Figure 5. Sirt3 knockdown increases intracellular ROS, mainly of mitochondrial origin, in preimplantation embryos.

(A and B) Injection of Sirt3 siRNA increased intracellular ROS levels, as estimated by CM-H₂DCFDA fluorescence intensity. This increase was abolished by NAC (A) and stigmatellin (B), but was only partially decreased by apocynin (B). Embryos were injected with control or Sirt3 siRNA at the pronuclear stage and were cultured with or without NAC for 72 hours. To identify the major origin of increased intracellular ROS, embryos were treated with apocynin or stigmatellin for 30 minutes before CM-H₂DCFDA staining. Quantitative data of fluorescence intensity, obtained using ImageJ, were standardized by dividing each value by the average value of the control group in each experiment. Data are derived from 3 (A) or 4 (B) independent experiments. Statistical assessments were performed by applying Games-Howell test. *P < 0.05; **P < 0.001. (C and D) Representative images of CM-H₂DCFDA fluorescence in embryos analyzed in A and B, respectively. Scale bars: 100 μm.