Spermatozoa cryopreservation alters pronuclear formation and zygotic DNA demethylation in mice

This study was conducted to investigate the effects of spermatozoa cryopreservation on DNA demethylation in mouse zygotes. Global methylation was studied in zygotes fertilized with cryopreserved sperm by immunostaining, and relative transcript abundance of Tet3, a key gene responsible for zygotic DNA demethylation, was examined by real-time quantitative polymerase chain reaction. Fresh sperm group served as control. Results indicated spermatozoa cryopreservation decreased fertilization rate (68.2% vs. 86.9%; P < 0.01) and delayed pronuclear formation (P < 0.05), compared with the control group. The percentages of embryos developed to cleavage and blastocyst stages in the freezing group (52.9% and 66.8%, respectively) were lower (P < 0.01 and P < 0.05, respectively) than those of the control group (83.4% and 81.1%, respectively). Furthermore, embryos obtained from cryopreserved sperm had higher relative methylation levels (P < 0.05) and less Tet3 mRNA concentrations (P < 0.01) in advanced pronuclear stages. Hence, we reported that spermatozoa cryopreservation disturbed the Tet3-mediated DNA demethylation progression in the zygotic paternal genome, which could be detrimental to the development of early mouse embryos, and most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm.