多氯联苯对小鼠生殖细胞印迹基因DNA甲基化的影响及其跨代传递

Epidemiological investigation showed that PCBs (polychlorinated biphenyls), one species of environmental pollutants, had been a threat to human reproduction. Studies on animals had proved that PCBs not only have a significant reproductive toxicity on parental mice, but also can be inherited trans-generationally. Furthermore, our recent study proved that DMR methylation patterns of imprinted genes Snrpn in oocytes of offspring mice had been altered by PCBs exposure. However, it is still unclear about the epigenetic mechanism underlying trans-generational inheritance of reproductive toxicity caused by PCBs exposure. PCB118, a typical PCB, is used in this study to build three PCB exposure models which are in the period of mouse pre-gestation, gestation and lactation respectively, and then get F1, F2 and F3 offspring mice. Five imprinted genes Peg3, Snrpn, H19, Igf2 and Igf2r, which are related to reproductive toxicity in germ cells, were chosen, and molecular biological technique was utilized to analyse their methylated states of differentially methylated regions (DMRs) in mouse gametes (F0 to F3) and the expression of relevant proteins in order to understand the molecular mechanisms. This study aims to illustrate the correlation between the effects of trans-generational inheritance of reproductive toxicity and DNA methylation of imprinted gene in gametes, and to provide molecular understanding for prevention and treatment of infertility and birth defects caused by environmental pollutants.

流行病学调查发现环境污染物多氯联苯(polychlorinated biphenyls，PCBs)已经威胁到了人类的生殖和繁衍，我们和他人的研究均证实PCBs对亲代小鼠可产生明显的生殖毒性，并且可导致生殖毒性的跨代传递，最近我们研究还发现PCBs可导致子代小鼠卵母细胞印迹基因Snrpn DMR甲基化水平的改变。目前尚不了解PCBs造成生殖毒性跨代传递的表观遗传学机制。本研究以PCBs的典型代表物PCB118作为染毒物质，制备三类染毒模型，获得F1到F3代小鼠。选取与PCBs生殖毒性具有相关性的5个印迹基因，利用分子生物学技术,重点研究PCB118对小鼠生殖细胞中这5个印迹基因的表达和DMR甲基化水平的影响及其机制，初步探索PCBs生殖毒性的跨代传递与生殖细胞中印迹基因DNA甲基化之间的相关性，为由环境污染物引起的不孕不育和出生缺陷的预防和治疗奠定基础。

本项目在多氯联苯PCBs对小鼠生殖细胞印迹基因DNA甲基化的影响及其跨代传递方面进行了深入细致的研究。清楚地阐明了PCB118导致小鼠生殖细胞印迹基因DNA甲基化水平发生的变化及其分子机制；明确了孕期暴露PCB118对亲代和子代产生的生殖毒性与印迹基因Peg3、Snrpn、H19、Gtl2及Igf2r DMR甲基化模式改变之间的相关性，证明了PCB118对小鼠生殖细胞印迹基因DMR的影响可以跨代传递的猜想。. 亲代孕期暴露PCBs对子代小鼠生殖细胞印迹基因DNA甲基化水平的研究发现：子代雄性以及雌性小鼠的器官发育受到干扰，子代小鼠的生殖细胞质量降低，精子畸形率显著上升，卵母细胞的体外成熟率和凋亡率呈现剂量依赖性下降。子代雄鼠睾丸组织中，DNA甲基转移酶家族中的Dnmt1、Dnmt3a、Dnmt3b、Dnmt3l基因以及表观遗传调控因子Uhrf1的相对表达水平显著性降低。DNMT1和DNMT3A的蛋白表达水显著性下降，子代雄鼠胚胎期和成年期睾丸组织的5-甲基胞嘧啶水平显著性下降。子代小鼠精子中H19和Gtl2基因DMR区的甲基化水平显著性降低；子代小鼠卵母细胞中Snrpn，Peg3以及Igf2r的相对表达水平显著性升高，印迹基因Snrpn，Peg3以及Igf2r的DMR区甲基化水平显著性下降。子代小鼠卵母细胞中，DNA甲基转移酶Dnmt1、Dnmt3a、Dnmt3l以及表观遗传调控因子Uhrf1的相对表达水平显著性降低，同时去甲基化酶Tet3的相对表达水平显著性升高。子代小鼠卵母细胞中5-甲基胞嘧啶的水平显著性降低而5-羟甲基胞嘧啶的水平显著性升高。所做工作得到同行专家的认可和较高评价，发表在Archives of Toxicology的工作入选第九次全国毒理学大会优秀论文。. 本课题首次研究了多氯联苯对生殖细胞中印迹基因DMR甲基化并发现毒物（与人体乳汁残留浓度相当的PCBs）对生殖细胞中印迹基因DMR甲基化的影响能以配子为信息载体进行跨代传递,导致后代小鼠生殖系统损伤以及表观遗传修饰的改变。为环境污染物，尤其是多氯联苯引起的生殖系统疾病的预防和治疗提供了重要的参考依据。