Transmission Of Mitochondrial DNA Research Articles

Rapid Mitochondrial DNA Segregation in Primate Preimplantation Embryos Precedes Somatic and Germline Bottleneck

The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.

Barriers to Male Transmission of Mitochondrial DNA in Sperm Development

Across the eukaryotic phylogeny, offspring usually inherit their mitochondrial genome from only one of two parents: in animals, the female. Although mechanisms that eliminate paternally derived mitochondria from the zygote have been sought, the developmental stage at which paternal transmission of mitochondrial DNA is restricted is unknown in most animals. Here, we show that the mitochondria of mature Drosophila sperm lack DNA, and we uncover two processes that eliminate mitochondrial DNA during spermatogenesis. Visualization of mitochondrial DNA nucleoids revealed their abrupt disappearance from developing spermatids in a process requiring the mitochondrial nuclease, Endonuclease G. In Endonuclease G mutants, persisting nucleoids are swept out of spermatids by a cellular remodeling process that trims and shapes spermatid tails. Our results show that mitochondrial DNA is eliminated during spermatogenesis, thereby removing the capacity of sperm to transmit the mitochondrial genome to the next generation.

Mitochondrial DNA disease: new options for prevention

Very recently, two papers have presented intriguing data suggesting that prevention of transmission of human mitochondrial DNA (mtDNA) disease is possible. [Craven, L., Tuppen, H.A., Greggains, G.D., Harbottle, S.J., Murphy, J.L., Cree, L.M., Murdoch, A.P., Chinnery, P.F., Taylor, R.W., Lightowlers, R.N. et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature, 465, 82-85. Tachibana, M., Sparman, M., Sritanaudomchai, H., Ma, H., Clepper, L., Woodward, J., Li, Y., Ramsey, C., Kolotushkina, O. and Mitalipov, S. (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 461, 367-372.] These recent advances raise hopes for families with mtDNA disease; however, the successful translational of these techniques to clinical practice will require further research to test for safety and to maximize efficacy. Furthermore, in the UK, amendment to the current legislation will be required. Here, we discuss the clinical and scientific background, studies we believe are important to establish safety and efficacy of the techniques and some of the potential concerns about the use of these approaches.

Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm

Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear-mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8-cell stage (P>0.05). However, buffalo iSCNT embryos did not develop beyond the 16-cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8- to 16-cell stage (P>0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P<0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8-16-cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear-mitochondrial interactions.

Selective Transmission of Mitochondrial DNA Occurs in Individual Flies

Selective Transmission of Mitochondrial DNA Occurs in Individual Flies

Two modes of mitochondrial DNA transmission in mice: Maternal inheritance and rapid segregation

哺乳動物には，2種類のゲノム，核DNAとミトコンドリアDNA（mtDNA）が存在している．mtDNAの遺伝様式は，母性遺伝と急調分離に代表されるように，核DNAとは大きく異なる． mtDNAは古くから母性遺伝をすることが信じられてきた．なぜなら，哺乳動物では卵に存在するmtDNAコピー数が精子よりも103-4倍多く，極微量の精子由来mtDNAが次世代へと伝達されることが考えにくかったためである．しかし，当時の技術ではこの極微量の“精子由来のmtDNA”が検出されず，父親由来のmtDNAが子孫へ伝達している可能性が残されていた．その後，高感度なPCR法を用いることで，マウス精子のmtDNAが前核期後期までに消失することが示され，幾つかの特殊な事例を除き，mtDNAは完全に母性遺伝することが，実験的に証明された．形態学的解析からもマウスでは受精時に卵細胞質内に侵入した精子由来ミトコンドリアが2細胞期までにほぼ消失する様子が観察されており，ユビキチン‐プロテアソーム分解系の関与が報告されている． 一方，急調分離とはmtDNAの状態がヘテロプラズミーからホモプラズミーへ速やかに移行する遺伝現象である．mtDNAは体細胞では103-4コピー存在しており，核DNAの約1-10倍も変異を起こしやすい．このことから，ヘテロプラズミーの方がホモプラズミーより起こりやすいことが想定されるが，実際はその逆で一つの細胞に複数のmtDNA分子種が存在することは極めて稀である．このホモプラズミー維持の機構が「ボトルネック効果」であり，これまで「雌性生殖系の細胞中のmtDNA数が極端に減少する」としたモデルが広く受入れられてきた．ところが，実際にmtDNA分子数を我々が測定するとそのコピー数に極端な減少は無く，ホモプラズミーの維持はコピー数減少以外の機構によってmtDNAの分離単位の実効数が小さくなるためと考えられる．

