Figure 2: Compromised maternal mitochondria in fzo-1(tm1133) oocytes compete with paternal mitochondria for autophagosomes.

(a–f) Analysis of colocalization of LGG-1 autophagosomes with MTR-stained paternal mitochondrial clusters in zygotes. Zygotes from mating of MTR-stained males with unstained hermaphrodites right after sperm entry were fixed and stained with an antibody to LGG-1. Arrowheads indicate MTR-stained paternal mitochondrial clusters, some of which were colocalized with LGG-1 aggregates. Scale bar represents 10 μm. (g) Colocalization analysis using Mander's overlap coefficient (MOC). MOC (with thresholds) represents the degree of colocalization between MTR-stained paternal mitochondrial clusters (Red) and LGG-1 aggregates (Green). The significance of difference between different mating experiments was determined by unpaired t test. **P<0.01. ‘n.s.’ indicates no significant difference. 15 zygotes from each mating were scored. (h) Oligomycin treatment suppresses the defect of delayed PME caused by loss of maternal fzo-1. Quantification of MTR-stained paternal mitochondrial clusters in 64-cell and>100-cell stage cross-fertilized embryos from the indicated crosses grown on regular Nematode Growth Medium (NGM) plates or NGM plates with the DMSO control or 150 μg ml⁻¹ oligomycin was performed as in Fig. 1g. In all crosses, MTR-stained males were mated with unstained hermaphrodites. Data shown are mean±s.e.m. (n=10). The significance of difference between different mating experiments was determined by unpaired t test. *P<0.05, **P<0.01. ‘n.s.’ indicates no significant difference. (i) PCR-based assays to monitor persistent uaDf5 paternal mtDNA in cross-fertilized embryos. Upper panel, a schematic diagram depicts the C. elegans mitochondrial genome and the uaDf5 deletion. The primers used in the nested PCR assays to amplify the uaDf5 deletion and the expected sizes of PCR products are indicated. These primers failed to amplify wild-type mtDNA (expected size of 3861, bp) under the PCR conditions used. Bottom panel, the results of PCR analysis of 32-cell and comma stage cross-fertilized embryos from mating of MTR-stained uaDf5/+ males with unstained fzo-1(tm1133) or N2 hermaphrodites grown on NGM plates containing either the DMSO control or 150 μg ml⁻¹ oligomycin. Each PCR reaction was prepared from eight cross-fertilized embryos. In all panels, fzo-1(tm1133) and drp-1(tm1108) alleles were used.