218 Description of mitochondrial DNA methylation and transcription throughout bovine early development

Growing evidence has been showing how important the relationship is between metabolism, mitochondrial function, and epigenetics during early embryonic development. In this study, our goal was to characterise changes in mitochondrial DNA (mtDNA) methylation profile at the initial stages of bovine development, and possibly show correlations between the presence of epigenetic modifications and the functionality of the mtDNA. For that, bovine oocytes were obtained from abattoir ovaries and submitted to IVM, IVF, and in vitro culture. Groups of 10 oocytes or 5 embryos (n = 3 replicates/group) were collected for analysis at various stages (immature oocyte, IM; mature oocyte, MII; zygote, ZY; 4-cells, 4C; 16-cells, 16C, and blastocysts, BL). Total DNA (including mtDNA) was extracted. Part of the DNA was used for library preparation (NEBNext Enzymatic Methyl-seq Kit; New England Biolabs) followed by high coverage whole genome sequencing to detect cytosine methylation levels. Then, looking for possible correlations between methylation and mtDNA transcription, the remaining extracted DNA was used for absolute quantification (Droplet Digital PCR) of transcripts of genes selected by the strand position and/or their functions (ND5, ND6, CYTB, tRNA-Phe, and tRNA-Gln). For statistics, total methylation (total methylated reads ÷ total number of reads) and the number of transcripts were compared for each gene among stages using ANOVA followed by Tukey’s (P < 0.05). Pearson correlation coefficients were also calculated between number of transcripts and total methylation. The quantity of mitochondrial transcripts for each gene varied a lot among stages. In general, significantly higher amounts were found at MII and ZY, followed by an intense drop at 4C, 16C, and BL. Methylation profile also varied, but in the opposite direction. Significantly higher methylation levels were observed mainly at 4- and 16-cell stage for ND5, ND6, CYTB, and tRNA-Phe. This result helps explain the negative correlations observed between methylation and the number of transcripts for the same genes. Interestingly, the strongest correlations were observed at 16C (> −0.8). This stage coincides with the embryonic genome activation (EGA) phase, reinforcing a possible regulatory role played by cytosine methylation in the transcription of mitochondrial genome. Variations in methylation profile were also observed according to the strand (light or heavy) and the context (CG, CHG, or CHH), suggesting that cytosine modifications are not randomly placed. This is a first description of mitochondrial methylation changes occurring during initial stages of bovine embryonic development. Our results indicate that methylation might be working to regulate the number of mtDNA transcripts at a local level. This is particularly important around the time of EGA to fine-tune the events that sustain mitochondrial function and metabolic changes at this time.

This research was supported by FAPESP 2019/25982-7.