Human Reproduction Update Advance Access originally published online on March 31, 2006
Human Reproduction Update 2006 12(4):401-415; doi:10.1093/humupd/dml011
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Transmission of mitochondrial DNA following assisted reproduction and nuclear transfer
The Mitochondrial and Reproductive Genetics Group, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
1 To whom correspondence should be addressed at: The Medical School, The University of Birmingham, Birmingham B15 2TT, UK. E-mail: j.stjohn.1{at}bham.ac.uk.
Submitted on November 25, 2005; accepted on February 28, 2006
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Abstract
IntroductionMitochondria are the organelles responsible for producing the majority of a cell’s ATP and also play an essential role in gamete maturation and embryo development. ATP production within the mitochondria is dependent on proteins encoded by both the nuclear and the mitochondrial genomes, therefore co-ordination between the two genomes is vital for cell survival. To assist with this co-ordination, cells normally contain only one type of mitochondrial DNA (mtDNA) termed homoplasmy. Occasionally, however, two or more types of mtDNA are present termed heteroplasmy. This can result from a combination of mutant and wild-type mtDNA molecules or from a combination of wild-type mtDNA variants. As heteroplasmy can result in mitochondrial disease, various mechanisms exist in the natural fertilization process to ensure the maternal-only transmission of mtDNA and the maintenance of homoplasmy in future generations. However, there is now an increasing use of invasive oocyte reconstruction protocols, which tend to bypass mechanisms for the maintenance of homoplasmy, potentially resulting in the transmission of either form of mtDNA heteroplasmy. Indeed, heteroplasmy caused by combinations of wild-type variants has been reported following cytoplasmic transfer (CT) in the human and following nuclear transfer (NT) in various animal species. Other techniques, such as germinal vesicle transfer and pronuclei transfer, have been proposed as methods of preventing transmission of mitochondrial diseases to future generations. However, resulting embryos and offspring may contain mtDNA heteroplasmy, which itself could result in mitochondrial disease. It is therefore essential that uniparental transmission of mtDNA is ensured before these techniques are used therapeutically.
Key words: assisted reproduction technology / mitochondrial DNA / nuclear transfer
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Introduction |
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Mitochondria are found in all eukaryotic cells and are essential to cellular function because of their ability to generate ATP, the cell’s major source of energy. They comprise a double membrane with the outer membrane separating the mitochondrial matrix from the cytoplasm of the cell, whereas the inner membrane forms the cristae. Mitochondrial cristae are the site of the electron transfer chain (ETC), the final stage in cellular respiration where oxidative phosphorylation (OX-PHOS) takes place (Sherratt, 1991
). This aerobic process allows further metabolism of the products of anaerobic glycolysis and the citric acid cycle to produce carbon dioxide and water, with the subsequent release of 32 molecules of ATP. This is a far more economical return than provided by anaerobic generation of ATP (Pfeiffer et al., 2001). The ETC consists of five enzyme complexes, each containing multiple polypeptide subunits (Figure 1). Electrons flow through the first four complexes of the ETC releasing protons from the mitochondria which set up the mitochondrial membrane potential. Pumping these protons back into the mitochondria through the fifth complex results in the generation of ATP.
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The importance of ATP in oocytes and early embryos has been demonstrated in cattle, as assessed by morphology. Here, the higher quality oocytes contained significantly higher levels of ATP than poorer quality oocytes (Stojkovic et al., 2001). Those containing the highest ATP levels also progressed to produce a significantly higher proportion of blastocysts. A similar pattern has been demonstrated in human oocytes, with those containing at least 2
mol ATP at fertilization being more capable of embryonic development and implantation (Van Blerkom et al., 1995). The correlation between mitochondrial distribution in individual blastomeres and blastomere ATP content further emphasizes the importance of OX-PHOS for early preimplantation development (Van Blerkom et al., 2000).
