Simultaneous inactivation of cps-6 and allo-1 showed a synergistic effect on sperm mitochondria stabilization.

(A) After orthogonal projection of the z-stacks from embryos imaged with a confocal microscope, the region of interest corresponding to the embryo was delimited and a threshold applied to remove background noise. The relative amount of sperm mitochondria per embryo corresponds to the sum of fluorescence intensity from each stack. (B) Quantification of the HSP-6::GFP labeled sperm-derived mitochondria (as % of the 1-cell stage) from the 2-cell stage to 100-cell stage. (C) Maximum intensity projections of confocal images of HSP-6::GFP sperm-derived mitochondria (green) and nuclear DNA (blue) in 2-cell and late stage (3-fold) in control and cps-6(tm3222); allo-1(tm4756) double mutant and (D) quantification of HSP-6::GFP labeled sperm mitochondria (as % of the 1-cell stage). (E) PCR amplification of paternally transmitted mitochondrial DNA (uaDf5) and wild-type (+) mitochondrial haplotypes in 2 to 64-cell stage embryos, L2 and adults offspring and in F2 adults in the cps-6(tm3222); allo-1(tm4756) double mutant. Wilcoxon test p-values: *p<0.05; **p<0.01; ***p<0.001. Scale bar: 10µm.

Inactivation of maternal and paternal specific factors did not prevent sperm-derived mitochondria degradation.

(A) Quantification of the HSP-6::GFP labeled sperm-derived mitochondria (as % of the 1-cell stage) from the 2-cell to 100-cell stage in the cps-6(tm3222); allo-1(tm4756) and cps-6(tm3222); fndc-1(rny14); phb-2(RNAi); allo-1(tm4756) mutants. (B) Quantification of the HSP-6::GFP labeled sperm-derived mitochondria (as % of the 1-cell stage) at the 2-cell and 3-fold stage in cps-6(tm3222); allo-1(tm4756) and cps-6(tm3222); fndc-1(rny14); phb-2(RNAi); allo-1(tm4756) mutants. (C) Quantification of sperm mitochondria labeled with CMXRos in the embryos from the indicated crosses. (D) Maximum intensity projections of confocal images of HSP-6::GFP sperm-derived mitochondria (green) and nuclear DNA (blue) in 2-cell and late stage (3-fold) in cps-6(tm3222); fndc-1(rny14); phb-2(RNAi); allo-1(tm4756) mutant. (E) PCR amplification of paternally transmitted (uaDf5) and wild-type (+) mtDNA haplotypes in 2-64-cell stage embryos, L2, adults offspring and in F2 adults from the indicated crosses. (F) Quantification of the HSP-6::GFP labeled sperm-derived mitochondria (as % of the 1-cell stage) from the 2-cell to 16-cell stage from crosses with feminized adults with males fndc-1(rny14), or cps-6(tm3222); fndc-1(rny14) or cps-6(tm3222); fndc-1(rny14); phb-2(RNAi) mutants. Wilcoxon test p-values: *p<0.05; **p<0.01; ***p<0.001. Scale bar: 10µm.

No poly-ubiquitylation detected on sperm-derived mitochondria in control nor in the absence of ALLO-1, CPS-6, PHB-2 and FNDC-1.

(A, B) Maximum intensity projections of representative control and cps-6(tm3222); fndc-1(rny14); phb-2(RNAi); allo-1(tm4756) mutant early and bean stage embryos labeled with Pan-TUBEs (green) and for (A) MOs (red) and (B) sperm-derived mitochondria (red). (C) Quantification of the percentage of MOs and (D) sperm-derived mitochondria labeled with Pan-TUBEs in control and cps-6(tm3222); fndc-1(rny14); phb-2(RNAi); allo-1(tm4756) mutants embryos at the indicated developmental stages. Wilcoxon test p-values: *p<0.05; **p<0.01; ***p<0.001. Scale bar : 10µm.

PINK-1 can target sperm-derived mitochondria in the absence of ALLO-1.

(A) Quantification of the HSP-6::GFP labeled sperm-derived mitochondria (as % of the 1-cell stage) from the 10-cell to 100-cell stage and (B) in the late stage (3-fold stage). (C) Maximum intensity projections of confocal images of sperm-derived mitochondria (green) and nuclear DNA (blue) in 2-cell and late stage (3-fold) in embryos from the indicated crosses. Wilcoxon test p-values: *p<0.05; **p<0.01; ***p<0.001. Scale bar : 10µm.

Putative model of the mechanisms involved in the degradation of sperm-derived mitochondria in C. elegans.

No deleterious effects on allo-1(tm4756) and cps-6(tm3222) simple and double mutants embryos.

(A) Quantification of the progeny per worm and (B) quantification of the embryonic lethality. Student test (A) and Wilcoxon (B) test p-values: *p<0.05; **p<0.01; ***p<0.001.