Alpha-ketoglutarate modulates the reprogramming of mitochondrial and nuclear DNA methylation during the earliest stages of the bovine preimplantation embryo development

Abstract During the preimplantation embryonic development, metabolic and epigenomic remodeling are crucial events that can be significantly impacted even by minor environmental changes. Right after fertilization, nuclear deoxyribonucleic acid (DNA) undergoes demethylation, reaching the lowest levels at the 8–16 cell stage, marking the onset of mitochondrial metabolism and genome activation. This coincides with “de novo” DNA methylation events guiding cell lineage differentiation at the blastocyst stage. High levels of α-ketoglutarate (AKG) in cancer and pluripotent stem cells promote a more undifferentiated state, acting as a cofactor for ten-eleven translocation (TET) demethylases. In this study, we examined the metaboloepigenetic adaptations from AKG supplementation during the in vitro culture in a bovine model. Bovine embryos were produced in vitro and culture until Day 4 in the presence or not of 4 mM of AKG. After AKG discontinuation, embryos remained in culture until Day 7 for blastocyst analysis. AKG during the early stages of development led to the expected increase in the levels of TET family enzymes, resulting in a decrease in nuclear 5mC levels. Additionally, the blastocysts derived from the AKG group exhibited reduced mitochondrial activity in the inner cell mass, along with increased methylation levels of mitochondrial DNA. Integrative analysis of DNA methylation and transcription revealed low correlation between these two omics, with a higher effect of AKG treatment on DNA methylation than the ribonucleic acid content. These results indicate that minor metabolic alterations early in development, such as AKG supplementation, have a significant impact on the embryo’s metabolism and molecular control, compromising the epigenetic reprogramming.