Rapid, Selective Digestion of Mitochondrial DNA in Accordance With the matA Hierarchy of Multiallelic Mating Types in the Mitochondrial Inheritance of Physarum polycephalum

Corresponding author: Y. Moriyama, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-601, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan., kk17527{at}mail.ecc.u-tokyo.ac.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Although mitochondria are inherited uniparentally in nearly all eukaryotes, the mechanism for this is unclear. When zygotes of the isogamous protist Physarum polycephalum were stained with DAPI, the fluorescence of mtDNA in half of the mitochondria decreased simultaneously to give small spots and then disappeared completely 1.5 hr after nuclear fusion, while the other mitochondrial nucleoids and all of the mitochondrial sheaths remained unchanged. PCR analysis of single zygote cells confirmed that the loss was limited to mtDNA from one parent. The vacant mitochondrial sheaths were gradually eliminated by 60 hr after mating. Using six mating types, the transmission patterns of mtDNA were examined in all possible crosses. In 39 of 60 crosses, strict uniparental inheritance was confirmed in accordance with a hierarchy of relative sexuality. In the other crosses, however, mtDNA from both parents was transmitted to plasmodia. The ratio of parental mtDNA was estimated to be from 1:1 to 1:10^-4. Nevertheless, the matA hierarchy was followed. In these crosses, the mtDNA was incompletely digested, and mtDNA replicated during subsequent plasmodial development. We conclude that the rapid, selective digestion of mtDNA promotes the uniparental inheritance of mitochondria; when this fails, biparental inheritance occurs.

MITOCHONDRIA are inherited strictly maternally in many species. The maternal inheritance of mitochondria was first reported in 1974 in horse-donkey hybrids (HUTCHISON et al. 1974 ). Related studies reached the same conclusion in the rat (HAYASHI et al. 1978 ; KROON et al. 1978 ), the pocket gopher Geomys pinetis (AVISE et al. 1979 ), the frog Xenopus laevis (DAWID and BLACKLER 1972 ), the fruit fly Drosophila melanogaster (REILLY and THOMAS 1980 ), and humans (GILES et al. 1980 ). Particularly in oogamous species, uniparental inheritance of mitochondria has been attributed to the small number of mitochondria in the male gamete. Although fertilized eggs are heteroplasmic (i.e., they contain mitochondria from both parents), a small population of mitochondria derived from the male gamete is segregated rapidly after repeated cell division. Consequently, most cells are thought to contain mitochondria from the female parent (DAWID and BLACKLER 1972 ; HUTCHISON et al. 1974 ; BIRKY 1995 ; ANKEL-SIMONS and CUMMINS 1996 ). However, the idea of segregation of parental mitochondrial DNA (mtDNA) has recently been challenged in several reports. Backcrosses between Mus musculus and M. spretus (an interspecific cross) yielded offspring in which a very small proportion of paternal mtDNA (0.01–0.1%) could be detected by sensitive PCR techniques (GYLLENSTEN et al. 1991 ). This promoted a reexamination, using PCR, of more common intraspecific crosses between mammals from which KANEDA et al. 1995 concluded that, in intraspecific crosses (M. musculus), the paternal mtDNA was eliminated by the two-cell stage.

Uniparental inheritance of mitochondria has also been reported in the isogamous protist Physarum polycephalum (KAWANO et al. 1987 ; KAWANO and KUROIWA 1989 ; MELAND et al. 1991 ). The life cycle of Physarum includes two distinct vegetative forms: the haploid amoeba and the diploid plasmodium. The haploid myxamoebae act as isogametes; individuals of different mating types pair and fuse to form diploid zygotes that develop into macroscopic, diploid plasmodia after repeated mitotic cycles without cell division. Thus, the segregation of parental mtDNA is not involved in uniparental inheritance. There are more than just two mating types of Physarum; the mitochondria are transmitted uniparentally in accordance with the relative sexuality determined by the mating-type locus matA, which has at least 13 alleles. The matA alleles can be ranked in a linear hierarchy to determine the loss of mtDNA (KAWANO and KUROIWA 1989 ; MELAND et al. 1991 ): matA7 > matA2 > matA11 > matA12 > matA1//matA15 > matA6 (matA1 and matA15 have not been tested against each other). The mitochondrial donor is generally the amoeba that possesses the dominant matA allele, and in each mating pair, one strain consistently acts as the mtDNA donor, although this strain does not always act as the donor when combined in other mating pairs. MELAND et al. 1991 suggested that the elimination of the mtDNA from one parent is completed within 36 hr of mating. These facts suggest that the universal phenomenon of uniparental inheritance of mitochondria requires a species-specific recognition system by which the zygote cytoplasm identifies and eliminates mitochondria or mtDNA from one parent.

