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Degradation of Paternal Mitochondria by Fertilization-Triggered Autophagy in C. elegans Embryos

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Science  25 Nov 2011:
Vol. 334, Issue 6059, pp. 1141-1144
DOI: 10.1126/science.1210333

Abstract

The mitochondrial genome is believed to be maternally inherited in many eukaryotes. Sperm-derived paternal mitochondria enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism responsible for this clearance has been unknown. Here, we show that autophagy, which delivers cytosolic components to lysosomes for degradation, is required for the elimination of paternal mitochondria in Caenorhabditis elegans. Immediately after fertilization, sperm-derived components trigger the localized induction of autophagy around sperm mitochondria. Autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genome remain even in the first larval stage. Thus, fertilization-triggered autophagy is required for selective degradation of paternal mitochondria and thereby maternal inheritance of mitochondrial DNA.

It is widely believed that mitochondrial DNA (mtDNA) is maternally inherited in many eukaryotes despite the fact that paternal mitochondria enter into the ooplasm after fertilization in most species (1, 2). One possible explanation is that the paternal mtDNA is simply diluted with an excess copy number of oocyte mtDNA and hardly detected in embryos (3). Another is that the paternal mtDNA and mitochondria themselves are selectively degraded and eliminated from the embryonic cytoplasm. Cytologically, sperm-derived mitochondria packed in the midpiece disappear during early embryogenesis, typically by the eight-cell stage in mice (4). Active digestion of the paternal mtDNA in embryos was also observed (5, 6). Involvement of the ubiquitin-proteasome system has been suggested in mammals (7); however, the mechanisms responsible for the clearance of paternal mitochondria from the embryonic cytoplasm still largely remain unknown. Macroautophagy (referred as autophagy hereafter) is another major degradation system of cytoplasmic proteins and organelles in which a portion of the cytoplasm is sequestered into autophagosomes and targeted to lysosomes (811). In mice, autophagy is essential for preimplantation development by supporting a normal rate of protein synthesis (12). In Caenorhabditis elegans, autophagy is required for dauer formation and P-granule degradation (13, 14), but its function in early development had not been addressed.

In C. elegans, oocytes of the hermaphrodite gonad are arrested in meiotic prophase I. After the mature oocyte is ovulated and travels to the spermatheca, which contains sperm, immediate fertilization results. Subsequently, embryos move to the uterus, complete meiosis, and start zygotic development (fig. S1A) (15, 16). Males can inseminate the hermaphrodites, and male sperm is predominantly used to fertilize oocytes (17). Worm sperm lacks a flagellum (midpiece and tail structures) but contains 50 to 70 tightly packed mitochondria around the nucleus (fig. S1B) (18). To monitor the fate of sperm-derived mitochondria in embryos, males were incubated in the presence of MitoTracker Red (MT) to stain sperm mitochondria and mated with nonlabeled hermaphrodites (fig. S1). The paternal condensed pronuclear DNA and MT-labeled paternal mitochondria clustering around it were detected in the very early embryo (Fig. 1A). Sperm-derived mitochondria then became scattered throughout the cytoplasm, keeping a condensed granular appearance in the pronuclear-fusion stage. These paternal MT signals gradually disappeared, and most signals were entirely undetectable by the 16-cell stage (Fig. 1, B to E). In two- and four-cell stage embryos, sperm-derived mitochondria seemed to be randomly inherited into most blastomeres. We also labeled sperm mitochondria with heat shock protein-6–GFP (green fluorescent protein; mtGFP) and observed similar results (fig. S2).

Fig. 1

Disappearance of paternal mitochondria in C. elegans early embryos. (A to D) Wild-type hermaphrodites were mated to wild-type males labeled with MT (red). F1 embryos were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). In all fluorescent images, dotted lines indicate outline of embryos, and projected images of confocal z-stacks are shown. MT signals outside embryos are unfertilized male sperms. p, paternal pronuclear DNA; m, maternal pronuclear DNA; SP, spermatheca. Scale bar indicates 10 μm. (E) Time course of paternal mitochondria disappearance. The areas showing sperm-derived MT signals were quantified in each embryo. Error bars indicate SD.

