Maternal Inheritance of Mouse mtDNA in Interspecific Hybrids: Segregation of the Leaked Paternal mtDNA Followed by the Prevention of Subsequent Paternal Leakage

Corresponding author: Hiromichi Yonekawa, Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, Tokyo, 113, Japan, yonekawa{at}rinshoken.or.jp (E-mail).

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F₁ hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10⁸-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F₁ hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.

ANIMAL mitochondrial DNA (mtDNA) has been generally believed to be inherited strictly maternally (AVISE 1991 ; BIRKY 1995 ). However, the possibility remains that failure to find the paternal mtDNA is simply due to a technical problem: the contribution of sperm mtDNA is extremely small compared to oocyte mtDNA. Recent studies have demonstrated the presence of leaked paternal mtDNA, that is, biparental transmission of mtDNA, particularly in interspecific hybrids between closely related species in several genera such as Mytilus (ZOUROS et al. 1994 ; RAWSON et al. 1996 ),¹ Drosophila (KONDO et al. 1990 ) and Mus (GYLLENSTEN et al. 1991 ; KANEDA et al. 1995 ).

In the genus Mus, for example, GYLLENSTEN et al. 1991 observed paternal mtDNA by the use of PCR techniques in mouse strains congenic for mtDNA that were generated by 8–26 generations of successive backcrossing of female hybrids between Mus musculus and Mus spretus to male M. spretus or male M. musculus ; the leaked paternal mtDNA accumulated constantly in all tissues of every individual examined to the extent of 0.1–0.01%. Moreover, the amount of leaked paternal mtDNA increased slightly with additional backcross generations, and its uniform distribution in overall tissues persisted stably even after 2–14 generations of subsequent sister-brother matings. Based upon these observations, they proposed that leakage of a small amount of paternal mtDNA occurred constantly in the inheritance of animal mtDNA, and that the apparent discrepancy of absence (HAYASHI et al. 1978 ) or presence (GYLLENSTEN et al. 1991 ) of paternal mtDNA leakage must be due to the low resolution of sperm mtDNA detection procedures, mainly Southern blot analysis or ethidium bromide staining, that were used in these studies.

However, these findings seem inconsistent with our recent observations that the sperm-derived mtDNA disappears rapidly and completely in the early pronucleus stage of embryogenesis in interspecific hybrid F₁ mice, and that leakage is not always observed even in interspecific F₁ embryos between M. musculus and M. spretus (KANEDA et al. 1995 ). Furthermore, it has been generally considered that coexistence of wild-type and mutant mtDNA within individuals–-heteroplasmy–-is not stable in mammals due to stochastic mtDNA segregation (HAYASHI et al. 1983 ), and that mtDNAs segregate rapidly in a few generations resulting in producing individuals with homoplasmic mtDNA (HAUSWIRTH and GILLHAM 1978; LAIPIS 1982). These observations are not consistent with continuous leakage of sperm mtDNA and stable transmission to the following generations (GYLLENSTEN et al. 1991 ), and warrant careful reexamination.

In the present work, we use F₁ hybrids produced by the interspecific cross between female M. musculus and male M. spretus to examine whether sperm-derived mtDNA that escapes the presumptive exclusion mechanism in the fertilized egg can be transmitted constantly and uniformly to various tissues, and whether it can accumulate further in subsequent backcrosses of the F₁ hybrid female to male M. spretus.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mouse strains and crosses:
Mice of the inbred strain C57BL/6J (B6, Mus musculus) and of Mus spretus were purchased from CLEA (Osaka, Japan) and The Jackson Laboratory (Bar Harbor, ME), respectively. The mtDNA congenic strain B6.mt ^Jpn was established by backcrossing females carrying mtDNA of Japanese Mus musculus molossinus from donor strain ddY to B6 males (KANEDA et al. 1995 ). We crossed female M. musculus with male M. spretus to make interspecific F₁ hybrids. However, reciprocal crosses, for example, female M. spretus with male M. musculus, have not succeeded yet. N₂ hybrids were isolated by backcrossing F₁ females to male M. spretus [(M. musculus C x M. spretus F) F₁ C x M. spretus F] or B6.mt ^Jpn [(M. musculus C x M. spretus F) F₁ C x B6.mt ^Jpn F] by in vitro fertilization.

