Alcohol-Induced Alterations in Sperm Noncoding RNAs Persist After One Month Cessation and Correlate with Indicators of Epididymal Mitochondrial Dysfunction I

Background Chronic preconception paternal alcohol use modifies the sperm epigenome, inducing fetoplacental growth defects in the offspring of exposed males. A crucial outstanding question in the field of paternal epigenetic inheritance concerns the resilience of the male reproductive tract and the germline's capacity to recover and correct sperm-inherited epigenetic errors after stressor withdrawal. Objectives We set out to determine if measures of the sperm-inherited epigenetic program revert to match the control treatment one month after withdrawing daily alcohol treatments. Materials and Methods Using a voluntary access model, we exposed C57Bl6/J males to 10% alcohol for ten weeks, withdrew alcohol treatment for four weeks, and used RNA sequencing to examine gene expression patterns in the caput section of the epididymis. We then compared the abundance of sperm small RNA species between treatments. Results In the caput section of the epididymis, chronic alcohol exposures induced changes in the transcriptional control of genetic pathways related to mitochondrial function, oxidative phosphorylation, the generalized stress response (EIF2 signaling), and Sirtuin signaling. Subsequent analysis identified region-specific, alcohol-induced changes in mitochondrial DNA copy number across the epididymis, which correlated with increases in the mitochondrial DNA content of alcohol-exposed sperm. Notably, in the corpus section of the epididymis, increases in mitochondrial DNA copy number persisted one month after alcohol cessation. Analysis of sperm noncoding RNAs between Control and Alcohol-Exposed males one month after alcohol withdrawal revealed a ~100-fold increase in mir-196a, a microRNA induced as part of the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-driven cellular antioxidant response. Discussion and Conclusion Our data reveal that alcohol-induced epididymal mitochondrial dysfunction and differences in sperm noncoding RNA content persist after alcohol withdrawal. Further, differences in mir-196a and sperm mitochondrial DNA copy number may serve as viable biomarkers of adverse alterations in the sperm-inherited epigenetic program. RNA seq on EtOH treated and EtOH ceased isolated caput portions of the epididymis

背景 孕前父系长期酒精暴露可改变精子表观基因组，导致暴露雄性的子代出现胎儿胎盘生长缺陷。当前父系表观遗传研究领域尚存一个关键未决问题：雄性生殖道的恢复能力，以及生殖细胞系在应激撤除后修复精子携带的表观遗传错误的能力。

研究目的 本研究旨在明确：在每日酒精暴露撤除一个月后，精子携带的表观遗传程序相关指标是否可恢复至与对照组匹配的水平。

材料与方法 本研究采用自愿摄入模型，将C57Bl6/J雄性小鼠暴露于10%酒精溶液中10周，随后撤除酒精干预4周；通过RNA测序（RNA-seq）检测附睾头段的基因表达模式，并比较不同干预组间精子小RNA的丰度差异。

结果 慢性酒精暴露可导致附睾头段中与线粒体功能、氧化磷酸化、整体应激应答（EIF2信号通路）以及沉默信息调节因子（Sirtuin）信号通路相关的遗传通路转录调控发生改变。后续分析发现，酒精暴露可引发附睾不同区段出现特异性的线粒体DNA拷贝数变化，该变化与酒精暴露组精子的线粒体DNA含量升高相关。值得注意的是，在附睾体段，线粒体DNA拷贝数的升高在酒精撤除一个月后仍持续存在。对酒精撤除一个月后的对照组与酒精暴露组雄性小鼠的精子非编码RNA进行分析后发现，mir-196a的表达量升高约100倍；该微小RNA（microRNA）是核因子E2相关因子2（Nrf2）介导的细胞抗氧化应答过程中诱导产生的。

讨论与结论 本研究数据显示，酒精诱导的附睾线粒体功能异常以及精子非编码RNA含量差异在酒精撤除后仍持续存在。此外，mir-196a表达差异与精子线粒体DNA拷贝数差异或可作为精子携带的表观遗传程序发生不良改变的可靠生物标志物。本研究对酒精处理及酒精撤除后分离得到的附睾头段样本开展了RNA测序。