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

项目摘要/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，a 一组其他基因，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 广泛表达的转录、翻译和蛋白质稳定性调节因子。尽管这些显然很重要 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 进程仍然有限。这是由于这种染色体的不寻常的基因组结构，几十年来它一直在呈现 它能抵抗任何目标攻击。随着TALEN和CRISPR/CAS9技术的出现 染色体的基因功能直接成为可能。 Y染色体结构的怪异也使其难以测序。小鼠的Y染色体序列是 最终在2014年被定义，也就是小鼠基因组剩余部分被确定12年后。当鼠标按下Y顺序时 在Y短臂上发现了两个新的单拷贝基因：Prssly(Y连锁丝氨酸类蛋白水解酶)和 Teyorf1(睾丸表达，Y染色体开放阅读框架1)。这些基因被归类为“获得性基因” 被认为是男性生育基因的候选者。这两个基因都不是靶点，它们的功能 仍然不为人知。 在初步数据中，我们发现Prsly和Teyorf1的表达仅限于睾丸减数分裂后生殖细胞。我们也 分析了PRSSLY和TEYORF1预测的蛋白质结构和功能，发现两个预测的蛋白质相似 已知在精子发生中起作用的蛋白质。PRSSLY属于多肽酶S1家族的蛋白质 包括丝氨酸蛋白酶PRSS21和PRSS55，它们对精子活力和精子使卵母细胞受精的能力至关重要。TEYORF1 属于Claudin蛋白家族，其成员被报道对细胞的组装和维持很重要 血-睾丸屏障，一种对正常精子发生至关重要的组织屏障。 Y染色体的获得、睾丸的特异性表达以及与已知生殖功能的蛋白质的相似性 这促使我们提出了一种假设，即Prsly和Teyorf1在男性生殖中发挥作用。我们将解决这一问题 追求两个特定目标的假设： 目的1：建立Prsly和Teyorf1基因敲除(KO)小鼠和Prsly和Teyorf1报告基因标签敲入(KI)小鼠 CRISPR/Cas9系统和受精卵电穿孔。 目的2：描述这些小鼠的表型，重点评估生育能力、精子参数、精子功能。 体外，以及精子发生的进展。 结合这些结果，可以确定Prsly和Teyorf1是否在精子发生和生育中发挥作用。这个 KI小鼠的发展将为未来对这些因素的机制研究打开大门。该项目将对 关于Y染色体基因功能及男性生育遗传基础的认识。它还将有助于讨论 关于Y染色体的进化和获得性Y基因的重要性。

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.