DNA preparation from adult tissues:
Twelve tissues (see Table 1) were removed from the adult F₁ mouse killed by ethyl ether anesthesia. The tissues were minced with a fresh disposable razor blade in a fresh disposable plastic dish (FALCON 1008) in a clean bench to prevent contamination by M. spretus mtDNA. Samples were placed in PCR buffer/nonionic detergents and Proteinase K [50 mM KCl, 10 mM Tris-HCl (pH 8), 1.5 mM MgCl₂, 0.1% gelatin, 0.45% NP-40, 0.45% Tween-20, and 100 µg/ml Proteinase K], and incubated at 37° overnight. Total DNA was purified from the lysate by phenol-chloroform extraction and then precipitated in 70% ethanol. Each of the steps was done in a fresh disposable tube (FALCON 2059). The DNA was dissolved in distilled water to the concentration of about 1 µg/µl.

DNA preparation from a single unfertilized egg and a single two-cell-stage embryo:
Unfertilized eggs were collected from oviducts of the F₁ female mice superovulated by pregnant mare's serum gonadotropin and human chorionic gonadotropin (HOGAN et al. 1986 ). The two-cell-stage embryos of the N₂ generation were obtained by in vitro fertilization of unfertilized eggs from the female F₁ hybrids with sperm from M. spretus or B6.mt ^Jpn (KANEDA et al. 1995 ). Each egg or embryo was placed into a 0.5 ml plastic microfuge tube containing 10 µl of 100 µg/ml Proteinase K solution. After a period of 1 hr at 55° for digest proteins, samples were heated at 94° for 10 min to inactivate Proteinase K.

Purification of mtDNA:
mtDNA was prepared as described by YONEKAWA et al. 1978 . For further purification of mtDNA, we carried out a second round of CsCl-ethidium bromide density gradient ultracentrifugation.

Detection of M. spretus mtDNA using a nested-PCR system:
We synthesized two PCR primer pairs specific for M. spretus or M. m. molossinus mtDNA (SPR-1 or MTJ-1, COM-1; SPR-2 or MTJ-2, COM-2) in the D-loop region (KANEDA et al. 1995 ). The other two common primers (OL-1 and OL-2R) span the replication origin of the L strand, which is highly conserved between M. spretus and M. musculus (KANEDA et al. 1995 ).

Mixtures for PCR were set up with a TaKaRa PCR Amplification Kit recommended by the manufacturer (TaKaRa, Tokyo, Japan) without primer concentration (final concentration was 1 µM). The first round of PCR involved the outer, SPR-1 and COM-1 for 30 cycles of 94°, 1 min for denaturation, 45°, 1 min for annealing, 72°, 1 min for extension. The second round of PCR, which was run under the same conditions as the first, employed the inner primers SPR-2 and COM-2. For positive control of unfertilized eggs and embryos, common primers OL-1 and OL-2R were used under the same PCR conditions. We used 1 µl of the first PCR mixture as a template. After the second round of PCR, 20 µl of the reaction mixture was applied to a gel of 3% Nusieve agarose/1% agarose and electrophoresed in 1 x TAE buffer. Gels were stained with ethidium bromide (0.1 µg/ml) to detect PCR products.

Sequence analysis for PCR products:
PCR products were purified by Centricon-100 Concentrator columns (Amicon, Beverly, MA) or eluted from the gel by an Ultrafree C3HV spin column (Millipore, Bedford, MA). Direct DNA sequencing was performed using a Dye Terminator Cycle Sequencing Kit recommended by the manufacturer (Perkin Elmer, Norwalk, CT) and an automated DNA sequencer (ABI 373 DNA Sequencer).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To decide whether the leaked paternal mtDNA was distributed uniformly over all tissues, we tested mouse F₁ hybrids from the interspecific cross of female M. musculus and male M. spretus using primers SPR-1 plus COM-1 in the first round and SPR-2 plus COM-2 in the second round for PCR amplification. These primers have already been shown to detect a single sperm mtDNA of M. spretus even in the presence of a whole unfertilized egg of M. musculus (for details cf. MATERIALS AND METHODS). To test further the sensitivity and reliability of our nested-PCR technique in detecting a very small amount of the leaked paternal mtDNA of M. spretus, serially diluted mtDNA samples purified from liver of M. spretus were amplified. Consequently, this procedure could detect 0.01 fg M. spretus mtDNA, which corresponded to a few mtDNA molecules (Figure 1). Moreover, 0.01 fg M. spretus mtDNA was still detectable even in the presence of 1 µg M. musculus total DNA isolated from liver (Figure 2 and Figure 3), in which about 50 ng mtDNA was present. Therefore, we could find M. spretus mtDNA even in the presence of >10⁸ times excess of M. musculus mtDNA, suggesting that the sensitivity of our PCR technique is sufficient to detect paternal mtDNA in the 10³- to 10⁴-fold x excess of maternal mtDNA in single embryos (PIKO and MATSUMOTO 1976 ; HECHT et al. 1984 ).

