PIWI/piRNA在哺乳动物精子形成及男性不育中的功能机制

piRNAs (PIWI-interacting RNAs) are a class of germline-specific small non-coding RNAs that were identified in 2006. In mammals, piRNAs are mainly expressed in testes, and are essential to spermatogenesis. Recent studies have greatly advanced our understanding of how piRNAs are processed and how PIWI/piRNAs function in worms, flies, and fish. However, the function and mechanisms of PIWI/piRNAs in spermatogenesis and human male infertility remained largely unclear. In addition, development of post-meiotic male germ cells, termed as spermiogenesis, in animals is known as a complex differentiation and morphological process. Due to the complexity of spermiogenesis and the lack of an adequate in vitro cell culture system for studying post-meiotic differentiation, the molecular mechanisms underlying this dramatic differentiation process have remained largely unknown. Our recent studies established the high efficient lentiviral testis transduction system, which allowed us to be able to manipulate gene expression in haploid spermatids and examine the roles and mechanisms of specific genes in spermiogenesis. With this tool, we recently showed that pachytene piRNAs, in complex with MIWI and deadenylase CAF1, mediate massive mRNA degradation in late spermatids in mouse. We also found that mouse PIWI protein MIWI and piRNAs, after they fulfill their function in spermiogenesis, is eliminated by in late stages of spermatids by a feedforward loop via APC/C-ubiquitin pathway. Most recently, we identified ubiquitination-deficient Mutations in Hiwi (Human Piwi) from azoospermia patient and demonstrated such mutations cause male infertility by impairing Histone-to-Protamine exchange during spermiogenesis. In this research plan, we will explore the roles and mechanisms of PIWI/piRNA machinery in haploid spermatid development, including acrosome and flagellum formation, chromatin remodeling, nuclear condensation, and sperm DNA methylation. We will further screen potential gene mutations in PIWI/piRNA pathways in azoospermia patients, and study the function and mechanisms of the identified mutations in male infertility. The obtained results may bring new insights into key regulatory pathways governing spermatid development and spermatozoa formation, and also may provide molecular target(s) for the diagnosis and therapeutics of male infertility.

哺乳动物中piRNA(piR)及结合蛋白PIWI在睾丸组织高表达，为精子发生必需，但目前对PIWI/piR通路在哺乳动物精子发生中的功能机制还了解不多。减数分裂后的单倍体精子细胞发育过程称为精子形成，是最复杂的细胞分化和形态变化过程之一，由于缺乏合适的研究系统，目前对此过程认识还极其有限。我们的前期研究建立了可高效干预小鼠精子细胞基因表达的慢病毒睾丸转导系统，揭示了PIWI/piR指导精子形成后期mRNA大规模降解、PIWI蛋白降解的分子机制及其降解异常致精子形成缺陷等。本项目将深入研究PIWI/piR在哺乳动物精子细胞顶体发生、染色质重构、细胞核压缩、基因组DNA甲基化修饰等精子细胞发育过程关键事件中的功能机制，鉴定临床无精症患者中潜在的piR/PIWI通路基因突变，研究相关突变在无精症发生中的功能机制。项目研究获得结果将揭示哺乳动物精子形成的调控机制，并为相关男性不育症诊治提供分子靶。

男性不育是目前全球性的医学问题及社会问题。由于精子发育过程的独特性及其复杂性，目前对于生性细胞的发育调控及男性不育的致病原因还不甚了解，导致临床诊断和治疗策略极其有限。本课题组长期致力于PIWI/piR在精子发生及男性不育中的作用的研究。前期工作发现生精细胞发育后期，PIWI/piR介导mRNA大规模清除及PIWI/piR协同清除的分子机制。在此基础上，本项目通过一系列深入研究，发现生精细胞发育早期，PIWI/piRNA机器通过与转录起始因子eIF3F互作介导精子细胞发育必需基因的翻译激活（Cell 2019A）；而PIWI蛋白泛素化修饰降解异常导致精子发育后期及组蛋白-鱼精蛋白交换受阻，并最终导致男性不育（Cell 2017）。以上研究结果，首次报道了piRNA对蛋白质编码基因的双向调控功能，解析了PIWI/piRNA通路异常与人类男性不育的关系，系统揭示了PIWI/piRNA机器在哺乳动物生精细胞发育调控及男性不育中的功能机制。项目研究紧密结合基础研究与临床疾病，为深入了解精子发育调控机制及相关男性不育症的临床诊治提供新的靶点和思路，具有广阔的应用前景。.在本项目的支持下，已发表研究论文7篇（Cell 2017, 2019A, 2019B; Nucleic Acids Res 2018; Nature Cell Biol 2020; Mol Cell 2020; Cancer Res 2017），综述论文2篇，申请国家发明专利1项。人才培养方面，项目负责人先后获得“上海市巾帼创新奖”、“谈家桢生命科学创新奖”、“全国妇幼健康科学技术”一等奖，入选中组部“万人计划”等多项奖励。此外，项目组培养了6位博士研究生、1位硕士研究生毕业取得学位、1位出站博士后。