Mitochondrial DNA and the Mammalian Oocyte

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Mitochondrial DNA and the Mammalian Oocyte Eric A. Shoubridge and Timothy Wai Department of Human Genetics, Montreal Neurological Institute McGill University, Montreal, Quebec H3A 2B4, Canada

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction mtDNA: Structure, Organization, Replication, and Expression Origin and Development of the Germ Line Transmission of mtDNA in the Female Germ Line OXPHOS, Atresia, and Oocyte Quality mtDNA Copy Number in the Oocyte Male Transmission of mtDNA Clinical Perspectives Conclusions and Future Prospects Acknowledgments References

In mammals, mitochondria and mitochondrial DNA (mtDNA) are transmitted through the female germ line. Mature oocytes contain at least 100,000 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 developmental bottleneck for the transmission of mtDNA. Ultrastructural investigations of primordial germ cells show that they contain approximately 10 mitochondria, suggesting that mitochondrial biogenesis is arrested during early embryogenesis, and that the mitochondria contributing to the germ cell precursors are simply apportioned from those present in the zygote. Thus, as few as 0.01% of the mitochondria in the oocyte actually contribute to the oVspring of the next generation. Mitochondrial replication restarts in the migrating primordial germ cells, and mitochondrial numbers steadily increase to a few thousand in primordial oocytes. Genetic evidence from both heteroplasmic mice and human pedigrees suggests that segregation of mtDNA sequence variants is largely a stochastic process that occurs during the mitotic divisions of the germ cell precursors. This process is essentially complete by the time the primary oocyte population is diVerentiated in fetal life. Analysis of the distribution of pathogenic mtDNA mutations in the oVspring of carrier mothers shows that risk of inheriting a pathogenic mutation increases with the

Current Topics in Developmental Biology, Vol. 77 Copyright 2007, Elsevier Inc. All rights reserved.

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proportion in the mother, but there is no bias toward transmitting more or less of the mutant mtDNAs. This implies that there is no strong selection against oocytes carrying pathogenic mutations and that atresia is not a filter for oocyte quality based on oxidative phosphorylation capacity. The large number of mitochondria and mtDNAs present in the oocyte may simply represent a genetic mechanism to ensure their distribution to the gametes and somatic cells of the next generation. If true, mtDNA copy number, and by inference mitochondrial number, may be the most important determinant of oocyte quality, not because of the eVects on oocyte metabolism, but because too few would result in a maldistribution in the early embryo. ß 2007, Elsevier Inc.

I. Introduction ATP produced by oxidative phosphorylation (OXPHOS) is essential for the normal function of most eukaryotic cells. This process requires the activity of five multimeric enzyme complexes located in the inner mitochondrial membrane. Electron transport along complexes I–IV of the respiratory chain creates an electrochemical gradient for protons, which is used to drive the synthesis of ATP from ADP and inorganic phosphate by complex V, ATP synthase. The subunits of the OXPHOS complexes are encoded by both the nuclear and mitochondrial (mtDNA) genomes. Of the approximately 80 structural subunits of OXPHOS, 13 are mtDNA encoded, and all are essential for function. Mitochondria and mtDNA are maternally inherited in mammals, and as mitochondria cannot be made de novo, but rather only elaborated from other mitochondria, all of our mitochondria ultimately derive from those in one of our mother’s oocytes. Paternal mitochondria containing mtDNA are present in the zygote; however, they are rapidly degraded in the preimplantation embryo by a process that remains poorly understood. Mutations in mtDNA in humans are an important cause of a group of multisystem disorders whose birth prevalence has been estimated at 1:5000 (Chinnery et al., 2000a; Thorburn, 2004). An understanding of the mechanisms of transmission and segregation of mtDNA in the female germ line is important for genetic counseling and clinical management of patients aVected by these disorders. Further, the introduction of new reproductive technologies, such as cytoplasmic transfer and direct sperm injection, have forced a reevaluation of the potential contribution of exogenous or paternally derived mitochondria to the next generation. Here we review the current knowledge in these areas.

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II. mtDNA: Structure, Organization, Replication, and Expression mtDNA is a double‐stranded circular DNA molecule of approximately 16.5 kb in all mammals in which it has been sequenced (Fig. 1). The two stands are referred to as heavy (H) and light (L), reflecting their behavior in density gradients. Mammalian mtDNA codes for 13 polypeptides, all of which are subunits of the enzyme complexes of the OXPHOS system, and 22 tRNAs and 2 rRNAs, which constitute part of the dedicated mitochondrial translation machinery. The genome is exceedingly compact; there are no introns, and there is only one noncoding (control) region of approximately 1 kb that contains the replication origin for leading strand synthesis (OH), and the promoters for transcription of the H‐ and L‐strands. The mtDNA copy number in somatic cells is generally in the range of 103–104 copies per cell, packaged in a DNA‐protein structure called the nucleoid at approximately 2–10 copies per nucleoid (Legros et al., 2004; Satoh and Kuroiwa, 1991). Gametes are a notable exception: mature oocytes have approximately 105 mtDNAs (Piko and Taylor, 1987) and sperm about 102 (Hecht et al., 1984). Investigation of the protein constituents of the yeast mitochondrial nucleoid by mass spectrometry has revealed a large number of proteins, some of which have dual functions in nucleoid maintenance and tricarboxylic acid cycle activity (Chen et al., 2005; Kaufman et al., 2000). The mitochondrial nucleoid in higher eukaryotes is reported to contain TFAM, a mitochondrial transcription factor, single‐stranded binding protein, Twinkle (a helicase), and at least four additional inner membrane proteins (Bogenhagen et al., 2003; Garrido et al., 2003). TFAM is a basic protein of the HMG box family that is thought to package mtDNA (Alam et al., 2003). Decreasing TFAM levels results in loss of mtDNA; likewise, cells devoid of mtDNA contain no detectable TFAM, leading to the suggestion that mtDNA levels are controlled by TFAM (Ekstrand et al., 2004). The mechanism of mtDNA replication has been the subject of recent controversy. The conventional strand‐displacement model developed over the past two decades by Clayton and colleagues (Clayton, 1991; Shadel and Clayton, 1997) was suggested to be an artifact of biased incorporation of ribonucleotides into the L‐strand (Yang et al., 2002), and a coupled strand‐ synchronous mechanism was proposed in its stead (Bowmaker et al., 2003; Holt et al., 2000). In the strand‐displacement model, leading strand synthesis of the H‐strand starts from OH in the control region and proceeds about two‐ third the way around the molecule until a second origin (OL) is exposed, allowing synthesis of the L‐strand to proceed. The alternative model, based largely on evidence from 2‐D agarose gels, proposes that replication is strand‐ coupled, originating from a broad zone around OH (Bowmaker et al., 2003).