Recently, several studies reported selective destruction, rather than segregation, of sperm mitochondria in the zygote, particularly in mammalian cells (KANEDA et al. 1995 ; SUTOVSKY et al. 1999 , SUTOVSKY et al. 2000 ). The possible involvement of ubiquitin in the destruction of sperm mitochondria in fertilized cow and monkey eggs was suggested. Conversely, in chloroplast inheritance, it has been demonstrated that fluorescent chloroplast nucleoids derived from the male (mt^-) parent disappear after zygote formation in the isogamous green algae Chlamydomonas reinhardtii (KUROIWA et al. 1982 ; NISHIMURA et al. 1999 ). Unfortunately, however, the behavior of mtDNA before destruction is difficult to detect microscopically because of the small copy number and molecular size of mammalian mtDNA. In our work, to investigate the mechanism for eliminating mitochondria from one parent, we observed the fate of mitochondria and mt-nucleoids (complexes of mtDNA and proteins) throughout the mating of Physarum. The mitochondria and mtDNA of Physarum are easily observed by phase-contrast and epifluorescence microscopy. The mitochondria are well developed and contain 20–40 63-kb mtDNA molecules, which are highly organized by proteins into a large rod-shaped mitochondrial nucleoid in each mitochondrion (KUROIWA 1982 ; TAKANO et al. 2001 ). We observed the rapid, selective digestion of the mtDNA from one parent during early zygote development of Physarum. The uniparental inheritance of mitochondria seems to be promoted by this rapid, selective digestion of mtDNA.

Some articles have reported that biparental inheritance of mtDNA does occur (KONDO et al. 1990 ; GYLLENSTEN et al. 1991 ; ZOUROS et al. 1994 ; KANEDA et al. 1995 ; RAWSON et al. 1996 ). In particular, GYLLENSTEN et al. 1991 detected paternal mtDNA by PCR in interspecific mitochondrial congenic mice. Since the paternal contribution was only 0.01–0.1%, these authors suggested that earlier failures to detect paternal mtDNA were due to the low sensitivity of the assays used. The situation remains ambiguous, however, because many reported cases of paternal transmission involve interspecific rather than intraspecific hybrids. Since matings in nature by definition occur mostly within species, it is important to examine whether mtDNA is also biparentally transmitted in intraspecific hybrids. In this study, we used all possible crosses between 16 strains with matA1, matA2, matA11, matA12, matA15, or matA16 alleles to demonstrate that digestion of mtDNA from one parent is highly selective and thorough, in accordance with the matA hierarchy of multiallelic mating types. In 21 of the 60 possible crosses, however, uniparental mtDNA inheritance did not occur, and mtDNA from both parents was transmitted to plasmodia at varying frequencies. Since the rapid, selective digestion of mtDNA in the recessive mitochondria was incomplete, leakage of paternal mtDNA occurred.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and culture:
The amoebal strains of P. polycephalum used in this study are listed in Table 1. Myxamoebae were cultured on PGY plates (0.5% glucose, 0.05% yeast extract, 2 mM MgSO₄, and 1.5% agar in 25 mM potassium phosphate buffer, pH 6.6) at 23° with live bacteria (Klebsiella aerogenes) for food. Zygote formation was induced on SM-30 mating plates (30 mM citrate buffer, pH 4.5, 10 mM MgSO₄, and 1.5% agar) at 23°. For efficient crossing, myxamoebae must carry different matA alleles, and in each mating pair, one strain consistently acts as the mitochondria donor. However, the dominant strain does not necessarily act as the mitochondria donor in other combinations; the donor in each pair is determined by the respective matA alleles.

Plasmodium formation:
About 3 days after zygote formation, small agar blocks carrying young plasmodia were cut from the mating plates and transferred to malt extract agar (MEA) plates (KAWANO et al. 1987 ) for further growth at 23°.

Microscopic observation and fluorometry:
To observe the mitochondria clearly, cells were fixed with 8% formaldehyde in 10x PBS (pH 11) containing 0.01% Tween 20 on SM-30 mating plates. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), and a coverslip was placed over the stained sample. Photographs were taken with a BX62 Olympus (Tokyo) epifluorescence microscope equipped with a c4742 CCD camera (Hamamatsu Photonics, Shizuoka, Japan) and an Aquacosmos system. The length of mt-nucleoids and the relative mtDNA fluorescence were determined using the same system.

Electron microscopy:
Samples were fixed with 1% osmium tetroxide in PBS, pH 7.6 for 6 hr at 4°. They were then dehydrated in a graded ethanol series and embedded in Spurr's resin (SPURR 1969 ). Ultrathin sections (0.06–0.09 µm) were cut with a glass knife on an ultramicrotome (Leica Ultracut UCT; Leica Mikrosysteme, Vienna) and mounted on Formvar-coated copper grids. The sections were stained with 3% uranyl acetate for 10 min at room temperature and lead citrate (0.13 M lead nitrate, 0.2 M trisodium citrate dehydrate) for 5 min at room temperature and then examined with an electron microscope (H-7600; Hitachi, Tokyo).

Isolation of single cells:
A single amoeba or zygote was isolated from the SM-30 mating plate under a phase-contrast microscope (IMT-2; Olympus) using a capillary system that is typically used for microinjection (MO-202; Narishige, Tokyo). Single cells were transferred to individual microtubes containing 10 µl 1x PCR buffer, 0.5% Tween 20, and 2 µg/ml proteinase K. The samples were incubated overnight at 37° to digest proteins, and heated to 95° for 5 min to inactivate proteinase K. Each sample was divided into two tubes and used directly as template DNA for PCR.

