Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation

By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels.Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.During mammalian fertilization, two sets of genomes from male and female gametes join together and then undergo large-scale reprogramming to restore the totipotency. However, although reside in the same zygotic cytoplasm, the paternal and maternal genomes are reprogrammed in different ways. It is well known that several epigenetic modifications are involved in the reprogramming events [1,2].The first described epigenetic modification is DNA methylation. DNA methylation at CpG dinucleotides is associated with the repression of gene transcription and is essential for mammalian development [3]. In mouse zygotes, the paternal genome undergoes active DNA demethylation shortly after fertilization, while the maternal genomic DNA remains methylated throughout the first mitosis [4,5]. Although the active demethylation of paternal genome has been observed in several mammalian species [6], with the same immunostaining approach no paternal DNA demethylation can be detected in sheep, rabbit and goat zygotes [7-9]. Therefore, the paternal demethylation even