Exogenous METTL7A reduces mitochondrial stress and decreases superoxide level of bovine pre-implantation embryos. (A) Experimental scheme of injecting METTL7A IVT-RNA into one blastomere of 2-cell embryos. (B) Immunostaining analysis of F-actin, mitochondrial stress marker (HIF1a), and METTL7A-6xHisTag in METTL7A^OE blastomeres and control. The arrow points to condensed chromatin and degradation of actin filament, scale bar = 50 μm. After 12 h of injection, METTL7A^OE blastomeres developed through 1 or 2 cell cycles, while non-injected blastomeres were arrested (n = 5, 4/5 or 80%) with described phenotype. (C) Experimental scheme of zygotic injection and western blot analysis. (D and E) Western blot analysis of Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA, a marker for mitochondrial respiratory activity) in METTL7A^OE (n = 6) blastocysts compared to control (n = 7). (F) The immunostaining analysis of superoxide level measured by MitoSox green in METTL7A^OE embryos and control at 8-cell (n = 13 embryos for both groups) and blastocyst (n = 10 embryos for both groups) stage, scale bar, 50 μm. (G and H) The quantification of superoxide level in METTL7A^OE embryos and control at 8-cell (G) and blastocyst stage (H). (I and J) The GSH level METTL7A^OE embryos and control at 8-cell (I, n = 6 for both groups) and blastocyst stage (J, n = 3 for both groups).