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Mitochondrial DNA |
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Components of the ETC are encoded by both chromosomal and mitochondrial DNA (mtDNA), with all components being required for efficient function of the ETC. The human mitochondrial genome is a double-stranded circular DNA consisting of 16 569 base pairs (Anderson et al., 1981
). Using a slightly different genetic code to that of nuclear DNA, mtDNA encodes 13 polypeptides involved in the ETC, 22 transfer (t)RNAs and 2 ribosomal (r)RNAs (Anderson et al., 1981). MtDNA codes for seven subunits of nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND; complex I), one component of complex III, three subunits of cytochrome C oxidase (complex IV) and two subunits of ATP synthase (complex V). All other components of the ETC are encoded by the nuclear genome (Figure 1). The mitochondrial genome is highly compact, with no introns contained between the coding regions (Anderson et al., 1981) and some of the coding regions even overlap (ATPase 6 and ATPase 8, ND4 and ND4L; Anderson et al., 1981). Others lack termination codons, with these sequences being provided by post-transcriptional polyadenylation (Ojala et al., 1981). Despite its small size in comparison with the nuclear genome, the host cell depends on the mitochondrial genome for aerobic respiration as much as the mitochondria rely on proteins encoded by the host cell’s nuclear DNA. In fact, as much as one third of the total DNA content of an oocyte consists of mtDNA (Piko and Matsumoto, 1976). Somatic cells have between 103 and 104 copies of mtDNA, with 2–10 copies per organelle (Satoh and Kuroiwa, 1991), correlating to each particular cell’s requirement for OX-PHOS. For example, it has been reported that human subcutaneous fat cells contain more mtDNA copies than peripheral blood mononuclear cells (Gahan et al., 2001) and that liver tissue contains more mtDNA than various muscle tissues (Wiesner et al., 1992). |
MtDNA transcription and replication |
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Transcription of mtDNA occurs following interaction between nuclear-encoded regulatory proteins and regions within the D-loop of mtDNA (Figure 2). Requirements for transcription include the mitochondrial RNA polymerase (Tiranti et al., 1997
), mitochondrial transcription factor A (TFAM; Fisher and Clayton, 1985; 1988), and one of the recently identified TFB1M and TFB2M (Falkenberg et al., 2002; Table I). Once initiated, transcription generates a polycistronic precursor RNA transcript, allowing co-ordinated transcription of all genes on the same strand. Excision of the polycistronic precursor by endonucleases produces precursor rRNAs and tRNAs, which are then processed further to allow them to translate the precursor mRNAs (Ojala et al., 1981).
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The replication of mtDNA is also dependent on nuclear-encoded regulatory proteins (Table I). The replication process begins with light strand transcription from the light strand promoter region to the second conserved sequence block (CSB) within the D-loop region, producing an RNA sequence complementary to the light strand DNA (Xu and Clayton, 1995). Binding of the mtDNA-specific polymerase gamma (PolG) to the resulting RNA–DNA hybrid allows replication of the heavy strand to begin from the origin of heavy strand replication (OH) contained within the D-Loop (Figure 2). After the replication of approximately two thirds of the genome, the formation of the new heavy strand exposes the origin of light strand replication (OL) on the heavy strand parent. Light strand replication is then able to commence in the opposite direction.
As mtDNA transcription and replication, and therefore cellular ATP-generating capacity, are so dependent on proteins encoded by the nuclear genome, mutations in these genes can result in mtDNA depletion syndromes (reviewed by Suomalainen and Kaukonen, 2001). Unlike the vast majority of mtDNA diseases, these diseases can be transmitted in a Mendelian fashion and include chronic progressive external ophthalmoplegia (CPEO; Van Goethem et al., 2001; Graziewicz et al., 2004) and Alpers syndrome (Naviaux et al., 1999). This effect is also mimicked by nucleoside antiretroviral analogues such as 3'-azido-3'-deoxythymidine (AZT) and 2'3'-dideoxycytidine (ddC). For example, men treated with these drugs for 12 months had increased numbers of deleted mtDNA molecules in their sperm samples (White et al., 2001). Furthermore, although heterozygous PolG knockout mice develop normally, despite slightly reduced mtDNA levels, homozygous knockouts have severe mtDNA depletion and do not survive beyond embryonic day 8.5 (Hance et al., 2005).
TFAM plays an important role in both transcription and replication of the mitochondrial genome. It is therefore not surprising that low levels of TFAM have been associated with a variety of mitochondrial depletion syndromes, such as infantile mitochondrial myopathy (Poulton et al., 1994), familial mtDNA-associated liver disease (Spelbrink et al., 1998), fatal childhood myopathy (Larsson et al., 1994), skeletal muscle and mitochondrial encephalomyopathy disorders (Siciliano et al., 2000) and ocular myopathy, exercise intolerance and muscle wasting (Tessa et al., 2000). In contrast, there is a decrease in TFAM expression localized to the mitochondria during spermatogenesis. This has been proposed as a functional mechanism for specifically reducing mtDNA copy number in maturing sperm and therefore paternal mtDNA transmission to future generations, in both the human (Larsson et al., 1997) and the mouse (Larsson et al., 1996). This arises at the late spermatocyte/early spermatid stage where an isoform is expressed lacking the mitochondrial targeting sequence. In the mouse, TFAM was also detected in the nucleus of developing sperm cells, indicating a possible further role as a nuclear transcription factor (Larsson et al., 1996). The significance of TFAM expression as a major regulator of mtDNA copy number is exemplified in homozygous knockout mouse embryos that are completely depleted of mtDNA and do not survive after embryonic day 10.5 (Larsson et al., 1998).
