Extended Data Fig. 4: Mitochondria are acidified during OZT; DRP-1 and FUNDC1 are necessary for mitochondrial acidification.
From: Programmed mitophagy at the oocyte-to-zygote transition promotes lineage endurance
a,b, Top: Confocal fluorescence images of mitochondria endogenously tagged with both eGFP and mKate2 in the oocyte and zygote. Scale bar, 10 µm. Bottom: Magnifications of boxed regions. Scale bar, 1 µm. Blue arrows indicate non-acidified mitochondria with robust green and red fluorescence. Yellow arrows indicate acidified mitochondria, where red fluorescence dominates. Mitochondria marked by arrowheads are magnified in (b). Scale bar, 1 µm. Green-to-red (G/R) ratio of the overall –2 oocyte mitochondria was normalized to be 1, and the relative G/R values of marked regions in (b) are denoted. c, Quantification of overall mitochondrial eGFP/mKate2 ratio in –2 oocytes and zygotes, revealing mitochondrial acidification in zygotes. Average eGFP/mKate2 ratio of –2 oocytes was normalized to be 1. Data represent mean ± s.d. (n = 25 animals) from three biological replicates. P value using a two-tailed Student’s t-test. d, Quantification of eGFP/mKate2 ratio in whole oocyte mitochondrial network and in the eGFP-diminished mitochondrial regions noted in zygotes. Average G/R ratio of the overall –2 oocyte mitochondrial network was normalized to be 1. Relatively decreased G/R ratio of these zygotic mitochondria suggests they are acidified. Data represent mean ± s.d. (n = 24 animals) from three biological replicates. P values using a two-tailed Wilcoxon test. e, Semi-automated quantification of the number of acidified mitochondria (mitolysosomes) in –2 oocytes and zygotes quantified using the ‘mitoQC counter’ plugin. Data represent mean ± s.d. (n = 26 animals per strain) from three biological replicates. P values using a two-tailed Wilcoxon test. f–i, Mitochondrial acidification visualized and quantified in drp-1 RNAi-treated animals, similar to a-e. Scale bars, 10 µm (top) and 2 µm (bottom) (f), 2 µm (g). Data represent mean ± s.d. from three biological replicates. P values using a two-tailed Student’s t-test (n = 22 animals) (h) and a two-tailed Mann–Whitney test (n = 16 animals) (i). j–m, Mitochondrial acidification visualized and quantified in fndc-1 mutant animals. Scale bars, 5 µm (top) and 1 µm (bottom) (j), 2 µm (k). Data represent mean ± s.d. from three biological replicates. P values using a two-tailed Student’s t-test (n = 25 animals) (l) and a two-tailed Mann–Whitney test (n = 16 animals) (m). In all panels, blue arrows indicate non-acidified mitochondria, and yellow arrows indicate acidified mitochondria. ‘G/R ratio’ denotes the eGFP/mKate2 fluorescence ratio.
