Changes in rat sperm DNA methylation induced by age and by perinatal exposure to brominated flame retardant flame retardants 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47)

Advanced paternal age at fertilization has been suggested to be a risk factor for neurodevelopmental, psychiatric and other disorders in offspring. One emerging hypothesis suggests that altered offspring phenotype is linked with age-related accumulation of epigenetic changes in the sperm of fathers. Given that paternal age is increasing in the developed world, understanding aging-related epigenetic changes in sperm is needed as well as environmental factors that modify such changes. In this study, we characterize age-dependent changes in sperm DNA methylation profiles between young pubertal (postnatal day (PNDs) 65) and mature (PND120) Wistar rats. We also analyze these changes in rats exposed perinatally to 0.2 mg/kg of ubiquitous environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). Reduced representation bisulfite sequencing (RRBS) libraries were prepared from caudal epididymal sperm DNA and differentially methylated regions (DMRs; ≥ 10x coverage depth, ≥ 3 CpGs per cluster, ≥ 5% methylation change, q < 0.05) were identified via MethPipe package. We identified 21 and 9 exposure-related DMRs in sperm collected on PND65 and PND120, respectively. Two DMRs overlapped between the two time-points. This is the first study to demonstrate that environmentally-relevant perinatal exposure to PBDE results in long-lasting changes in sperm DNA methylation. In control animals, 5,319 age-dependent DMRs were identified, with 99.3% DMRs hypermethylated in mature animals compared to young pubertal rats. These age-related DMRs were enriched for functional categories essential for embryonic development, such as pattern specification, forebrain and sensory organ development, and the Wnt pathway. In BDE-47 exposed rats, sperm DNA methylation was higher in young pubertal and lower in mature animals when compared to controls, which resulted in a significant attenuation in the number of age-dependent DMRs (N = 189) identified in the exposed group. In conclusion, our results indicate that the natural aging process has profound effects on sperm methylation levels and this effect may be modified by environmental exposures. Moreover, our results further support the role of sperm DNA methylation as a likely mechanism by which advanced paternal age is associated with adverse offspring health and development.

受孕时父亲的高龄已被认为是子代发生神经发育、精神及其他疾病的风险因素。当前一项新兴假说提出，子代表型改变与父亲精子中随年龄积累的表观遗传变化（epigenetic changes）相关。鉴于发达国家中父亲生育年龄呈上升趋势，因此亟需阐明精子中与衰老相关的表观遗传变化，以及可调控此类变化的环境因素。本研究对青春期早期（出生后第65天，PND65）与成熟（出生后第120天，PND120）Wistar大鼠的精子DNA甲基化（DNA methylation）谱中的衰老依赖性变化进行了表征。同时，本研究还分析了围产期暴露于0.2 mg/kg普遍存在的环境外源性化学物2,2',4,4'-四溴二苯醚（2,2',4,4'-tetrabromodiphenyl ether, BDE-47）的大鼠的此类变化。本研究从附睾尾精子DNA中构建简化代表性亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）文库，并通过MethPipe软件包鉴定差异甲基化区域（differentially methylated regions, DMRs；筛选标准为测序深度≥10×、每个簇包含≥3个CpG位点、甲基化变化幅度≥5%、q值<0.05）。研究分别在PND65与PND120采集的精子中鉴定出21个和9个与暴露相关的DMRs，其中两个DMRs在两个时间点存在重叠。本研究首次证实，环境相关剂量的围产期多溴二苯醚（PBDE）暴露会导致精子DNA甲基化发生持久改变。在对照组大鼠中，共鉴定出5319个衰老相关DMRs，其中99.3%的DMRs在成熟大鼠中相较于青春期早期大鼠呈现高甲基化状态。此类衰老相关DMRs显著富集于胚胎发育必需的功能类别，包括模式形成、前脑及感觉器官发育，以及Wnt信号通路（Wnt pathway）。与对照组相比，BDE-47暴露组的青春期早期大鼠精子DNA甲基化水平更高，而成熟大鼠则更低，这导致暴露组中鉴定出的衰老相关DMRs数量显著减少（N=189）。综上，本研究结果表明，自然衰老过程会对精子甲基化水平产生显著影响，且该影响可被环境暴露所调控。此外，本研究结果进一步支持了精子DNA甲基化作为潜在机制，可解释父亲高龄与子代不良健康及发育结局之间的关联。