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Aminoglycoside-induced deafness

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Figure 1 Mitochondrial disease phenotypes associated with mutations in mtDNA‐encoded genes. Human mtDNA is a circular genome coding for 37 genes. The structural and rRNA genes are shown as shaded bars: COI‐III, subunits of cytochrome c oxidase (complex IV); ND1–5, subunits of NADH CoQ reductase (complex I); ATP6,8, subunits of ATP synthase (complex V); Cyt b, cytochrome b subunit of CoQ‐cytochrome c reductase (complex III). The tRNA genes are depicted as solid circles using the single‐letter amino acid code. The major clinical phenotypes associated with mutations in the individual genes are shown in boxes next to the position of the gene. The extent of the common deletion, which is associated with Kearns–Sayre syndrome and removes five tRNA genes, is indicated by lines inside the genome. CPEO, chronic progressive external ophthalmoplegia; LS, Leigh syndrome; PECM, progressive encephalomyopathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes; MERRF, myoclonus epilepsy and ragged red fibers; LHON, Leber’s hereditary optic neuropathy; NARP, neuropathy, ataxia, and retinitis pigmentosa; MILS, maternally inherited Leigh syndrome.

A study in which atomic force microscopy was used to examine mtDNA replicative intermediates in mouse liver was entirely consistent with the strand‐displacement model, and did not show any evidence of the theta structures that would be predicted from the strand‐coupled model (Brown

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et al., 2005). The same study also provided evidence for alternative L‐strand origins, and it was suggested that their presence, in addition to the propensity for branch migration in replicating DNA, might account for the patterns observed by 2‐D gel electrophoresis that were interpreted as evidence for strand‐coupled replication (Brown and Clayton, 2006). Replication is catalyzed by a distinct nuclear‐encoded polymerase, the

‐DNA polymerase, and leading strand synthesis at OH is primed by a short piece of RNA generated by transcription from the L‐strand promoter. Thus, replication of the mitochondrial genome is linked in some way to the expression of mtDNA genes. Both models require a primase to prime replication of the L‐strand, and although a primase activity has been recovered from human mitochondria (Wong and Clayton, 1985), its molecular identity remains unknown. Phylogenetic analysis of eukaryotic homologues of the Twinkle helicase suggests that it might retain a mitochondrial primase function, although the domains associated with this function are not well conserved in mammals (Shutt and Gray, 2006). mtDNA copy number varies widely from cell to cell and is tightly regulated in a cell‐specific fashion; however, replication of mtDNA is not tightly coupled to the cell cycle (Clayton, 1991). Thus, during mitosis some templates may replicate more than once, others not at all. This behavior, coupled with the random distribution of mtDNAs to daughter cells at cytokinesis, provides a mechanism for the segregation of mtDNA sequence variants. It is important to point out that replicative segregation of mtDNA also occurs in postmitotic cells, as mtDNA replication and turnover is an ongoing process throughout their lifetime (Gross et al., 1969). mtDNA is transcribed as three polycistronic units: the entire H and L strands and the two rRNAs, by a single subunit, phage‐like RNA polymerase (Shadel and Clayton, 1997). The rRNAs are transcribed 10–60 times more frequently than the entire H‐strand, a process controlled by a specific termination factor that binds to both the transcription initiation site and a termination site located 30 to the 16S rRNA in the gene coding for tRNALeu(UUR) (Fernandez‐Silva et al., 1997; Martin et al., 2005). Mitochondrial transcription has been reconstituted in vitro and requires only the presence of two transcription factors, TFAM and TFBM (either B1 or B2), and the single‐subunit RNA polymerase (Falkenberg et al., 2002; Gaspari et al., 2004). Maturation of the mitochondrial transcripts requires an RNase P activity, and it is thought that the tRNA genes, which are interspersed between many of the protein‐coding genes, act as signal sequences in this process (Ojala et al., 1981). Mitochondrial translation occurs on a dedicated apparatus located in the mitochondrial matrix that resembles that of prokaryotes (Spremulli et al., 2004). Mitoribosomes are sensitive to antibiotics that inhibit prokaryotic translation, but they are remarkable in that they have a much higher protein/ rRNA ratio (O’Brien, 2002). In fact, almost half of the mitochondrial

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ribosomal proteins have no obvious orthologues with either prokaryotic ribosomal proteins or those in cytoplasmic ribosomes. The initiation, elongation, and termination factors necessary for mammalian mitochondrial translation have all been cloned, but the regulation of translation remains poorly understood (Spremulli et al., 2004). All of the proteins involved in the replication and expression of mtDNA are encoded in the nuclear genome and must be targeted to the mitochondria.