Germline Transmission of Mitochondrial DNA in the Mouse.

In mammals, mitochondria and mitochondrial DNA (mtDNA) are transmitted through the female germline. Mature oocytes contain nearly 200,000 copies of mtDNA, organized at 1-2 copies per organelle. Despite the high genome copy number, mtDNA sequence variants are observed to segregate rapidly between generations, and this has led to the concept of a genetic bottleneck for the transmission of mtDNA. Here, we demonstrate that a subgroup of replicating genomes in the early post-natal ovary is responsible for the rapid segregation of mtDNA and show that the rate of segregation of mtDNA can be accelerated when mtDNA copy number is further reduced in heteroplasmic germline-specific knockout mouse models, yet very extreme reductions in germ cell mtDNA content seem to cause female-specific infertility. Low copy number embryos can be fertilized and proceed through preimplantation development yet fail to develop normally after implantation. Tracking the developmental outcome of embryos containing a range of mtDNAs points to a developmental threshold of about 50,000 copies of mtDNA in the oocyte Thus, we advance the hypothesis that the large number of mitochondria and mtDNAs present in the oocyte represent a genetic mechanism to ensure their distribution to the gametes and somatic cells of the next generation. If true, mtDNA copy number may be the most important determinant of oocyte quality not because of the effects on oocyte metabolism, but because too few would result in a maldistribution in the embryo. (platform)

Relative quantification of the M and F mitochondrial DNA types in the blue mussel Mytilus edulis by real-time PCR

Doubly uniparental inheritance (DUI) is an exceptional mode of mitochondrial DNA (mtDNA) transmission that has been reported in some bivalve species. In species with DUI there are two types of mtDNA, one being transmitted from the female parent to both daughters and sons through the egg (F type), and the other being transmitted from the male parent to sons only through the sperm (M type). Females are normally homoplasmic, having the F type in somatic tissues and eggs, whereas in males both the F and M types are found in the somatic tissues, but the M type is the exclusive type in spermatozoa. However, since the early description of DUI, the M type has been detected in the somatic tissue of females. In a recent study, the M type was detected in all Mytilus galloprovincialis females analysed. We describe here a real-time PCR assay for relative quantification of F and M types in the adductor muscle of M. edulis males and females. Dissociation curve analysis showed a single peak for the F and M primer pairs. The quantitative real-time PCR (qPCR) assay had a mean inter- and intraassay coefficient of variation of ,5%. We observed that in the majority of M. edulis females (75%) the M type is absent in the adductor muscle and when present it is in low amounts. The M/F ratio in the adductor muscle of M. edulis females ranged from 0.00002 and 0.00005, whereas in M. edulis males the M/F ratio ranged from 0.005 to 1.667. The qPCR assay developed in the present study may be used for the relative quantification of the M and F types in M. edulis from the East Atlantic region, but may also be used in M. galloprovincialis from the same region.

