Effects of doxorubicin on sperm DNA methylation in mouse models of testicular toxicity

Highlights: • Epigenetic influences of male germline by doxorubicin-induced testicular toxicity. • DNMT3a and DNMT3b were decreased in the testes of testicular toxicity model mice. • Major effect of testicular toxicity on sperm DNA was hypomethylation. • These changes also occurred in a group without histopathological damage in testis. Testicular toxicity is a frequent adverse effect of cancer chemotherapy that has no effective clinical biomarker. To find new biomarkers, we focused on epigenetic mechanisms in the male germline. We investigated the DNA methylation status of the male germline during testicular toxicity induced by doxorubicin (DXR), a widely used anticancer agent. We established mouse models of early stage testicular toxicity and testicular pre-toxicity by the administration of 0.2 mg/kg and 0.02 mg/kg DXR, respectively, twice weekly for 5 weeks. Histological analysis showed sparse abnormalities in testicular tissue; however, western blotting analysis revealed reduced testicular expression levels of DNA methyltransferases DNMT3a and DNMT3b in both DXR-treated groups. Interestingly, comprehensive sperm DNA methylation analysis using Methyl-CpG binding domain protein-enriched genome sequencing revealed that hypomethylation was the most frequent change induced by DXR. These findings suggest that sperm DNA methylation status may be used as an early diagnostic marker for testicular changes not detected by conventional toxicity analysis.