III. Origin and Development of the Germ Line The emergence and development of the female germ line in mammals is a complex and dynamic process involving the interaction of the soma and the germ cells. Germ line development begins with the specification of the primordial germ cells (PGCs), which are induced in the primitive ectoderm of the pregastrulating embryo. In the mouse embryo, PGCs are first observed as a founding cluster of approximately 50 cells appearing posterior to the primitive streak between the endoderm and mesoderm of the ventral part of the amniotic fold. These large, round cells were first identified by the intense plasma membrane‐staining activity of tissue‐nonspecific alkaline phosphatase (TNAP), which still remains the de facto histochemical marker for PGCs (Chiquoine, 1954; Ginsburg et al., 1990). Beginning at E6.0, the earliest events of specification were investigated by single‐cell transcriptional profiling of nascent PGCs, leading to the identification of two genes, originally termed Fragilis and Stella (Saitou et al., 2002; Tanaka and Matsui, 2002), that are believed to define PGC competence prior to germ cell fate determination. Further investigation of Fragilis (also referred to as IFITM3), revealed a cluster located on human chromosome 11 containing two other family members, IFITM1 and ‐2 (Tanaka et al., 2005). IFITM1 expression mediates the repulsive activity that drives the migration of PGCs from the mesoderm to endoderm, and while the role of IFITM3 is less clear, it seems that its activity is important in the regionalization of PGCs to the posterior endoderm. After E7.0, signaling from the overlying ectoderm by BMP4/BMP8b (Lawson et al., 1999; Ying et al., 2001) to the precursor population of nascent PGCs is absolutely necessary for further specification of germ cell fate. Mice lacking BMP4 function are unable to produce PGCs, while BMP8b null mice have a severely reduced number of germ cells. Following PGC specification, these cells emigrate from the posterior primitive streak while expressing a subset of well‐characterized and spatiotemporally specific markers of the germ cell lineage. In addition to the IFITMs and Stella, Oct4, which belongs to the POU family of transcription factors, is required to maintain the pluripotency of the germ cell lineage. A transgenic mouse in which the PGC‐specific promoter elements of Oct4 are used to drive

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expression of a GFP reporter has served as a useful tool to track the movements of PGCs in vivo (Molyneaux et al., 2001; Szabo et al., 2002; Yoshimizu et al., 1999). At E8.5, the PGCs reside largely in the stalk of the allantois and the yolk sac splanchnopleure at the posterior end of the primitive streak. By E9.5, the PGCs have increased in number when they exit dorsally and become embedded in the wall of the hindgut endoderm, at that point there are approximately 200 TNAP‐positive cells (Ginsburg et al., 1990). Molyneaux et al. have used time‐lapse movies to show that the motility of PGCs is constant while migrating from the allantois, during which time cell division occurs every 16 hours (Molyneaux et al., 2001; Tam and Snow, 1981). From E9 until gonad colonization, the PGCs develop pseudopodia and other structures capable of ameboid movement (Buehr, 1997), suggesting an active migration through the dorsal mesentery to the genital ridge. However, the proliferation and motility of these cells are not exclusively a cell‐ autonomous process. On the contrary, the somatic cells adjacent to the PGCs also play a role in maintaining the survival of PGCs as they migrate to their final destination. It has been suggested that the invaginating hindgut of the embryo facilitates PGC migration by sweeping the PGCs passively during gastrulation (Buehr, 1997). PGCs apparently do not migrate independently, but rather form intercellular cytoplasmic processes, creating an extensive network (Fox et al., 1981). At E10.5, the gonads begin to form by proliferation of somatic cells at the genital ridge. PGCs begin to leave the hindgut endoderm and move along the dorsal mesentery toward the paired thickenings on the ventromedial surface of the mesonephros. At E11.5, the coalescence of germ and somatic cell leads to the formation of paired gonadal primordia. All the PGCs reach the gonad beginning around E12–13 in the mouse and E35 in humans (Wartenberg et al., 1998), at that time these cells are termed oogonia in females. In mice, the number of oogonia has been estimated at 4000–11,000 per gonad. Sexual dymorphism becomes apparent morphologically as these cells proceed along sex‐specific diVerentiation paths that are influenced by signaling events initiated by adjacent somatic cells of the mesonephros (Godin et al., 1990; Tam and Snow, 1981). Concomitant with these events is the proliferation of oogonia until they become oocytes. These germ cells enter meiosis at around E13.5 in the mouse when expression of markers such as TNAP and Oct4 are downregulated. Concomitant with meiotic arrest at the diplotene stage, the oocyte surrounds itself with a layer of pregranulosa cells of somatic origin, creating a primordial follicle. Primary follicles arise once the proliferating granulosa cells assume a cuboidal shape and the zona pellucida synthesis begins. Oocyte growth rates to this point are rapid, with follicles increasing from 40 (primary follicles) to 140 mm preovulatory follicles found in humans. The establishment of the oocyte pools is a function of three parameters: (1) the number of PGCs that reach the gonad, (2) the mitotic activity of dividing

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Figure 2 Diagrammatic representation of changes in the number of mitochondria during development of the female germ line. An estimate of the number of mitochondria is indicated at each stage of germ line development. A genetic bottleneck for the transmission of mtDNA occurs in the primordial germ cells. Development to the mature oocyte involves at least a 10,000‐fold increase in the number of mitochondria. It is not known how mtDNA copy number

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oogonia, and (3) the attrition of oogonia during oogenesis. The dogma that female germ line stem cells become extinct at birth has been challenged (Johnson et al., 2004), but the interpretation of the data in that study has been seriously questioned (Gosden, 2004). Remarkably, little is known about the metabolic activity or molecular biology of mitochondria during PGC specification and migration, and oogenesis. Ultrastructural studies of human PGCs show that the number of mitochondria increases from about 10 in premigratory PGCs to about 200 in oogonia, and to several thousand by the diplotene stage of meiosis where they arrest (Jansen and de Boer, 1998). There are no data on the fate of mtDNA copy number during this period of development. There are approximately 4500 cells in an E7 mouse embryo and 14,000 cells in an E7.5 embryo (Snow, 1977). If the approximately 100,000 mitochondria in the zygote were simply apportioned to all cells in the early embryo, and there were no mitochondrial replication, PGCs (and somatic cells) would contain between 7 and 22 mitochondria, remarkably similar to what has been reported from transmission EM studies.

