Minimum Y gene complement necessary for successful ART

描述(由申请人提供)：大多数Y染色体编码基因可能在精子的产生或功能中起作用。然而，目前还不清楚这些基因是否提供了基本的生精功能，或者只是加强了生精过程。这项应用的目标是确定与辅助生殖技术(ART)成功繁殖兼容的最低Y染色体基因要求。这一应用的假设是，只要两个或三个基因的Y基因互补就足以产生能够参与受精的雄配子(圆形精子细胞或精子)，如果通过注射进入卵母细胞的话。在初步数据中，我们提供的证据表明，在小鼠中，只存在两个Y编码基因，Sry和Eif2s3y，允许形成睾丸，进行精原细胞增殖，并完成减数分裂以产生圆形精子细胞。我们认为，进一步增加一个Zfy拷贝就足以产生一些精子，尽管头部形态不正常。我们还表明，通过ICSI和试管受精的辅助生殖，能够从Y染色体缺陷的不育和不育男性中产生活的后代。在本应用程序中，我们将重点分析具有有限Y基因互补的各种小鼠模型：(1)Y短臂几乎完整但完全缺乏Y长臂基因的雄性；(2)没有Y长臂基因且已知Y短臂基因仅限于Sry、Eif2s3y、Zfy的单一拷贝和Rbmy拷贝数减少的雄性；以及(3)仅具有Eif2s3y和Sry的雄性。在特定的目标1中，我们将产生这些具有有限Y基因互补的男性，并更准确地定义生精细胞在哪个阶段停止或变得异常。我们将对睾丸进行组织学分析，通过免疫染色确认特定生精细胞类型的存在，并检查顶体发育作为生精阶段的标志。在特定目标2中，我们将使用来自这些模型的精子和/或圆形精子细胞进行ICSI和/或ROSI。我们将观察注射后早期受精事件，获取胚胎，并产生活后代。我们将对后代进行基因分型，以测试哪些精子基因型成功地支持了受精和胚胎发育。这一应用的意义在于，它将促进对Y染色体编码基因在精子发生和精子功能中作用的了解，也将为使用ART治疗与Y基因缺陷相关的人类不育提供有价值的信息。公共卫生相关性：在这项应用中，我们寻求确定与通过ART(ICSI和ROSI)产生具有受精能力的精子相容的最低Y基因补体。这项研究将通过确定哪些Y基因提供基本的生精功能，而不仅仅是加强生精过程，从而加深对Y染色体编码基因功能的理解。该应用对那些利用ART治疗男性不育的人也具有意义。

DESCRIPTION (provided by applicant): It has been argued that most Y chromosome coded genes are likely to have roles in sperm production or function. However, it is not clear if these genes provide essential spermatogenic function or just potentiate the spermatogenic process. The goal of this application is to determine the minimum Y chromosome gene requirement that is compatible with successful reproduction by assisted reproductive technologies (ART). The hypothesis of this application is that a Y gene complement of as few as two or three genes is enough to enable the production of male `gametes' (round spermatids or sperm) that are capable of participating in fertilization if delivered into the oocytes via injection. In preliminary data we provide evidence that in the mouse the presence of only two Y-coded genes, Sry and Eif2s3y, allows formation of testes, ongoing spermatogonial proliferation, and completion of meiosis to generate round spermatids. We suggest that further addition of one copy of Zfy is sufficient to allow the production of some sperm, albeit with morphologically abnormal heads. We also show that assisted reproduction by ICSI and IVF enable the generation of live offspring from subfertile and infertile males with Y chromosome deficiencies. In this application we will focus on analyzing various mouse models with limited Y gene complements: (1) males with an almost intact Y short arm but complete absence of Y long arm genes; (2) males with no Y long arm genes and the known Y short arm genes limited to Sry, Eif2s3y, a single copy of Zfy, and a reduced number of copies of Rbmy; and (3) males with only Eif2s3y and Sry. In Specific Aim 1 we will generate these males with limited Y gene complements and define more precisely at what stage of spermiogenesis cells arrest or become abnormal. We will perform histological analysis of the testes, confirm the presence of specific spermatogenic cell types by immunostaining, and examine acrosome development as a marker of spermiogenic stage. In Specific Aim 2 we will use sperm and/or round spermatids from these models for ICSI and/or ROSI. We will observe early post-fertilization events after injection, obtain embryos, and produce live offspring. We will genotype progeny to test which sperm genotypes were successful in supporting fertilization and embryo development. The significance of this application is that it will advance the understanding of the role of Y chromosome encoded genes in spermatogenesis and sperm function; it will also provide valuable information for those using ART to treat human infertility associated with Y gene deficiencies. PUBLIC HEALTH RELEVANCE: In this application we seek to determine the minimum Y gene complement that is compatible with the generation of sperm competent in fertilization via ART (ICSI and ROSI). The study will add to the understanding of the functions of Y chromosome coded genes by defining which Y genes provide essential spermatogenic function rather than just potentiating the spermatogenic process. The application also has significance for those utilizing ART to treat male infertility.