DNA isolation:
Approximately 20 mg of amoeba cells grown on the PGY plates or plasmodium harvested on the MEA plates for 4 days was transferred to a microcentrifuge tube and suspended in 500 µl of 10x Tris/saline EDTA (100 mM Tris-HCl, pH 8, 150 mM NaCl, 100 mM EDTA) containing 2% SDS and 0.5 mg/ml proteinase K. After incubation of the suspension at 37° for 2 hr, 1 ml of saturated NaI with distilled water was added, and the lysate was incubated at 0° for 30 min. The lysate was then centrifuged at 20,000 x g for 10 min at 4°, the surface debris was removed, and 5 µl of Glassmilk from a Gene Clean II kit (BIO 101, Vista, CA) was added. The Glassmilk was washed and the DNA eluted according to the manufacturer's directions. The eluted DNA was used as the template for PCR.

Detection of parental mtDNA types:
According to the restriction fragment length polymorphism analyses, the mtDNA genotypes of the amoebae used in this study are classified into M-, W-, T-, and N-types (see Table 1). Unlike the M-type, the mtDNA of the W-, T-, and N-types has a 2-kb deletion (SAKURAI et al. 2000 ). This difference was exploited to detect mtDNA from single cells by semi-nested PCR (see Fig 3C). The DNA from a single cell was separated into two subsamples and each was amplified using the specific primers for either the M-type or the other types (W, T, or N) of mtDNA. As one complete round of PCR was insufficient to detect the mtDNA from a single cell, a second round was performed with seminested primers. The primer sequences were as follows:

1. F₁, 5'-TACCCTGTATATGGAACAG-3';

   
   

   View larger version (191K): In this window In a new window Download PPT slide   Figure 1. Loss of mt-nucleoids during zygote formation in P. polycephalum. (A–H) Merged images from phase-contrast and fluorescence microscopy. (A) Myxamoebae. (B) Zygote. (C–H) Timing of mt-nucleoid loss. (C and F) Fused cell. (D and G) Uninucleate zygote just after nuclear fusion. (E and H) Uninucleate zygote 1.5 hr after nuclear fusion. Bars: A–E, 5 µm; F–H, 1 µm.

   
   

   View larger version (71K): In this window In a new window Download PPT slide   Figure 2. Sequential images of mt-nucleoid loss. (A) Fluorescent image showing the simultaneous loss of half the mt-nucleoids in the zygote. (B–E) Representative images of mt-nucleoid loss. Phase-contrast and fluorescent images are merged. (B) Mitochondrion and its nucleoid in a uninucleate zygote just after nuclear fusion. (C and D) Beginning and end of mt-nucleoid loss. (E) Mitochondrion completely lacking an mt-nucleoid. Bars: A, 5 µm; B–E, 1 µm.

   
   

   View larger version (42K): In this window In a new window Download PPT slide   Figure 3. Detection of parental mtDNA from a single cell by PCR. (A and B) Before and after isolation of a single zygote from a mating plate with a capillary tube. (C) Scheme for detecting parental mtDNA by semi-nested PCR. The primer sets used are marked by half arrows (see MATERIALS AND METHODS). (D) Inheritance of parental mtDNA, as detected in single zygotes at several developmental stages of AI35 x U41. Three representative samples are shown for each stage of development. Bar, 30 µm; arrows: (A) zygote; (B) after isolation of the zygote.

2. F₂, 5'-GAATTGATAGAAGAACTCAGAAGAGG-3';

3. MR, 5'-GGTCCCCAAATATTTCTTATAGAATATGC-3';

4. TR, 5'-TGCTTCCATAATTGCATCGT-3'.

PCR reaction mixtures were prepared with ExTaq DNA polymerase (Takara, Otsu, Japan) according to the manufacturer's instructions in a final volume of 50 µl. The first and second rounds of PCR included 35 cycles at 94° for 0.5 min, at 54° for 0.5 min, and at 72° for 1 min. M-type mtDNA was amplified from the sample in one of the paired tubes with primers i and iii for the first round of PCR and with primers ii and iii for the second round. A 1-µl sample of the mixture from the first round of PCR was used as template for the second round. T-type mtDNA was amplified from the remaining tube with primers i and iv for the first round of PCR and with ii and iv for the second round. The lengths of the fragments amplified with the second pairs of primers from the M- and T-type mtDNA were expected to be 673 and 526 bp, respectively. The T-type-specific primer also amplified a fragment of 3 kb from the mtDNA of M-type. However, preliminary experiments showed that this 3-kb fragment was not amplified from a mixture of mtDNA from two parents, due to competition with the 526-bp fragment from the T-type.

Estimation of the ratio of parental mtDNA with a PCR matrix:
To estimate the ratio of mtDNA from each parent in the plasmodium, a PCR matrix of different PCR cycles and different template ratios was made using purified M- and W-type mtDNA. The DNA was amplified by PCR with primer sets i + iii or i + iv from AI35 (M-type) or DP246 (W-type) and then purified with a GFX PCR DNA and gel band purification kit (Amersham Pharmacia Biotech) as recommended by the manufacturer. The copy numbers of each product were estimated from the DNA concentration, and the two products were mixed at ratios of 1:10⁹–10⁹:1. Using such template mixtures, PCR was carried out for 30 sec at 94°, 30 sec at 55°, and 1 min at 72°, for 20, 25, 30, and 35 cycles with primer sets ii + iii or ii + iv. The products amplified with the two primer sets using the same template ratios were loaded in one lane and electrophoresed together. The electrophoresis patterns are shown in Fig 7. The parental mtDNA in the plasmodium was detected by PCR using plasmodial DNA isolated with Glassmilk from a Gene Clean II kit as the template. For PCR, 1 µl of 0.01x, 0.1x, and 1x template solution was used. PCR was performed with primer sets ii + iii or ii + iv for 20, 25, 30, and 35 cycles of 30 sec at 94°, 30 sec at 55°, and 1 min at 72°. The ratio of mtDNA from the parents in the plasmodium was estimated by comparing this PCR pattern with the PCR matrix.