The replication of embryonic mtDNA, at least in the mouse, does not occur during early preimplantation development (Piko and Taylor, 1987). However, a small window of replication at the two-cell stage was recently reported (McConnell and Petrie, 2004). The mtDNA molecules present in the mature oocyte at fertilization are therefore largely relied upon to provide sufficient ETC components to allow OX-PHOS activity during the preimplantation phase of embryonic development. In order to meet this demand, mtDNA is amplified during oocyte maturation within the ovary, with a 45-fold increase being reported in cattle from the primordial germ cells stage to pre-ovulating oocytes (Smith and Alcivar, 1993), resulting in a mean of 260 000 copies being present in mature bovine oocytes (Michaels et al., 1982). Similar amplifications have been reported in other species. For example, human premigratory primordial germ cells possess approximately 10 mitochondria per cell (Jansen and De Boer, 1998). Mature mouse oocytes contain mean mtDNA copy number values between 119 000 (Piko and Taylor, 1987) and 159 000 (Steuerwald et al., 2000). In the human, copy number ranges from 10 000 to 700 000 (Almeida-Santos et al., 2006), with other reported mean values of 314 000 (Steuerwald et al., 2000) and 193 000 (Reynier et al., 2001).
Oocytes with higher copy numbers of mtDNA are known to be associated with improved fertilization rates. For example, human oocytes failing to fertilize because of poor sperm quality, and therefore assumed to be of good quality, had significantly higher mtDNA copy number than poor quality oocytes which were fertilized by good quality sperm (mean 255 000 versus 152 000 copies; Reynier et al., 2001). Furthermore, low-quality oocytes from patients with a variety of ovarian disorders contained an average of 100 000 mtDNA molecules compared with an average of 256 000 copies in oocytes from healthy individuals (May-Panloup et al., 2005). We have also recently reported similar results in porcine (El Shourbagy et al., 2006) and human (Almeida-Santos et al., 2006) oocytes. However, we also saw significantly different copy number between arrested two-cell human embryos and those that developed further (Almeida-Santos et al., 2006). A putative threshold number of mtDNA molecules has been proposed for the mature unfertilized oocyte to allow embryo development to take place after fertilization. In the mouse, this has been predicted at approximately 100 000 copies per oocyte (Piko and Taylor, 1987), with a similar value being appropriate for the pig (El Shourbagy et al., 2006).
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The relevance of mtDNA copy number threshold to fertilization outcome and embryo development |
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The number of mtDNA copies per organelle is indicative of the number of mitochondria in an oocyte. In the mouse, each mitochondrion has just one copy (Piko and Taylor, 1987
). Consequently, the minimum number of mtDNA copies would indicate the minimum number of mitochondria required for fertilization to take place. These mitochondria would support embryonic development to cavitation, after which glycolysis becomes increasingly important in many species (human – Leese et al., 1993; pig – Sturmey and Leese, 2003; cow – Thompson et al., 1996; rat – Brison and Leese, 1991; mouse – Houghton et al., 1996). Furthermore, this minimum number may be essential as it has been hypothesized that a threshold level of ATP is required to energize cell division (Brenner et al., 2000) and may also synchronize mitochondrial dynamics. The distribution of mitochondrial networks throughout the cell varies with different stages of the cell cycle (Margineantu et al., 2002). Mature and immature oocytes also have distinct patterns of mitochondrial distribution (Stojkovic et al., 2001; Nishi et al., 2003). Those oocytes with mtDNA copy number lower than the required threshold may be unable to form the networks required for developmental competence. In support of this, developmental competence appears to be related to the numbers of mitochondria present (El Shourbagy et al., 2006).
