泛素E3连接酶HUWE1维持精原干细胞的作用机制研究

Spermatogonial stem cells (SSCs) undergo self-renewal and differentiation to maintain spermatogenesis. Defects of which would lead to male infertility and cancers. Previous studies have identified extrinsic factors that promote the self-renewal of SSC. However, the intrinsic factors that regulate SSC self-renewal and their underlying mechanisms remain largely unknown. Recent studies have suggested the involvement of ubiquitin-proteasome system in the establishment and maintenance of SSC. Our preliminary study showed that HUWE1, a ubiquitin E3 ligase, plays an essential role in the maintenance of SSC. Germ cell-specific knockout of HUWE1 in mice results in male infertility and degeneration of SSC. Knockout of HUWE1 in primary spermatogonial culture or spermatogonial cell line result in cell degeneration through neither apoptosis nor senescence pathway. Interestingly, knockout of HUWE1 provokes hyperactivated DNA damage response (DDR) without altering the expression of the majority of known HUWE1 substrate involved in the DDR and apoptosis. These results prompt us to hypothesize that HUWE1 targets a new set of substrate(s) to modulate the DDR and maintenance of SSC. .We aim to test this hypothesis with the following specific aims: 1) To characterize the degeneration of HUWE1 knockout SSC; 2) To identify the substrate(s) of HUWE1 by ubiquitinome analysis; and 3) To elucidate the mechanism underlying the maintenance of SSC by HUWE1 and its downstream substrate(s). The results of the proposed study will provide novel insight into our understanding of the self-renewal and maintenance of SSC that is crucial for the production of sperm. The study on the role of HUWE1 in SSC may uncover new mechanism underlying male infertility and provide promising targets for the development of infertility diagnosis and treatment.

精原干细胞透过自我更新和分化来维持精子发生。有关调控出现紊乱会影响精子发生，导致男性不育及睾丸癌等病症。然而，调节精原干细胞自我更新的细胞内因子及其机制尚不清晰。最近的研究表明，泛素化蛋白酶体系统可能参与精原干细胞的形成及维持。我们的初步数据还表明，当中的泛素化连接酶HUWE1是维持精原干细胞所必须的。 HUWE1生殖细胞特异敲除小鼠的表形为不育及精原干细胞死亡。在元代培养的精原干细胞和细胞株中敲除HUWE1亦会导致细胞通过非凋亡非衰老机制死亡。重要的是，敲除HUWE1引起剧烈的DNA损伤反应，但已知的、与DNA损伤反应及凋亡有关的大部份HUWE1目标蛋白的表达未见异常。这些观察促使我们推测HUWE1作为细胞内因子，通过诱导一个或多个未知的目标蛋白的泛素化来调节DNA损伤反应，维持精原干细胞的自我更新。本项目将利用元代精原干细胞培养及泛素化蛋白组学的方法对以上假设进行研究。研究结果将阐明调控精原干细胞自我更新的分子机制，亦将对其临床应用有着指导意义。

精原干细胞（SSCs）进行自我更新和分化以维持精子发生。这些过程的缺陷会导致男性不育和癌症。之前的研究已经找出一系列促进SSC自我更新的外在因素。但迄今为止，调节SSC自我更新的内在因素及其潜在机制仍有待阐明。最近的一项研究表明，泛素-蛋白酶体系统可能参与SSC的建立和维持。在这项研究中，我们表明，泛素E3连接酶HUWE1在SSC的维持中起着至关重要的作用。HUWE1调节组蛋白H2Ax的水平和DNA损伤反应来维持SSC。丧失HUWE1活性会导致SSC通过有丝分裂灾难（mitotic catastrophe）死亡及不育。HUWE1小分子抑制剂BI8622处理可诱导SSC中的有丝分裂灾难，与基因敲除／敲减的表现相似。BI8622处理后的蛋白质组学研究在三个不同的时间点鉴定出多于 200种差异表达的蛋白。信号通路分析揭示了凋亡机制可能与早期的有丝分裂灾难相似，但在细胞死亡期间有独特的信号通路。我们亦进一步探索了BI8622作为靶向精原干细胞的新型男性避孕药的潜在应用。为了将药物送递到睾丸，我们参与开发了一套人工智能的演算法来设计新型水凝胶。我们已经开展了研究，探讨新设计水凝胶的胶凝能力和支持Sertoli细胞生长的能力。总之，我们的研究结果揭示了维持SSC的新机制，并探讨了有关结果在男性避孕药开发中的转化潜力。