View larger version (23K): In this window In a new window Download PPT slide   Figure 4. Changes in the number of mitochondria derived from each parent in a single cell during early plasmodial development. The number of mitochondria containing mtDNA in a cell () and the number of mitochondria that lost mtDNA (•) are shown in A and B in different ranges of the y-axis. All measurements are shown as mean ±SD. A t-test was used to estimate the significance of differences in the numbers of mitochondria between one stage and a previous stage (P = 0.01).

View larger version (138K): In this window In a new window Download PPT slide   Figure 5. Elimination of mitochondria that lost mtDNA during zygote development. (A–C) Merged images from phase-contrast and fluorescence microscopy. (A) Zygote 24 hr after mating. (B) Zygote 36 hr after mating. (C) Zygote 48 hr after mating. (D) Ultrastructural change in the mitochondria. Arrow, disordered, degraded, destroyed mitochondrion. Arrowhead, mitochondrion that contains mt-nucleoid (MN). Bars: A–C, 5 µm; D, 500 nm.

View larger version (71K): In this window In a new window Download PPT slide   Figure 6. Transmission pattern of mtDNA in four representative crosses. Parental mtDNA was detected from five plasmodia in each cross of (A) AI35 x TU41, (B) AI16 x TU41, (C) AI35 x DP246, and (D) AI16 x DP246.

View larger version (85K): In this window In a new window Download PPT slide   Figure 7. PCR matrix of different PCR cycles from template solution containing different copy numbers of the two mitochondrial DNA types. PCR was performed with strain-specific primers from template solution containing different copy numbers of the two mtDNA types for 20, 25, 30, and 35 cycles. Each mtDNA sample was amplified separately using different primer sets and applied to one lane. The upper fragment (673 kb) is the M-type product, and the lower fragment (526 kb) is the W-type.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Visualization of loss of mtDNA during zygote formation:
Myxamoebae are uninucleate cells that act as isogametes in crosses. In our serial observation of mating, syngamy occurred soon after mixing two myxamoebae strains with different matA alleles. Cell nuclei fused 2 hr after syngamy, and the resultant diploid nucleus divided repeatedly in the absence of cell division to form a multinucleate, diploid plasmodium. Phase-contrast observations of myxamoebae clearly revealed the elliptical mitochondria. After DAPI staining, cell nuclei and mt-nucleoids emitted bright blue-white fluorescence. Each amoeba contained 15 mitochondria before mating, and each mitochondrion contained a long rod-shaped mt-nucleoid at its center (Fig 1A). Zygote formation was induced on a mating plate by mixing two strains of different mating types. Surprisingly, about half of the total mitochondria in zygotes lacked an mt-nucleoid, although the shape of the mitochondria was consistent with the amoebal stage (Fig 1B). To investigate the loss of mt-nucleoids, the behavior of mitochondria during zygote formation was analyzed throughout the course of mating. Soon after two myxamoebae fused, a full complement of 30 parental mitochondria mixed together. Each mitochondrion was characterized by the presence of a fluorescent mt-nucleoid (Fig 1C and Fig F). The fused cells formed a uninucleate zygote as a result of nuclear fusion 2 hr after mating. The fluorescent mt-nucleoid persisted in every mitochondrion until this stage (Fig 1D and Fig G). However, 1 hr after nuclear fusion, mt-nucleoid fluorescence completely disappeared in about half of the mitochondria in the zygote (Fig 1E and Fig H).

The process of mtDNA loss:
Rapid loss of mt-nucleoids is expected to occur within an hour of nuclear fusion. We investigated this stage of mating in detail and found that mt-nucleoid fluorescence diminished synchronously to small spots, regardless of the position of the mitochondria in the zygote (Fig 2A). Within another 30 min, the mt-nucleoid fluorescence in these mitochondria disappeared completely. Mitochondria were arranged according to the time course of mt-nucleoid loss, as shown in Fig 2B–E. In half of the mitochondria, the long rod-shaped mt-nucleoid present in each mitochondrion just after nuclear fusion (Fig 2B) disappeared, starting from both ends of the mt-nucleoid 1 hr after nuclear fusion (Fig 2C). The fluorescence of each mt-nucleoid grew fainter and was rapidly reduced to a single small spot (Fig 2D). This spot was consistently located at the margin of the mitochondrion, and it disappeared completely without any other apparent major changes in the mitochondrion (Fig 2E). These observations suggest the presence of two types of mitochondria in the uninucleate zygote: those in which the mt-nucleoid is lost and those in which it persists. A mechanism for the uniparental inheritance of mitochondria could be proposed if it is established that lost mt-nucleoids originate from a single parent.