In sea urchin oocytes, mitochondria act as calcium sinks (Eisen and Reynolds, 1985). The Ca2+ wave pacemaker that is necessary for the completion of meiosis at fertilization is maintained by mitochondrial uptake of Ca2+ (Dumollard et al., 2003). Failure to maintain the Ca2+ wave pacemaker results in apoptotic cell death of the oocyte (Liu et al., 2001). Therefore, oocytes with low levels of ATP production, because of low levels of mitochondria and mtDNA, may be unable to maintain the Ca2+ wave pacemaker and complete meiosis, and instead, undergo apoptosis. We have previously hypothesized that oocytes without sufficient wild-type mtDNA, and therefore the capacity to generate ATP would not normally be ovulated (St. John, 2002). This is reflected in the low numbers of mtDNA observed in degenerate oocytes (mean = 44 629 ± 40 729; Almeida-Santos et al., 2006). Consequently, the use of superovulation protocols during IVF allows these low-quality oocytes to undergo the maturation process and the inadequacies of insufficient mtDNA molecules to be observed (St. John, 2002).
Mitochondrial morphology changes after oocyte maturation to the inactive state (Bavister and Squirrell, 2000). This may be because of the lack of mtDNA replication and the import of nuclear-encoded regulatory factors from the cytoplasm. However OX-PHOS is still functional during early embryo development, and mitochondria are dynamic organelles, moving to provide ATP where it is most needed (Stojkovic et al., 2001). Staining of mitochondria has shown that during embryo development, they cluster around the nucleus of blastomeres during the cleavage stages, providing the energy required for cell division processes such as spindle formation. However, when cell division is not taking place, they tend to spread throughout the cytoplasm of the cell (Stojkovic et al., 2001; Sun et al., 2001; Wilding et al., 2001).
Equal segregation of mitochondria following cell division reduces the possibility of resulting tissues of the offspring lacking the ability to produce sufficient ATP through OX-PHOS. However, disproportionate numbers of mitochondria among blastomeres has been reported in the pig (El Shourbagy et al., 2006) and human (Van Blerkom et al., 2000; Lin et al., 2004). This may result in some blastomeres with reduced ATP-generating capacity. If this occurs early on in development, blastomeres may fragment, resulting in embryo arrest or less competent blastocysts with fewer cell numbers. In the human, mitochondrial morphology changes back to the active state at the eight-cell stage, coinciding with the activation of the embryonic genome (Sathananthan and Trounson, 2000). Furthermore, during cavitation, shortly after embryonic genome activation, there is an increased dependence on glycolysis in most mammalian species (see Van Blerkom et al., 2002 for discussion). This would suggest that nuclear embryonic genome activation provides a signal to the mitochondria to reduce OX-PHOS activity. Increased use of glycolysis may allow embryonic cells that are no longer able to generate sufficient ATP through OX-PHOS to survive for long enough to enable production and import of nuclear-encoded regulatory factors necessary for the onset of mtDNA replication post-implantation (El Shourbagy et al., 2006). Indeed, studies in pigs using OX-PHOS inhibitors have indicated that the inhibition of OX-PHOS function at compaction can improve embryo viability (Machaty et al., 2001). This hypothesis is supported by reports of glycolysis rather than OX-PHOS metabolism occurring in other cell types with a functioning nuclear genome but low mtDNA copy number, including somatic cells being depleted of their mtDNA (King and Attardi, 1989). However, ATP production through glycolysis is far less efficient than through OX-PHOS. It is therefore vital that sufficient mtDNA molecules are present at fertilization to maintain embryo survival until mtDNA replication resumes.
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The relevance of mtDNA replication to maternal transmission of mtDNA |
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MtDNA is further unlike nuclear DNA as it does not follow the normal Mendelian pattern of inheritance but is maternally inherited (Giles et al., 1980
). Owing to its size, a mature oocyte can contain far more mitochondria in its cytoplasm than the fertilizing sperm cell. Coupled with replication of the mitochondrial genome during oocyte maturation in the ovary, this results in an oocyte : sperm mtDNA ratio of 1000:1 (Ankel-Simons and Cummins, 1996), with sperm mtDNA contributing approximately only 100 molecules at fertilization (Hecht and Liem, 1984).
