，但在理解生育能力和 人的不育。