IV. Transmission of mtDNA in the Female Germ Line Most mammals have a single mtDNA sequence variant in all of their cells, a condition referred to as mtDNA homoplasmy. The relative rarity of mtDNA heteroplasmy (the occurrence of more than one sequence variant in an individual) and the high degree of population polymorphism suggest that new mtDNA sequence variants are rapidly segregated in maternal lineages. This seemed paradoxical given the high mtDNA copy number (
105) in mature oocytes, and the relatively small number of cell divisions in the development of the female germ line, and suggested the existence of a genetic bottleneck for the transmission of mitochondria and mtDNA (Fig. 2). The concept of a bottleneck was first proposed by Hauswirth and Laipis (1982) to explain the rapid segregation of an mtDNA D‐loop sequence variant in several maternal lineages of Holstein cows. Complete switching of the same allelic variant was observed in a single generation in 40% of the mother–daughter pairs examined in another study (Koehler et al., 1991). Segregation of many diVerent pathogenic mtDNA sequence variants has also been observed in a large number of human pedigrees; however, it is

changes during germ line development, but it is thought that there are 1–2 copies per mitochondrion in the mature oocyte. The segregation of mtDNA sequence variants is essentially complete by the time the primary oocytes are diVerentiated in fetal life. The numbers at the left indicate the approximate numbers of cells in the mouse.

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rarely as rapid as that observed in the D‐loop of cows (Chinnery et al., 2000b; Jenuth et al., 1996). The mtDNA copy number is not known in either PGCs or oogonia; however, it has been speculated that mtDNA copy number may be 1 copy per organelle at all stages of gametogenesis (Jansen and de Boer, 1998). The ultrastructural evidence clearly demonstrates a physical bottleneck in the number of mitochondria in the PGC population; however, it is possible that mtDNA replication is not coupled to mitochondrial biogenesis prior to specification of the PGC population such that each mitochondrion in the PGCs contains multiple mtDNAs. Replication of mtDNA does not appear to restart until after the blastocyst stage (Piko and Taylor, 1987; Thundathil et al., 2005), although one study suggests that mtDNA replication occurs during a very narrow window (one‐ to two‐cell stage) of development in the preimplantation embryo, albeit without a change in mtDNA copy number (McConnell and Petrie, 2004). Thus, it is likely that the number of copies of mtDNA in PGCs lies somewhere between 10 and 100. The 10,000‐fold reduction in organelle number from the zygote to the PGCs may simply be the result of a failure to restart zygotic replication of mitochondria, but whether that is the result of the absence of some key factors or mitochondrial biogenesis is actively repressed remains unknown. Studies of transmission of polymorphic mtDNA sequence variants in heteroplasmic mice using single‐cell PCR on PGCs and oocytes (Jenuth et al., 1996) have shown that segregation is essentially complete by the time the primary oocyte population is diVerentiated. Surprisingly, little intercellular variation in the degree of mtDNA heteroplasmy was found among individual PGCs sampled from individual embryos (although it must be said that the number of PGCs analyzed was quite small). These data imply that the PGCs contain a more or less representative sample of the mitochondria in the zygote and that the segregation of mtDNA sequence variants occurs by replicative segregation during the expansion of the oogonial population. The observation that the mean level of heteroplasmy in a large sample of oVspring from single mothers was not significantly diVerent than the level of heteroplasmy in the mother suggests that mtDNA segregation between generations occurs principally by random genetic drift (Jenuth et al., 1996). Studies of homopolymeric tract heteroplasmy in humans are consistent with the mouse studies, showing that segregation has occurred by the time oocytes are mature (Marchington et al., 1997). An attempt was made to estimate the eVective number of segregating units of mtDNA in the germ line using a population genetic model that takes into account the number of mitotic divisions in the development of the germ line, but assumes a constant mtDNA copy number during gametogenesis (Jenuth et al., 1996). While the latter is clearly not the case (mtDNA copy number steadily increases during germ line development), the analysis at least provides a

