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

The function and regulatory mechanisms of 5-methylcytidine (m5 C) 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 m5 C levels decreased in oligoasthenospermia. The m5 C levels were upregulated through the treatments of 5-methylcytidine. The testicular morphology and sperm concentrations were improved via upregulating m5 C. The cytoskeletal regenerations of testis and sperm were accompanying with m5 C treatments. m5 C treatments improved T levels and reduced FSH and LH levels. The levels of ROS and MDA were significantly reduced through m5 C treatments. RNA sequencing analysis showed m5 C 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 m5 C 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. m5 C may be a potential therapeutic agent for oligoasthenospermia.