Detection of parental mtDNA from a single cell by PCR:
To determine the parental origin of the lost mt-nucleoids, parental mtDNA in single cells was analyzed during zygote formation using semi-nested PCR. A single gamete or zygote was isolated under a phase-contrast microscope using a microinjection capillary (Fig 3A and Fig B), and its mtDNA was amplified by PCR. Two amoebal strains of different mating types were used, AI35 (matA2; mtDNA, M-type) and TU41 (matA12; mtDNA, T-type). Unlike M-type, the mtDNA of the T-type has a 2-kb mtDNA deletion, so that mtDNA specific for the M- and T-types can be distinguished with PCR primers (Fig 3C). The results from three representative samples at each developmental stage are shown in Fig 3D. When AI35 and TU41 were crossed, the parental mtDNA coexisted in the fused cell and was detectable in the uninucleate zygote just after nuclear fusion. However, 1.5 hr after nuclear fusion, no parental mtDNA from TU41 was detected, and this was correlated with the loss of mt-nucleoids. Thus, mt-nucleoid loss appears to be the result of the selective digestion of mtDNA from one parent. Such selective digestion may account for uniparental inheritance of mitochondria.

Fate of mitochondria lacking mtDNA during plasmodial development:
To investigate the fate of mitochondria that lost mtDNA, mitochondria in the developing zygote were observed from 0 to 60 hr after mating. As the zygote of myxomycetes undergoes repeated nuclear division without cell division after mating, all of the mitochondria derived from the parents are kept in a single cell during zygote development. The total number of mitochondria in a single cell was counted at 8, 12, 24, 36, 48, and 60 hr after crossing AI35 and TU41 (Fig 4). AI35 and TU41 had 14 and 16 mitochondria per cell, respectively. There were 32 mitochondria in the zygote, 16 of which lost mtDNA by 8 hr after mating. As expected, the number of mitochondria with mtDNA increased >25-fold to 430 by 60 hr after mating as a result of repeated mitochondrial fission (Fig 4A). By contrast, the number of mitochondria without mtDNA remained at 16 until 36 hr after mating (Fig 4B). Then, the number decreased to 3 in a single cell by 48 hr, and all were lost by 60 hr after mating (Fig 4B).

Since the decrease in mitochondria lacking mtDNA might have been due to mitochondrial sheath destruction, including destruction of the outer and inner membranes and cristae, mitochondrial morphology was examined during early zygote development. Morphological changes were examined by light and electron microscopy during the early stages of zygote development to plasmodium. The fused, diploid nucleus divided to form a binucleate zygote (a small plasmodium). At 24 hr after mating, no morphological changes were observed by phase-contrast microscopy in mitochondria lacking mtDNA (Fig 5A). The plasmodium became 50 µm in diameter and had many nuclei 36 hr after mating (Fig 5B). The vacant mitochondrial sheaths remained visible at this time, and no degraded mitochondrial sheaths were observed. By 48 hr after mating, almost all of the vacant mitochondria had been eliminated (Fig 5C).

Although phase-contrast microscopy suggested that those that remained were unchanged morphologically (Fig 5C), electron microscopy clearly revealed ultrastructural changes in the inner membrane by 36 hr after mating. Fig 5D represents two typical mitochondria (left and right) at this stage. The left mitochondrion preserves an electron-dense mt-nucleoid at the center of the matrix and has well-developed tubular cristae. Almost all of the mitochondria at this stage were of this type. In contrast, the mitochondrion on the right has no mt-nucleoid and has collapsed cristae. Almost the entire mitochondrion was an electron-transparent region, although some parts retained double membranes and tubular cristae, enabling their detection using phase-contrast microscopy (Fig 5B and Fig D). Mitochondrial degradation was observed by 36 hr after mating, and the elimination of the mitochondrial sheath was completed by 60 hr after mating. Compared with the rapid and selective digestion of mtDNA from one parent within 0.5 hr at 3 hr of mating, elimination of the vacant mitochondria derived from one parent was a lengthy procedure.

Mitochondrial inheritance according to the matA hierarchy:
In Physarum, there are multiple mating types, and the mitochondrial inheritance mode is determined by alleles matA1–matA16, which are ranked in a linear hierarchy with respect to mitochondrial inheritance (KAWANO et al. 1987 ; KAWANO and KUROIWA 1989 ; MELAND et al. 1991 ). We investigated whether selective digestion of mtDNA occurred in accordance with the relative sexuality of matA. To determine whether the mtDNA from TU41 could survive when crossed with a second strain with a lower matA rank, AI16 (matA1; mtDNA, M-type) was crossed with TU41. Since AI16 and AI35 have the same parent (DEE 1960 ), they have the same mitochondrial genotype (Table 1). Consequently, crosses of these strains with TU41 also permitted examination of the relationship between digestion selectivity and mtDNA molecules. In crosses of AI16 with TU41, mt-nucleoid loss occurred in the same manner as in the original AI35 x TU41 cross. In the plasmodium of AI35 x TU41, mtDNA of TU41 was lost, as previously described (Fig 3D and Fig 6A). In the plasmodium of AI35 x TU41, however, the TU41 mtDNA survived, and the lost mt-nucleoids were of AI16 origin (Fig 6B). The results show that the digestion of parental mtDNA from one strain is highly selective and is in accord with the matA hierarchy.