To test the possible involvement of autophagy in the degradation of sperm-derived mitochondria, we first monitored autophagosome formation in embryos that maternally express GFP–LGG-1. LGG-1 is a homolog of LC3 and Atg8 that is necessary for autophagosome formation and also serves as a marker for autophagosome membranes (8, 9, 11). In oocytes, several small GFP–LGG-1–labeled puncta were scattered throughout the cytoplasm (Fig. 2A). After fertilization, strong accumulation of GFP–LGG-1 was detected on vesicular structures clustering in one pole of the early embryos (Fig. 2B, arrow). We found that these GFP–LGG-1–positive structures were distributed around the paternal pronuclear DNA in the posterior pole region (fig. S3, C and D). The GFP–LGG-1 puncta started to disperse into the cytoplasm as the paternal pronucleus expanded (fig. S3E). The majority of GFP–LGG-1–positive puncta disappeared by the 16-cell stage (Fig. 2B, +3 and +4 embryos, and fig. S3G). The second induction of GFP–LGG-1–positive puncta was observed in embryos around the 32- to 64-cell stage, and many smaller GFP–LGG-1–positive puncta appeared in whole embryos (Fig. 2B, +5 and +6 embryos, and fig. S3H). The GFP signal finally disappeared in the late-stage embryos. We confirmed that the behavior of endogenous LGG-1 is similar to that of GFP–LGG-1 (Fig. 2, C and D, and fig. S3, J to M). These results demonstrate the dynamic regulation of LGG-1–positive autophagosome formation in C. elegans early embryos. The induction of autophagy was comparable in self- and cross-fertilized embryos.

Fig. 2

LGG-1–positive autophagosomes are induced in the one-cell-stage embryo just after fertilization. (A and B) GFP–LGG-1 in live intact animals. An arrow indicates accumulation of GFP–LGG-1 in the very early embryo. (A′ and B′) Differential interference contrast (DIC) images. Scale bars, 20 μm. (C and D) lgg-1(tm3489) (C) and wild-type (WT) (D) embryos were stained with an antibody against LGG-1 (green) and DAPI (blue). One-cell-stage embryos in meiois II are shown. (D′) An enlarged image of the boxed area in (D). PB, extruded polar body. Scale bars, 10 μm.

Autophagosome formation is regulated by a cascade of Atg proteins (8, 9, 11). Induction of GFP–LGG-1–positive puncta at the one-cell stage was strongly inhibited in unc-51(e369) (atg1 and ULK1 homolog), atg-18(gk378), and atg-7(RNAi) (where RNAi indicates RNA interference) mutant embryos (fig. S3, N to Q), suggesting that this induction of autophagy is regulated by the canonical autophagic pathway.

The localized induction of autophagy around the paternal pronuclear DNA led to the assumption that this autophagosome formation is triggered directly by sperm entry. To test this possibility, we used temperature-sensitive mutants, spe-9 and spe-11 (Fig. 3). spe-9 encodes a sperm protein required for normal fertilization. The spe-9(hc52ts) mutants produce sperms that cannot fertilize oocytes, resulting in accumulation of unfertilized eggs in the uterus (19). In spe-9(hc52ts) mutant eggs, the first induction of GFP–LGG-1–positive autophagosomes was not observed (Fig. 3B). spe-11 encodes a sperm protein essential for normal embryogenesis but not for fertilization (20). Even in the spe-11(hc77ts) mutant, GFP–LGG-1–positive autophagosomes were induced around the paternal pronuclear DNA (Fig. 3C). Prior studies have reported that the spe-11 mutation increases the frequency of polyspermy (21). In such polyspermic embryos, autophagosomes were induced around each paternal pronuclear DNA (Fig. 3E). These results suggest that penetrated sperm components trigger localized autophagy induction. GFP-LGG-1–positive structures were visible as early as meiotic metaphase II (fig. S3C). However, meiotic cell cycle arrest at metaphase I by emb-27(RNAi) did not affect on autophagosome induction, suggesting that autophagy is induced independently of the onset of anaphase I (Fig. 3D).