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basis for comparison, although it will tend to overestimate the number of segregating units, as the rate of mtDNA segregation is inversely related to copy number. Using this model, the number of segregating units of mtDNA was estimated at 200 in the mouse (Jenuth et al., 1996), in broad agreement with estimates obtained from human pedigrees segregating pathogenic point mutations, and nearly identical to that obtained by direct evaluation of heteroplasmy in the oocytes from a patient carrying a pathogenic tRNA mutation (Brown et al., 2001). However, it does not seem that all mutations are treated equally in the germ line. Two studies have investigated oocytes or early embryos from mothers carrying the T8993C neuropathy, ataxia, and retinitis pigmentosa (NARP) mutation in the ATP6 gene, and have shown almost complete segregation of this mutation, suggesting that the bottleneck for transmission of this mutation might be extraordinarily narrow (Blok et al., 1997; SteVann et al., 2006). Consistent with extreme skewing of heteroplasmy in the germ line in these cases, a disproportionate number of reports of apparently de novo mutations in the ATP6 gene have appeared (White et al., 1999). The pattern of transmission of mtDNAs has also been analyzed in a large number of pedigrees segregating six of the most common pathogenic mtDNA point mutations (Fig. 1): A8344G (MERRF, mitochondrial encephalomyopathy, and ragged red fibers), A3243G (MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke‐like episodes), T8993C (NARP), T8993G (NARP), G11778A (LHON, Leber’s hereditary optic neuropathy), and G3460A (LHON) (Chinnery et al., 2000b). If the transmission of pathogenic mtDNAs was stochastic, then the proportions of mutant mtDNAs in the oVspring of carrier mothers should be symmetrically distributed about the value in the transmitting female; in other words, the proportion of mutant mtDNAs would be just as likely to increase as decrease. Transmission of the pathogenic mutation was not diVerent from that predicted by a stochastic model for two of the mutations, and for three of the mutations there was a significant increase in mutant mtDNAs in the oVspring. Only one showed a very small shift toward the wild‐type sequence. It has not been possible to construct animal models segregating specific pathogenic mtDNA, as a method to transform mammalian mitochondria has remained elusive. This problem has been circumvented in one instance by transferring naturally occurring large‐scale mtDNA deletions to one‐cell embryos using enucleated cytoplasts as the transfer vehicle (Inoue et al., 2000). In humans, these large deletions are usually associated with progressive external ophthalmoplegia (PEO) or Kearns–Sayre syndrome (KSS), both of which are nearly always sporadic diseases (DiMauro and Schon, 2003). The mice were able to transmit the deleted species of mtDNA at high levels (greater than 80% mutant mtDNAs in some animals) through three generations, clearly showing that there is no barrier to the transmission of pathogenic mtDNA mutations in this model, and no loss of oocytes with

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high proportions of mtDNA deletions. By contrast, the risk of transmitting a large‐scale mtDNA deletion in humans is only about 4% (Chinnery et al., 2004; SteVann et al., 2006). It is not known why the human and mouse diVer in their ability to transmit these particular mtDNA mutations, but it likely reflects the fact that humans with high proportions of large‐scale mtDNA deletions have a severe clinical phenotype and rarely reproduce. The above data, while still limited, suggest that the transmission of polymorphic and pathogenic mtDNA sequence variants in mammals is primarily a stochastic process and that the presence of a genetic bottleneck in the PGCs results in more rapid sorting out of new mutations than expected is based on the high genome copy number in the zygote.

V. OXPHOS, Atresia, and Oocyte Quality Most of the oocytes that are formed during fetal life die by atresia. About half die before birth and the process of wastage is continuous throughout life. Thus, in humans, of the roughly 106 primary oocytes present at birth, about 3  105 remain at sexual maturity, and 103 at menopause (Perez et al., 2000). An evolutionary argument has been advanced, suggesting that mitochondrial function might be the basis for atresia (Krakauer and Mira, 1999). The argument is based on the strong correlation between the size of the genetic bottleneck for mtDNA and the fraction of atretic cells determined in a number of diVerent animal species. Mitochondrial function has also been suggested to be an important contributor to oopause and oocyte aging (Jansen, 2000; Jansen and de Boer, 1998), and the introduction of mitochondria into rodent oocytes can reduce the number undergoing apoptosis in vitro (Perez et al., 2000). However, several lines of evidence argue against the idea that any of these eVects are based on the capacity for energy production by OXPHOS in the oocyte. First, mtDNA diseases are a significant medical problem. Full‐term, apparently normal babies, with very high proportions of pathogenic mtDNA mutants, are born to heteroplasmic carrier mothers. These babies only become symptomatic after birth when there is increased demand for OXPHOS and an upregulation of mitochondrial biogenesis. The simplest interpretation of these facts is that stringent selection for OXPHOS function does not exist, either during oogenesis or at any stage of fetal life. If atresia were a filter for OXPHOS capacity, it would eVectively prevent the transmission of oocytes with mtDNA mutations because the mutations would trigger a suicide response at some stage of oogenesis. Second, it is very diYcult to imagine how new germ line mutations could be selected against at any stage of oogenesis because they would initially be rare. The expression of a pathogenic phenotype due to an mtDNA mutation usually depends on reaching a critical

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threshold of mutant mtDNAs before a biochemical or clinical abnormality is observable. These thresholds can range from 30 to 85% of mutant mtDNAs (Boulet et al., 1992; Macmillan et al., 1993), so it is unlikely that a small proportion of mutant mtDNAs could be selected against. Even if there were a mechanism to select against such mutants, the number of oocytes harboring such mutations would be orders of magnitude less than the numbers of oocytes that die by atresia. Finally, atresia that occurs late in development is initiated by the somatic granulosa cells (Perez et al., 2000), and it is diYcult to see how the capacity for OXPHOS in the oocyte would initiate an apoptotic cascade in the surrounding follicular cells. What about mitochondrial function and oocyte aging? A number of studies have investigated the occurrence of mutations in aging oocytes, usually using as a marker the presence of the so‐called common deletion associated with KSS (Schon et al., 1989). Although these studies have generally been able to detect this mutation in some fraction of oocytes, the proportion of mtDNAs aVected has always been very small, and there is not a reproducible correlation with maternal age (Barritt et al., 1999; Brenner et al., 1998; Chan et al., 2005; Hsieh et al., 2002; Keefe et al., 1995). It is probably premature to completely discard the notion that some aspect of mitochondrial function is important in oocyte quality, but there is currently no compelling evidence to suggest that this correlates with the capacity for OXPHOS.