To confirm the strictly uniparental inheritance at the PCR level in any combination of mating type, the transmission patterns of mtDNA were examined in all possible crosses between the strains listed in classes A and B in Table 1. The six strains ranked in class A are progeny of a x i and have either matA1 or matA2. Their mtDNA is M-type. The strains ranked in class B have different origins and have matA11, matA12, matA15, or matA16, depending on their origin. Since they have W-, T-, or N-type mtDNA, the mtDNA transmission pattern can be detected when they are crossed with class A strains. The mtDNA of parents was detected in plasmodia 10 days after mating by using PCR for 35 cycles. In 39 of 60 possible crosses, strict uniparental inheritance was confirmed (Table 2). For example, the mtDNA of AI35 (M-type) was transmitted in AI35 x TU41, while the mtDNA of TU41 (T-type) was transmitted in AI16 x TU41, as shown in Fig 6A and Fig B. These results are arranged in Table 2. Strict uniparental inheritance of mtDNA occurred in all of the crosses of matA1 and -2 strains with matA11 and -12 strains. The mtDNA of matA11 and -12 strains (N-type) was transmitted in the crosses with matA1 strains, but was not transmitted in the crosses with matA2 strains. These results are in accord with the matA hierarchy: matA2 > matA11 > matA12 > matA1. The matA2 strains were mitochondrial donors when they were crossed with matA16 strains. In 21 of 60 crosses, however, mtDNA from both parents was detected (Table 2). For example, when AI35 was crossed with DP246, the mtDNA of AI35 (M-type) was transmitted to the plasmodium (Fig 6C). However, when AI16 (M-type) was crossed with DP246 (W-type), uniparental transmission of mtDNA did not occur: Instead, mtDNA from both parents was transmitted to plasmodia (Fig 6D). Such biparental inheritance of mtDNA occurred only in the crosses matA1 x matA15, matA1 x matA16, and matA2 x matA15.

Ratio of parental mtDNA in biparental inheritance of mtDNA:
We examined the mtDNA type of every plasmodium at 10 days after mating by PCR with 35 cycles, as shown (Fig 6). With this number of PCR cycles, it is possible to detect the presence/absence of either parental mtDNA, but it is not possible to estimate the ratio of parental mtDNA, because the PCR product is saturated under this condition. Therefore, to estimate the ratio of parental mtDNA in the 21 crosses that inherited mtDNA biparentally, the PCR efficiency of mixtures of the two different kinds of mtDNA (M- and W-types) in copy-number ratios of 1:10⁹–10⁹:1 was examined at 20, 25, 30, and 35 PCR cycles using different primer sets for the M- and W-types. The two PCR products using the different primer sets were loaded in one lane. The results were arranged in a matrix, as shown in Fig 7, to show the PCR efficiency with different numbers of cycles and different ratios of the two templates. This efficiency matrix can detect at least 1 x 10⁵ molecules of mtDNA and can detect mtDNA from one genotype in a 10⁵–10⁰ times excess of mtDNA from the other. Therefore, using this PCR matrix, we estimated the ratios of parental mtDNA in 1 µl of 0.01x, 0.1x, and 1x template DNA solution that was isolated from plasmodium 10 days after mating for 20, 25, 30, and 35 cycles of PCR (Table 2). There were biased ratios in the parental mtDNA for the 21 crosses that inherited mtDNA biparentally. For example, the AI5 x DP246 plasmodium, which had inherited mtDNA biparentally, contained 10^-3 as much mtDNA from AI5 as from DP246 (Table 2). Conversely, AI16 x DP246 contained mtDNA from both parents in equal amounts, according to the PCR matrix. Equal biparental inheritance occurred in 5 of the 21 crosses, including AI16 x DP246. The mtDNA genotypes of each plasmodium were abbreviated using <<, <, =, or > according to the ratio of mtDNA from the parents (Table 2), and these results are arranged in Table 3 according to mating types. The bias of parental mtDNA in the crosses matA1 x matA15, matA1 x matA16, and matA2 x matA15 seems to obey the matA hierarchy. Furthermore, in matA1 x matA15, 5 of 12 crosses (AI5 x DP89, AI39 x DP89, AI2 x DP248, AI5 x DP248, and AI39 x DP248) showed exceptional uniparental inheritance of mtDNA, resulting in matA1 > matA15.

Biased biparental inheritance caused by partial mtDNA loss:
To investigate the mechanism of the biased biparental inheritance in particular crosses, we observed the behavior of mitochondria during plasmodium formation with DAPI-fluorescence and phase-contrast microscopy. In the crosses that showed biased biparental inheritance, e.g., AI5 x DP246, the disappearance of fluorescent mt-nucleoids was observed after mating in about half the mitochondria. However, the digestion of mtDNA in the recessive mitochondria from AI16 was not complete 24 hr after mating; very faint, small spots, representing fluorescent mt-nucleoids, persisted in some mitochondria (Fig 8A&NDASH;C). Surprisingly, 24–36 hr after mating, the persistent mt-nucleoids seemed to increase gradually in size due to mtDNA replication (Fig 8, D–F).

View larger version (149K): In this window In a new window Download PPT slide   Figure 8. Incomplete digestion of mtDNA in a plasmodium showing biparental inheritance of mtDNA. (A–F) Merged images from phase-contrast and DAPI fluorescence microscopy of whole cells. (A–C) Binucleate zygote 24 hr after mating. (D–F) Multinucleate zygote 36 hr after mating. B and E are enlargements of areas in A and D, respectively. Bars: A and D, 10 µm; B and E, 5 µm.

