139 PERTURBATION OF THE DYNAMICS OF DNA METHYLATION IN THE PATERNAL GENOME FOLLOWING IN VITRO FERTILIZATION IN CATTLE: THE SECRETS OF HEAT STRESS EFFECTS

Abstract

Ejaculates collected from breeding bulls during the summer season under tropical or subtropical conditions are often reported to have a lower fertilization potential. We recently reported that sperm cells at post-meiotic stages of development were more susceptible to heat stress (scrotal insulation technique). In post-meiotic stages of sperm cell development, extensive incorporation of histone variants and hyperacetylation confirms unstable chromatin. The unstable forms of chromatin are more vulnerable to heat stress and affect sperm DNA-protamine condensation. By using a special stain and nuclear morphometric assessment, Chromomycin A₃ and Fourier harmonic analysis, respectively, we observed protamine-deficient spermatozoa with altered chromatin condensation in the heat-stressed ejaculates. In a recent study, we investigated the patterns of global DNA methylation in pronuclear development and fertilization potential of such altered chromatin condensed spermatozoa. To this end, 1239 in vitro-matured oocytes were fertilized with spermatozoa of 3 groups (i.e. standard IVF control, non-heat-stressed control, and heat-stressed spermatozoa). Data were analysed by means of ANOVA. The results showed that heat-stressed spermatozoa with altered chromatin condensation perturb the dynamics of DNA methylation reprogramming in the paternal pronucleus (i.e. disordered the active demethylation followed by a de novo methylation pattern during one cell stage). In addition, there was a tendency for a decrease in the size of both paternal and maternal pronuclei developed after fertilization with heat-stressed spermatozoa in comparison with standard IVF control spermatozoa, leading to lower fertilization rates (54 v. 82%). Future studies should focus on the mechanisms of perturbed DNA demethylation in the paternal genome after in vitro fertilization of oocytes with spermatozoa having altered chromatin condensation.