Fig. 3

Induction of LGG-1–positive autophagosomes depends on fertilization. Localization of GFP–LGG-1 in embryos defective in fertilization or early embryogenesis was observed in live animals. (A) Mock RNAi; (B) spe-9(hc52ts) at 25°C; (C) spe-11(hc77ts) at 25°C; (D) emb-27(RNAi). Arrows indicate the cluster of GFP–LGG-1–positive autophagosomes. (E) A spe-11(hc77ts) embryo resulting from polyspermy (left). A fluorescent image is merged with a corresponding DIC image (right). Scale bars, 10 μm.

We next investigated whether these autophagosomes participate in the degradation of paternal mitochondria. We found that, in the early embryo, the GFP–LGG-1–positive autophagosomes and MT-labeled paternal mitochondria were closely associated with each other and partially overlapped (Fig. 4A, upper). During the pronuclear-fusion stage and the first mitosis, these organelles spread into the cytoplasm and remained associated or colocalized with each other (Fig. 4A, lower). Both signals disappeared by the 16-cell stage, following a similar time course (Fig. 1 and fig. S3). Similar observations were made by using sperm-derived mtGFP and endogenous LGG-1 (fig. S4, A to F). Autophagosomes appeared to specifically associate with paternal mitochondria among the total mitochondrial population (fig. S5). We also assessed whether paternal mitochondria are eventually targeted to the lysosomes. In one-cell-stage embryos, paternal mitochondria were localized to vesicles labeled with GFP–RAB-7, a marker of lysosomes and late endosomes (fig. S4G) (22). In addition, disappearance of MT-labeled sperm mitochondria was inhibited in rab-7(ok511) null mutant embryos (23), supporting lysosome-dependent degradation of paternal mitochondria (Fig. 4B, middle).

Fig. 4

Paternal mitochondria are degraded by autophagy. (A) Paternal MT-labeled mitochondria colocalize with GFP–LGG-1–positive autophagosomes in one-cell-stage embryos. (B) WT or indicated mutant hermaphrodites were mated to MT-labeled WT males. Embryos were fixed and stained with DAPI in (A) and (B). (C) lgg-1(tm3489) hermaphrodites were self-fertilized (left) or crossed with MT-labeled lgg-1(tm3489) males (right). F1 L1 larvae were observed. Merged images of green and red channels are shown in which autofluorescence of lysosome-like gut granules is seen as yellow. Arrowheads indicate remaining paternal mitochondria. Scale bars, 10 μm. (D) Schematic representation of mtDNA and polymerase chain reaction (PCR) primers used. (E and F) Paternal mtDNA was marked with a uaDf5 deletion, and its inheritance to the next generation was examined in the lgg-1(+) (E) or lgg-1 null (F) background. Total DNA prepared from single worm [adult (A) or L1] or 10 L1 larvae was examined by PCR.

