Table 1.
The list of primers used to validate DMRs in ZY, 4C, 16C and IVP blastocyst groups using bisulfite sequencing.
Fig 1.
Differentially methylated regions (DMRs) in ZY, 4C, 16C and IVP blastocyst groups.
Red and green bars indicate the number of hypermethylated and hypomethylated genomic regions, respectively. The magnitude the DMRs stretched between 1.5 and 2.3 absolute fold changes in ZY and 4C groups, between 1.5 and 4.6 in the 16C group and between 1.5 and 2.6 folds in IVP group with reference to VO group.
Fig 2.
The circos plot representing the overall methylation levels in the IVP blastocyst group.
The mean p-values of 5 Mbp windows are indicated along with the 100 most significant DMRs. Positive and negative fold-changes represent the level of hypermethylation and hypomethylation in IVP blastocysts.
Fig 3.
Venn diagram depicting differentially methylated genomic regions specific in ZY, 4C, 16C or IVP blastocyst group or genomic regions stably hypermethylated or hypomethylated in two or more blastocyst groups.
Symbols, ↑ and ↓ indicate hypermethylation and hypomethylation, respectively. DMRs = the number of differentially methylated regions. The gene lists under the column target genes indicate only representative ones.
Table 2.
Differentially methylated CpG islands in ZY blastocyst group.
Table 3.
Differentially methylated CpG islands in 4C blastocyst group.
Table 4.
Differentially methylated CpG islands in 16C blastocyst group.
Table 5.
Differentially methylated CpG islands at the promoter and proximal promoter regions in IVP blastocyst group.
Fig 4.
Differentially methylated CpG islands in ZY, 4C, 16C and IVP blastocyst groups.
(A), The relative proportion of long, short and intermediate CpG islands with respect to the total differentially methylated CpG islands. (B), The relative proportion of hypermethylated or hypomethylated small, long and intermediate CpG islands in ZY, 4C, 16C and IVP blastocyst groups. Black and white bars indicate hypermethylation and hypomethylation, respectively. (C), The relative proportion of CpG islands and non-CpG islands with respect to the total probeset (EDMA array) or relative to the total differentially methylated probes. The red dotted line represents the baseline of the ratio when all probes are taken into account. The probe is assumed to be in CpG shore, CpG shelves and Open Sea if the fragment the probe is located 1–2 kbp, 2–4 kbp and > 4 kbp away from the nearest CpG island, respectively. (D), Enrichment ratios of hypermethylated non-CpG islands DMRs with respect to their distance to the nearest CpG islands. Positive log2 enrichment indicates increased proportion of hypermethylated region and negative log2 enrichment values depict higher proportion of hypomethylated genomic regions at Open-Sea, CpG-shore or CpG-shelf.
Fig 5.
Differentially methylated bovine repetitive elements in different blastocyst groups.
(A), The number of differentially methylated long-terminal-repeat (LTR), short-interspersed repetitive elements (SINEs), long-interspersed repetitive elements (LINEs), low-complexity repetitive elements and simple repeats in ZY, 4C, 16C and IVP blastocyst groups. (B), Enrichment analysis indicating enrichment ratio of hypermethylated LTR, SINEs, LINEs, low-complexity repetitive elements or simple repeats of in ZY, 4C, 16C and IVP blastocyst groups. Positive log2 enrichment values indicate higher proportion of hypermethylated LTR, SINEs, LINEs, low-complexity repetitive elements or simple repeats.
Fig 6.
Genomic localization of differentially methylated regions.
(A), The proportion of DMRs falling within the promoter, proximal promoter, exonic, intronic or distal promoters in ZY, 4C, 16C and IVP blastocyst groups. (B), Enrichment ratio of hypermethylated regions in gene body and promoter sites in ZY, 4C, 16C and IVP blastocyst groups. Positive log2 enrichment indicates higher proportion of the hypermethylated probes at the proximal promoter, promoter, distal promoter, intron or exon regions. The proximal promoter, promoter and distal promoter regions are defined as the first 1 kbp, 5 kbp and 50 kbp from the transcription start site, respectively and probes which are not within the "Distal promoter" distance were classified as "no nearby gene".
Fig 7.
Functional annotation of genes targeted by differentially methylated region in ZY (A), 4C (B) and 16C (C) blastocyst groups.
Numbers in parenthesis indicate the number of genes affected by DMRs.
Fig 8.
Functional annotation of genes targeted by differentially methylated regions in IVP blastocyst group.
Numbers in parenthesis indicate the number of genes affected by the DMRs.
Fig 9.
Heatmaps demonstrating inverse relationship between the patterns of gene expression and DNA methylation profile in ZY (A), 4C (B) and 16C (C) blastocyst groups.
DMRs = differentially methylated regions, DEGs = differentially expressed genes, Region = Genomic region of the DMRs. Ppromoter = proximal promoter.
Fig 10.
Heatmaps displaying inverse relationship between the hypermethylated genomic regions and downregulated gene expression patterns in IVP blastocyst group.
Region = genomic Region of the DMRs. Ppromoter = proximal promoter.
Fig 11.
Heatmaps displaying inverse relationship between the hypomethylated genomic regions and increased expression pattern of their corresponding genes in IVP blastocyst group.
DMRs = differentially methylated regions, DEGs = differentially expressed genes Region = genomic regions of the DMRs. Ppromoter = proximal promoter.
Fig 12.
Validation of selected hypermethylated (A) and hypomethylated (B) genomic regions in ZY, 4C, 16C and IVP blastocyst groups using bisulfite sequencing.
The numbers on the top of the bars correspond to the EDMA probe IDs. Bars are presented as mean ± SEM.
Fig 13.
The qPCR validation result of selected candidate differentially expressed genes in IVP (A) and 4C (B) blastocyst groups.