The kinetic of early cleavages influence the DNA methylome status of bovine embryos

DNA methylation is an epigenetic control mechanism essential during embryonic development, directing the differentiation of cell lineages and preventing regression to an undifferentiated state. Based on the fact that embryos with different development kinetics present differences in gene expression, this study aimed to characterize the differences between fast and slow embryos regarding the methylome status. For that, COCs from slaughterhouse ovaries were subjected to IVM for 22-24 h, IVF using sexed semen (female) for 18 h and IVC for 7 days. After 40 hours of insemination, embryos were classified as fast (four or more cells) or slow (2 or 3 cells) remaining in culture until the blastocyst stage (40 blastocysts per group in 4 replicates). These embryos were analyzed by EmbryoGENE Methylation DNA Array (EDMA). Briefly, the DNA extracted from 10 embryos was fragmented with MseI restriction enzyme and the fragments were extracted using methylation-sensitive digestion and ligation-mediated amplification PCR- (LMA-PCR). Hybridization was performed according to the manufactureru0027s instructions (Agilent Technologies). It was considered as differentially methylated regions (DMRs) fragments with methylation differences with P 1.5 between groups. From probes used for microarray, 9082 were expressed above background only in fast blastocysts (FBL), 20670 in slow blastocysts (SBL) and 47713 were observed in both groups. FBL presented 7976 DMRs and SBL presented 3608 DMRs. While fast embryos presented more hypermethylations in DMRs distributed throughout the genome, such as introns, exons, promoter and repeat elements, slow embryos presented more methylation of various densities (High - FBL: 17; SBL: 35; Intermediate - FBL: 81; SBL: 143 and Low - FBL: 31; SBL: 68) focused on CpG islands independent of length (Long - FBL: 14; SBL: 41; Intermediate - FBL: 79; SBL: 152 and Small - FBL: 36; SBL: 53). With Ingenuity Pathway Analysis 25 pathways from FBL and SBL were identified with differences in DNA methylation. These pathways include DNA damage, repair and replication, development disorder, cell morphology, gene expression, post-transcriptional modifications, metabolic diseases, lipid metabolism, post-translational modifications and embryonic development (33-35 genes observed in each pathway). Hypermethylation differences were also observed in genes related to pluripotency (NANOG, OCT4), epigenetic control (DNMT3A, HDAC8, MECP2) and embryonic development (EGFR, FGF8, IGFR1). In conclusion, the DNA methylation profile is different between embryos with fast or slow kinetics of development, thus influencing the phenotype and response to the environment.