Paternal obesity alters the sperm epigenome and and is associated with changes in the placental transcriptome and its cellular composition [ChIP-seq]

Growing evidence point towards a strong contribution of paternal factors in placental health with implications in adult-onset complex disease risk. We have recently demonstrated that paternal diet-induced obesity alters sperm histone methylation and is associated with metabolic disturbances in the next generation. Diet-sensitive epigenetic regions in sperm were found at genes involved in trophectoderm and placental development, and corresponded to epigenetic and gene expression profiles in these tissues. We sought to investigate whether paternal diet-induced obesity before conception can alter the placental transcriptome, and whether differential gene expression corresponds with sperm obesity-associated epigenetic signatures. C57BL6/J males were fed either a control or high-fat diet for 10 weeks beginning at 6 weeks of age. They were then bred to control-fed C57BL6/J females to induce pregnancies and E14.5 placentas were collected. RNA-sequencing was performed (n=4 per group per sex) to detect sex-specific transcriptional changes associated with paternal diet. At necropsy, sperm was collected for chromatin immunoprecipitation followed by sequencing (ChIP-seq; n=3 per group) targeting histone H3 lysine 4 tri-methylation (H3K4me3) to detect obesity-induced changes in H3K4me3 enrichment. There were sex-specific differentially expressed genes in placentas with some overlapping with promoters showing obesity-associated sperm epimutations. A deconvolution analysis using single-cell RNA-seq data from mouse E14.5 placenta (Han et al., Cell, 2018) revealed significant differences in trophoblast subtype proportions in placentas derived from HFD sires. This study highlights a previously underappreciated role of the placenta at the origin of paternally-induced metabolic disturbances in offspring. Overall design: Examination of sperm H3K4me3 in control or high-fat fed sires (n=3 per group), examination of E14.5 placenta total RNA derived from control or high-fat fed sires (n=4 per sex per group)

越来越多的研究证据表明，父系因素对胎盘健康具有显著的调控作用，且与成年起病复杂疾病的患病风险密切相关。我们此前的研究证实，父系饮食诱导的肥胖会改变精子的组蛋白甲基化修饰，并与子代的代谢紊乱存在显著关联。研究人员在与滋养外胚层（trophectoderm）和胎盘发育相关的基因中，发现了精子内对饮食敏感的表观遗传区域，且这些区域与上述组织中的表观遗传谱及基因表达谱高度契合。本研究旨在探究孕前父系饮食诱导的肥胖是否会改变胎盘转录组（transcriptome），以及差异基因表达是否与精子中肥胖相关的表观遗传标记存在对应关系。

实验方案如下：将6周龄的C57BL6/J雄性小鼠分为两组，分别喂食正常饮食（control diet）与高脂饮食（high-fat diet, HFD），持续干预10周；随后将这些雄性小鼠与喂食正常饮食的C57BL6/J雌性小鼠合笼以建立妊娠模型，收集胚胎发育第14.5天（E14.5）的胎盘组织。对胎盘组织开展RNA测序（RNA-sequencing），每组每性别的样本量为4，以检测与父系饮食相关的性别特异性转录变化；剖检时收集精子样本，针对组蛋白H3赖氨酸4三甲基化（H3K4me3）进行染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq，每组样本量为3），以检测肥胖诱导的H3K4me3富集水平变化。

研究结果显示：胎盘中存在性别特异性的差异表达基因，其中部分基因的启动子区域与精子中肥胖相关的表观遗传突变存在重叠；利用小鼠E14.5胎盘的单细胞RNA测序数据（Han等，《Cell》，2018）进行细胞反卷积分析（deconvolution analysis），发现高脂饮食组父系来源的胎盘中，滋养层细胞（trophoblast）亚型的占比存在显著差异。

本研究揭示了胎盘在父系诱导的子代代谢紊乱起源中此前未被充分重视的关键作用。
整体实验设计：① 检测正常饮食与高脂饮食喂食的父鼠精子中的H3K4me3修饰（每组n=3）；② 检测正常饮食与高脂饮食喂食的父鼠所产子代的E14.5胎盘总RNA（每组每性别n=4）