We measured the length and fluorescence intensity of mt-nucleoids stained with DAPI in cells 24 and 36 hr after mating and in mature plasmodium, using an epifluorescence microscope equipped with a CCD camera and fluorometric software. The values of their major axes (in micrometers) and fluorescence intensities (rfiu; relative fluorescent intensity unit) are arranged in a scatter plot in Fig 9. At 24 hr after mating, 18% of the mitochondria were vacant (0 µm, 0 rfiu), and 69% had well-developed mt-nucleoids (0.5–1.8 µm, 0.4–2.2 rfiu), but some mitochondria contained small mt-nucleoids that emitted very faint fluorescence (0.1–0.25 µm, 0–0.5 rfiu). These very small mt-nucleoids were generated by incomplete mtDNA digestion. Such small mt-nucleoids were rarely observed 36 hr after mating (Fig 8, D–F), because they appear to become larger (0.25+ µm, 0.5+ rfiu). During plasmodial development, mtDNA is replicated, and the size of these surviving mt-nucleoids increases, as shown in Fig 9.

View larger version (33K): In this window In a new window Download PPT slide   Figure 9. Scatter plots of fluorescence intensity vs. mt-nucleoid length at 24 and 36 hr after mating and in plasmodia in AI5 x DP246. The relative fluorescence intensity of mt-nucleoids stained with DAPI was plotted against the length of the major axis. Vacant mitochondria that had lost their mt-nucleoid were plotted at the origin of the coordinate axes.

At 36 hr after mating, the vacant mitochondria that had lost mt-nucleoids completely remained at the points of origin. In the plasmodia, the vacant mitochondria were completely eliminated, and the surviving mt-nucleoids and mitochondria were indistinguishable from each other (0.4–1.8 µm, 0.3–1.9 rfiu). However, the biased biparental inheritance of mtDNA suggests that the incompletely digested mtDNA was of uniparental origin and that the normal copy number in mitochondria was restored during plasmodial development. These results suggest that complete digestion of each mt-nucleoid in mitochondria is needed to destroy and eliminate the mitochondrial sheath. Incomplete digestion of mtDNA enables biased biparental inheritance.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Rapid, selective digestion of mtDNA causes uniparental inheritance of mitochondria:
Mitochondrial inheritance is thought to be predominantly uniparental in nearly all eukaryotes. The combination of mainly uniparental inheritance and frequent mutation invites great interest in mtDNA as an indicator of evolutionary relationships (INGMAN et al. 2000 ). However, the mechanism behind uniparental inheritance has been unclear. In this study, we observed the rapid and simultaneous loss of mt-nucleoids in about half of the mitochondria during an early stage of zygote maturation (Fig 1 and Fig 2). Molecular analysis of a single cell showed that mt-nucleoid loss coincided with the uniparental inheritance of mitochondria (Fig 3); after mt-nucleoid loss, mtDNA from only one of the two parents was detected by PCR. These results indicate that the lost mt-nucleoids were of uniparental origin. The loss of mtDNA has also been shown in higher plants and algae (KUROIWA and HORI 1986 ; CORRIVEAU and COLEMAN 1991 ; NAGATA et al. 1999 ). This loss of mtDNA organized in mt-nucleoids occurs before fertilization in the mature generative cell inside a pollen grain or in the male gamete before fertilization. In Physarum, however, mt-nucleoid loss occurs in the zygote after mating. Although mitochondria from both parents were well mixed in the zygote, mtDNA from the mitochondrial recipient strain was digested synchronously and completely, while that from the mitochondrial donor strain was completely protected from digestion. The mt-nucleoid loss progressed synchronously after nuclear fusion (Fig 2). This suggests that a nuclease, or nuclease activation signal, is synchronously transported from the cytoplasm to targeted mitochondria within a very limited period. The digestion of mtDNA seemed to be independent of the mtDNA sequence, since mtDNA with the same sequence were digested in the AI35 x TU41 cross, but not in the AI16 x TU41 cross (Fig 6). The results of reciprocal crosses in which one strain (TU41) played a dual role in uniparental inheritance, acting as a recipient in one cross (AI35 x TU41) and a donor in another (AI16 x TU41), confirmed that mtDNA of uniparental origin are not destined to be digested before mating. The uniparental inheritance of mitochondria seems to involve mechanisms that recognize the origin of mtDNA and promote the selective digestion of mtDNA from one parent in the zygote.

After the complete digestion of mtDNA, the number of mitochondria that contained mtDNA increased greatly, whereas the number of mitochondria that lost mtDNA remained unchanged until 36 hr after mating. After another 24 hr, they were lost completely (Fig 4). Degraded or disintegrated mitochondria were not observed directly at these stages by phase-contrast microscopy (Fig 5, A–C). In Saccharomyces cerevisiae and C. reinhardtii, it is well known that organelles derived from both parents fuse. However, the decrease in the number of mitochondria that lost mtDNA was not due to fusion with mitochondria that contained mtDNA, since no fused mitochondria were observed at these stages in Physarum. Moreover, the strains used here do not have an mF plasmid, which is known to promote mitochondrial fusion in P. polycephalum (KAWANO et al. 1993 ).

