5- methylcytidine effectively improves spermatogenesis recovery in busulfan-induced oligoasthenospermia mice.

The function and regulatory mechanisms of 5-methylcytidine (mC) in oligoasthenospermia remain unclear. In this study, we made a mouse model of oligoasthenospermia through the administration of busulfan (BUS). For the first time, we demonstrated that mC levels decreased in oligoasthenospermia. The mC levels were upregulated through the treatments of 5-methylcytidine. The testicular morphology and sperm concentrations were improved via upregulating mC. The cytoskeletal regenerations of testis and sperm were accompanying with mC treatments. mC treatments improved T levels and reduced FSH and LH levels. The levels of ROS and MDA were significantly reduced through mC treatments. RNA sequencing analysis showed mC treatments increased the expression of genes involved in spermatid differentiation/development and cilium movement. Immunofluorescent staining demonstrated the regeneration of cilium and quantitative PCR (qPCR) confirmed the high expression of genes involved in spermatogenesis. Collectively, our findings suggest that the upregulation of mC in oligoasthenospermia facilitates testicular morphology recovery and male infertility via multiple pathways, including cytoskeletal regeneration, hormonal levels, attenuating oxidative stress, spermatid differentiation/development and cilium movement. mC may be a potential therapeutic agent for oligoasthenospermia.