Transgenerational transcriptomic and DNA methylome profiling of mouse fetal testicular germline and somatic cells after exposure of pregnant mothers to tributyltin, a potent obesogen

Obesogens such as tributyltin (TBT) are xenobiotic compounds that promote obesity, in part by distorting the normal balance of lipid metabolism. The obesogenic effects of TBT can be observed in directly exposed (F1 and F2 generations) and also subsequent generations (F3 and beyond) that were never exposed. To address the effects of TBT exposure on germ cells, we exposed pregnant transgenic OG2 mouse dams (F0), which specifically express EGFP in germline cells, to an environmentally relevant dose of TBT throughout gestation through drinking water. When fed with a high fat diet (HFD), F3 male offspring of TBT-exposed F0 dams (TBT-F3) accumulated much more body fat than did Control-F3 males. TBT-F3 males also lost more body fluid and lean compositions than did Control-F3 males. Expression of genes involved in transcriptional regulation or mesenchymal differ-entiation was upregulated in somatic cells of TBT-F1 (but not TBT-F3) E18.5 fetal testes, and promoter-associated CpG islands were hyper-methylated in TBT-F1 somatic cells. Global mRNA expression of protein-coding genes in F1 or F3 fetal testicular cells was unaffected by F0 exposure to TBT; however, expression of a subset of endogenous retroviruses was significantly affected in F1 and F3. We infer that TBT may directly target testicular somatic cells in F1 testes to irreversibly affect epigenetic suppression of endogenous retroviruses in both germline and somatic cells. Overall design: Mouse E18.5 fetal testicular cells were separated to germline cells (P4 and P5 populations) and somatic (P6) cells and then subjected to RNA-sesq (transcriptomic profiling) or MBD-seq (DNA methylation analysis).

致肥胖物（obesogens）如三丁基锡（tributyltin, TBT）是一类可诱发肥胖的外源性化合物，其部分作用机制为扰乱脂质代谢的正常稳态。三丁基锡的致肥胖效应不仅可在直接暴露的F1、F2子代中观测到，也可在未直接暴露的F3代及后续子代中显现。为探究三丁基锡暴露对生殖细胞的影响，本研究将妊娠期内特异性在生殖系细胞中表达增强型绿色荧光蛋白（EGFP）的转基因OG2小鼠孕鼠（F0代），在整个妊娠期通过饮水暴露于环境相关剂量的三丁基锡。以高脂饮食（HFD）喂养时，经TBT暴露的F0代孕鼠所产的F3代雄性子代（TBT-F3）的体脂积累量显著高于对照组F3代（Control-F3）雄性小鼠；同时，TBT-F3雄性小鼠的体液与瘦体重流失量也多于Control-F3雄性小鼠。在TBT-F1（而非TBT-F3）胎龄18.5天（E18.5）小鼠胎儿睾丸的体细胞中，参与转录调控或间充质分化的基因表达水平上调；且TBT-F1体细胞中，与启动子关联的CpG岛呈现高甲基化状态。F1或F3代胎儿睾丸细胞中蛋白编码基因的整体mRNA表达水平不受F0代TBT暴露的影响；然而，内源性逆转录病毒的部分亚群的表达在F1和F3代中均受到显著调控。本研究推测，三丁基锡可能直接靶向F1代睾丸中的体细胞，不可逆地干扰生殖系与体细胞中内源性逆转录病毒的表观遗传抑制作用。总体实验设计：将胎龄18.5天的小鼠胎儿睾丸细胞分离为生殖系细胞（P4与P5群）与体细胞（P6群），随后分别进行RNA测序（原文笔误为RNA-sesq，标准写法为RNA-seq）与甲基结合域测序（MBD-seq，用于DNA甲基化分析）。