Unique insights into maternal mitochondrial inheritance in mice

Abstract

In animals, mtDNA is always transmitted through the female and this is termed "maternal inheritance." Recently, autophagy was reported to be involved in maternal inheritance by elimination of paternal mitochondria and mtDNA in Caenorhabditis elegans; moreover, by immunofluorescence, P62 and LC3 proteins were also found to colocalize to sperm mitochondria after fertilization in mice. Thus, it has been speculated that autophagy may be an evolutionary conserved mechanism for paternal mitochondrial elimination. However, by using two transgenic mouse strains, one bearing GFP-labeled autophagosomes and the other bearing red fluorescent protein-labeled mitochondria, we demonstrated that autophagy did not participate in the postfertilization elimination of sperm mitochondria in mice. Although P62 and LC3 proteins congregated to sperm mitochondria immediately after fertilization, sperm mitochondria were not engulfed and ultimately degraded in lysosomes until P62 and LC3 proteins disengaged from sperm mitochondria. Instead, sperm mitochondria unevenly distributed in blastomeres during cleavage and persisted in several cells until the morula stages. Furthermore, by using single sperm mtDNA PCR, we observed that most motile sperm that had reached the oviduct for fertilization had eliminated their mtDNA, leaving only vacuolar mitochondria. However, if sperm with remaining mtDNA entered the zygote, mtDNA was not eliminated and could be detected in newborn mice. Based on these results, we conclude that, in mice, maternal inheritance of mtDNA is not an active process of sperm mitochondrial and mtDNA elimination achieved through autophagy in early embryos, but may be a passive process as a result of prefertilization sperm mtDNA elimination and uneven mitochondrial distribution in embryos.

Keywords: assisted reproductive technologies; mitophagy; paternal inheritance.

Fig. 1.

Live cell fluorescence imaging of GFP-LC3 and sperm mitochondria in early embryos. GFP-LC3 congregated to sperm mitochondria immediately after fertilization, but dissociated at the four-cell stage. (Scale bars: 20 µm.)

Fig. 2.

Live cell fluorescence imaging of sperm mitochondria (red) in early embryos. Sperm mitochondria disaggregated and became restricted to only one blastomere during one-cell to four-cell stages. Sperm mitochondria distributed in several cells after the eight-cell stage and could be detected until morula stages. (Scale bars: 20 µm.)

Fig. 3.

Single copy of paternal (C57/6j) mtDNA could be selectively amplified and distinguished from female (BALB/c) mtDNA of an oocyte. Lane 1, PCR product of BALB/c mtDNA could not be digested with Tth111I restriction enzyme and was 520 bp; lane 2, PCR product of C57/6j mtDNA was digested to 383- and 157-bp segments by Tth111I restriction enzyme; lanes 3–6, PCR products amplified from single copy of C57/6j mtDNA (as a result of Poisson distribution effect, C57/6j mtDNA was detected in lane 6 but not in lanes 3–5); lanes 7–10, PCR products amplified from single copy of C57/6j mtDNA and a BALB/c oocyte mtDNA were digested with Tth111I restriction enzyme (as a result of Poisson distribution effect, C57/6j mtDNA was detected only in lanes 9 and10).