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. 2023 Feb 23;10(12):2204794. doi: 10.1002/advs.202204794

Figure 7.

Figure 7

Mitochondrial dysfunction in H3.3‐K4M oocytes. A) Immunofluorescence staining of TOM20 in H3.3‐WT and H3.3‐K4M GV oocytes. Scale bar = 10 µm. B) The fluorescence intensity quantification of TOM20 in H3.3‐WT and H3.3‐K4M GV oocytes. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, **p < 0.01. C) H3.3‐WT and H3.3‐K4M GV oocytes were labeled with Mito‐Tracker Red to visualize active mitochondria for examination of quantification and localization. Scale bar = 10 µm. D) The fluorescence intensity quantification of Mito‐Tracker Red in H3.3‐WT and H3.3‐K4M GV oocytes. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, ** p <0.01. E) Representative images of mitochondrial membrane potential assessed by JC‐1 dye. Scale bar = 10 µm. F) Histogram showing the JC‐1 aggregates/monomers fluorescence ratio. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, ***p < 0.001. G) The relative mtDNA copy numbers in H3.3‐WT and H3.3‐K4M GV oocytes. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, ****p < 0.0001. H) Quantitative analysis of the relative ATP levels from H3.3‐WT and H3.3‐K4M GV oocytes. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, ****p < 0.0001. I) H3.3‐WT and H3.3‐K4M GV oocytes were labeled with DCFH‐DA to visualize ROS level. Scale bar = 10 µm. Right panel shows the quantification of fluorescence intensity of ROS in H3.3‐WT and H3.3‐K4M oocytes. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, **p < 0.01. J) Relative expression of Nrf2, Sod2, Gsr, and Gpx1 in H3.3‐WT and H3.3‐K4M oocytes by qRT‐PCR. Data are presented as Mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001. K) H3.3‐WT and H3.3‐K4M oocytes were labeled with Annexin‐V‐FITC to visualize apoptosis level. Scale bar = 10 µm. Right panel shows the percent of Annexin‐V‐FITC positive oocytes from H3.3‐WT and H3.3‐K4M mice. Data are presented as Mean ± SD, n = 20 GV oocytes derived from each genotype, **p < 0.01. L) Relative expression of pro‐apoptotic related genes in H3.3‐WT and H3.3‐K4M oocytes by qRT‐PCR. Data are presented as Mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.