Whole genome bisulfite sequencing of nicotine-exposed sperm reveals differences in global DNA methylation levels, which would partially reverse following cessation

DNA methylation, a pivotal epigenetic modification, regulates the expression of numerous genes during spermatogenesis and influences health outcomes in offspring. We have conducted whole genome bisulfite sequencing (WGBS) to explore the effects of nicotine exposure on global sperm DNA methylation patterns. Previous studies have shown that nicotine exposure increased DNA methylation status in sperm, whereas the detailed mapping of global DNA methylation patterns remains elusive. In addition, whether the DNA methylation changes induced by nicotine may be reversible upon cessation is still unclear. Here, we demonstrated the effects of nicotine exposure on global sperm DNA methylation patterns and verified the reversibility of this phenomenon, providing new insights into the mechanisms by which smoking affects male fertility and the long-term health of offspring. Overall design: Three-week-old C57BL/6J male mice were divided into three groups: (1) Control group (C), which received normal drinking water; (2) Nicotine-exposed group (N), which received drinking water with 200 µg/mL nicotine for 8 weeks; and (3) Nicotine withdrawal group (NW), which received normal drinking water for an additional 5 weeks following 8 weeks of nicotine exposure. Followed by treatment, sperm was extracted, and WGBS was performed on total sperm DNA.