More detailed observation by electron microscopy revealed that degradation of the mitochondrial inner membrane occurred without any changes in size of mitochondria 36 hr after mating (Fig 5D). Morphological changes that do not result in changes in the overall size of mitochondria have also been noted during apoptosis and necrosis (LEMASTERS et al. 1998 ; SCORRANO et al. 2002 ). In Physarum, it is likely that the inner membrane of mitochondria that lose mtDNA is disrupted, and then the empty, nonfunctional mitochondria are removed, probably by lysosomes. In hamsters, rats, and bovines, degradation of sperm mitochondria has been observed during the early stage of embryonic development (SZOLLOSI 1965 ; HIRAOKA and HIRAO 1988 ; KANEDA et al. 1995 ; SUTOVSKY et al. 1999 , SUTOVSKY et al. 2000 ). SUTOVSKY et al. 1999 , SUTOVSKY et al. 2000 reported that sperm mitochondria were ubiquitinated before fertilization and subsequently destroyed in the mammalian egg. They insisted that the destruction of mitochondria invites uniparental inheritance of mitochondria. The candidate ubiquitin substrate was proposed to be prohibitin, an integral protein of the inner mitochondrial membrane. It is reasonable to assume that modification and destruction of the mitochondrial membrane plays an important role in mitochondrial inheritance. In Physarum, however, such ubiquitination before gamete fusion is unlikely, since the gamete has only relative sexuality, which is determined by the matA allele of the mating partner. As the choice of a fusion partner is random, the mitochondria or mtDNA cannot be primed before mating. At least the integrity of the mitochondrial membrane is conserved just before the digestion of mtDNA, as the digestion of mtDNA in dividing mitochondria occurs (Fig 1B). We believe that prohibitin ubiquitination may perform an important role in the destruction of the inner mitochondrial membrane after mtDNA digestion.

Incomplete digestion of mtDNA causes biased biparental inheritance of mitochondria:
In Physarum, the inheritance mode of mtDNA is determined by the mating-type locus matA, which has at least 13 alleles. We examined the recognition and digestion of mtDNA among multiple mating types, using PCR for 35 cycles of DNA from plasmodia 10 days after mating. Of the eight possible combinations of six mating types, five (39 of possible 60 crosses) showed strict uniparental inheritance of mitochondria in accordance with the relative sexuality of matA (Fig 6, Table 3). Conversely, mtDNA from both parents was transmitted in three combinations of matA (21 crosses).

To estimate the ratio of parental mtDNA readily, we made PCR matrices that showed the efficiency of the PCR reaction depending on the ratio of parental mtDNA. Our PCR matrix method is very useful for treating many samples to estimate the ratio of parental mtDNA. The ratio ranged from 1:10^-4 to equal amounts (Table 2 and Table 3), and one of the parental mtDNA genotypes always dominated in accordance with the matA hierarchy. In the zygotes of these crosses, the digestion of mtDNA was incomplete (Fig 8, A–C). The ratios of mtDNA from parents, listed in Table 2, may depend on the effectiveness of the digestion of mtDNA. The aberrant mitochondria with small mt-nucleoids (Fig 8C and Fig F) become normal sized by replicating their mtDNA (Fig 9). The temporary reduction and subsequent replication of mtDNA can explain the biased biparental inheritance of mtDNA from parents in Physarum.

The presence of leaked paternal mtDNA, particularly in interspecific hybrids between closely related species, occurs in several genera, such as Mytilus (ZOUROS et al. 1994 ; RAWSON et al. 1996 ), Drosophila (KONDO et al. 1990 ), and Mus (GYLLENSTEN et al. 1991 ; KANEDA et al. 1995 ), and in humans (SCHWARTZ and VISSING 2002 ). In such cases, the selection or digestion of mtDNA from one parent in the zygote might fail. The biased biparental inheritance of mtDNA in Physarum suggests that the destruction of mitochondria from a strain lower in the hierarchy never occurs unless the mtDNA is digested completely. If the destruction of mitochondria rather than the digestion of mtDNA were the critical mechanism of mitochondrial inheritance, strict uniparental inheritance of mitochondria would occur even though complete digestion of mtDNA failed.

Our results suggest that complete digestion of mtDNA is needed for the destruction of mitochondria and that uniparental inheritance of mitochondria is directly caused by the rapid and selective digestion of the mtDNA from one parent. The mechanisms for recognizing the parental origin of mtDNA and for promoting the selective digestion of mtDNA are unknown. We are now investigating mitochondrial nuclease(s) that are nuclear DNA coded and involved in the recognition and digestion of mtDNA from one parent. The isolation and characterization of nuclease(s) should explain the leakage of paternal mtDNA to subsequent generations.

	ACKNOWLEDGMENTS

We thank T. Kuroiwa (Graduate School of Science, University of Tokyo) for helpful discussions and A. Hirata (Graduate School of Frontier Science, University of Tokyo) for advice on electron microscopy. We also thank S. Matsunaga (Graduate School of Engineering, University of Osaka) for helpful technical advice. This study was supported by grants for Scientific Research in Priority Areas (no. 13440246 to S.K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Manuscript received December 16, 2002; Accepted for publication March 13, 2003.

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