5 Age-dependent changes in DNA methylation levels of spermatozoa and the relationship to their motility and fertility in Japanese Black bulls

Predicting the fertility of semen before AI enables to avoid economic loss. Recently, it has been reported that sperm DNA methylation levels on specific CpG sites correlate with the conception rate after AI in Japanese Black bulls (Takeda et al. 2021 J. Reprod. Dev. 67, 99–107). However, it is unclear how the DNA methylation levels affect the sperm fertility. In this study, we aimed to investigate whether the DNA methylation levels relate to the sperm motility and the developmental competence of embryos following IVF. Since preliminary experiments confirmed that freezing procedure did not affect the DNA methylation levels, frozen semen from 16 Black Japanese bulls aged 16 to 131 months were used. A total of 49 samples, including samples taken from the same individual at different ages, were subjected to DNA methylation analysis. The DNA methylation levels on three CpG sites (CpG-F3, CpG-F4, and CpG-F5) reported to correlate with the conception rate after AI were evaluated using the COBRA method. Based on the DNA methylation analysis, hypo- and hypermethylated spermatozoa were selected and applied to the motility analysis at 0 and 3 h after thawing using the Sperm Motility Analysis System (SMAS, DITECT, Tokyo, Japan). To assess in vitro development, embryos fertilized with hypo- and hypermethylated spermatozoa were cultured for 7 days under time-lapse monitoring system taking photographs of the embryos at 15-min intervals during the culture period. The total cell number and apoptotic status of the blastocysts were also determined by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. All data obtained from more than three replicates was analysed using a Student’s t-test. The DNA methylation levels on CpG-F4 and CpG-F5 sites tended to increase with age of bulls and then remained at constant levels. In the samples we examined in this study, a significant increase in these levels was observed after ~50 months of age. In contrast, DNA methylation level on a CpG-F3 site was constant regardless of age. For sperm motility, hypermethylated spermatozoa maintained total motility, linear velocity, curvilinear velocity, and mean velocity after 3 h of thawing, whereas hypomethylated spermatozoa significantly decreased these parameters. However, no significant differences in the developmental parameters were observed between embryos fertilized with hypo- and hypermethylated spermatozoa. These results suggest that the DNA methylation levels on CpG-F4 and CpG-F5 sites in spermatozoa does not relate directly to their fertility, but the hypomethylation may be one of the characteristics of spermatozoa produced by immature bulls, as well as low motility persistence. In conclusion, the DNA methylation levels on CpG-F4 and CpG-F5 sites of spermatozoa could be one of the indicators to estimate the maturity of bulls.