基因组印记在p,p’-DDE致跨代雄性生殖毒性中的作用及叶酸的干预研究

Environmental hormones exposures are important factors with adverse effects on male reproductive function. p,p’-Dichlorodiphenoxydichloroethylene (p,p’-DDE) is the primary metabolite of organochlorine pesticide DDT with strong persistence. It is also a kind of typical environmental hormones found in environment and organism. p,p’-DDE could induce transgenerational male reproductive toxicity. However, the exact mechanism is still unclear. This study will analyze the possible mechanism of genomic imprinting in the transgenerational male reproductive toxicity. With the method of methylated DNA immunoprecipitation sequencing, methylated DNA immunoprecipitation qualitative PCR, bisulfite sequencing PCR and genome-wide microarray analyses, we will explore the effect of p,p’-DDE on the epigenetic transgenerational inheritance of altered transcriptome and epigenome of Sertoli cell and sperm and the possible interventional effect of folic acid. After horizontal and vertical comparison, we will analyze the relationship between methylated modification and gene expression, and explore the effect of altered germline imprinting on the somatic cells. From the perspective of epigenetics, this study will demonstrate the possible mechanism underlying male reproductive impairment induced by p,p’-DDE exposure and the possible interventional method. This study will provide new theory basis for the altered epigenetic effect induced by other environmental chemicals and the intrauterine source of chronic diseases.

环境激素暴露是男性生殖健康受损的重要因素。典型环境激素p,p’-DDE是有机氯农药DDT在环境和生物体内最持久、浓度最高的代谢产物，具有跨代雄性生殖毒性，分子机制不明确。本研究通过横向（对照、p,p’-DDE组和叶酸干预组）和纵向（F1代、F2代和F3代）比较，采用MeDIP-Seq、MeDIP-qPCR、亚硫酸氢钠测序、全基因组表达芯片等方法，分析F0代孕鼠p,p’-DDE暴露致雄性F1-F3代成熟精子、支持细胞基因组印记缺陷、表达改变及甲基供体叶酸的干预效应，分析甲基化修饰与基因表达改变、生殖细胞与体细胞印记改变之间的关系，探讨基因组印记在p,p’-DDE致跨代雄性生殖毒性中可能机制。本研究从表观遗传学层面阐明p,p’-DDE暴露出生子代风险增加的原因及可能阻断途径，为有机氯污染物致雄性生殖毒性防治开拓思路，并为其他环境激素的表观遗传效应及复杂疾病的宫内源性研究提供新的理论依据。

p,p’-DDE是有机氯农药DDT在环境和生物体内最持久、浓度最高的主要代谢产物，具有雄性生殖毒性，但分子机制尚不明确。本研究发现，p,p’-DDE可上调F1-F3代雌雄比例，下调F3代睾丸脏器系数，诱导雄性F1代胎鼠和乳鼠睾丸细胞凋亡，损伤支持细胞和生精细胞的紧密连接，引起睾丸曲细精管面积下降、含有长型精子曲细精管比例下调、活动精子数减少、精子穿透力水平下降、雄性子代生育力下降，并通过雄性生殖细胞系跨代传递到F3代，叶酸可干预p,p’-DDE引起的精子质量下降和睾丸细胞凋亡。.p,p’-DDE宫内暴露可下调F1和F2胎鼠睾丸DNA重新和维持甲基化转移酶DNMT3A和DNMT1的转录，下调雄性F1代精子的基因组甲基化水平，引起3917个基因低甲基化，137个高甲基化，F2代精子1102个基因低甲基化，874个基因高甲基化，其中F1代20-30%区别甲基化基因可传递到F2代，包括778个低甲基化和41个高甲基化基因。F1和F2代交叉的区别甲基化基因中有70%富集在内含子和外显子，可富集多个精子发生和精子活力、睾丸支持细胞-生精细胞紧密连接、支持细胞-支持细胞紧密连接。此外，p,p’-DDE可下调雄性F1-F3代精子细胞父性印记基因Igf2、Gtl2和H19 DMR甲基化水平，但对母性印记基因Snrpn、Peg3甲基化影响不明显，叶酸可干预p,p’-DDE引起的印记缺陷。.本研究从表观遗传学层面阐明p,p’-DDE暴露出生子代风险增加的原因，为有机氯污染物致雄性生殖毒性防治开拓思路，并为其他环境激素的表观遗传效应及复杂疾病的宫内源性研究提供新的理论依据。