VI. mtDNA Copy Number in the Oocyte The idea that mitochondrial function is an important determinant of oocyte quality has led to a recent barrage of studies trying to demonstrate that oocyte mtDNA content, and by inference OXPHOS capacity during early embryogenesis, is a marker for female infertility. In this light, several groups have suggested that oocytes from women with premature ovarian failure or from older donors have a reduced ability to generate viable embryos, owing to a reduced mtDNA copy number. This argument could have some merit, not because of reduced copy number impairs OXPHOS capacity in the oocyte in an important way, but rather because reduced mtDNA copy number (and the associated reduced mitochondrial numbers) in the zygote would be predicted to aVect the distribution of mitochondria in the cells of the early embryo at a period when mitochondrial biogenesis is slowed or arrested. These studies have generated several estimates of mtDNA copy number in presumably healthy metaphase II human oocytes ranging from 50,000 to 1,500,000 copies (Barritt et al., 2002; Chan et al., 2005; May‐Panloup et al., 2005; Reynier et al., 2001; Steuerwald et al., 2000). The range in the estimates of mtDNA copy number (30‐fold) is at least tenfold greater than earlier measurements made by

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Southern blot in murine and bovine oocytes (Michaels et al., 1982; Piko and Taylor, 1987). There are a number of possible explanations for the variability in the estimates of mtDNA copy number in human oocytes as compared to those in other mammals. First, it may simply reflect the fact that the estimates from mice and cows are derived from animals with a more homogeneous nuclear genetic background than the human population. Second, the observed variability may reflect real biological variation in the sample of oocytes used in these studies. For obvious ethical reasons, it is seldom possible to harvest oocytes from normal healthy women for research purposes. Invariably, researchers must turn to in vitro fertilization (IVF) clinics to recuperate ooctyes from women who have given informed consent to donate their surplus oocytes. As a result, the oocyte quality and donor fertility are likely subject to stratification. Thus, it seems unlikely that oocytes harvested from this cohort accurately reflect random sampling from a healthy population. A third and more disconcerting cause of the inter‐ and intrastudy variation in mtDNA copy number variability may have to do with the use of the quantitative real‐time PCR platform that all the recent studies have used to estimate copy number. Real‐time PCR is a method of simultaneous DNA amplification and quantification. After each round of amplification, the DNA is quantified by measuring a fluorescent signal that is generated immediately following the synthesis of DNA, as it intercalates with a fluorescent dye such as SYBR Green. Tracking the fluorescence as the reaction progresses, it is possible to monitor the early cycles of the log–linear phase of amplification. In theory, there exists an inverse relationship between the quantity of starting material and the fluorescence generated during the log–linear phase of amplification. This is reported to the user as a Ct value, which represents the PCR cycle number at which the fluorescence is detectable above a predefined threshold. Using a standard curve generated from serial dilutions of a PCR product or plasmid of known concentration, real‐ time PCR machines can automatically extrapolate unknown starting concentrations from Ct values and provide the user with an absolute starting template number. Under the appropriate conditions, real‐time PCR quantitation of nucleic acid can be a powerful and sensitive platform, rivaling all other detection methods including hybridization techniques (i.e., Southern blots, Northern blots) and traditional endpoint PCR analyses. Nevertheless, real‐time PCR does has its own Achilles heel. Most calculations make the fundamental assumption that PCR eYciency of the amplicon of interest is constant over time and has the same value in all studied samples (i.e., that the amplification eYciency is equal to 2 such that each amplicon is doubled after every cycle). Largely ignored have been the reports that amplification eYciencies can vary over a range of 1.8–2.0 (Ramakers et al., 2003). When this variation in PCR eYciency is taken into account, a real

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tenfold diVerence can turn up as any value between 0.7 and 210 (Ramakers et al., 2003). Thus, without the proper postrun analyses that take into account variation in amplification eYciency, it becomes diYcult to accurately determine sample concentrations, even with a serially diluted standard curve, and hence derive meaningful conclusions from these data. It is not clear that any of the studies on single oocytes have analyzed their data with a rigor that would allow one to conclude that the data reflect real biological variation in the populations that have been sampled. Ramakers et al. (2003) designed an easily implemented, and since widely used, method for the assumption‐free analysis of quantitative real‐time PCR data that uses an empirically determined PCR eYciency in the calculation of starting sample concentration. Briefly, the program uses a sliding ‘‘window‐ of‐linearity’’ function to determine the linear phase of amplification and corresponding amplification eYciency for individual samples and performs a linear regression to determine the starting concentration of each sample. To accurately determine mtDNA content in single oocytes, we suggest the implementation of this program in conjunction with a complementary secondary technique like nucleic acid hybridization (slot blots) on pooled oocytes for the purposes of corroboration. Until such studies are done, we suggest that the reported variation in mtDNA copy number in human oocytes be treated with caution.

