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例之一。分子遗传学技术揭示的导致男性不育的最常见原因是Y染色体的缺失。由于Y染色体不存在遗传重组，因此不可能进行传统的位置定位和男性生殖细胞发育相关Y基因的克隆。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有关。在小鼠中，大多数负责精原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)被转基因基因救援法发现；然而，最近的数据表明，在这个缺失区间内的其他基因参与了生精过程。然而，进一步的转基因挽救实验变得非常复杂，因为需要生产具有Y染色体缺失背景的多个转基因小鼠。尽管进行了多次尝试，但使用常规ES细胞技术的Y染色体基因打靶并未导致小鼠的活体突变。我们建议通过产生短发夹状RNA转基因小鼠来靶向Y染色体基因的表达。在所提出的实验中，shRNA转基因表达的激活和随后的RNA干扰是由cre-重组酶控制的，从而允许转基因生殖系传输和在不同的生精阶段进行特定的基因靶向。这种方法还允许以单拷贝和多拷贝Y染色体基因为靶点，并严格控制RNAi的特异性。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。本项目的总体目标是通过生产和分析shRNA转基因小鼠的生殖特性来表征Y染色体基因的生精功能。为此，我们设计了两个特定的目标：1.制备以Y染色体短臂上的8个基因为靶点的条件性shRNA转基因小鼠。2.在雄性生殖细胞分化的不同阶段，我们将分析Y染色体基因抑制对shRNA转基因激活的男性精子发生的影响。从这些研究中获得的信息将成为未来对男性不育的准确遗传诊断、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中的关键作用是生殖生物学的基本范式之一。我们建议通过靶向下调Y染色体基因来研究新型转基因小鼠的精子发生和生殖细胞更新。

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.