人PIWI蛋白（HIWI）泛素化修饰缺陷致男性不育的功能机制研究

Asthenozoospermia/azoospermia is a major cause for male infertility in human. Its pathogenesis is varied and complicated. Piwi proteins that form complexes with their associated piRNAs, a class of animal germ-cell specific small non-coding RNAs, protect the stability and integrity of germ-cell genome by preventing the activity of mobile genetic elements, which are essencial to gametogenesis in animal. In mammals, both Piwi proteins and piRNAs are specifically expressed in male germ cells, while mutations in Piwi/piRNA pathway in mice leads to male sterility but appears to not affect the fertility of females; however, it has been unclear whether mutations in Piwi genes affect human fertility. Our recent studies revealed that piRNAs trigger MIWI ubiquitination and removal by APC/C-ubiquitin pathway in mouse late stages of spermatids. Moreover, MIWI degradation in turn leads to piRNA elimination, suggesting a feed-forward mechanism for coordinated removal of the MIWI/piRNA complex at a specific developmental stage. Importantly, inhibition of MIWI degradation either by knock-down of APC/C component or by expressing mutant form of MIWI that is resistant to APC/C-mediated ubiquitination prevented formation of mature sperms, implicating that proper temporal regulation of the MIWI/piRNA metabolism is essential for normal spermiogenesis. More interestingly, we identified three D-box mutations of Hiwi gene from asthenozoospermia/ azoospermia patients, providing the first evidence that mutations in Piwi genes may be critical to spermatogenesis in human. In this research plan, we propose two aims. First, we will further investigate the correlation between ubiquitination-resistant Hiwi mutations and male sterility. Second, we will construct knock-in and transgenic mouse models that express the identified Hiwi D-box mutants and study the molecular mechanisms of male sterility caused by ubiquination-deficient PIWI mutants. Data generated from this project will reveal the role and mechanism of proper temporal regulation of the PIWI/piRNA metabolism in mammalian spermiogenesis, bring new insights into molecular information for asthenozoospermia/azoospermia, and provide molecular targets for diagnosis and treatment of male sterility.

少弱/无精是男性不育的主要原因，其病理复杂多变。哺乳动物Piwi蛋白及其结合的piRNA在生精细胞中特异表达，沉默基因组的转座元件；Piwi失活致小鼠雄性不育，但Piwi突变是否影响人类生殖还未见报道。我们最近发现，小鼠piRNA在精子形成后期触发其结合蛋白PIWI（MIWI）经APC/C-泛素化降解，在后期精子细胞中稳定MIWI阻断精子发生，显示适时降解MIWI对精子生成至关重要。后续研究发现，少弱/无精症病人Piwi基因（Hiwi）存在拮抗泛素化的遗传性突变，首次提示Piwi突变与男性不育相关。在本申请项目中，我们将构建knock-in和转基因小鼠模型，深入研究HIWI降解异常与男性不育的关系，揭示PIWI泛素化修饰缺陷阻断哺乳动物精子形成的分子机制。项目研究获得结果将揭示PIWI/piRNA代谢调控在动物精子形成中的功能机制，为精子生成障碍提供分子信息，并为男性不育症诊治提供分子靶。

已有的证据表明，Piwi/piRNA通路是动物生殖细胞特异性的一条拮抗基因组转座遗传元件、维持生殖细胞基因组稳定性和完整性的小RNA通路，对动物生殖细胞发育及个体生殖至关重要，但目前对该通路是否具有沉默转座元件以外的功能还知之甚少，对该通路缺陷与男性不育症之间的联系了解更是有限。在该项目支持下，我们研究了小鼠piRNA及其结合蛋白MIWI（小鼠PIWI）对睾丸组织mRNA稳定性的影响，并探索了HIWI（人PIWI）蛋白突变在无精症发生中的功能机制，主要获得了以下研究结果，包括：1）从无精患者中鉴定了拮抗HIWI（人源PIWI）蛋白泛素化修饰的D-box突变，并通过Knock-in小鼠模型发现，此类突变阻碍精子变形过程中的组蛋白-鱼精蛋白交换并导致精子形成异常及雄性不育；2）发现小鼠piRNA在与靶mRNA高度配对时，可指导MIWI利用其slicer活性对靶mRNA直接切割降解，并发现部分piRNA介导的靶mRNA降解对于小鼠精子形成至关重要，揭示了piRNA除了沉默转座元件以外，还具有调控生殖细胞编码基因表达的新功能。此外，还深入揭示了miRNAs在肿瘤发生发展及肿瘤细胞代谢等过程中的功能机制。.共发表了项目资助标注论文5篇（其中3篇IF>10.0），包括Cell (accepted; IF:28.7)、Cancer Res (2017, 77(1):100-111; IF:8.55)、Oncogene (2016, 35(46):6015-6025; IF:7.93)、Cell Res (2015, 25(2):193-207; IF:14.81)和EMBO J (2015, 34(21):2671-2685; IF:10.43),申请发明专利一项（申请号201610525017）。此外，在项目执行期间，培养了3名博士研究生毕业取得博士学位，1名硕士研究生毕业取得硕士学位。