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. 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.