VII. Male Transmission of mtDNA There are only rare exceptions to the general rule of uniparental inheritance of organellar genomes, either mitochondrial or chloroplast (Birky, 1995), and it has been suggested that this nearly universal phenomenon may have evolved to limit the spread of selfish genetic elements (Hurst et al., 1996). The lack of a paternal contribution of mtDNA has been attributed to a variety of factors, including dilution and active elimination. That mtDNA content of the mammalian male gamete is estimated to be 3 orders of magnitude less than its female counterpart has caused some to invoke a simple dilution of paternal mtDNA in the oocyte at fertilization as the mechanism that makes transmission to the next generation unlikely. While this may be the most parsimonious explanation for the paucity of paternal mtDNA transmission in mammals, others have invoked a mechanism of active exclusion whereby active destruction of paternal mitochondria (which are present in the sperm midpiece that penetrates the oocyte during fertilization) in the zygote lead to uniparental inheritance of mitochondria and their genome (Sutovsky et al., 1999). The surveillance system that targets paternal mtDNA can apparently be bridged in interspecific crosses as shown in studies with mice (Gyllensten et al., 1991; Kaneda et al., 1995). Gyllensten et al. discovered that successive

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backcrossing between species of mice for up to 26 generations can yield animals harboring low levels of paternally derived mtDNA in all of their tissues. In a similar study using the same interspecific crosses between Mus musculus domesticus  M. spretus, paternal mtDNA was detected by PCR analysis in early embryos and in about half of all oVspring generated from these crosses (Shitara et al., 1998). Unlike the previous report, the distribution of paternal mtDNA in tissues obtained from these positive oVspring was nonuniform. Less than 10% of hybrid females generated from this interspecific cross possessed paternal mtDNA in their ovaries, and none of the unfertilized oocytes from these animals were positive for the presence of paternal mtDNA. These observations lead to the notion that sperm from closely related species or subspecies may perchance escape the normal surveillance mechanisms put in place to ensure strict matrilineal transmission of mtDNA. Interestingly, no paternal mtDNA was detected in any of the N2 backcross animals (tested by examining embryos obtained by IVF), suggesting some barrier to transmission even in F1 hybrid animals, and that the genetic factors responsible act as either a dominant or codominant maternal trait. Other mammalian examples of paternal transmission (also referred to as paternal leakage) of mtDNA have been reported in sheep (Zhao et al., 2004) using similar PCR‐based methods. For instance, Zhao et al. demonstrated the occurrence of leakage in two related hybrid families (out of a total of 48) produced by Dorset crossed to Small‐Tail Han sheep. Taken together, these results suggest that the molecular targets for paternal mtDNA elimination in intraspecific crosses is a nuclear‐encoded molecule(s) associated with the sperm midpiece. It has been widely accepted that spermatozoa are translationally silent, but a study has challenged this dogma (Gur and Breitbart, 2006). Gur and Breitbart showed that labeled amino acid incorporation was completely blocked by mitochondrial translation inhibitors, but not by a cytoplasmic 80s ribosomal inhibitor. This suggests that mammalian sperm translates nuclear‐encoded proteins by mitochondrial‐type ribosomes that are important in the fertilization of the oocyte. Sutovsky et al. have suggested that mitochondria are earmarked for destruction by the addition of ubiquitin tags (Sutovsky, 2003; Sutovsky et al., 1999). The ubiquitination of sperm occurs in the male reproductive tract at the secondary spermatocyte/round spermatid stage, is masked during epididymal passage (possibly by disulfide bond cross‐linking), and reappears after fertilization (Sutovsky et al., 2000). Although markers of sperm mitochondria and anti‐ubiquitin antibodies colocalize by immunofluorescence in early embryos, there is no direct evidence that the mitochondria themselves are tagged. The destruction of sperm mitochondria could, however, be prevented by the injection of anti‐ubiquitin antibodies, or an antagonist of proteosome activity (Sutovsky et al., 2000), suggesting that ubiquitination is an important signal in the degradation pathway of paternal mitochondria. However, it is unclear

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how the nuclear genes products required for ubiquitin‐mediated protein degradation can be diVerentially recognized in closely related species by the oocyte proteosome. More recently, it was shown that the paternal mitochondrial genome in fish is actively removed prior to the purported degradation of sperm mitochondria. Using optical tweezers to remove intact sperm from within freshly fertilized fish eggs, Nishimura et al. (2006) showed that paternal mtDNA was in fact actively degraded well before the disappearance of the paternal mitochondria. While the elimination of paternal organellar DNA seems to be a conserved mechanism used to ensure matrilineal transmission (Nishimura et al., 2006), the nature of such a nucleolytic mechanisms remains a mystery. Currently, there are no data on how paternal mtDNA is actively eliminated from the mammalian oocyte. A single case of paternal transmission of a pathogenic mtDNA mutation was reported in an individual with a severe muscle myopathy (Schwartz and Vissing, 2002). This case prompted other investigators to search for paternal transmission retrospectively in a large group of patients (Filosto et al., 2003; Taylor et al., 2003). However, no evidence of paternal transmission was found in these studies, suggesting that it is likely to be an extremely rare event in human biology.

VIII. Clinical Perspectives Our current understanding of the transmission and segregation of pathogenic mtDNA mutations can be applied in the clinic in two major areas: counseling women who are carriers about recurrence risks and preventing the transmission of high proportions of mutant mtDNAs to their children. Although in principle, it ought to be possible to calculate recurrence risks for these diseases, there are a number of complicating issues. First, the distribution of mutant mtDNAs among tissues is rarely uniform, so it is generally not possible to infer the distribution in the germ cells from a blood or tissue sample. Second, the bottleneck ensures that there will be a range of heteroplasmy in the oocyte population, which is likely to be mutation dependent. Third, the threshold behavior of pathogenic mutations makes it diYcult to precisely predict the severity of the clinical phenotype, especially at intermediate levels of heteroplasmy. The Melbourne group has conducted the most thorough analysis of the prospects for genetic counseling in a large number of pedigrees segregating the ATP6 (8993) gene mutations associated with NARP (White et al., 1999). This analysis showed that the recurrence risk increased with the mutant load in the mother, as predicted from a stochastic model of transmission, but the confidence intervals on the risk estimate of having an aVected child were extremely large, demonstrating that the risk of

