Role of Y chromosomal genes in male fertility

描述（申请人提供）：Y染色体在性别决定和男性生育中的关键作用是生殖生物学的基本范式之一。分子遗传技术揭示的男性不育症的最常见原因是Y染色体的缺失。由于缺乏Y染色体的基因重组，因此不可能对负责男性生殖细胞进展的Y基因进行传统的定位定位和克隆。对人类和小鼠Y染色体的缺失分析表明，有几个关键区域与男性生育能力有关。在小鼠中，大多数负责原精子维持和生殖细胞分化的基因位于Y染色体的短臂上。其中一个基因（Eif2s3y）通过转基因基因拯救法得到鉴定；然而，最近的数据表明，在这个缺失间隔内的其他基因参与了生精过程。然而，由于需要在Y染色体缺失的背景下生产具有多个转基因的小鼠，因此进一步的转基因拯救实验变得非常复杂。尽管多次尝试使用传统的胚胎干细胞技术进行Y染色体基因靶向，但并未导致活体小鼠突变。我们建议通过生产短发夹RNA转基因小鼠来靶向Y染色体基因的表达。在本实验中，shRNA转基因表达的激活和随后的RNA干扰由cree -recombinase控制，从而允许转基因种系传播和特定基因靶向不同的生精阶段。这种方法也允许靶向单拷贝和多拷贝Y染色体基因，严格控制RNAi特异性。我们的主要假设是，位于Y染色体上的基因有助于男性生殖细胞的维持和分化。本课题的总体目标是通过制备和分析shRNA转基因雄性的生殖特性来表征Y染色体基因的生精功能。为了实现这一目标，我们设计了两个具体目标：以Y染色体短臂中的8个基因为靶点，制备条件shRNA转基因小鼠。2. 我们将通过在雄性生殖细胞分化的不同阶段激活shRNA转基因来分析Y染色体基因抑制对雄性精子发生的影响。从这些研究中获得的信息将作为未来研究男性不育症的准确遗传诊断、与Y染色体缺失相关症状的基于知识的病因治疗以及避孕研究的基础。公共卫生相关性：Y染色体在性别决定和男性生育能力中的关键作用是生殖生物学的基本范式之一。我们拟对Y染色体基因靶向下调的新型转基因小鼠进行精子发生和生殖细胞更新的研究。

DESCRIPTION (provided by applicant): The critical role of Y chromosome in sex determination and male fertility is one of the basic paradigms in reproductive biology. The most frequent cause of etiopathogenetic male infertility revealed by molecular genetic techniques is a deletion of the Y chromosome. The absence of genetic recombination of the Y chromosome makes it impossible to perform traditional positional mapping and cloning of the Y genes responsible for male germ cell progression. Deletion analysis of the human and mouse Y chromosome suggested several critical regions involved in male fertility. In mice most of the genes responsible for spermatogonial maintenance and germ cell differentiation are located on the short arm of the Y chromosome. One of such genes (Eif2s3y) was identified by transgenic gene rescue approach; however more recent data indicate that the additional genes within this deletion interval participate in spermatogenic progression. Further transgenic rescue experiments however become highly complicated as a production of mice with multiple transgenes on Y chromosome deletion background is required. Despite numerous attempts Y chromosome gene targeting using conventional ES cell technology did not result in live mouse mutant. We suggest here to target Y chromosome gene expression by production of mice with short hairpin RNA transgene. The activation of shRNA transgene expression and subsequent RNA interference in the proposed experiments is controlled by cre-recombinase, thus allowing transgene germ line transmission and specific gene targeting at different spermatogenic stages. This approach also allows targeting both single and multiple copy Y chromosomal genes, with a strict control of RNAi specificity. Our main hypothesis is that the genes located on the Y chromosome contribute to male germ cell maintenance and differentiation. The overall objective of current project is to characterize the spermatogenic functions of the Y chromosome genes through production and analysis of reproductive characteristics of shRNA transgenic males.To address this goal we designed two specific aims: 1. To produce conditional shRNA transgenic mice targeting eight genes from the short arm of the Y chromosome. 2. We will analyze the effect of Y chromosome gene suppression on spermatogenesis in males with shRNA transgene activated at different stages of male germ cell differentiation. Information obtained from these studies will serve as a basis for future studies in accurate genetic diagnostics of male infertility, the knowledge-based etiological therapy of symptoms associated with Y chromosome deletion, as well as contraceptive research. PUBLIC HEALTH RELEVANCE: The critical role of Y chromosome in sex determination and male fertility is one of the basic paradigms in reproductive biology. We propose to study the spermatogenesis and germ cell renewal in novel transgenic mice with targeted down-regulation of the Y chromosomal genes.