Minimum Y gene complement necessary for successful ART

描述（由申请人提供）：有观点认为，大多数Y染色体编码基因可能在精子产生或功能中发挥作用。然而，目前尚不清楚这些基因是否提供了必要的生精功能或只是加强生精过程。本申请的目的是确定与辅助生殖技术（ART）成功生殖相容的最低Y染色体基因要求。本申请的假设是，少至两个或三个基因的Y基因互补物足以使得能够产生雄性“配子”（圆形精子细胞或精子），如果通过注射递送到卵母细胞中，所述雄性“配子”能够参与受精。在初步数据中，我们提供的证据表明，在小鼠中，只有两个Y-编码基因，Sry和Eif 2s 3 y的存在下，允许睾丸的形成，持续的精原细胞增殖，并完成减数分裂产生圆形精子细胞。我们认为，进一步添加一个Zfy拷贝足以允许产生一些精子，尽管具有形态异常的头部。我们还表明，通过ICSI和IVF辅助生殖能够从Y染色体缺陷的低生育力和不育男性中产生活的后代。在本申请中，我们将集中分析具有有限Y基因互补的各种小鼠模型：（1）具有几乎完整的Y短臂但完全不存在Y长臂基因的雄性;（2）不具有Y长臂基因且已知Y短臂基因限于Sry、Eif 2s 3 y、Zfy的单拷贝和Rbmy的拷贝数减少的雄性;和（3）仅具有Eif 2s 3 y和Sry的雄性。在特定目标1中，我们将产生这些具有有限Y基因互补的男性，并更精确地定义精子发生细胞停滞或变得异常的阶段。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，我们将使用这些模型中的精子和/或圆形精子细胞进行ICSI和/或ROSI。我们将在注射后观察早期受精后事件，获得胚胎并产生活的后代。我们将对后代进行基因分型，以测试哪些精子基因型成功支持受精和胚胎发育。这项应用的意义在于，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理解;它还将为那些使用ART治疗与Y基因缺陷相关的人类不育症的人提供有价值的信息。公共卫生相关性：在本申请中，我们试图确定与通过ART（ICSI和ROSI）产生有受精能力的精子相容的最小Y基因互补。这项研究将增加对Y染色体编码基因的功能的理解，通过定义哪些Y基因提供必要的精子发生功能，而不仅仅是促进精子发生过程。该应用对于那些利用ART治疗男性不育症的人也具有重要意义。

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.