136 DNA Methylation Status and Transcript Profile of Genes Associated with Lipid Metabolism, Cell Survival, and Pluripotency in Bovine Blastocysts with Different Kinetics of Development

Abstract

During in vitro production (IVP), blastocysts can be differentiated based on the kinetics of early cleavage. These groups present distinct patterns of global DNA methylation, an epigenetic characteristic generally responsible for suppression (presence of methylation) or activation (absence of methylation) of genes from different biological pathways. This work investigated the DNA methylation and mRNA levels of genes related to embryo development and viability. For this purpose, bovine embryos underwent IVP using conventional protocols. After 40 h of insemination, embryos were classified as FBL (fast cleavage: 4 cells or more) or SBL (slow cleavage: 2 or 3 cells), remaining in culture until blastocyst stage. Sexed semen was used to prevent differences due to sex, even without statistical differences in male:female ratio already reported. Blastocysts (40 per group) were analysed by EmbryoGENE Methylation DNA Array (Ispada et al. 2016 Proc. 49th SSR: 181) and later analysed through BioMark™HD (Fluidigm Corp., South San Francisco, CA, USA) for the transcripts profile. The PPIA gene was used as endogenous control for ΔCt calculation and submitted to Student’s t-test. Genome-wide DNA methylation analysis identified 47,713 methylated regions (7976 hypermethylated in FBL and 3608 hypermethylated in SBL). Fast embryos presented more hypermethylations distributed throughout the genome, such as introns, exons, promoter and repeat elements, whereas hypermethylation were more present in CpG islands in slow embryos. Differentially methylated regions were clustered by means of biological processes and the most affected pathways were related to lipid metabolism and cell differentiation and survival. Regarding the gene expression analysis, all results are presented in FBL in relation to SBL. Of genes involved in lipid metabolism, ACSL3, ELOVL6, PPARA, and FADS, previously identified as hypermethylated genes, were down-regulated, whereas PPARG and PTGS2 showed no statistical difference; SCD and FASN, although hypomethylated, were also down-regulated, and ACSL6, which did not differ in DNA methylation status, was down-regulated. Of genes involved in survival/death, BAX, HSPA1A, BID, NFE2L2, and GPX1 were hypermethylated; however, the first 2 were up-regulated, BID was down-regulated, and the last 2 were not statistically different. Although CASP9, TXNRD1, and FOXO3 were all hypomethylated, only CASP9 was up-regulated. Also, DDIT3 was down-regulated and NOS2 was up-regulated, although they did not differ in DNA methylation between groups. Of genes involved in cell differentiation, POU5F1 and SALL4 were both hypermethylated, but only the POU5F1 was down-regulated; NANOG, which did not differ in DNA methylation status between groups, was also down-regulated. In conclusion, although we did not find correlation in DNA methylation and RNA levels for all genes evaluated, the chosen pathways were indeed different between groups, which could lead to their potential suppression/activation and affect embryo viability. Also, this lower correlation may be a result of the influence of other epigenetic mechanisms differently activated between groups.