During the first cleavages of bovine embryos, the DNA demethylation (active and passive) occurs and will be responsible for the production of totipotent cells, until around 8-16 cells. Then the DNA of these embryos must be methylated again until, at the blastocyst stage, when its blastomeres are classified as pluripotent and differentiated as belonging to the internal cell mass or trophectoderm. The enzymes responsible for the active DNA demethylation, ten-eleven translocation (TET), are known to use ±-ketoglutarate, a metabolite of the TCA cycle, as a co-substrate. Other metabolites from the energy metabolism, such as succinate and malate, can reduce TET enzymes activity and prevent DNA and histone demethylation. The direct relation between the levels of these metabolites from the TCA cycle and presence of epigenetic marks had already being demonstrated in somatic and pluripotent stem cells. However, for embryos, the effect of these metabolites and alteration in their levels in epigenetic marks is just now being elucidated. At the moment, as part of the proposed postdoctoral project, the effect of succinate/±-ketoglutarate ratio during embryonic culture was demonstrated by alterations in DNA methylation levels in embryos at day 4 and day 7 of development. Thus, the hypothesis of this work is that, since metabolites from the TCA cycle can affect the DNA demethylation and methylation wave, they are going to affect the RNA profile of blastocysts due to alterations in genome accessibility. To test this hypothesis, bovine embryos cultured in the presence of 4 mM of ±-ketoglutarate or 4 mM of succinate will be collected in the blastocyst stage for inner cell mass retrieved. The inner cell mass collected will be subjected to ATAC-seq and RNA-seq analysis to identify the genomic regions accessible for transcription and transcripts profile, respectively.
News published in Agência FAPESP Newsletter about the scholarship: