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.

精子发生和有生育能力精子的发育涉及复杂的 睾丸、附睾和雌性生殖道中的突起。这些生理过程 精子形成和发育的过程如果不是不可能的话， 体外易于遗传操作的小鼠模型对于理解 雄性配子的功能。 在识别对细胞凋亡至关重要的信号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 附睾和雌性生殖道中精子的细胞内pH和钙水平。 许多基因敲除方法靶向参与代谢的信号蛋白， 第二信使的作用导致男性不育。然而，正常的机械基础 以及精子功能受损的情况仍不清楚。 我们发现，蛋白磷酸酶PP 1 β 2，这是两个旁系同源物之一， 一个基因Ppp 1cc，在睾丸中高表达，存在于精子中。另一个参数PP 1 - 1是 在脑和几种体细胞、组织和细胞中表达。另外两个基因编码 PP 1 β和PP 1 β同种型。这四种PP 1同种型在它们之间高度保守， 跨越物种。同种型PP 1 β 2仅存在于胎盘哺乳动物中，表明其在哺乳动物中是必需的。 它在哺乳动物精子功能的独特功能中的作用。值得注意的是，来自其他国家的精子 物种含有蛋白磷酸盐同种型PP 1 β 1、PP 1 β 2或PP 1 β 3之一。有针对性的敲门声 Ppp 1cc导致男性不育。PP 1 β 2的转基因表达，而不是PP 1 β 1，由 睾丸特异性启动子恢复Ppp 1cc基因敲除小鼠的生育能力，突出了 PP 1的要求在这个建议中，我们将检查我们生成的鼠标线，其中 通过Crispr/Cas9编辑Ppp 1cc基因，以在睾丸中单独表达PP 1 β 1。测定 携带PP 1 β 1的小鼠和精子的功能受损应导致关键蛋白的鉴定 对正常的精子功能至关重要。 男性不育是造成约10%的不育夫妇。识别 男性不育症的病因和治疗方法有限。许多因素会影响男性不育： 主要因素可能是环境或遗传。这种携带非哺乳动物的小鼠模型 精子中的PP 1亚型不仅在理解精子中PP 1的重要性方面， PP 1 β 2在所有哺乳动物中的存在，而且在理解生育力的生化基础方面， 男性不育症