6 ALTERED CHROMATIN CONFORMATION IN BOVINE SPERMATOZOA PERTURBS DYNAMIC DNA METHYLATION IN THE MALE PRONUCLEUS AFTER FERTILIZATION IN VITRO

Abstract

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.