MtDNA is usually homoplasmically transmitted, i.e. only one mtDNA genotype being present (Monnat et al., 1985). Occasionally, two or more mtDNA genotypes can be present, often because of either a pathological or a non-pathological rearrangement, resulting in heteroplasmy, as in the oocytes of women harbouring a mtDNA disease (Blok et al., 1997). MtDNA has a much higher mutation rate than nuclear DNA (Wallace et al., 1987). This is a consequence of the production of mutagenic free radicals within the mitochondria, which are further confounded by limited mtDNA repair mechanisms (Yakes and Van Houten, 1997). Furthermore, mtDNA is not stabilized with histones as is the case for nuclear DNA, although TFAM is now thought to be involved in mtDNA packaging (Alam et al., 2003). Despite the high mutation rate, mtDNA has been highly conserved throughout evolution, and when it arises, mtDNA heteroplasmy tends to revert back to homoplasmy sometimes within one generation (Koehler et al., 1991).
MtDNA replication does not occur in synchrony with nuclear DNA replication, and therefore some mtDNA genomes are not replicated during one-cell cycle, whereas others are copied many times (Sazer and Sherwood, 1990). It has been reported that there is a preferential replication of those mtDNA molecules that are closer to the nucleus (Davis and Clayton, 1996), perhaps because of the faster diffusion of nuclear-encoded regulatory factors. The introduction of exogenous mtDNA into zygotes either close to the nucleus or at the periphery influences the amount of exogenous mtDNA observed at the blastocyst stage, with higher proportions arising from karyoplast injections (Meirelles and Smith, 1998). Consequently, the position of mitochondria in the early embryo may play a role in determining which mtDNA genomes are selected for amplification.
Contradictory data have recently shown that mtDNA replication is not just restricted to mitochondria in proximity to the nucleus (Magnusson et al., 2003). Here, it was demonstrated that all components of the replication machinery were available in sufficient quantities throughout the cell, allowing replication to take place throughout the entire cytoplasm. This included PolG and TFAM. The previously reported preferential labelling of mtDNA near cell nuclei was suggested to be because of denser aggregation of mitochondria around nuclei (Magnusson et al., 2003). MtDNA replication throughout the cell cytoplasm is supported by a lack of evidence for selection against pathogenic mtDNA mutations in oogenesis or early embryonic development (reviewed by Shoubridge, 2000).
When mtDNA replication resumes following implantation (Piko and Taylor, 1987), each cell type of the developing embryo contains a few copies of mtDNA, as indicated by the few mitochondria present in undifferentiated embryonic stem cells (ESCs; Sathananthan et al., 2002; St. John et al., 2005a). Consequently, the combination of segregation followed by the amplification of selected templates produces an mtDNA purification process (Hauswirth and Laipis, 1982), which is important for the maintenance of mtDNA homoplasmy. If blastomeres inherit mutated genomes or are depleted of mitochondrial genomes, then the resulting adult tissues would be deficient in OX-PHOS capacity, which could result in the onset of mitochondrial disease (Wallace, 1992).
There are many diseases caused by mutations in mtDNA and symptoms of these diseases generally appear in tissues highly dependent on OX-PHOS (Wallace, 1992). These include Leber’s hereditary optic neuropathy (LHON), which can result from mutations in the gene for the NADH-Q oxidoreductase component of complexes I and III (Brown et al., 1992). Myoclonic epilepsy with ragged-red fibres (MERRF) is also transmitted by maternal inheritance and results in myoclonus, epilepsy and ataxia. This can result from mutations in mitochondrially encoded tRNA genes (Hanna et al., 1995; Nakamura et al., 1995; Nishigaki et al., 2003; Mancuso et al., 2004) and protein genes (Naini et al., 2005). As well as point mutations, some mitochondrial diseases are caused by large-scale deletions to the genome. The most well known in this group is Kearns–Sayre Syndrome (KSS), which includes symptoms of PEO, pigment retinopathy and disorders of cardiac conduction (Klopstock et al., 1995; Lertrit et al., 1999; De Block et al., 2004). The clinical phenotypes resulting from mtDNA mutations are dependent on the proportion of mutated mtDNAs (Wallace, 1992). In the case of LHON, >60% mutant mtDNA load is required before the disease phenotype presents (Chinnery et al., 2001). In some mitochondrial diseases, including a case of MERRF, over 85% mutant mtDNAs need to be present before symptoms are observed (Boulet et al., 1992).