We further examined the effect of mutation or depletion of autophagy regulators on the disappearance of paternal mitochondria. The lgg-1(tm3489) null mutant homozygotes reached adulthood and produced an almost normal number of self-fertilized eggs. However, 36% of the eggs did not hatch, and 59% of the embryos hatched but were lethal at the L1 larval stage, suggesting that autophagy is not essential for fertilization but is required for the completion of normal embryogenesis and survival of L1 larvae (fig. S6). When hermaphrodites of the lgg-1(tm3489) mutant were mated with MT-labeled wild-type males, paternal mitochondria remained in the embryos beyond the 16-cell stage (n > 20) (fig. S7A). A similar retardation of the disappearance of paternal mitochondria was observed when unc-51(e369), atg-18(gk378), or atg-7(RNAi) mutant hermaphrodites were mated with wild-type males (Fig. 4B and fig. S7A). Conversely, lgg-1(tm3489) mutation in males did not affect clearance of paternal mitochondria in embryos, suggesting that maternally provided autophagic activity is required for effective degradation of sperm-derived mitochondria in the early embryo (fig. S7B). On the other hand, even in embryos from the lgg-1(tm3489) hermaphrodites, paternal mitochondria were observed as late as at the lima-bean stage but eventually disappeared at later stages. We assumed that the wild-type lgg-1 gene provided by the sperm was expressed and may have restored the autophagic activity in late embryos. Supporting this possibility, crossing of lgg-1(tm3489) hermaphrodites and wild-type males resulted in viable F1 embryos (fig. S6). To generate lgg-1(tm3489) homozygous embryos, MT-labeled lgg-1(tm3489) males were crossed with lgg-1(tm3489) hermaphrodites. In lgg-1(tm3489) homozygous F1 embryos, MT-labeled paternal mitochondria were detected in late embryos and even in L1 larvae, showing that autophagy is required for elimination of paternal mitochondria (Fig. 4C and fig. S8, A to C). The same results were obtained by using sperm-derived mtGFP (fig. S7, C to F′). In mutant larvae, MT-labeled mitochondria were distributed throughout various tissues. To further detect traces of paternal mtDNA in F1 offspring, we used a large deletion allele of mtDNA (uaDf5) (Fig. 4D). In hermaphrodites, uaDf5 is stably inherited in a heretoplasmic state with the wild-type mtDNA (24). When paternal mtDNA was marked with uaDf5, it was not normally transmitted to the next generation (Fig. 4E). In contrast, in the lgg-1(tm3489) background, paternally provided uaDf5 was detected in F1 offspring, demonstrating inheritance of paternal mtDNA (Fig. 4F and fig. S8D).

We also examined whether sperm mitochondria were ubiquitinated as reported in mammals (7). In sperm, no obvious staining was detected by using an antibody against polyubiquitin (fig. S9A). Instead, punctate structures giving a strong polyubiquitination signal appeared around paternal pronuclear DNA in early embryos (fig. S9B). We found that these structures did not directly overlap with paternal mitochondria but colocalized with a marker of membranous organelles (MOs), specialized vesicular structures in sperms (fig. S9D) (25). These ubiquitinated MOs were also engulfed by GFP–LGG-1–positive autophagosomes. MOs and paternal mitochondria seemed to be engulfed together or independently by autophagosomes (fig. S9E).

We have shown that autophagy is essential for the effective elimination of paternal mitochondria. Our results also suggest that autophagic degradation of paternal mitochondria could be the mechanism of maternal inheritance of mtDNA. Autophagosomes induced upon fertilization were localized and appeared to selectively engulf sperm components, which is reminiscent of the selective autophagic degradation of damaged mitochondria and invading bacteria in mammals (26, 27). Paternal mitochondria remaining in the lgg-1 mutant retained the characteristic granular morphology and did not increase in number during embryogenesis (figs. S7 and S8), implying that they are proliferation- and fusion-inactive. Such qualitative difference may underlie selective elimination of paternal mitochondria. The paternal mitochondria and/or mtDNA could be heavily damaged by reactive oxygen species before fertilization (28). Selective elimination of paternal mitochondria may prevent the spreading of potentially deleterious mitochondria to the whole population. In mice, autophagy is also up-regulated immediately after fertilization (12), suggesting that fertilization-triggered autophagy is a conserved phenomenon in animals.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1210333/DC1

Materials and Methods

Figs. S1 to S9

Table S1

References (2939)

References and Notes

  1. Acknowledgments: We thank K. Sato and other members of Sato’s laboratory for technical assistance and discussions; Y. Ohsumi for discussions; S. Mitani, Y. Kohara, and the Caenorhabditis Genetic Center for supplying strains and an antibody. This research was supported by a Grant-in-Aid for Young Scientists (A), Japan Society for the Promotion of Science; a Grant-in-Aid for Scientific Research on Priority Areas “Protein Community” and Scientific Research on Innovative Areas “Intracellular Logistics,” Ministry of Education, Culture, Sports, Science, and Technology (MEXT); the GCOE Program, MEXT. This research was also supported by the Uehara Memorial Foundation (to K.S.) and the Naito Foundation (to M.S.).
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