DNA甲基化在介导应激诱发的男性生育力下降跨代遗传中的分子机制研究

In recent years, the decline in male fertility and rise in male infertility became a global problem. Researches had revealed that the environment stresses could affect sperm quality, resulted in declining in fertility and then been transmitted to offspring. It was reported that the epigenetic mechanisms played important roles in this process. Our previous study had shown that chronic restraint stress could induce decline in male mouse fertility, and this phenotype could be transmitted to F2 offspring. Further analysis revealed that transgenerational inheritance of the acquired trait was closely related with the inheritance of variations in sperm DNA methylation. However, it was still unclear that when and how these heritable DNA methylation variations “escaped” from the epigenetic reprogramming process during the offspring embryonic development. Here, we plan to figure out these questions through profiling the DNA methylome of the offspring embryos in different stages. Then, we will verify the mechanism by epigenome editing using CRISPR/dCas9 system which fused with epigenetic modifiers. In addition, the DNA methylation patterns of the differentially methylated regions which related with declining fertility will be identified in oligozoospermia or azoospermia patients. These study will provide the theoretical basis for expounding the underlying mechanisms of transgenerational inheritance of the environmental-factors induced acquired phenotypes, and provide candidate targets for infertility diagnosis and therapy.

近年来，男性生育力下降和不育症上升成为一个全球性问题。研究表明环境应激可影响精子发育从而导致生育力下降并可传递到子代，而表观遗传机制在其中起重要作用。我们前期研究发现，慢性应激诱发雄性小鼠生育力下降并可传递到F2代，这种获得性性状的跨代遗传与精子DNA甲基化变化跨代遗传密切相关。但可遗传DNA甲基化变化如何逃脱子代胚胎发育重编程过程从而实现跨代遗传的？这个问题仍未阐明。我们拟通过绘制子代胚胎发育不同时期DNA甲基化图谱，阐明可遗传DNA甲基化变化位点逃脱子代胚胎发育重编程过程的时期和方式。利用CRISPR/dCas9融合表观修饰因子对可遗传位点进行表观基因组编辑，进一步验证DNA甲基化跨代遗传分子机制。最后筛选部分与生育力异常相关的差异DNA甲基化位点并在少弱精症患者精子中进行验证。本项目为揭示环境诱发的男性生育力下降跨代遗传分子机制提供坚实的理论基础，并为男性生育力异常诊疗提供潜在靶点。

父本生活经历会通过配子影响子代健康，表观遗传提供了一种潜在机制。然而，在子代胚胎发育和精子发生过程中存在表观修饰重编程，这成为表观遗传最大障碍。到目前为止，父本精子表观修饰改变如何携带“环境记忆”跨过子代胚胎发育重编程过程仍不清楚。在此，我们利用一个长期束缚应激小鼠模型研究父本长期心理应激效应代间和跨代遗传的潜在机制。我们发现，应激可导致生育力下降、血糖代谢紊乱和行为异常父系遗传。对两个分组（对照组和应激组）三个连续世代（F0，F1 和F2）共计18份精子样本进行全基因组甲基化测序发现，长期心理应激诱导的差异甲基化区域（DMRs）大约有11.36%可以代间遗传，0.48%可以跨代遗传。功能分析发现，这些DMRs相关基因与心理应激反应相关，而且子代组织继承这些DMRs可以传递父本生理异常。更重要的是，我们发现这些DMRs是通过擦除后重建的模式，而不是不擦除的模式，跨过子代胚胎发育重编程的，但是它们的重建比例在原条期（E7.5）发生了变化。随后，我们对父本精子进行了sncRNA测序发现，长期心理应激诱导父本精子中tsRNA、miRNA和rsRNA失调可能与精子DMRs的发生和父系遗传有密切关系。这些结果表明精子表观修饰变化参与父本长期心理应激暴露效应的代间和跨代遗传并阐明了可能的机制。此外，我们不仅利用外源因子（环境应激等）诱导的表观修饰改变研究跨代遗传（被动模型），还利用内源因子诱导的表观修饰改变研究跨代遗传（主动模型）。利用人源化大鼠模型研究人源DNA甲基转移酶DNMT3A在大鼠睾丸中超表达介导的精子DNA甲基化变化及遗传。分析发现在转基因去除的情况下，变化的DNA甲基化位点仍可以传递给子代，即表观修饰遗传不依赖于转基因的传递。这些结果为男性生育力下降及男性不育的诊治提供参考靶点，为干预和阻断心理应激诱导的男性生育力下降及其遗传提供理论依据。