高雄激素通过TET3调控卵母细胞去甲基化致子代糖代谢改变的传代机制研究

Adverse environmental exposure during gamete development predisposes offspring to adult metabolic diseases, the mechanism is not yet clear. Using in vitro fertilization-embryo transplant (IVF-ET) mice model, our team previously found out that offspring of oocyte exposed to high androgen level showed impaired glucose tolerance and abnormal insulin levels, oocyte exposed to high androgen demonstrated significantly altered methylation level in insulin signaling pathway and up-regulated TET3 expression. In this project, we will continue to analyze the RRBS results to predict candidate genes in insulin signaling pathway, use BSP/Mspl-QPCR to detect the methylation/hydroxymethylation levels and immunofluorescence to detect the expression of these candidate genes. Also, we plan to obtain the pancreatic islets of the offspring to verify the methylation and expression of candidate genes, in order to determine the target genes playing key roles in the intergenerational transmission. Next we will use mice islet cell line to knock down or overexpress the target genes to observe the islet function, and also to conduct IVF-ET using oocyte knocked down or overexpressed the target genes to observe the phenotype of the offspring. Further, we will perform ChIP to detect the combination of TET3 promoter and androgen receptor, knock down TET3 to investigate the global and target genes’ methylation/hydroxymethylation levels, and use CpG free luciferase plasmid to detect the effect of hydroxymethylation alteration to target gene expression. We hope to reveal the mechanism of the intergenerational transmission of the methylation modification from high androgen exposed oocyte to the offspring, also to provide theory basis for the original intervention of diabetes.

父母配子阶段的不良暴露增加子代代谢疾病的风险。前期我们利用小鼠体外受精（IVF-ET）模型发现，卵母细胞高雄激素暴露出生子代出现糖耐量及胰岛素水平异常，RRBS测序提示高雄卵子中胰岛素通路相关甲基化水平改变且TET3高表达。本项目将深入分析测序结果预测胰岛素通路差异候选基因，BSP/Mspl-QPCR验证甲基化/羟甲基化水平，免疫荧光检测表达。利用高雄IVF小鼠子代胚胎及成年后胰岛验证候选基因甲基化及表达，筛查出传代效应的靶基因；胰岛细胞系敲减/过表达靶基因观察胰岛功能改变；通过显微注射敲减/过表达卵母细胞靶基因后行IVF-ET，观察子代表型是否得到纠正。进一步ChIP探索雄激素对TET3的转录调控；卵子敲减TET3观察整体及靶基因羟甲基化改变；双荧光素酶报告质粒分析靶基因羟甲基化改变对其表达的影响。从而揭示TET3介导的高雄暴露卵母细胞甲基化修饰的传代机制，为糖尿病源头干预提供理论基础。

父母配子阶段的不良暴露增加子代代谢疾病的风险，妊娠晚期高雄激素造模的小鼠其雌性子代连续三代（F1-F3）均出现脂代谢异常。本项目前期已建立卵母细胞高雄激素暴露后体外受精-胚胎移植小鼠模型，我们发现母亲孕前高暴露显著增加子一代成年期糖耐量异常的风险，同时，高雄子代还表现出血脂水平异常，其中雄性成年子代的表型改变最为显著。我们在前期高雄暴露卵母细胞RRBS测序的基础上进行生信分析，结果显示高雄暴露卵母细胞的差异甲基化基因与胰岛素代谢通路相关，进一步我们选择了甲基化水平差异显著的pcx, idh3a, ogdh, pttg1, versican基因作为候选，利用MSP和5-hmC DIP-qPCR技术，发现高雄暴露显著下调pcx, idh3a, versican基因启动子区5mC水平，上调versican基因启动子区的5hmC水平。下一步，我们获取高雄暴露的子一代胚胎期及雄性子一代成年期胰岛细胞，检测靶基因的表达及甲基化水平。结果显示pcx, idh3a, versican在子一代胎鼠及成年雄性的胰岛细胞中表达均显著上调，值得关注的是，versican基因启动子区在胚胎及成年后胰岛细胞中均表现出显著的5mC水平下调及5hmc水平上调，这些印记与高雄卵母细胞中versican的甲基化改变一致，提示我们母细胞高雄暴露所致的子代甲基化/去甲基化异常可能持续至子代成年期，可能为代谢性疾病的配子源性提供表观遗传学机制。