Dynamic Methylation of DNA During Fertilization in Cattle: the Role of Chromatin Condensation of the Sperm

Epigenetics play an important role in mammalian embryo development. Sperm chromatin condensation is crucial for epigenetic reprogramming in paternal genome soon after fertilization, which is required for totipotent zygotic development. We have recently reported that heat stress on bulls alters sperm chromatin condensation. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming as epigenetic marks in the paternal pronucleus and the early embryo development potential after fertilization of oocytes with altered chromatin condensed spermatozoa. Dynamics of DNA methylation reprogramming were immunocytochemically investigated using anti-5-methylcytosine (5mC) antibody in zygotes collected at three different time points, i.e. 12, 18 and 24 hours post insemination (hpi). A total of 1239 oocytes were fertilized and 5mC fluorescence intensities of the paternal pronuclei (n= 223, ≥ 60 in each group) were measured by ImageJ and data were analyzed by ANOVA. DNA methylation pattern in the paternal pronucleus after fertilization of oocytes with heat-stressed spermatozoa 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. Moreover, heat-stressed spermatozoa, having altered chromatin condensation showed a highly reduced (P < 0.01) fertilization rate compared to non-heat-stressed or normal IVF control spermatozoa (53.7% vs. 70.2% or 81.5%, respectively). In conclusion, altered sperm chromatin condensation due to heat stress perturbs dynamics of DNA methylation reprogramming in the paternal genome and early embryo development which may hamper nuclear totipotency and embryo survival.