PINK-1 mitophagy is specifically required for <i>daf-2</i> reproductive span.

(A) mtRosella is silenced in the germline. Image of the dissected germline of the WMB1119 strain with Ppie-1TOMM-20::Rosella insert. Image was captured on the Nikon eclipse Ti at 60x magnification. The mCherry and FITC channels are superimposed Z-stacks taken at 0.7μm steps. (B) Mating capability of all genotypes used in this study when young hermaphrodites are mated with fog-2 males. (C) Late-mating capability in pink-1 vs. N2. Day 7 adults are mated with young males. (D) Lifespan of pink-1(tm1779) (n = 62) is unchanged compared to N2 (n = 69). (PDF)</p

Representative Z-stack of mitochondria in the -1 oocyte of Day 7 adult <i>daf-2(e1370)</i>, 60x.

(AVI)</p

Statistical data tables.

(A) Reproductive span, (B) Late-mating, and (C) Lifespan statistical information. Statistics were performed on Prism. Mean reproductive/alive was performed on OASIS. (DOCX)</p

Fission, not fusion, is required for <i>daf-2</i> extended reproductive longevity.

(A) Representative images of young daf-2;drp-1 and daf-2.Scale bar 20μm. (B) Representative images of aged daf-2(e1370) left and daf-2(e1370);fzo-1(tm1133) germlines. Scale bar 50μm. (C) Reproductive spans of daf-2 vs. daf-2;drp-1 (Fig 3D) including N2 and drp-1 genotypes. (D) Reproductive spans of daf-2 vs. daf-2;fzo-1 (Fig 3E) including N2 and fzo-1 genotypes. (PDF)</p

Fission and fusion are necessary for normal reproductive function with age.

(A) Mitochondrial morphology in mature oocytes of N2, drp-1(tm1108), and fzo-1(tm1133) in Day 3 adults. Representative images highlight the different morphologies in N2 (elongated), drp-1(tm1108) (elongated), and fzo-1(tm1133) (punctate). White-dotted boxes are enlarged (right) of the morphology in the -1 oocyte (I) and the -2 oocyte (II). Images in A were processed using the same parameters across the entire image (i.e. adjusting for brightness and contrast). (B) Blind scoring results of mitochondrial morphology in the -1 oocyte in Day 3 adults of N2 (n = 34), drp-1(tm1108) (n = 29), and fzo-1(tm1133) (n = 29). (C) Mitochondrial length measurements. Each dot represents 1/3 measurements from the -1 oocyte of 20–25 individual hermaphrodites in each genotype. Reproductive spans of mated drp-1(tm1108) (D, n = 96–109) and fzo-1(tm1133) (E, n = 70–89). (F) Embryonic lethality in N2, drp-1, and fzo-1. Representative data from days 3, 4, and 5 of adulthood are shown due to high rates of matricide after day 5 (n = 11–13). 2-way Anova. (G-H) Oocyte quality declines with age in drp-1(tm1108). Representative oocyte quality images in Day 1 (G) and Day 6 (H) adults of N2 (left panel) and drp-1(tm1108) (right panel). Highlighted features include: embryos in the uterus (white arrows), the -1 oocyte (black asterisk), oocyte quality-decline phenotypes (yellow text and arrows), uterine mass (yellow dotted outline). (I) Quantification of oocyte quality decline phenotypes in N2 (n = 64) and drp-1(tm1108) (n = 51). (J-K) Oocyte quality declines in fzo-1(tm1133). Representative oocyte quality images in Day 1 (J) and Day 6 (K) adults of N2 (left panel) and fzo-1(tm1133) (right panel). Highlighted features same as H. Scale bars 20μm. (L) Quantification of oocyte quality decline phenotypes in N2 (n = 38) and fzo-1(tm1133) (n = 34). Chi-square test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.</p

Representative Z-stack of mitochondria in the -1 oocyte of Day 7 adult <i>daf-2(e1370)</i>, 100x.

(AVI)</p

<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.</p

Mitochondrial morphology in N2 and <i>daf-2</i> oocytes and muscle.

(A) Day 1 adult mitochondrial morphology images in the -1 oocytes of N2 (left 2 panels) and daf-2(e1370) (right 2 panels). (B) Day 7 adult mitochondrial morphology images in the -1 oocytes of N2 (left 2 panels) and daf-2(e1370) (right 2 panels). In A and B images were taken on the Nikon eclipse Ti at 60x magnification. (C) Day 7 adult mitochondrial morphology images in the muscle of N2 (left 2 panels) and daf-2(e1370) (right 2 panels). In C images were taken on the scanning confocal Nikon A1 at 60x. All images are of mitochondria stained with ATP5α to mark membranes. (PDF)</p

Representative Z-stack of mitochondria in the -1 oocyte of Day 7 adult N2, 100x.

(AVI)</p

Mitochondrial dynamics are specifically required for reproduction function.

(A) Lifespans are not affected by loss of drp-1(tm1108) (n = 81, N2 n = 81) (B) fzo-1(tm1133) (n = 80, N2 n = 83). (C) Progeny count of mated N2 (n = 12), drp-1 (n = 13) and fzo-1 (n = 12) after mating (L4-Day 1) and after mating confirmation (Day 1-Day 2). Day 3, 4 and 5 of adulthood were scored. 2-way ANOVA. (PDF)</p