69 BLASTOCYSTS DEVELOPED FROM EMBRYOS THAT SPENT UP TO 2-CELL STAGE IN VIVO EXHIBITED MASSIVE DNA METHYLATION DYSREGULATION INCLUDING IMPRINTED GENES AND DNA METHYLTRANSFERASES

Suboptimal culture condition before minor or major genome activation is believed to affect the quality and the transcriptome landscape of the resulting blastocysts. Thus, we hypothesised that exposure of bovine embryos to suboptimal culture condition before minor embryonic genome activation could affect the genome methylation patterns of the resulting blastocysts. Therefore, here we aimed to investigate the genome wide DNA methylation patterns of blastocysts derived from embryos developed up to 2-cell stages in vivo followed by in vitro culture. For this, Simmental heifers were superovulated and artificially inseminated. The 2-cell stage embryos were then flushed using a state-of-the-art nonsurgical endoscopic early-stage embryo flushing technique and in vitro cultured until the blastocyst stage. The DNA methylation patterns of these blastocysts were then determined with reference to blastocysts derived from embryos developed completely under in vivo condition. For this, the genomic DNA isolated from each blastocyst group were fragmented, and unmethylated genomic regions were cleaved using methylation sensitive restriction enzymes. The samples were then amplified using ligation mediated PCR and labelled either with Cy-3 or Cy-5 dyes in a dye-swap design using the ULS Fluorescent genomic DNA labelling kit (Kreatech Biotechnology) and hybridized on an EmbryoGENE DNA Methylation Array as described previously (Saadi 2014 BMC Genomics 15, 451; Salilew-Wondim 2015 PLoS ONE 10, e0140467). Array hybridization was performed for 40 h at 65°C, and 4 hybridizations were preformed to represent 4 biological replicates. The slides were scanned using Agilent’s High-Resolution C Scanner (Agilent Technologies, Santa Clara, CA, USA), and Agilent’s Feature Extraction software (Agilent Technologies) was used to extract data features. Differentially methylated regions with fold change =1.5 and P-value < 0.05 were identified using linear modelling for microarray and R software. The results have shown that including imprinted genes (PEG3, IGF1, RASGRF1, IGF2R, GRB10, SNRPN, and PLAGL1) and DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), a total of 10,388 genomic regions were differentially methylated, of which 6393 genomic regions were hypermethylated in blastocysts derived from 2-cell flush compared with the complete vivo group. In addition, comparative analysis of the current DNA methylation data with our previous transcriptome profile data have shown that including DNMT3A, CTSZ, ElF3E, and PPP2R2B, the expression patterns of 603 genes was inversely correlated with the methylation patterns. Moreover, canonical pathways including gap junction, adherens junction, axon guidance, focal adhesion, and calcium signalling were affected by differentially methylated regions. Therefore, this study indicated that exposure of embryos to suboptimal culture condition before embryonic genome activation would lead to a massive dysregulation of methylation pattern of genes involved in developmentally relevant pathways in the resulting blastocysts.

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