<i>daf-2</i> oocytes display punctate mitochondria.

daf-2 have an elongated reproductive span and maintain punctate morphology in oocyte mitochondria throughout reproduction. (A) daf-2(e1370) reproductive span in hermaphrodites (n = 96–121) mated with males (Luo et al. 2010). † indicates loss of hermaphrodites due to high rates of matricide by bagging. (B) daf-2(e1370) (n = 115) and N2 (n = 110) late-mating capability at Day 7 adult. Error bars represent 95% confidence interval. (C) Representative oocyte quality images. N2 (left panel) and daf-2(e1370) (right panel) at Day 1 and Day 8 adult. Black asterisks mark the -1 oocyte, white arrows highlight developing embryos, yellow dotted outline marks uterine mass. Scale bars represent 20μm. (D) Blind scoring of 4 categories of oocyte quality decline in daf-2(e1370) and N2 mated Day 8 hermaphrodites (n = 27–30). (E-F) Representative images of mitochondrial morphology using anti-ATP5α to mark mitochondrial membranes. Day 1 and Day 7 adults in N2 (E), and daf-2(e1370) (F). Scale bar 20μm. White-dotted boxes are enlarged sections of the -1 oocyte. Images were processed using the same parameters across the entire image (i.e. adjusting for brightness and contrast). (G-J) Quantification of mitochondrial morphology in the -1 oocyte. Z-stacks were blindly-scored into 3 categories: punctate, mixed, or elongated. Mitochondrial morphology does not change with age in N2 (G, n = 117–83) and daf-2(e1370) (H, n = 107–118). daf-2 have more -1 oocytes with punctate morphology at Day 1(I) and Day 7 (J). Three independent replicates were imaged, scored, and pooled for graphs G-J. Because blind scoring must be compared against the opposing genotype, G and H used the same blind scoring data as I and J. Chi-square test. (K) Mitochondrial length of N2 and daf-2 was measured using a subset of the same fixed samples used for blind scoring. Each dot represents the average length of 3 individual mitochondrion in one -1 oocyte. One-way Anova. (L) Muscle mitochondria of Day 7 adults using the ATP5a antibody. Images were taken on the confocal. Scale bar 5μm. (M) Muscle mitochondrial length was measured from the same samples used to characterize oocyte mitochondria. Each dot represents 1/10 mitochondrion in 6 individual worms for each genotype. Student t-test. ****p ≤ 0.0001.