View larger version (61K): In this window In a new window Download PPT slide   Figure 1. Determination of the minimal detectable content of M. spretus mtDNA under our PCR amplification conditions. M, molecular weight standard ( x 174/HincII digests); N, negative control (distilled water). The numerals 1, 10-1, 10-2, and 10-3 give the amounts (in fg) of purified spretus mtDNA. Primer pairs specific to M. spretus mtDNA were SPR-1 plus COM-1 for the first round of PCR, and SPR-2 plus COM-2 for the second round.

View larger version (45K): In this window In a new window Download PPT slide   Figure 2. Detection of paternal mtDNA in 12 tissues of interspecific F1 hybrids from the cross of female M. musculus and male M. spretus. Primer pairs specific for paternal mtDNA were SPR-1 plus COM-1, and SPR-2 plus COM-2. M, molecular weight standards ( x 174/HincII digests); P, positive control (a mixture of 0.01 fg mtDNA purified from M. spretus liver and 1 µg total DNA isolated from M. musculus liver); N, negative control (1 µg total DNA isolated from M. musculus liver); 1–12, 1 µg total DNA isolated from various tissues of an F1 individual (1, liver; 2, stomach; 3, intestine; 4, pancreas; 5, lung; 6, kidney; 7, spleen; 8, blood; 9, heart; 10, skeletal muscles; 11, brain; 12, ovary). In the case of female number 14 in Table 1, an M. spretus-specific 170-bp fragment was found in liver and skeletal muscles. Sequence analysis proved that the 170-bp fragment was derived from M. spretus, while faint bands occasionally observed between 335–391 bp did not have mouse mtDNA sequences.

View larger version (65K): In this window In a new window Download PPT slide   Figure 3. Search for the paternal mtDNA in a single unfertilized egg of F1 hybrid and in a single embryo of N2 hybrid. Primer pairs for the first round of PCR were SPR-1 + COM-1. For the second round, they were either specific (S = SPR-2 + COM-2) or common (C = OL-1 + OL-2). S, PCR products using specific primers, which exclusively amplify M. spretus mtDNA and give rise to an M. spretus mtDNA-specific 170-bp fragment; C, PCR products using common primers, which amplify mtDNA of M. musculus and M. spretus and give rise to a 325-bp fragment; M, molecular weight standards ( x 174/Hinc II digests). (A) Segregation of the leaked paternal mtDNA in unfertilized eggs of F1 hybrids (M. musculus x M. spretus) containing paternal mtDNA in their ovaries. P, a mixture of 0.01 fg purified M. spretus mtDNA and 1 µg M. musculus total DNA isolated from liver as a positive control; N, 1 µg M. musculus total DNA isolated from liver as a negative control; 1, ovary with M. spretus mtDNA in F1 hybrids; 2 and 3, a single unfertilized egg isolated from a F1 female with ovaries containing M. spretus mtDNA. (B) Absence of the paternal mtDNA in N2 embryos obtained from backcrossing of female F1 hybrids to male M. spretus. P, a mixture of a single sperm of M. spretus and a single two-cell-stage embryo of M. musculus as a positive control; N, a single two-cell-stage embryo of M. musculus as a negative control; 1 and 2, a single two-cell-stage N2 embryo isolated by backcrossing [(M. musculus x M. spretus) F1 x M. spretus].