Mitochondrial DNA Transmitted from Sperm in the Blue MusselMytilus galloprovincialisShowing Doubly Uniparental Inheritance of Mitochondria, Quantified by Real-Time PCR

Doubly uniparental inheritance (DUI) of mitochondrial DNA transmission to progeny has been reported in the mussel, Mytilus. In DUI, males have both paternally (M type) and maternally (F type) transmitted mitochondrial DNA (mtDNA), but females have only the F type. To estimate how much M type mtDNA enters the egg with sperm in the DUI system, ratios of M type to F type mtDNA were measured before and after fertilization. M type mtDNA content in eggs increased markedly after fertilization. Similar patterns in M type content changes after fertilization were observed in crosses using the same males. To compare mtDNA quantities, we subsequently measured the ratios of mtDNA to the 28S ribosomal RNA gene (an endogenous control sequence) in sperm or unfertilized eggs using a real-time polymerase chain reaction (PCR) assay. F type content in unfertilized eggs was greater than the M type in sperm by about 1000-fold on average. M type content in spermatozoa was greater than in unfertilized egg, but their distribution overlapped. These results may explain the post-fertilization changes in zygotic M type content. We previously demonstrated that paternal and maternal M type mtDNAs are transmitted to offspring, and hypothesized that the paternal M type contributed to M type transmission to the next generation more than the maternal type did. These quantitative data on M and F type mtDNA in sperm and eggs provide further support for that hypothesis.

Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells

Mitochondrial DNA (mtDNA) encodes key proteins associated with the process of oxidative phosphorylation. Defects to mtDNA cause severe disease phenotypes that can affect offspring survival. The aim of this review is to identify how mtDNA is replicated as it transits from the fertilized oocyte into the preimplantation embryo, the fetus and offspring. Approaches for deriving offspring and embryonic stem cells (ESCs) are analysed to determine their potential application for the prevention and treatment of mtDNA disease. The scientific literature was investigated to determine how mtDNA is transmitted, replicated and segregated during pluripotency, differentiation and development. It was also probed to understand how the mtDNA nucleoid is regulated in somatic cells. mtDNA replication is strictly down-regulated from the fertilized oocyte through the preimplantation embryo. At the blastocyst stage, the onset of mtDNA replication is specific to the trophectodermal cells. The inner cell mass cells restrict mtDNA replication until they receive the key signals to commit to specific cell types. However, it is necessary to determine whether somatic cells reprogrammed through somatic cell nuclear transfer, induced pluripotency or fusion to an ESC are able to regulate mtDNA replication so that they can be used for patient-specific cell therapies and to model disease. Prevention of the transmission of mtDNA disease from one generation to the next is still restricted by our lack of understanding as to how to ensure that a donor karyoplast transferred to an enucleated oocyte is free of accompanying mutant mtDNA. Techniques still need to be developed if stem cells are to be used to treat mtDNA disease in those patients already suffering from the phenotype.

The role of mitochondrial DNA alterations in esophageal squamous cell carcinomas

The study objective was to evaluate the roles of mitochondrial DNA alterations in esophageal squamous cell carcinoma, with emphasis on the changes in the copy number and D310 variants of mitochondrial DNA. Paired samples microdissected from esophageal muscles, noncancerous esophageal mucosa, cancerous esophageal squamous cell carcinoma nests, and metastatic lymph nodes of 72 patients with esophageal squamous cell carcinoma were subjected to DNA extraction. The copy number and D310 variants of mitochondrial DNA were determined by quantitative real-time polymerase chain reaction and direct sequencing, respectively. Fifty-six patients (77.8%) with somatic D310 mutations had lower survival probability (P = .027). From noncancerous esophageal mucosa to cancerous esophageal squamous cell carcinoma nests and metastatic lymph nodes, the D310 variants were decreased from 2.2 to 1.7 and 1.5, respectively, with a trend to homoplasmy (P = .0009). Concurrently, the mitochondrial DNA copy number was increased from 0.159 to 0.192 and 0.206, respectively, (P = .024), especially in cigarette smokers (P = .014) and heavy wine drinkers (P = .005). Notably, a decrease in D310 variants (1.5, P < .001) and an increase in the incidence of the homoplasmic D310 pattern (P = .005) were observed in the matched esophageal muscle tissues. Among the 56 esophageal squamous cell carcinoma cancer nests with somatic D310 mutations, 51 (91.1%) had D310 variants in association with their corresponding noncancerous esophageal mucosa, including 36 (64.3%) fully related and 15 (26.8%) partially related pairs. We demonstrated that somatic D310 mutations and increase in the copy number of mitochondrial DNA are of clinical importance in esophageal squamous cell carcinoma. We also propose a model of DNA instability and clonal expansion during the carcinogenesis and progression of esophageal squamous cell carcinoma from the viewpoint of mitochondrial DNA transmission.

