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Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

Salilew-Wondim D, Fournier E, Hoelker M, Saeed-Zidane M, Tholen E, Looft C, Neuhoff C, Besenfelder U, Havlicek V, Rings F, Gagné D, Sirard MA, Robert C, A Shojaei Saadi H, Gad A, Schellander K, Tesfaye D - PLoS ONE (2015)

Bottom Line: However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions.In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups.Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, 53115 Bonn, Germany.

ABSTRACT
Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0140467.g002: 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.

Mentions: In this study, we investigated the DNA methylation landscape of blastocysts developed in vivo after embryos were cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) stage and blastocysts developed completely under in vitro condition (IVP) with reference to the blastocysts developed completely under in vivo condition (VO). For this, the blastocysts genomic DNA was digested using the MseI restriction enzyme followed by adapter ligation, methyl-sensitive restriction enzymes and ligation mediated PCR genomic amplification. The abundance of each fragment obtained from ligation mediated amplification in each sample was then measured using EmbryoGENE DNA Methylation Array (EDMA). Probes with signal intensities higher than the background signal in each comparison (ZY vs. VO, 4C vs. VO, 16C vs. VO and IVP vs. VO) were analyzed for further analysis. Accordingly, from a total of > 400K probes, the signal intensity of 196871, 199265, 255254, 248453 and 227075 probes in ZY, 4C, 16C, IVP and VO groups, respectively exceeded the background signal and 174460 probes were commonly detected in all sample groups (S1 Fig). Differentially methylated regions were identified by fitting intensity data using a linear model [27]. Probes that exhibited fold-changes of ≥ log21.5 at p < 0.05 in each comparison were considered differentially methylated regions (DMRs). Thus, differential methylated region analysis indicated that longer exposure of preimplantation embryos to the in vitro culture conditions increased DNA methylation profile alteration in the resulting blastocysts (Fig 1). A systematic increase in both hypermethylated and hypomethylated genomic regions were identified from the ZY to the IVP group, with hypermethylated loci outpacing that of the hypomethylated ones in 4C and 16C blastocyst groups. Furthermore, 42, 75, 69 and 51.8% of the total DMRs in the ZY, 4C, 16C and IVP blastocyst groups, respectively were hypermethylated and the rest were hypomethylated. Apart from these, the DNA methylation profiles identified in ZY, 4C, 16C and IVP blastocyst groups were evenly distributed across all chromosomes without showing any significant chromosome specific effects (Fig 2, S2–S4 Figs).


Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

Salilew-Wondim D, Fournier E, Hoelker M, Saeed-Zidane M, Tholen E, Looft C, Neuhoff C, Besenfelder U, Havlicek V, Rings F, Gagné D, Sirard MA, Robert C, A Shojaei Saadi H, Gad A, Schellander K, Tesfaye D - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4633222&req=5

pone.0140467.g002: 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.
Mentions: In this study, we investigated the DNA methylation landscape of blastocysts developed in vivo after embryos were cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) stage and blastocysts developed completely under in vitro condition (IVP) with reference to the blastocysts developed completely under in vivo condition (VO). For this, the blastocysts genomic DNA was digested using the MseI restriction enzyme followed by adapter ligation, methyl-sensitive restriction enzymes and ligation mediated PCR genomic amplification. The abundance of each fragment obtained from ligation mediated amplification in each sample was then measured using EmbryoGENE DNA Methylation Array (EDMA). Probes with signal intensities higher than the background signal in each comparison (ZY vs. VO, 4C vs. VO, 16C vs. VO and IVP vs. VO) were analyzed for further analysis. Accordingly, from a total of > 400K probes, the signal intensity of 196871, 199265, 255254, 248453 and 227075 probes in ZY, 4C, 16C, IVP and VO groups, respectively exceeded the background signal and 174460 probes were commonly detected in all sample groups (S1 Fig). Differentially methylated regions were identified by fitting intensity data using a linear model [27]. Probes that exhibited fold-changes of ≥ log21.5 at p < 0.05 in each comparison were considered differentially methylated regions (DMRs). Thus, differential methylated region analysis indicated that longer exposure of preimplantation embryos to the in vitro culture conditions increased DNA methylation profile alteration in the resulting blastocysts (Fig 1). A systematic increase in both hypermethylated and hypomethylated genomic regions were identified from the ZY to the IVP group, with hypermethylated loci outpacing that of the hypomethylated ones in 4C and 16C blastocyst groups. Furthermore, 42, 75, 69 and 51.8% of the total DMRs in the ZY, 4C, 16C and IVP blastocyst groups, respectively were hypermethylated and the rest were hypomethylated. Apart from these, the DNA methylation profiles identified in ZY, 4C, 16C and IVP blastocyst groups were evenly distributed across all chromosomes without showing any significant chromosome specific effects (Fig 2, S2–S4 Figs).

Bottom Line: However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions.In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups.Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, 53115 Bonn, Germany.

ABSTRACT
Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

No MeSH data available.


Related in: MedlinePlus