This procedure was applied to examine the tissue distribution of leaked paternal mtDNA in 12 different tissues from 38 individual F₁ hybrids (3 to 12 wk old). No M. spretus mtDNA was observed in any tissues of more than 55% of the F₁ hybrids, whereas some tissues of the remaining 45% F₁ hybrids showed an M. spretus mtDNA-specific fragment with molecular size of 170 bp (Figure 2, Table 1). The 170-bp PCR product was confirmed to be M. spretus mtDNA by the use of diagnostic polymorphic sequences identified either by direct sequencing or by digestion with Mse I, which gives rise to spretus-specific fragments with 87, 49, 20, and 14 bp (KANEDA et al. 1995 ). However, the leaked paternal mtDNA was not distributed uniformly in tissues of the 45% positive F₁ hybrids: presence of the paternal mtDNA was limited to only one to three tissues in most positive F₁ hybrids, and it was distributed randomly to various tissues (Table 1). Although one male, F₁ number 14, possessed paternal mtDNA in 6 out of 12 tissues, this presumably could be due to contamination of negative tissues by blood cells with paternal mtDNA (Table 1).

To study whether leaked paternal mtDNA in female F₁ hybrids can be transmitted through the germ line to the following generations during repeated backcrossing (GYLLENSTEN et al. 1991 ), we obtained another 91 female F₁ hybrids by the same combination of interspecifc cross of female M. musculus and male M. spretus. Unfertilized eggs were then collected from 91 F₁ females (3–24 eggs/F₁ female individual) by superovulation, followed by examination for the presence of leaked paternal mtDNA in their ovaries. Only 6 out of 91 F₁ females possessed paternal mtDNA in their ovaries (Figure 3A and Table 2). We then examined the presence of paternal mtDNA in 78 unfertilized eggs collected from these six females. The results showed that no paternal mtDNA could be observed in any of the 78 unfertilized eggs (Figure 3A and Table 2), suggesting that transmission of leaked paternal mtDNA to the next generation through the female germ line is an extremely rare phenomenon, if it occurs at all.

Finally, we examined the possibility that paternal mtDNA leakage was not limited to the first interspecific crosses but was repeated in subsequent backcross generation, as proposed previously (GYLLENSTEN et al. 1991 ). If paternal mtDNA is present in the N₂ embryos produced by backcrossing the female F₁ hybrids to male M. spretus, the strongest possibility is that it is derived from the newly introduced sperm by backcrossing, because the leaked paternal mtDNA in F₁ hybrids is unlikely to be transmitted to following generations through the female germ lines (Table 2). For testing this, we collected unfertilized eggs from ovaries of the F₁ hybrids (females M. musculus x male M. spretus) and fertilized them in vitro with sperm of M. spretus to generate N₂ embryos. Of 53 [(M. musculus C x M. spretus F) F₁ C x M. spretus F] N₂ embryos we isolated, none showed paternal mtDNA (Figure 3B and Table 2). Moreover, we tested to detect paternal mtDNA in [(M. musculus C x M. spretus F) F₁ C x B6.mt ^Jpn F] N₂ embryos, but we could not detect any paternal mtDNA (0/50; see Table 2). This observation suggests that complete exclusion of paternal mtDNA already has occurred during fertilization in the first backcross, as was the case in interspecific crosses between mouse subspecies that we reported previously (KANEDA et al. 1995 ). Therefore, it is highly unlikely that paternal mtDNA is continuously introduced into the eggs through the repeated backcrossing.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we used a sensitive technique to examine the transmission of sperm mtDNA. For identification of the leaked paternal mtDNA, we used PCR techniques that can detect a few molecules of paternal mtDNA (Figure 1). Moreover, paternal mtDNA could not be diluted by selective replication in the early embryo, because the mtDNA content remains constant from the one-cell to the blastocyst stage (PIKO and MATSUMOTO 1976 ). Thus, the results clearly showed that the proportion of leaked paternal mtDNA is not high enough to be distributed to all tissues in the F₁ hybrid individuals of interspecific cross (Table 1), nor to be transmitted stably to the next generation through the female germ line (Table 2). Moreover, the paternal mtDNA leakage was restricted to the first interspecific cross, and it did not spill over to subsequent backcrossing (Table 2).