Role‐reversal of gender‐associated mitochondrial DNA affects mitochondrial function in Mytilus edulis (Bivalvia: Mytilidae)

Mussel species of the genus Mytilus possess an unusual system of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance. They are characterized by the presence of two highly divergent gender-associated mtDNA genomes (often with>20 and>10% divergences in DNA and amino acid sequences, respectively) that are inherited either maternally (F mtDNA) or paternally (M mtDNA). Females are typically homoplasmic for the F mtDNA and males are heteroplasmic with the F mtDNA being most common in all tissues except the gonad that is dominated by the M mtDNA. Collectively, males are polymorphic for two classes of M mtDNAs known as the "standard male" and "recently masculinized" M types (SM and RM, respectively). The coding portions of the RM mtDNA genome differ from the SM mtDNA by as much as the maternally inherited F mtDNA genome differs from the SM type. Because the SM and RM types exhibit considerable amino acid sequence divergence, we hypothesized that these differences could affect mitochondrial respiratory chain complex enzyme activities. To test this hypothesis, the activity of the major mitochondrial respiratory chain complexes (complexes II, I+III and IV) as well as the activity of citrate synthase were measured on gonad samples from males containing either the SM or RM mtDNA. Our data demonstrate that the mitochondrial subunits encoded by the RM mtDNA are associated with higher enzymatic activities than the gene products of the SM mtDNA.

Phylogenetics and biogeography of the broad-nosed bats, genus Platyrrhinus (Chiroptera: Phyllostomidae)

The Neotropical broad-nosed bats, genus Platyrrhinus, represent a well-defined monophyletic group of 14 recognized species. A recent study of morphological characters confirmed Platyrrhinus monophyly and species diagnosis, but offered little support to their intra-specific relationships. We conducted phylogenetic analyses of the genus, using dense taxonomic sampling in combination with four gene sequences representing both mitochondrial and nuclear DNA transmission systems. Our aim was to elucidate the phylogenetic structure among species, using the resulting 3341 bp of DNA. Maximum parsimony, maximum likelihood, and Bayesian inference analyses produced similar topologies that confirm the monophyly of the genus Platyrrhinus and strongly support many previously unrecognized groups. Paraphyly of Platyrrhinus helleri and the unclear position of P. brachycephalus in the clades were also apparent in the data. Our biogeographical analysis suggests a Brazilian Shield origin for Platyrrhinus, followed by subsequent radiations of lineages in the Amazon Basin and Andes. Secondary dispersal from Amazonian and Andean centers is responsible for the Platyrrhinus inhabiting the Guianan Shield and the Pacific lowlands and Central America, respectively.

Analysis of transmission of sperm mitochondrial DNA in Drpsophila

Analysis of transmission of sperm mitochondrial DNA in Drpsophila

DOUBLY UNIPARENTAL INHERITANCE (DUI) OF MITOCHONDRIAL DNA INDONAX TRUNCULUS(BIVALVIA: DONACIDAE) AND THE PROBLEM OF ITS SPORADIC DETECTION IN BIVALVIA

Mitochondrial DNA is transmitted maternally in metazoan species. This rule does not hold in several species of bivalves that have two mtDNA types, one that is transmitted maternally and the other paternally. This system of mitochondrial DNA transmission is known as doubly uniparental inheritance (DUI). Here we present evidence of DUI in the clam Donax trunculus making Donacidae the sixth bivalve family in which the phenomenon has been found. In addition, we present the taxonomic affiliation of all species in which DUI is currently known to occur and construct a phylogeny of the maternal and paternal genomes of these species. We use this information to address the question of a single or multiple origins of DUI and to discuss whether failed attempts to demonstrate the presence of DUI in several bivalve species might be due to problems of detection or to genuine absence of the phenomenon.

