Do we need Y chromosome for successful reproduction?

描述（由申请人提供）：男性不育症影响5-10%的人口。与男性不育相关的一个主要因素是Y染色体缺失，但我们对Y染色体上特定基因的需求及其在精子产生/功能中的作用的理解仍然很差。Y染色体基因可能提供生精功能或只是促进生精过程。我们的长期目标是在辅助生殖技术（ART）的背景下定义小鼠中Y染色体编码基因的功能，作为模拟人类Y连锁不育病例的一种方式。我们已经确定，只有两个Y染色体基因，睾丸决定基因Sry和精原细胞增殖因子Eif 2s 3 y的生产所需的男性配子能够参与辅助受精。在初步数据中，我们表明，男性与Y补体限制为Sry和Eif 2s 3 y精子发生停滞，不产生成熟的精子。前体单倍体生殖细胞（精子细胞）是罕见的，往往异常。然而，通过圆形精子细胞注射（ROSI），我们成功地产生了可行的，健康的，可育的后代。这为患有广泛的Y基因丢失和导致无精子症的男性提供了希望。在这里，我们的具体目标是确定即使没有这个最小Y基因的贡献，ART是否可以实现。我们将测试的假设，Y染色体补体可以完全消除，同时保留生产功能的男性配子。在目标1中，我们将测试Sox 9（Sry的下游效应物）的转基因激活是否将有效地取代Sry功能，以及Sry到Sox 9的取代是否影响精子发生和生育力，直接测试这些基因在成熟性腺中的未知功能。我们还将确定是否Eif 2s 3x，Eif 2s 3 y的X编码同源物，可以取代精原细胞增殖中的Eif 2s 3 y功能。我们将产生和表征Eif 2s 3x转基因小鼠，并评估Eif 2s 3x的过表达是否可以挽救XOSry小鼠中的精原细胞增殖停滞。在目标2中，我们将产生没有任何Y基因但具有Eif 2s 3x过表达和Sox 9转基因激活的雄性，以及具有一个Y基因保留和另一个替换的雄性。我们将研究这些基因的存在如何影响精子发生的进程。我们还将测试这些雄性动物的精子发生是否能够使生殖细胞在ART中发挥作用，以及这些后代是否正常。在目标3中，我们将尝试使用新的“主动转基因”方法和超声介导的基因递送在体内Eif 2s 3 y基因转移来拯救成熟男性睾丸中的精原细胞停滞。我们的研究将促进对以下问题的理解：（1）性别决定的主要参与者Sry和Sox 9在成熟性腺中的作用;（2）性染色体基因的作用（Eif 2s 3 y和Eif 2s 3x）在精子发生起始中的作用;以及（3）广泛的Y基因丢失与成功的ART的兼容性。我们的结果应该转化为加强与Y染色体相关的人类不育症的治疗删除。!

DESCRIPTION (provided by applicant): Male infertility affects 5-10% of the population. A major factor associated with male infertility is Y chromosome deletions, yet our understanding of the requirement for specific genes on the Y chromosome and of their roles in sperm production/function is still poor. Y chromosome genes may provide essential spermatogenic functions or just potentiate the spermatogenic process. Our long-term goal is to define the function of Y chromosome encoded genes in mice in a context of assisted reproduction technologies (ART) as a way to model human Y- linked infertility cases. We have established that only two Y chromosome genes, testis determinant gene Sry and spermatogonial proliferation factor Eif2s3y are required for production of male gametes capable of participating in assisted fertilization. In Preliminary Data we show that males with a Y complement limited to Sry and Eif2s3y have spermatogenesis arrest and do not produce mature sperm. The precursor haploid germ cells (spermatids) are rare and often abnormal. Nevertheless, with round spermatid injection (ROSI) we succeeded in producing viable, healthy, and fertile progeny. This offers a promise to men with extensive Y gene loss and resulting azoospermia. Here, our specific goal is to define whether ART can be achieved even without this minimum Y gene contribution. We will test the hypothesis that the Y chromosome complement can be eliminated entirely while retaining production of functional male gametes. In Aim 1 we wil test if transgenic activation of Sox9, a downstream effector of Sry, will effectively replace Sry function and whether Sry-to-Sox9 replacement affects spermatogenesis and fertility, testing directly for the as yet unknown function of these genes in mature gonads. We will also establish if Eif2s3x, an X encoded homologue of Eif2s3y, can replace Eif2s3y function in spermatogonial proliferation. We will generate and characterize mice transgenic for Eif2s3x, and assess whether overexpression of Eif2s3x can rescue spermatogonial proliferation arrest in XOSry mice. In Aim 2, we will produce males without any Y genes but with overexpression of Eif2s3x and with transgenic activation of Sox9, as well as males with one Y gene retained and the other replaced. We will investigate how the presence of these genes affects spermatogenesis progression. We will also test if spermatogenesis in these males enables development of germ cells functional in ART, and whether such produced offspring are normal. In Aim 3, we will attempt to rescue spermatogonial arrest in testes of mature males with in vivo Eif2s3y gene transfer using novel 'active transgenesis' approach and ultrasound mediated gene delivery. Our studies will advance the understanding of (1) the roles that key players of sex determination (Sry and Sox9) play in mature gonads; (2) the roles of sex chromosome genes (Eif2s3y and Eif2s3x) in the initiation of spermatogenesis; and (3) the compatibility of extensive Y gene loss with successful ART. Our results should translate to enhance treatment of human infertility associated with Y chromosome deletions. !