GYLLENSTEN et al. 1991 proposed the persistent transmission of the leaked paternal mtDNA to subsequent generations, based on their experiments with individuals isolated after 8–26 generations of successive backcrossing of F₁ female hybrids between Mus musculus and Mus spretus. For stable propagation of paternal mtDNA to the next generations, the paternal must be constantly present at least in ovaries of the hybrid females. However, our results showed that this was not the case: the leaked paternal mtDNA was randomly distributed to various tissues (Table 1) and was observed in ovaries only in 6.6% of the F₁ hybrid females (Table 2). Moreover, paternal mtDNA was not found in any unfertilized eggs, even when they were collected from ovaries shown to possess leaked paternal mtDNA (Table 2).

In mammals, a "bottleneck effect"–-that the mtDNA copy number decreases through the female germ line–-has been theoretically assumed (HAUSWIRTH and LAIPIS 1982 ; ASHLEY et al. 1989 ). Recent experimental evidence using artificial mtDNA heteroplasmic mice suggests that a segregation unit during oogenesis is thought to be 200 copies ( JENUTH et al. 1996 ). PIKO and MATSUMOTO 1976 suggested that a single egg had 10⁵ mtDNA copies. Therefore, if one paternal mtDNA molecule was transmitted to a germ cell and if it proliferated with the identical replication efficiency during oogenesis, a mature egg must have at least 10⁵/200 = 500 molecules of paternal mtDNA. Therefore, the leakage of paternal mtDNA in a single egg must be detected by our PCR method, as it can detect 0.01 fg paternal mtDNA consisting of a few molecules of mtDNA (Figure 1). Because the proportion of the sperm-derived mtDNA in fertilized eggs is extremely small (10^-3 to 10^-4) (PIKO and MATSUMOTO 1976 ; HECHT et al. 1984 ), it is reasonable to suppose that the number of cells with the paternal mtDNA in the F₁ hybrids also would be small due to stochastic segregation of mtDNA during cell division (BIRKY 1995 ). Our observations are inconsistent with stable transmission of leaked paternal mtDNA (GYLLENSTEN et al. 1991 ), but they are consistent with various lines of previous experimental evidence that suggest the occurrences of the stochastic segregation of heteroplasmic mtDNA, leading to rapid recovery of homoplasmic mtDNA in Holstein cows (HAUSWIRTH and LAIPIS 1982 ; ASHLEY et al. 1989 ), in mice with exogenously introduced mtDNA (JENUTH et al. 1996 ), and in somatic cell hybrids (HAYASHI et al. 1983 ).

We further addressed the question of whether the paternal mtDNA leakage could continue in the subsequent backcrossing of the female F₁ hybrids to male M. spretus or M. musculus (B6.mt ^Jpn). We carried out in vitro fertilization to produce N₂ embryos and showed that no sperm-derived mtDNA was detected in any N₂ embryos (Table 2). These observations suggest the presence of a system(s) in egg cytoplasm of interspecific F₁ hybrids that can recognize and eliminate sperm mtDNA from M. spretus or M. musculus. Thus, paternal mtDNA leakage could occur only in the first interspecific cross. Our previous work (KANEDA et al. 1995 ) suggested that there are some systems, probably controlled by nuclear DNA-encoded factors, that exclude sperm mitochondria in egg cytoplasm. These systems could not completely recognize and exclude sperm mitochondria of different species (Table 2), but could recognize those of the same species and cause their complete degeneration, resulting in the exclusion of sperm mtDNA of the same species before the early pronucleus stage in interspecific F₁ embryos (KANEDA et al. 1995 ). Therefore, complete exclusion of sperm mtDNA in N₂ embryos (Table 2) can be explained by assuming that alleles of the factors involved in the system for sperm exclusion mtDNA behave in a dominant or codominant way.

We did not further repeat backcrossing for testing paternal mtDNA leakage, because we thought it was less likely that paternal mtDNA leaks in subsequent generations as the nuclear genome of the progeny progressively becomes closer to paternal type, whereas its mtDNA remains of the maternal type. Thus, repeated backcrossing results in the same mating combination as that of intraspecific crossing where paternal mtDNA is not allowed to leak (KANEDA et al. 1995 ). It was proposed that paternal mtDNA accumulated through successive backcrossing, based on the observations that 0.01–0.1% of paternal mtDNA was present in all tissues in mice of the congenic strain obtained after 8–26 generations of backcrossing the interspecific hybrids of M. musculus and M. spretus (GYLLENSTEN et al. 1991 ). However, our previous observations showed that mice of the congenic strain derived from the same progenitor stocks as those used by GYLLENSTEN et al. 1991 did not possess paternal mtDNA (KANEDA et al. 1995 ).