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recurrence is not insignificant at any level of heteroplasmy. This is a direct consequence of the genetic bottleneck. As mentioned previously, extreme skewing of heteroplasmy (suggesting a very narrow bottleneck) is often observed (but remains unexplained) in the germ line of individuals with mutations in the ATP6 gene in particular, so this might represent a worst case scenario for estimating recurrence risk. Although the experience is still limited, prenatal genetic diagnosis for these disorders seems promising. The fact that little, if any, segregation of mtDNA sequence variants occurs during fetal life (Jenuth et al., 1997), even those that are pathogenic (Harding et al., 1992; Matthews et al., 1994), suggests that sampling of any fetal tissue (chorionic villus, amniocytes) after establishment of pregnancy ought to provide a reliable indication of the overall mtDNA mutation load. Mosaicism for polymorphic mtDNA sequence variants has been reported in the placenta, but it was only apparent when very small tissue samples were analyzed (Marchington et al., 2006). It is also not known if all pathogenic mtDNA mutations will obey the same rules. A number of prenatal tests have been reported, mostly for the ATP6 mutation associated with NARP (reviewed in Jacobs et al., 2006). The utility of prenatal diagnosis for a broad spectrum of mtDNA mutations will likely only become apparent after years of clinical experience. The prospects for preimplantation genetic diagnosis are also bright. Because the individual’s mtDNA genotype is most likely determined by the relative proportions of wild‐type and mutant mtDNA in the oocyte, the mtDNA genotype of a blastomere sampled from an eight‐cell embryo (which should contain more than 10,000 copies of mtDNA), ought to be a random sample of the oocyte’s mtDNA complement and hence a good predictor of the mtDNA genotype of the fetus. A study using heteroplasmic mice has borne out this prediction (Dean et al., 2003), and confirmed earlier suggestions (Briggs et al., 2000) that the analysis can also be carried out on a polar body, without disturbing the embryo. There is at least one reported case where blastomere biopsy identified two homoplasmic wild‐type embryos and one homoplasmic mutant embryo from a mother carrying the ATP6 gene mutation associated with NARP (SteVann et al., 2006). Implantation of both wild‐type embryos resulted in the birth of a healthy baby. The introduction of new reproductive technologies (intracytoplasmic sperm injection, ICSI) has also given rise to concern that the normal surveillance mechanisms that target sperm for destruction may be abrogated and that some transmission of male mtDNA might occur. A number of studies have investigated this and to date no evidence for male transmission has been observed (Danan et al., 1999; Houshmand et al., 1997; Torroni et al., 1998). It must be said, however, that only blood has been examined in the individuals that have been born using this procedure, and questions remain about whether these studies had the sensitivity to detect paternal mtDNA at

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very low levels, so it may be premature to rule out the possibility of leakage of male mtDNA. Transfer of oocyte cytoplasm has been used in a number of instances in women with repeated implantation failure due to abnormal embryological development (Barritt et al., 2001). It is not, however, at all clear whether the introduction of exogenous mitochondria in these cases played any role in the establishment of a pregnancy. Some of the individuals that have been born as a result of this procedure are demonstrably heteroplasmic for the donor and recipient mtDNA haplotypes in blood and probably other tissues. This situation is analogous to male mtDNA leakage (although on a larger scale), and the consequences of such heteroplasmy are unknown. Although some surprising mtDNA segregation patterns have been observed in heteroplasmic mice (Jenuth et al., 1997), there is no indication that mixing two polymorphic mtDNA haplotypes can cause overt pathology (Battersby and Shoubridge, 2001).

IX. Conclusions and Future Prospects In mammals, all mitochondria and mtDNAs ultimately derive from those contained in an oocyte. Paternal mitochondria are actively eliminated from the early embryo by a process that remains ill‐defined, but which depends on unknown species‐specific targets recognized by components of the oocyte proteome. mtDNA haplotypes are thus propagated clonally along maternal lineages. The segregation of new germ line mtDNA mutations between generations is rapid and primarily stochastic, due to a genetic bottleneck for mtDNA in the PGCs. The eVect of this bottleneck is to rapidly fix neutral or near‐neutral polymorphisms in the population and to expose severe pathogenic mutations to selection. Investigating the mechanisms that produce the bottleneck, and possible variations in its size due to genetic modifiers or to OXPHOS defects, remain important future problems. In this context, it will be important to develop animal models that are segregating specific mtDNA point mutations. However, this will remain an extremely challenging problem until some robust method can be found to either transform mammalian mtDNA or identify and isolate cells containing naturally occurring mtDNA point mutations. Although oocytes contain a very large number of mitochondria, there is no evidence that this relates to a high demand for OXPHOS. The majority of mitochondria in an oocyte, early embryo, or fetus can harbor a pathogenic mutation without obviously aVecting prenatal development. The large number of mitochondria in the mature oocyte may in fact be a genetic, rather than a metabolic device, to ensure that mitochondria are apportioned to the PGCs before mitochondrial biogenesis restarts. The mechanisms that control mitochondrial biogenesis in the germ line and in early embryonic development

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remain completely unknown. The copy number of mtDNAs, and by association the number of mitochondria, might be an important determinant of oocyte quality because too small a number will result in maldistribution in the early embryo. It will be important to determine the independent eVects of varying mtDNA copy number and OXPHOS capacity on the development of the germ line, and the subsequent eVects on early embryonic development. The results of such investigations could have a major impact on our understanding of some causes of female infertility.

Acknowledgments Research in the author’s laboratory is supported by grants from the CIHR, NIH, Muscular Dystrophy Association (USA and Canada), and the March of Dimes. E.A.S. is a Senior Scientist of the CIHR and an International Scholar of the Howard Hughes Medical Institute. T.W. is supported by Canada Graduate Scholarship for Doctoral Research from the CIHR.

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