89 Embryonic metabolism orchestrates epigenetic mechanisms: What can we anticipate from the first cleavages?

Abstract

Intermediates of the energy metabolism (as acetyl Co-A) can donate their acetyl group to introduce acetylation in histones, establishing a relationship between metabolism and epigenetic control in somatic and embryonic stem cells. Embryos with different kinetics during the first cleavages also have alterations in epigenetic profile as well as in metabolism and energy substrate consumption during in vitro culture. The aim of this work was to verify if and how this relation between metabolism and epigenetic parameters was also presented in in vitro-produced bovine embryos. For that, we first characterised the pattern of H3K9ac, and the molecular pattern of enzymes involved with histone acetylation and acetyl-CoA production in female blastocysts derived from fast and slow cleavage embryos. To validate the results, we also produced bovine embryos cultured with an inhibitor of the pyruvate production, and consequently the acetyl Co-A generation to check if this could interfere in the H3K9ac pattern. For this, embryos were in vitro produced following standard protocol and classified at 40 hours post-insemination as fast (4 or more cells) or slow (2 cells) and collected at the blastocyst stage. Blastocysts were immunostained to H3K9ac and the fluorescence intensity of each nucleus was quantified using ImageJ and analysed by Student's t-test. For transcript quantitation, RNAseq data were accessed from a previous report using the same kinetics classification model (Milazzotto et al. 2016 Mol. Rep. Dev. 83, 324-336; https://doi.org/10.1002/mrd.22619) and analysed using limma-voom on Galaxy 3.38.3. To validate the results, bovine embryos were produced and cultured until Day 4 and then incubated until the blastocyst stage with different doses of iodoacetate (IA; 2 and 5 mM) to reduce the intracellular levels of acetyl CoA. These blastocysts were also assessed by H3K9 acetylation. Slow blastocysts presented higher fluorescence intensity for H3K9ac than fast blastocysts (fast 13.33 ± 0.37 AU vs. slow 38.14 ± 1.17 AU; P < 0.0001). Despite the fact that there were no differences in transcripts related to this acetylation (ELP3 and HAT2), slow blastocysts presented higher levels of transcripts for PDHB1 and PDHA1, responsible for acetyl-CoA production (PDHB1: fast 11.6 ± 0.2CPM vs. slow 13.1 ± 0.2 counts per million; P < 0.01 PDHA1: fast 12.6 ± 0.2 CPM vs. slow 13.2 ± 0.3 CPM; P < 0.01). The reduction of acetyl-CoA in blastocysts induced by IA led to lower levels of H3K9ac in 1 and 2 mM doses when compared with the control (control: 43.8 ± 0.7 AU; 1 mM: 34.7 ± 0.5 AU; 2 mM: 30.1 ± 0.6 AU; P < 0.0001). Interestingly, H3K9ac levels were similar for 5 mM IA and control group (5 mM: 41.2 ± 1.4; P > 0.05), suggesting a compensatory mechanism in extreme cases to maintain the histone acetylation. As far as we know, this is the first work that describes a relation between metabolism and epigenetics in bovine embryos. Although the pattern of genes related to acetylation seems to be unaltered, changes in acetyl Co-A production pathway exert an influence on H3K9ac status.

Grant support was provided by grant 2017/18384-0 and 2018/11668-6 from FAPESP and CAPES.