Although at present we cannot explain the entire discrepancy between GYLLENSTEN's observations (1991) and ours, the discrepancy is possibly because of a difference in DNA detection. GYLLENSTEN et al. purified mtDNA for detecting the leaked paternal mtDNA by PCR, but they did not examine purified mtDNA prepared from tissues of the maternal species as a negative control to exclude that contamination could occur during the complicated processes for mtDNA purification. In this study, we directly prepared total DNA from tissues or embryos omitting purification of mtDNA. In addition, total DNA prepared from tissues of the maternal species (M. musculus) was used as a negative control, and no paternal mtDNA was identified by our PCR techniques (Figure 3), suggesting our mtDNA detection method is reliable.

If females into which paternal mtDNA had leaked could subsequently transmit this DNA through the normal process of maternal mtDNA inheritance, then each individual eventually would be heteroplasmic for mtDNA derived from different ancestral males. However, it has already been shown, at least in mice (YONEKAWA et al. 1981 ), rats (HAYASHI et al. 1978 ), and humans (BROWN 1980 ), that population polymorphism of in-population mtDNA did not lead to heteroplasmic individuals. Subsequently, nucleotide sequence analysis of human mtDNA showed that mtDNA sequences within single individuals were extremely homogeneous (MONNAT et al. 1986). Furthermore, even in the rare case of individuals with mtDNA heteroplasmy in a maternal lineage of Holstein cows (HAUSWIRTH and LAIPIS 1982 ; ASHLEY et al. 1989 ) and in a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) (KOBAYASHI et al. 1991 ), no other mutations were observed between the heteroplasmic mtDNA molecules. Therefore, it is reasonable to suppose that the mtDNA heteroplasmy of these individuals is caused by a single mutation that occurred in the mtDNA population of their maternal germ line and not by paternal mtDNA leakage. These observations also support our idea that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring completely maternal mtDNA inheritance in mammals.

	FOOTNOTES

¹ To avoid confusion, we define biparental transmission as used here as the transmission that occurs when mtDNA derived from both female and male parents is transmitted to both female and male progeny. Thus, this does not mean the phenomenon observed in male progeny of Mitilus that is transmitted by both maternal and paternal lineage of mtDNA (14). Even in that case, however, paternal leakage in female progeny has been observed in interspecific hybrids but not in intraspecific hybrids.

	ACKNOWLEDGMENTS

We thank Dr. TAMIO HIRABAYASHI of the University of Tsukuba for his help and encouragement for this project and Dr. KIRSTEN FISCHER LINDAHL of the University of Texas Southwestern Medical Center for her critical reading of the manuscript. This work was supported in part by a grant from the Ministry of Education, Science, Sports (No. 09740570) and Culture of Japan and by the Sasakawa Scientific Research grant from The Japan Science Society.

Manuscript received July 23, 1997; Accepted for publication October 20, 1997.

	LITERATURE CITED

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				A. Sato, K. Nakada, M. Akimoto, K. Ishikawa, T. Ono, H. Shitara, H. Yonekawa, and J.-I. Hayashi Rare creation of recombinant mtDNA haplotypes in mammalian tissues PNAS, April 26, 2005; 102(17): 6057 - 6062. [Abstract] [Full Text] [PDF]

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				P. Sutovsky, G. Manandhar, T. C. McCauley, J. N. Caamano, M. Sutovsky, W. E. Thompson, and B. N. Day Proteasomal Interference Prevents Zona Pellucida Penetration and Fertilization in Mammals Biol Reprod, November 1, 2004; 71(5): 1625 - 1637. [Abstract] [Full Text] [PDF]

				A. Sato, K. Nakada, H. Shitara, H. Yonekawa, and J.-I. Hayashi In Vivo Interaction Between Mitochondria Carrying mtDNAs From Different Mouse Species Genetics, August 1, 2004; 167(4): 1855 - 1861. [Abstract] [Full Text] [PDF]