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Paternal mtDNA inheritance |
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There is increasing evidence demonstrating that maternal-only mtDNA transmission is not merely because of dilution of the paternal mitochondria beyond the level of detection. It has been suggested that paternal mtDNA is actively eliminated by ubiquitination in the oocyte cytoplasm and subsequent proteolysis during embryonic development (Sutovsky et al., 1999
; Nishimura et al., 2006). The elimination hypothesis is supported by reports demonstrating reduced paternal mtDNA content as embryonic development progresses (Cummins et al., 1997). Paternal mtDNA was detected only as far as the pronucleus stage following intra-specific mouse crosses compared with detection in neonates following inter-specific mouse crosses (Kaneda et al., 1995). This suggests that the elimination mechanism is species specific. However, the elimination mechanism is not only species specific but also tissue specific, as donor sperm mtDNA was eliminated, whereas exogenous liver mtDNA injected into the oocyte persisted at least until the neonatal period (Shitara et al., 2000).
The destruction of paternal mtDNA (reviewed in Sutovsky, 2003) and the presence of the genetic bottleneck should usually result in homoplasmic individuals. Recently, the accepted theory of uniparental mtDNA inheritance has been questioned. This is especially so in the case of a male patient presenting with a mitochondrial myopathy who was found to have inherited his muscle mtDNA from his father. However, the 2bp deletion giving rise to his myopathy was de novo (Schwartz and Vissing, 2002). This outcome is further supported by the evidence of sperm mtDNA persistence in human polyploid IVF-generated blastocysts (St. John et al., 2000). Paternal leakage of mtDNA has also been reported following inter-specific crosses in fruitflies (Kondo et al., 1990), mice (Gyllensten et al., 1991), honeybees (Meusel and Moritz, 1993), birds (Kvist et al., 2003), sheep (Zhao et al., 2004) and non-human primates (St. John and Schatten, 2004) and following intra-specific crosses in mussels (Zouros et al., 1992) and fruitflies (Kondo et al., 1992). The ubiquitin-related paternal mtDNA destruction process has therefore proven ineffective in these cases. It is as yet unknown how paternal mtDNA might escape the destruction process and how the paternal mtDNA might be preferentially replicated in a particular tissue to such an extent that it resulted in a mitochondrial disease (Schwartz and Vissing, 2002). In this latter instance, a considerable selective advantage must have taken place to out-compete the maternal source of mtDNA in the affected tissue.
A possible mechanism for bypassing the paternal mtDNA destruction process is the fusion of parental mitochondria followed by recombination of the two genomes. Recombination of mammalian mtDNA was originally thought not to arise, although it is known to occur in yeast. However, more recent evidence suggests that it may also occur in mammals. For example, Ono and colleagues created two respiration-deficient cell lines from patients with mitochondrial diseases caused by different mutations (Ono et al., 2001). Fusion of the two-cell lines resulted in morphology and respiratory enzyme activity returning to normal within 10–14 days. These authors proposed that the outcome resulted from mtDNA recombination and genome compensation. Mammalian mtDNA recombination has since been confirmed in human cells using PCR-based techniques (D’Aurelio et al., 2004) and recent sequence analysis of invertebrate and vertebrate species, including primates (Tsaousis et al., 2005). These authors suggested that recombination may have been previously missed because of the purification process during oocyte production prohibiting the generation of new haplotypes and their transmission to the next generation. Recombination of sperm and oocyte mtDNA is now thought to explain the presence of the 2bp deletion in the male myopathy patient (Kraytsberg et al., 2004).
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Invasive oocyte reconstruction protocols |
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The introduction of more advanced assisted reproductive technologies (ART) and their current use in the clinical setting and in animal production further confounds the unimaternal transmission of mtDNA. These technologies include (i) ICSI which is used clinically; (ii) cytoplasmic transfer (CT) which has been used clinically but is now, for example, banned in the United Kingdom and United States and (iii) germinal vesicle transfer (GVT), pronuclear transfer (PNT) and NT, which have not yet been used to treat humans. These more invasive ART protocols present more opportunities for the violation of the normal pattern of mtDNA inheritance. If uniparental mtDNA inheritance is not maintained following these techniques, offspring may harbour various forms of mtDNA heteroplasmy. Indeed, the introduction of these new technologies results in heteroplasmy being redefined. In this instance, either a wild-type molecule can co-exist with a rearranged molecule or two wild-type molecules may persist in the reconstructed oocyte.
