62 Comparison of N⁶-methyladenosine modification of mRNA during the bovine and murine oocyte to embryo transition

In recent years, the number of bovine embryos produced in vitro (IVP) has surpassed that of in vivo-derived embryos (IVD). However, the percentage of oocytes that produce a viable blastocyst remains low. A critical period during embryo development is the oocyte to embryo transition (OET), which is characterised by translation and degradation of maternally derived mRNAs and activation of transcription from the new embryonic genome. Therefore, the goal of our laboratory is to identify the mechanisms that regulate maternal and embryonic mRNA processing during the OET, and how disruptions may contribute to poor embryo development during IVP. In the current study, we compared changes in N⁶-methyladenosine (m6A), which is the most common RNA modification, during murine and bovine OET. Our hypothesis is that dynamic changes in expression of methyltransferase 3 (METTL3) may regulate the abundance and localization of global m6A modification of mRNA throughout the OET. Using our mouse model, mature (MII) oocytes were collected from superovulated CD-1 mice, in vitro fertilized, and zygotes were collected at early 1-cell (4hpi), 1-cell (8hpi), late 1-cell (12hpi), and 2-cell (24hpi). In our bovine model, we collected cumulus–oocyte complexes from ovaries that were ovariectomized from University of Nebraska–Lincoln research herd heifers, then performed in vitro maturation. The MII were collected or fertilized for 24 h, followed by collection of 1-, 2-, 4-, 6-, 8-, and 10-cell embryos up to 48 h in culture. In mice, METTL3 levels, measured by immunofluorescence and analysed by one-way ANOVA, increased (P < 0.0001) 4-fold at late 1-cell compared to MII, early 1-cell, and 1-cell, and decreased 2-fold at 2-cell. METTL3 was absent from pronuclei (PN) at early 1-cell, yet highly localised to both PN and nuclei at late 1-cell and 2-cell. Similarly, METTL3 was expressed at low levels in bovine MII, present in PN as early as 1-cell, and localised almost exclusively in the nuclei of cleavage-stage embryos. Next, we used an anti-m6A antibody to measure global m6A via immunofluorescence across timepoints and stages in both models. There was a 2-fold decrease (P < 0.01) in m6A from MII to early 1-cell and again to 1-cell, followed by a 3-fold increase (P < 0.001) at 2-cell during mouse OET. Conversely, in bovine we saw an overall decrease in global m6A, with a 2-fold decrease (P < 0.001) from MII to 1-cell, followed by another 2.5-fold decrease (P < 0.05) from 1-cell to 6-, 8-, and 10-cell stage. Across stages, nuclear m6A decreased significantly (P < 0.05) from the 1-cell stage to 8-cell stage. Interestingly, in bovine 1-cell embryos, m6A is highly localised within both PN, whereas we saw lack of m6A presence in murine 1-cell PN. These data lay the foundation for determining species-specific mechanisms of differential methylation of maternal and embryonic mRNAs, which may allow for translation or degradation of maternal mRNAs and stabilisation of embryonic mRNAs during the OET. Future studies will elucidate disruptions in m6A modification that may alter normal embryo development, specifically due to cell oxidative stress.