				J. C St John, R. E I Lloyd, E. J Bowles, E. C Thomas, and S. El Shourbagy The consequences of nuclear transfer for mammalian foetal development and offspring survival. A mitochondrial DNA perspective Reproduction, June 1, 2004; 127(6): 631 - 641. [Abstract] [Full Text] [PDF]

				J. C. St. John and G. Schatten Paternal Mitochondrial DNA Transmission During Nonhuman Primate Nuclear Transfer Genetics, June 1, 2004; 167(2): 897 - 905. [Abstract] [Full Text] [PDF]

				P. Sutovsky, T. C. McCauley, M. Sutovsky, and B. N. Day Early Degradation of Paternal Mitochondria in Domestic Pig (Sus scrofa) Is Prevented by Selective Proteasomal Inhibitors Lactacystin and MG132 Biol Reprod, May 1, 2003; 68(5): 1793 - 1800. [Abstract] [Full Text] [PDF]

				L. Kvist, J. Martens, A. A. Nazarenko, and M. Orell Paternal Leakage of Mitochondrial DNA in the Great Tit (Parus major) Mol. Biol. Evol., February 1, 2003; 20(2): 243 - 247. [Abstract] [Full Text] [PDF]

				S. Hiendleder, V. Zakhartchenko, H. Wenigerkind, H.-D. Reichenbach, K. Bruggerhoff, K. Prelle, G. Brem, M. Stojkovic, and E. Wolf Heteroplasmy in Bovine Fetuses Produced by Intra- and Inter-Subspecific Somatic Cell Nuclear Transfer: Neutral Segregation of Nuclear Donor Mitochondrial DNA in Various Tissues and Evidence for Recipient Cow Mitochondria in Fetal Blood Biol Reprod, January 1, 2003; 68(1): 159 - 166. [Abstract] [Full Text] [PDF]

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				M. E. Silliker, J. L. Liles, and J. A. Monroe Patterns of mitochondrial inheritance in the myxogastrid Didymium iridis Mycologia, November 1, 2002; 94(6): 939 - 946. [Abstract] [Full Text] [PDF]

				M. Schwartz and J. Vissing Paternal Inheritance of Mitochondrial DNA N. Engl. J. Med., August 22, 2002; 347(8): 576 - 580. [Full Text] [PDF]

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				N. Hattori, K. Kitagawa, S. Takumi, and C. Nakamura Mitochondrial DNA Heteroplasmy in Wheat, Aegilops and Their Nucleus-Cytoplasm Hybrids Genetics, April 1, 2002; 160(4): 1619 - 1630. [Abstract] [Full Text] [PDF]

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				S. T. Williams and N. Knowlton Mitochondrial Pseudogenes Are Pervasive and Often Insidious in the Snapping Shrimp Genus Alpheus Mol. Biol. Evol., August 1, 2001; 18(8): 1484 - 1493. [Abstract] [Full Text] [PDF]

				E. D. Ladoukakis and E. Zouros Direct Evidence for Homologous Recombination in Mussel (Mytilus galloprovincialis) Mitochondrial DNA Mol. Biol. Evol., July 1, 2001; 18(7): 1168 - 1175. [Abstract] [Full Text] [PDF]

				A. Ludwig, B. May, L. Debus, and I. Jenneckens Heteroplasmy in the mtDNA Control Region of Sturgeon (Acipenser, Huso and Scaphirhynchus) Genetics, December 1, 2000; 156(4): 1933 - 1947. [Abstract] [Full Text]

				H. Shitara, H. Kaneda, A. Sato, K. Inoue, A. Ogura, H. Yonekawa, and J.-I. Hayashi Selective and Continuous Elimination of Mitochondria Microinjected Into Mouse Eggs From Spermatids, but Not From Liver Cells, Occurs Throughout Embryogenesis Genetics, November 1, 2000; 156(3): 1277 - 1284. [Abstract] [Full Text]

P. Sutovsky, R. D. Moreno, J. Ramalho-Santos, T. Dominko, C. Simerly, and G. Schatten Ubiquitinated Sperm Mitochondria, Selective Proteolysis, and the Regulation of Mitochondrial Inheritance in Mammalian Embryos Biol Reprod, August 1, 2000; 63(2): 582 - 590. [Abstract] [Full Text]