Differentially methylated regions identified in bovine embryos are not observed in adulthood

Abstract

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.

Keywords: DMRs; cattle; epigenetics; methylome; reprogramming.

Figure 1. Representation of the KDM2A, KDM5A, KMT2D, and DOT1L gene structures, GC content, and CpG island prediction. Green bars represent the input sequence; below, blue lines represent introns, blue arrows represent exons, and orange arrows represent primer positions. The GC content and CpG islands are predicted for each gene. The graphs were generated using Geneious v2020.0.5 (Biomatters, Auckland, New Zealand).

Figure 2. DNA methylation profile of KDM2A gene in blood and sperm for control and low S/Co groups. (A) Blood samples, (B) Sperm samples, and (C) Comparative analysis of methylation by CpG sites between control and low S/Co in blood and sperm. Each line represents an individual DNA clone, and each circle represents a CpG dinucleotide. Black circles represent methylated cytosines and white circles represent unmethylated cytosines. The DNA methylation percentage for each animal (Bull 1, Bull 2, Bull 3, Bull 4, Bull 5, and Bull 6) is represented as mean ± standard deviation of the mean. Differences in DNA methylation among animals within the same group are shown by letters a and b (p < 0.05). (*) represents significant difference in the mean values for methylation of individual CpGs using Fisher's exact test (p<0.05). (n) represents the number of sequenced alleles of each sample.

Figure 5. DNA methylation profile of DOT1L gene in blood and sperm for control and low S/Co groups. (A) Blood samples, (B) Sperm samples, and (C) Comparative analysis of methylation by CpG sites between control and low S/Co in blood and sperm. Each line represents an individual DNA clone, and each circle represents a CpG dinucleotide. Black circles represent methylated cytosines and white circles represent unmethylated cytosines. The DNA methylation percentage for each animal (Bull 1, Bull 2, Bull 3, Bull 4, Bull 5, and Bull 6) is represented as mean ± standard deviation of the mean. Differences in DNA methylation among animals within the same group are shown by letters a and b (p < 0.05). (*) represents significant difference in the mean values for methylation of individual CpGs using Fisher's exact test (p<0.05). (n) represents the number of sequenced alleles of each sample.

Figure 4. DNA methylation profile of KMT2D gene in blood and sperm for control and low S/Co groups. (A) Blood samples, (B) Sperm samples, and (C) Comparative analysis of methylation by CpG sites between control and low S/Co in blood and sperm. Each line represents an individual DNA clone, and each circle represents a CpG dinucleotide. Black circles represent methylated cytosines and white circles represent unmethylated cytosines. The DNA methylation percentage for each animal (Bull 1, Bull 2, Bull 3, Bull 4, Bull 5, and Bull 6) is represented as mean ± standard deviation of the mean. Differences in DNA methylation among animals within the same group are shown by letters a and b (p < 0.05). (n) represents the number of sequenced alleles of each sample.

Figure 3. DNA methylation profile of KDM5A gene in blood and sperm for control and low S/Co groups. (A) Blood samples, (B) Sperm samples, and (C) Comparative analysis of methylation by CpG sites between control and low S/Co in blood and sperm. Each line represents an individual DNA clone, and each circle represents a CpG dinucleotide. Black circles represent methylated cytosines and white circles represent unmethylated cytosines. The DNA methylation percentage for each animal (Bull 1, Bull 2, Bull 3, Bull 4, Bull 5, and Bull 6) is represented as mean ± standard deviation of the mean. Differences in DNA methylation among animals within the same group are shown by letters a and b (p < 0.05). (*) represents significant difference in the mean values for methylation of individual CpGs using Fisher's exact test (p<0.05). (n) represents the number of sequenced alleles of each sample.

Figure 6. Percentage of methylation in KDM2A, KDM5A, KMT2D, and DOT1L genes (A) Comparison of DNA methylation levels between control and low S/Co groups for blood and sperm samples. (B) Comparison of DNA methylation levels in blood and sperm samples in the control and low S/Co groups, respectively. Numbers represent significant differences in the mean values for methylation using the Mann-Whitney test (p ≤ 0.05)