Unlike IVF, the ICSI procedure varies dramatically from the natural fertilization process. For example, the intact, non-capacitated, non-acrosome-reacted sperm is injected into the oocyte. There are also delays and alterations in post-fertilization processes following ICSI, including male pronucleus formation (Luetjens et al., 1999) and the calcium oscillations essential for the activation process (reviewed in Ludwig et al., 2001). This delayed response after sperm entry means that ICSI also has the potential to delay degradation of sperm mtDNA. Furthermore, couples who have male factor infertility and require ICSI are at a higher risk of using sperm containing abnormal mtDNA as some human mtDNA haplogroups are associated with high or reduced sperm motility (Ruiz-Pesini et al., 2000). This is demonstrated in those men harbouring large numbers of mtDNA deletions who are more likely to exhibit poorer semen quality (Lestienne et al., 1997; Reynier et al., 1997; St. John et al., 2001) as are those exhibiting point mutations (Spiropoulos et al., 2002). Despite the many diversions from the natural fertilization processes following ICSI, to date, it appears that sperm mtDNA is eliminated in offspring generated using this technique (Danan et al., 1999; Marchington et al., 2002). Furthermore, it appears to be eliminated before the blastocyst stage in ICSI-derived polyploid human embryos (St. John et al., 2000).
CT is an extension of ICSI and involves the injection of supplementary cytoplasm, as well as a sperm, into the oocyte (Figure 3). The rationale for using CT is that donor cytoplasm will contain supplementary, often younger, non-mutated mtDNA and will possibly contain other important cytoplasmic components to compensate for the defective cytoplasm in the oocyte (Cohen et al., 1997). In the human, it has resulted in higher pregnancy rates and live births (Cohen et al., 1997; Barritt et al., 2001). CT can also result in heteroplasmy as demonstrated by the analysis of various tissues of human CT offspring (Brenner et al., 2000). Consequently, it remains to be demonstrated whether the donor mtDNA out-competes the recipient mtDNA in the heteroplasmic offspring. We also need to ascertain whether the coupling of two distinct genomes accounted for the two cases of Turner’s syndrome, one electively aborted and the other spontaneously aborted, and the one case of pervasive development disorder (Barritt et al., 2001).
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To avoid the possible harmful effects of heteroplasmy in the offspring, cytoplasm from granulosa cells of the mother rather than cytoplasm from donor oocytes has been used (Kong et al., 2003; 2004). These are likely to have the same mtDNA sequence as the recipient oocyte. However, one possible problem with this procedure is that mtDNA deletions in granulosa cells are more common in older women (Seifer et al., 2002), i.e. those that are likely to have defective oocytes and require CT. Therefore, the treatment may be unsuccessful because of continued lack of wild-type mtDNA and may even result in the transmission of mitochondrial disease to the offspring that would not have occurred without CT. Another likely problem is that granulosa cells are fully differentiated, whereas newly fertilized oocytes are pluripotent. Cells at different stages of differentiation are known to be different in terms of their mitochondrial activity and content. For example, undifferentiated human (h) ESCs (Sathananthan et al., 2002; St. John et al., 2005a) and primordial germ cells (Jansen and De Boer, 1998) have been observed to contain low numbers of mitochondria. However, differentiated cells contain higher numbers of more active mitochondria, depending on OX-PHOS requirements of specific cell types (Moyes et al., 1998). Mitochondria donated from granulosa cells would therefore need to be ‘reprogrammed’ to match those of the oocyte. We have supplemented developmentally incompetent pig oocytes with pure populations of mitochondria from developmentally competent oocytes from maternal relatives. Interestingly, we observed improved fertilization outcome and the maintenance of unimaternal inheritance (El Shourbagy et al., 2006).
GVT and PNT have also been proposed as techniques to overcome problems associated with aged oocytes, whereby the GV or PN is transferred into an enucleated recipient oocyte. Ideally, the enucleated recipient should be from the same stage of development as the donor nucleus (Liu et al., 1999). In aged oocytes, there is likely to be a larger number and higher proportion of mutant mtDNA (Keefe et al., 1995). It has been hypothesized that mitochondria are important not only for the production of ATP but also for correct chromosomal segregation during meiosis (Schon et al., 2000). Furthermore, it has been suggested that aberrant mitochondrial function in aged oocytes may have a role in oocyte aneuploidy (Eichenlaub-Ritter et al., 2004), a common cause of infertility in older women (Munne et al., 1995; Dailey et al., 1996). The chances of success may therefore increase if the nuclear component of the aged oocyte was transferred into the enucleated ooplasm of a younger, healthier oocyte, rather than supplement the existing ooplasm. This transfer can be carried out at either the GV or the PN stage (Figure 3). Indeed, studies using human oocytes have shown GVT from aged oocytes to the enucleated ooplasm of young oocytes can overcome oocyte aneuploidy, with the majority of reconstructions having normal karyotypes (Zhang et al., 1999; Takeuchi et al., 2001). There may also be non-cytoplasmic components of the GV that influence chromosome segregation. This has been indicated in a series of mouse experiments mixing GVs and ooplasms of varying ages (Cui et al., 2005). A significantly greater number (57.1 %) of chromosomal abnormalities were found in reconstructions when the GV was transferred from older females to the enucleated ooplasm of younger females. This is in contrast to abnormalities found in reconstructions derived from the transfer of younger GVs into aged ooplasms (16.7 %).
