Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization

Abstract

Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

Fig. 1. Loss of cps-6 delays internal breakdown of paternal mitochondria following 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 in red, green, and blue, respectively. Dark aggregates and autophagosome membranes are indicated with blue and yellow arrowheads, respectively. Scale bars indicate 300 nm. (E) A histogram showing three classes of paternal mitochondria in different stage embryos 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 following fertilization to promote PME

(A) 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 P_sur-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. Means ± SEM; n=20 per cross. ** P < 0.0001 using unpaired Student's t-test. “n.s”, no significant difference. (E, 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 in parentheses indicate the numbers of immunogold particles scored. *** P < 0.0001 using Mann-Whitney U test. cps-6(tm3222) was used in all figures.

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

(A, B) DIC and fluorescence images of zygotes from the indicated crosses are shown. TMRE had equal access to maternal and paternal mitochondria in the zygote (B). Scale bars represent 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 using a Nikon SIM. Dash rectangles highlight the areas enlarged and shown below (D and F). Scale bars, 2 μm (C and E) and 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 the durations of cell divisions in the MS (B) and the P lineage (C) were scored in self-fertilized embryos (1-3) or cross-fertilized embryos from crosses (4-7) of the indicated genotypes. All males carried smIs42 and were stained with MTR to assist identification of zygotes (B and C). 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 < 0.001, * P < 0.05 using unpaired Student's t-test.