107 a-Ketoglutarate/succinate ratio alters TET2 quantity and cellular localisation in bovine embryos

Bovine embryos are great models to comprehend the metaboloepigenetic reprogramming that occurs during pre-implantation development. So far, the use of different metabolite analogues from the TCA cycle during the embryo culture resulted in alterations in mitochondrial and genomic DNA methylation levels that ultimately lead to differences in chromatin accessibility and gene expression. The present work aimed to investigate the role of TET2, a demethylase from the Ten-Eleven Translocation family, in controlling these alterations. For that, embryos were in vitro produced using standardised protocols and cultured in semi-defined medium (control [CO]) or treated with 4 mM dimethyl-a-ketoglutarate (aKG) or 4 mM dimethyl-succinate (SUC) from Day 0 to Day 4 of culture. Embryos were collected on Days 2, 4, and 7 of culture and stained with MitoTracker red CMX-ROS and Hoechst 33342 for mitochondria and nuclei identification, respectively, and immunofluorescence stained for TET2. Images were collected using confocal microscope TCS SP8 AOBS Tandem Scanner Leica for mitochondria colocalisation with TET2 (2D images), Leica Microsystems DM16000 B for total embryo and nucleus TET2 quantification (3D images with 43–67 z-stacks), and on both for negative control. The colocalisation between TET2 and mitochondria was calculated using the Coloc2 plugin on ImageJ (National Institutes of Health). All groups presented a progressive increase in colocalisation Pearson’s R value for TET2 and mitochondria through the development (CO: 0.46 ± 0.07, P < 0.0001; aKG: 0.41 ± 0.16, P = 0.0013; SUC: 0.48 ± 0.13, P < 0.0001). Also, lower colocalisation was observed in embryos from the SUC group at Day 2 (P = 0.0007) when compared with aKG (CO: 0.16 ± 0.4; aKG: 0.19 ± 0.06; SUC: 0.11 ± 0.03; Pearson’s R value), with no alterations observed between treatments for Day-4 and Day-7 embryos. When the total cellular amount of TET2 was quantified, only a reduction (P = 0.0004) was observed in SUC embryos at Day 2, in comparison with CO and aKG (CO: 976 ± 163; aKG: 1118 ± 359; SUC: 671 ± 149; AU). However, when only the nuclear TET2 was quantified, aKG presented higher levels at Day 2 (CO: 38 ± 8; aKG: 42 ± 9; SUC: 41 ± 9; P = 0.0435) and Day 7 blastocysts both on inner cell mass (CO: 59 ± 26; aKG: 75 ± 35; SUC: 59 ± 21; P = 0.0001) and trophectoderm (CO: 22 ± 7; aKG: 28 ± 21; SUC: 24 ± 8; P = 0.03), and lower levels of TET2 at Day 4 (CO: 64 ± 9; aKG: 58 ± 13; SUC: 64 ± 12 - P < 0.0001) when compared with CO. In conclusion, TET2 quantity and cellular localisation are influenced by the aKG/SUC ratio during the embryonic development. Moreover, TET2 appears to be an important regulator of genomic DNA methylation reprogramming for embryos treated with aKG. On the other hand, TET2 might be a regulator for mitochondrial DNA methylation in embryos treated with SUC during the first cleavages. Studies to understand the role of other DNA demethylases in these embryos are also necessary and currently in progress.

This research was supported by Sao Paulo Research Foundation 2018/11668-6 and 2019/25982-7.