(A) Fluorescence of mito-Dendra2 in a live sperm cell isolated from the cauda epididymis of a PhAM mouse. (B–F) Mito-Dendra2 in a 12 hr (B), 36 hr (C), 60 hr (D), 72 hr (E), and 84 hr embryo (F). In (B), note that mito-Dendra2 is circumscribed to a distinct rod-like structure. The mitochondria disperse in later embryos and are lost by 84 hr. (G) Quantification of the mito-Dendra2 signal (see Materials and methods) at 36, 60, 72, and 84 hr after fertilization. Each data point represents the mean of 15 embryos. Error bars indicate SD. (H) Representative maximum intensity projection images of maternal mitochondrial content versus paternal mitochondrial content over time. Embryos with mito-Dendra2-labeled maternal mitochondria were derived from crosses of wildtype males with homozygous PhAM females. Embryos with labeled paternal mitochondria were derived from crosses of wild-type females with homozygous PhAM males, whose sperm donate Dendra2-labeled mitochondria to the embryo upon fertilization. Embryos were cultured in vitro and imaged at the indicated time. Note that paternal Dendra2 signal decreases with time, whereas maternal Dendra2 signal does not. (I) Schematic of paternal mitochondrial elimination assay. Wildtype females are mated with PhAM males. One-cell embryos are microinjected in the perivitelline space with concentrated lentivirus targeting candidate genes. During in vitro culture, embryos are periodically imaged live and monitored for their ability to eliminate paternal mitochondria. (J) Representative images of embryos injected with lentivirus carrying nontargeting shRNA. The left three images show mito-Dendra2, phase-contrast, and mCherry signals at 60 hr; the right three images show the same as 84 hr. (K) Embryos injected with lentivirus carrying Atg3 shRNA. (L) Embryos treated with bafilomycin A1. (M) Embryos injected with lentivirus carrying Parkin shRNA. All scale bars are 10 μm.