Exploring the epigenetic landscape of spermatozoa: impact of oxidative stress and antioxidant supplementation on DNA methylation and hydroxymethylation. Exploring the epigenetic landscape of spermatozoa: impact of oxidative stress and antioxidant supplementation on DNA methylation and hydroxymethylation

Oxidative stress in the epididymal compartment of snGpx4-/-;Gpx5-/- male mice has been shown to increase the percentage of spermatozoa with 8OHdG positive staining (DNA oxidation). In these mice, the total amount of sperm nuclear 5-hydroxymethylCytosine (5hmC) is significantly higher than in wild type mice. As the transormation of the epigenetic mark 5-methyCytosine (5mC) into 5hmC is an oxidative process, normally driven by enzymes, this work was aimed to test whether the increase in 5hmC was driven by the oxidative stress. Mice were given an oral antioxidant supplementation (Fertilix®) for 14 days in their drinking water, a treatment previously shown by our group to decrease epididymal oxidative stress, shown by a significant decrease in the percentage of 8OHdG positive sperm cells. Genomic DNA was then extracted from cauda epididymsi spermatozao and sequenced, using EM-seq and EhM-seq to respectively determine 5mC and 5hmC bases in a reduced representation protocol. Overall design: Wild-type and snGpx4-/-;Gpx5-/- male mice were used in the study. They were given (Fx-samples) or not an oral antioxidant supplementation (Fertilix®) for 14 days in their drinking water. Cauda epididymal spermatozoa were then retrieved and their genomic DNA extracted. EM-seq and EhM-seq were performed for each individual in order to determine the occurence of 5mC and 5hmC, respectively.

已有研究证实，snGpx4-/-;Gpx5-/-雄性小鼠附睾区域的氧化应激可提升8-羟基脱氧鸟苷(8-hydroxy-2'-deoxyguanosine, 8OHdG)阳性染色精子的比例（即DNA氧化损伤）。该类小鼠的精子核总5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)含量显著高于野生型小鼠。由于表观遗传标记5-甲基胞嘧啶(5-methylcytosine, 5mC)向5hmC的转化是一类由酶介导的生理性氧化过程，本研究旨在验证5hmC水平升高是否由氧化应激驱动。

研究人员为小鼠饮用水中添加口服抗氧化剂Fertilix®，连续给药14天——该处理方案已被本团队前期证实可降低附睾氧化应激，具体表现为8OHdG阳性精子比例的显著降低。随后从附睾尾精子中提取基因组DNA，采用简化基因组测序策略，分别通过EM-seq与EhM-seq检测样本中5mC与5hmC碱基的分布情况。

总体实验设计：本研究纳入野生型及snGpx4-/-;Gpx5-/-雄性小鼠，将其分为两组：一组在饮用水中添加Fertilix®抗氧化剂（Fx-samples组），连续干预14天，另一组不做处理作为对照。随后收集各组小鼠的附睾尾精子并提取基因组DNA，对每只个体均开展EM-seq与EhM-seq测序，分别检测5mC与5hmC的发生丰度。