Do we need Y chromosome for successful reproduction?

描述（由申请人提供）：男性不育影响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，然而我们对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必要的生精功能或只是增强生精过程。我们的长期目标是在辅助生殖技术（ART）的背景下确定小鼠Y染色体编码基因的功能，作为模拟人类Y相关不孕症的一种方法。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Eif2s3y才能产生能够参与辅助受精的雄性配子。在初步数据中，我们发现Y补体仅限于Sry和Eif2s3y的男性精子发生阻滞，不能产生成熟精子。前体单倍体生殖细胞（精子）是罕见的，通常是不正常的。然而，通过圆形精子注射（ROSI），我们成功地产生了可存活的、健康的、可生育的后代。这给大量Y基因缺失导致无精子症的男性带来了希望。在这里，我们的具体目标是确定即使没有这种最小的Y基因贡献，ART是否也能实现。我们将测试一个假设，即Y染色体补体可以完全消除，同时保留功能性雄性配子的生产。在Aim 1中，我们将测试Sry的下游效应物Sox9的转基因激活是否会有效地取代Sry功能，以及Sry-to-Sox9的替代是否会影响精子发生和生育，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Eif2s3x是Eif2s3y的X编码同源物，是否可以取代Eif2s3y在精原细胞增殖中的功能。我们将产生并鉴定Eif2s3x转基因小鼠，并评估过表达Eif2s3x是否可以挽救XOSry小鼠的精原细胞增殖阻滞。在Aim 2中，我们将产生没有任何Y基因，但过度表达Eif2s3x和转基因激活Sox9的雄性，以及保留一个Y基因而替换另一个Y基因的雄性。我们将研究这些基因的存在如何影响精子发生的进程。我们还将测试这些男性的精子发生是否能使生殖细胞在抗逆转录病毒治疗中发挥作用，以及这些产生的后代是否正常。在aims 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传递，通过体内转移Eif2s3y基因来挽救成熟男性睾丸中的精原细胞阻滞。我们的研究将促进对(1)性别决定的关键参与者（Sry和Sox9）在成熟性腺中的作用的理解；(2)性染色体基因Eif2s3y和Eif2s3x在精子发生起始中的作用；(3)广泛的Y基因缺失与成功的ART的兼容性。我们的结果应该转化为加强治疗与Y染色体缺失相关的人类不孕症。！

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. !