In vitro processes alter the embryonic disc epigenome and transcriptome in the pre-implantation elongated bovine embryo

Abstract

The objective was to quantify the effect of in vitro procedures on the epigenome and transcriptome of the embryonic disc (ED) and extra-embryonic membranes (EEM) of day 15 in vitro produced (IVP) conceptuses compared to their in vivo (IVV) counterparts. IVP embryos (n = 7) were cultured serum-free until transfer at day 7, while IVV embryos (n = 9) were conceived through artificial insemination. Animals were flushed at day 15 of gestation, and sections of the ED and EEM underwent DNA and RNA extraction for whole-genome bisulfite or RNA sequencing. Raw fastq files were aligned to the ARS-UCD1.3 bovine genome. Processed data were integrated through a multi-omics approach based on machine learning to determine the key ontological terms that characterize each embryonic tissue lineage according to their methylome and transcriptome, followed by overrepresentation analyses (adjusted P-value < 0.05) of differentially methylated genes (DMG), differentially expressed genes (DEG), or genes that were both differentially methylated and differentially expressed in the ED or EEM of IVP compared to IVV conceptuses. Results demonstrated that identified critical ontological terms for the ED, such as somitogenesis, mesoderm formation, and gastrulation, were enriched among hypermethylated DMG, down-regulated DEG, and genes hypermethylated in the promoter and inhibited in expression in the ED of IVP embryos. Genes hypermethylated in the promoter and inhibited in expression in the EEM of IVP conceptuses were involved in epigenetic regulation. In conclusion, in vitro procedures alter the development of main lineage tissues in the pre-implantation embryo, even after interaction with the maternal environment.