PIWI/piRNA作用通路介导调控精子DNA损伤的研究

The main mission of the sperm is to have its genome DNA completely inherited to the next generation. Sperm DNA damage can lead to male infertility, miscarriage and birth defect. Therefore, the potential harm that sperm DNA damage posed to male reproductive and health of the offspring has become a major issue of concern in the field of reproductive medicine in need of urgent solution..Studies confirmed that testis-derived piRNA is involved in the development of germinal cells, silence of transposons, formation of heterochromatin, maintaining of the germ cell’s genetic integrity of rodent and Drosophila , and of great significance to the stable inheritance of genetic material.We found that the degree of damage to Sperm DNA of infertility patients was associated with the decrease of piRNA in the early experiment. Also, it is confirmed that the abnormality of piRNA’s pathway can cause Specific developmental defects in Drosophila axis through the regulation of Chk-2.Therefore, we speculated that piRNA might get involve in the regulation on sperm DNA damage. To confirm this hypothesis and explore its mechanism, we apply this project to study the expression of piRNA in testis and seminal plasma and explore the mechanism of regulation of PIWI/piRNA on sperm DNA damage. We hope that this research will offers some new targets for the treatment of human sperm DNA damage.

精子的主要使命是将其基因组DNA完整地遗传给下一代，若精子DNA损伤可导致男性不育、流产和出生缺陷，因此精子DNA损伤对于男性生殖和子代健康构成潜在危害已成为本领域亟需解决的重要课题。研究证实睾丸特异源性piRNA参与啮齿动物及果蝇生殖细胞的发育、转座子的沉默、异染色质的形成和生殖细胞DNA完整性的维持等过程，对遗传物质的稳定遗传具有重要意义。我们在前期试验中发现不育症患者精子DNA损伤程度与piRNA量减少相关，且有报道piRNA的作用途径异常可通过Chk-2作用通路导致果蝇轴线发育特异性的缺陷。因此，我们推测piRNA可能在人睾丸中参与调控精子DNA损伤，为证实此推断并研究其作用机制，本项目拟开展piRNA 在人睾丸和精浆中表达及DNA损伤的相关性研究，并通过对细胞周期阻滞相关信号检测，研究PIWI/piRNA 作用通路调节精子DNA损伤作用机制，为精子DNA损伤治疗提供新的靶点。

本研究发现piRNA与精子DNA碎片率、DNA双键断裂情况、精子染色质包装存在一定的相关性；不育男性患者精浆中piRNA的减少伴有精子质量的下降，以及DNA碎片率的升高，其配偶的妊娠结局较差，其受精率、卵裂率、优胚率以及临床妊娠率呈下降趋势；在细胞水平转染piRNA-000765 antagomir与agomir进行功能缺失性研究与过表达研究后发现，在不同时间点转染antagomir后piRNA-000765表达下降，并导致小鼠精原细胞GC-1与小鼠精母细胞GC-2的凋亡增多，细胞周期多阻滞于G2/M期，伴有PIWI蛋白的减少，而在不同时间点转染agomir后piRNA-000765表达增多，PIWI蛋白表达增多，推测piRNA的表达量与可能与细胞的DNA损伤情况、细胞周期发育阻滞及凋亡存在一定关系，主要可能为对DNA的保护。当 DNA 受到损伤时，开启了相应的检测点系统，这时细胞周期会发生延迟或阻滞现象，主要是使受损的 DNA 能及时的进行自我修复。DNA 损伤后可激活 ATM-Chk2-Cdc25 和 ATR-Chk1-Cdc25 两条途径，DNA 双链损伤后激活 ATM/CHK2 分支，DNA 单链损伤或者大量的损伤主要激活ATR/CHK1 旁路。通过将piRNA-000765 antagomir与agomir转染至GC-1细胞、GC-2后不同时间点DNA损伤检查点蛋白水平的检测，初步推测由于piRNA介导的DNA损伤可能主要通过ATM-Chk2-Cdc25途径参与了 G2/M 检查点信号通路的反应，其具体机制需要进一步深入研究。