The role Y chromosome genes Prssly and Teyorf1 in male reproduction.

Y 染色体基因 Prssly 和 Teyorf1 在男性生殖中的作用。

• 批准号: 10337013
• 负责人: Monika A Ward
• 金额: $ 7.25万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-27 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10337013
• 关键词: Address; Automobile Driving; Back; Biological Assay; Blood-Testis Barrier; C-terminal; CRISPR/Cas technology; Chromosome Structures; Collaborations; Data; Defect; Development; Distal; Effectiveness; Electroporation; Evolution; FLAG peptide; Fertility; Fertilization; Future; Gene Proteins; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genotype; Germ Cells; Homologous Gene; Immunoblotting; Immunofluorescence Immunologic; In Vitro; Investigation; Knock-in; Knock-in Mouse; Knock-out; Knowledge; Link; Maintenance; Male Infertility; Mediating; Medical; Morphology; Mus; Oocytes; Open Reading Frames; Partner in relationship; Peptide Hydrolases; Phenotype; Physiological Processes; Play; Protein Family; Proteins; RNA Interference; Reporter; Reporting; Reproduction; Research Personnel; Resistance; Role; Serine; Serine Protease; Side; Sperm Motility; Spermatogenesis; System; Technology; Terminator Codon; Testing; Testis; Tissues; Transgenes; Translations; Y Chromosome; arm; cell motility; cell type; claudin 3; deletion analysis; gene function; interest; male; male fertility; medical specialties; member; molecular sequence database; mouse genome; positional cloning; protein expression; protein structure function; reproductive; reproductive function; sex; sex determination; sperm cell; sperm function; spermatogenic epithelium structure; sry Genes; structural genomics; transcription activator-like effector nucleases; ward; zygote

Project Summary/Abstract The mammalian Y chromosome is a symbol of maleness as it encodes the Sry gene driving male sex determination, a battery of other genes thought to be involved in various aspects of male reproduction, and other genes playing roles of broadly expressed regulators of transcription, translation and protein stability. In spite of these clearly important functions, the knowledge linking the roles of specific Y chromosome genes to specific reproductive and other physiological processes remains limited. This is due to the unusual genomic structure of this chromosome, which for decades rendered it resistant to any targeting attempts. With the emergence of TALEN and CRISPR/Cas9 technologies addressing Y chromosome gene function directly became possible. The oddity of the Y chromosome structure made it also defiant to sequencing. The mouse Y chromosome sequence was finally defined in 2014, 12 years after the remainder of the mouse genome was characterized. When the mouse Y sequence was revealed two new single copy genes were identified on the Y short arm: Prssly (Y-linked serine-like protease) and Teyorf1 (testis expressed, chromosome Y open reading frame 1). These genes were classified as ‘acquired genes’ and were proposed to be the candidates for male fertility genes. Neither of these genes has been targeted and their function remains unknown. In Preliminary Data we show that Prssly and Teyorf1 expression is restricted to testicular postmeiotic germ cells. We also analyzed PRSSLY and TEYORF1 predicted protein structure and function and found that both predicted proteins resemble proteins that are known to play roles in spermatogenesis. PRSSLY belongs to the peptidase S1 family of proteins that include serine proteases PRSS21 and PRSS55 essential for sperm motility and sperm ability to fertilize oocytes. TEYORF1 belongs to the Claudin family of proteins members of which were reported important for assembly and maintenance of blood-testis barrier, a tissue barrier critical for normal spermatogenesis. Acquisition to the Y chromosome, testis specific expression, and resemblance to proteins of known reproductive functions prompted us to propose a hypothesis that Prssly and Teyorf1 play roles in male reproduction. We will address this hypothesis pursuing two specific aims: Aim 1: Generate Prssly and Teyorf1 knock-out (KO) and Prssly and Teyorf1 reporter tag knock-in (KI) mice using CRISPR/Cas9 system and zygote electroporation. Aim 2: Characterize phenotype of these mice focusing on assessment of fertility, sperm parameters, sperm function in vitro, and spermatogenesis progression. The combined results will allow to define whether Prssly and Teyorf1 play roles in spermatogenesis and fertility. The development of KI mice will open the door to future mechanistic investigations of these factors. The project will impact on understanding of Y chromosome gene function and genetic basis of male fertility. It will also contribute to discussions regarding the evolution of Y chromosomes and the importance of acquired Y genes.

项目概要/摘要 哺乳动物的 Y 染色体是男性的象征，因为它编码驱动男性性别决定的 Sry 基因， 一系列其他基因被认为参与男性生殖的各个方面，以及其他发挥作用的基因 尽管这些显然很重要，但广泛表达的转录、翻译和蛋白质稳定性调节因子。 功能，将特定 Y 染色体基因的作用与特定生殖和其他生理功能联系起来的知识 这是由于这条染色体的不寻常的基因组结构，几十年来一直受到限制。 随着针对 Y 的 TALEN 和 CRISPR/Cas9 技术的出现，它对任何靶向尝试都有抵抗力。 染色体基因功能直接成为可能。 Y染色体结构的奇特性也使其无法测序。 2014 年，即小鼠 Y 序列的其余部分被定性后的 12 年，它终于得到了定义。 结果显示，在 Y 短臂上发现了两个新的单拷贝基因：Prssly（Y 连接丝氨酸样蛋白酶）和 Teyorf1（睾丸表达，Y 染色体开放阅读框 1）这些基因被归类为“获得性基因”并被 被提议作为男性生育基因的候选者，这些基因及其功能都尚未被靶向。 仍然未知。 在初步数据中，我们还表明 Prssly 和 Teyorf1 的表达仅限于睾丸减数分裂后生殖细胞。 分析了 PRSSLY 和 TEYORF1 预测的蛋白质结构和功能，发现两种预测的蛋白质类似于 已知在精子发生中发挥作用的蛋白质属于肽酶 S1 蛋白质家族。 包括对精子活力和精子使卵母细胞受精的能力至关重要的丝氨酸蛋白酶 PRSS21 和 PRSS55。 属于 Claudin 蛋白质家族，据报道该家族成员对于组装和维持非常重要 血睾屏障，一种对正常精子发生至关重要的组织屏障。 获得 Y 染色体、睾丸特异性表达以及与已知生殖功能的蛋白质的相似性 促使我们提出一个假设，即 Prssly 和 Teyolf1 在男性生殖中发挥作用，我们将解决这个问题。 假设追求两个特定目标： 目标 1：使用 Prssly 和 Teyorf1 敲除 (KO) 小鼠以及 Prssly 和 Teyorf1 报告基因敲入 (KI) 小鼠 CRISPR/Cas9 系统和合子电穿孔。 目标 2：表征这些小鼠的表型，重点评估生育能力、精子参数、精子功能 体外和精子发生进展。 综合结果将有助于确定 Prssly 和 Teyorf1 是否在精子发生和生育能力中发挥作用。 KI 小鼠的开发将为未来研究这些因素的影响机制打开大门。 了解 Y 染色体基因功能和男性生育力的遗传基础也将有助于讨论。 关于 Y 染色体的进化和获得性 Y 基因的重要性。