双酚A对卵泡发育及胚胎着床的表观遗传干扰机制研究

As one of most common environmental endocrine disruptors, bisphenol A has caused increasing concerns about its influences on reproductive system. Recent researches suggested that bisphenol A might affect the reproductive function by disturbing epigenetic modifications. We have recently found that exposure to bisphenol A altered DNA methylation of HoxA10 in the endometrium of the mice models, which might account for the reduced endometrial receptivity. Based on our previous findings and protocols, this project aims to elucidate the detailed epigenetic mechanisms of bisphenol A disturbing reproductive function. The main measure include establishment of pregnant mice models, primary cell culture of human cumulus granulosa cells, tryphocytes, endometrial stromal cells, and endometrial glandular cells. Based on the above animal or cellular models, the epigenetic alternations caused by bisphenol A in the important reproductive events such as folliculogenesis and embryo implantation were evaluated, focusing on heomobox genes which play important roles in regulating individual development and differentiation. The possible measures include identification of key loci involved in epigenetic changes through methylation specific PCR or bisulfite sequencing, clarification of the upstream and downstream mechanisms and their biological effects using appropriate molecular or cellular biology techniques, analysis of the relationships between the epigenetic abnormality and the related clinical events. The expected results will contribute to the combination of environmental hygiene with clinical medicine, deepen the understanding of related diseases, provide new instructions for the clinical practice of reproductive medicine, and offer references for further improvement of health policy.

双酚A是最常见的环境内分泌干扰物，新近研究认为表观遗传干扰效应可能是其影响生殖功能的重要途径之一。我们前期研究发现：双酚A可引起小鼠同源盒基因HoxA10甲基化异常，进而影响其子宫内膜容受性。为了进一步阐明双酚A对生殖功能的影响及其调控机制，本项目拟在前期研究的基础上，从表观遗传学角度入手，针对卵子生成、胚胎着床等生殖过程的关键事件，以在个体发育分化中发挥重要作用的同源盒基因家族为重点，整合动物模型-人类原代细胞培养模型，采用甲基化特异性PCR、重亚硫酸盐测序等技术甄选表观遗传修饰异常的关键位点，运用各种蛋白质、核酸分析技术探究其上下游机制，揭示其引发的生物学效应，并借助于人类辅助生殖治疗的时间窗，进一步验证上述关键因子的表观遗传修饰异常与相应临床指标的关系。预期成果将促进环境卫生与临床医学的有机结合，深化对相关疾病的认识，为生殖医学的临床实践提供新的思路。

环境污染与不孕关系密切。双酚A（BPA）是一种典型的环境内分泌干扰物，其对生殖发育的影响已见报道，但BPA对生殖发育尤其是对卵子发生、胚胎着床等关键生殖事件的表观遗传学调控机制却不明确。此次课题我们围绕从卵细胞发育到胚胎着床到子宫内膜发生蜕膜化这一系列早孕期重要时间节点进行研究，通过建立多个体内外模型，观察到了双酚A（BPA）对生殖系统（子宫及卵巢）的直接毒性效应，并证实了BPA与卵巢储备功能下降之间的必然联系。围绕HOX基因家族探讨BPA干预生殖发育的内在分子机制，我们发现BPA可加速细胞凋亡相关信号通路分子表达，引起卵细胞发育相关基因表达失调，这可能是由组蛋白甲基化修饰酶EZH2介导HOXA7甲基化修饰异常这一分子调控机制所引起。而当卵细胞发育并进入胚胎着床环节，实验结果发现，BPA会通过ESR-HOXA10-ITGB3信号通路加速着床位点子宫内膜组织凋亡、降低黏附力，从而损伤子宫内膜容受性，减少胚胎着床，EZH2介导的HOXA10高甲基化很可能是其中关键调控机制之一。除此以外，EZH2、MLL1有望成为研究BPA影响子宫内膜蜕膜化的新靶标。本课题实验结果均证实了HOX基因家族甲基化修饰调控生殖发育的重要作用。并从中挖据出EZH2/MLL1作为HOX家族上游基因调控胚胎着床的关键地位。以上内容有望为临床不孕的诊治提供思路。