Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization

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Science  22 Jul 2016:
Vol. 353, Issue 6297, pp. 394-399
DOI: 10.1126/science.aaf4777
  • Fig. 1 Loss of cps-6 delays internal breakdown of paternal mitochondria after fertilization.

    (A to D, F to J) Tomographic slice images and corresponding 3D models of a mitochondrion in an N2 (A) or cps-6(tm3222) (F) spermatozoon or a paternal mitochondrion in an N2 embryo [(B) to (D)] or a cps-6(tm3222) embryo [(G) to (J)] at the indicated stages. 3D models of autophagosomes (AuPh) and endoplasmic reticulum (ER) are shown. Mitochondrial membranes, cristae, and aggregates are colored red, green, and blue, respectively. Dark aggregates and autophagosome membranes are indicated with blue and yellow arrowheads, respectively. Scale bars, 300 nm. (E) Histogram showing three classes of paternal mitochondria in embryos of different stages from the indicated N2 cross (n = 45) or cps-6(tm3222) cross (n = 56).

  • Fig. 2 CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to promote PME.

    (A) Diagram of C. elegans mtDNA, the uaDf5 deletion, primers used in the nested PCR assays, and sizes of PCR products in N2 and uaDf5/+ animals. (B and C) Hermaphrodites and MTR-stained males were mated as indicated. Males also carried smIs42, an integrated Psur-5sur-5::gfp transgene used to track cross progeny (see fig. S2A). A single unfertilized oocyte and a single cross-fertilized embryo or larva (MTR- or GFP-positive) at the indicated stage was analyzed by PCR. uaDf5/+ and N2 hermaphrodites were controls. (D) Quantification of MTR-stained paternal mitochondrial clusters in 64-cell embryos from the indicated crosses with MTR-stained males. Data are means ± SEM; n = 20 per cross. **P < 0.0001 (unpaired Student t test); n.s., not significant. (E and F) Five cross-fertilized embryos (E) or transgenic embryos (F) at approximately 100-cell stage from the indicated crosses were analyzed by PCR. (G to J) Representative immuno-EM images of mitochondria in N2 spermatozoa and paternal mitochondria in zygotes from the indicated cross. CPS-6–specific and PD-E2–specific immunogold particles are marked with arrowheads. Scale bars, 300 nm. (K) Histogram of the distances of 15-nm immunogold particles from the mitochondrial membrane, illustrating CPS-6’s movement after fertilization. Numbers of immunogold particles scored are shown in parentheses. ***P < 0.0001 (Mann-Whitney U test). cps-6(tm3222) was used in all figures.

  • Fig. 3 Depolarization of paternal mitochondria after fertilization and autophagosome formation on paternal mitochondria.

    (A and B) Differential interference contrast (DIC) and fluorescence images of zygotes from the indicated crosses. TMRE had equal access to maternal and paternal mitochondria in the zygote (B). Scale bars, 10 μm. (C to F) Zygotes from the indicated crosses with MTR-stained males were labeled with an antibody to LGG-1. Images were acquired with a Nikon SIM microscope. Dashed rectangles highlight the areas enlarged and shown below [(D) and (F)]. Scale bars, 2 μm [(C) and (E)], 0.5 μm [(D) and (F)].

  • Fig. 4 Delayed removal of paternal mitochondria increases embryonic lethality and cell division durations.

    (A to D) The embryonic lethality rate [(A) and (D)] and durations of cell divisions in the MS lineage (B) and P lineage (C) were scored in self-fertilized embryos (1 to 3) or cross-fertilized embryos from crosses (4 to 7) of the indicated genotypes. All males carried smIs42 and were stained with MTR to assist identification of zygotes [(B) and (C)]. Data are means ± SEM; n > 1000 embryos per cross at 25°C [(A) and (D)] and n = 3 embryos per cross at 20°C [(B) and (C)]. *P < 0.05, **P < 0.001 (unpaired Student t test).

Supplementary Materials

  • Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization

    Qinghua Zhou, Haimin Li, Hanzeng Li, Akihisa Nakagawa, Jason L.J. Lin, Eui-Seung Lee, Brian L. Harry, Riley Robert Skeen-Gaar, Yuji Suehiro, Donna William, Shohei Mitani, Hanna S. Yuan, Byung-Ho Kang, Ding Xue

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S8
    • Table S1
    • Caption for movie S1
    • References

    Images, Video, and Other Media

    Movie S1
    Tomographic volume of the area surrounded by the dash rectangle in fig. S1C''. Paternal mitochondria are indicated with arrowheads. No sign of autophagsome assembly is detected around paternal mitochondria with aggregates in their matrices.

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