Mitochondrial DNA Transmission and Transcription After Somatic Cell Fusion to One or More Cytoplasts

Following fertilization, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in a state of heteroplasmy. To determine whether the genetic diversity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, fetal tissues, and blood samples from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than fetal tissues. Phylogenic analysis of embryos generated using single cytoplasts divided the mitochondrial DNA sequence variants into three separate groups with various amounts of genetic divergence from the donor cell line. In heteroplasmic tissue and blood samples, the predominant mitochondrial DNA population was significantly more divergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids, and the ratios of mitochondrial DNA/mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may have an impact on more divergent intergeneric nuclear transfer and the use of this approach for the generation of embryonic stem cells.

Strong Purifying Selection in Transmission of Mammalian Mitochondrial DNA

There is an intense debate concerning whether selection or demographics has been most important in shaping the sequence variation observed in modern human mitochondrial DNA (mtDNA). Purifying selection is thought to be important in shaping mtDNA sequence evolution, but the strength of this selection has been debated, mainly due to the threshold effect of pathogenic mtDNA mutations and an observed excess of new mtDNA mutations in human population data. We experimentally addressed this issue by studying the maternal transmission of random mtDNA mutations in mtDNA mutator mice expressing a proofreading-deficient mitochondrial DNA polymerase. We report a rapid and strong elimination of nonsynonymous changes in protein-coding genes; the hallmark of purifying selection. There are striking similarities between the mutational patterns in our experimental mouse system and human mtDNA polymorphisms. These data show strong purifying selection against mutations within mtDNA protein-coding genes. To our knowledge, our study presents the first direct experimental observations of the fate of random mtDNA mutations in the mammalian germ line and demonstrates the importance of purifying selection in shaping mitochondrial sequence diversity.

SPERM MOTILITY IN MYTILUS EDULIS IN RELATION TO MITOCHONDRIAL DNA POLYMORPHISMS: IMPLICATIONS FOR THE EVOLUTION OF DOUBLY UNIPARENTAL INHERITANCE IN BIVALVES

Bivalves of the families Mytilidae, Unionidae, and Veneridae have an unusual mode of mitochondrial DNA (mtDNA) transmission called doubly uniparental inheritance (DUI). A characteristic feature of DUI is the presence of two gender-associated mtDNA genomes that are transmitted through males (M-type mtDNA) and females (F-type mtDNA), respectively. Female mussels are predominantly homoplasmic with only the F-type expressed in both somatic and gonadal tissue; males are heteroplasmic with the M-type expressed in the gonad and F-type in somatic tissue for the most part. An unusual evolutionary feature of this system is that an mt genome with F-coding sequences occasionally invades the male route of inheritance (i.e., a "role reversal" event), and is thereafter transmitted as a new M-type. Phylogenetic studies have demonstrated that the new or "recently masculinized" M-types may eventually replace the older or "standard" M-types over time. To investigate whether this replacement process could be due to an advantage in sperm swimming behavior, we measured differences in motility parameters and found that sperm with the recently masculinized M-type had significantly faster curvilinear velocity and average path velocity when compared to sperm with standard M-type. This increase in sperm swimming speed could explain the multiple evolutionary replacements of standard M-types by masculinized M-types that have been hypothesized for the mytilid lineage. However, our observations do not support the hypothesis that DUI originated because it permits the evolution of mitochondrial adaptations specific to sperm performance, otherwise, the evolutionarily older, standard M genome should perform better.

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.