Mitochondria are known to be involved in chromosome organization and movement. For example, the induction of mitochondrial damage in mouse oocytes prevented oocyte maturation, chromosomal segregation and spindle formation (Takeuchi et al., 2005). This damage was overcome by GVT of the karyoplast derived from a damaged oocyte into the ooplasm of a healthy enucleated oocyte. Cytogenetic analysis showed that 20/21 of these reconstructions had a normal number of chromosomes. Furthermore, performing ICSI on GVT reconstructions and transfer of the resulting two-cell embryos led to the generation of live mouse offspring. It has also been demonstrated that an intact mouse ooplasm could rescue mitochondrial-damaged karyoplasts induced by photoirradiation with 62% of these reconstructions maturing to metaphase II (Palermo et al., 2002). PNT is essentially the same procedure, except for the nuclear material being removed after fertilization. This technique has also been used in mice with considerable success as determined by the birth of live offspring (Meirelles and Smith, 1997; 1998; He et al., 2003; Sato et al., 2005). This procedure, however, involves the destruction of a zygote, which may restrict its usage because of ethical and moral considerations.
GVT and PNT have been proposed as techniques for treating mtDNA disease (Cummins 1998; Trounson, 2001). However, potential problems could arise as the transferred GV or PN is still surrounded by mitochondria, which will also be carried over into the donor ooplasm. These mitochondria remain close to the centre of the immature reconstruction at first but then disperse throughout the cytoplasm as maturation ensues (Fulka, 2004). Owing to segregation during embryogenesis, this provides an opportunity for karyoplast mitochondria to be present in all cell types of the offspring. Any disease-associated mitochondria transferred with the nuclear material may therefore continue to be transmitted.
It is further possible that, due to its initial proximity to the nucleus, karyoplast mtDNA would be preferentially replicated in the reconstructions. Indeed, fusion of enucleated somatic cells with mtDNA-depleted somatic cells can lead to the preferential expansion of one particular type of mtDNA such as the mutant molecule (Dunbar et al., 1995). Although, to our knowledge, there has been no analysis of mtDNA transmission following GVT, results are likely to be similar to those reported following PNT. In this instance, exogenous mtDNA accompanying the murine zygotic karyoplast is found in higher concentrations at blastocyst than mtDNA from transferred cytoplasts (Meirelles and Smith, 1998). This has been further highlighted where zygotic karyoplast transfer into zygotes resulted in varying amounts of mtDNA being transmitted to offspring (0–69%; Meirelles and Smith, 1997). Consequently, it would be difficult to predict to what extent a mutated or deleted mtDNA molecule would be selected. However, a recent report suggests that the varying levels of mtDNA deletion in mouse zygotes could be reduced to levels lower than those expected for the phenotypic onset of mtDNA disease when the zygotic nucleus is transferred into an enucleated healthy oocyte (Sato et al., 2005). The subsequent offspring also remained under the level for pathological onset even with the accumulative increase of the deletion with time. These authors state however that, in the human, this form of nuclear transplantation could not be applied to those mtDNA diseases where the pathogenic mutation had significant replicative advantage over wild-type mtDNA. This is most likely as in humans the prenatal period is 13 times longer than in the mouse, and this represents the window in which mutant mtDNA replication is most proliferative (Sato et al., 2005).
NT involves the injection of a donor nucleus or a whole cell into an enucleated recipient oocyte (Figure 3). Mostly commonly, either embryonic cell (EC) NT, using a blastomere, or somatic cell NT (SCNT) are performed. Resulting embryos have then been used to generate blastocysts and offspring in a variety of species (Campbell et al., 2005). However, NT is renowned for its low efficiency, with low proportions of embryos developing to the blastocyst stage (White et al., 1999) and even fewer progressing far enough to implant, survive pregnancy and produce live offspring (Wilmut et al., 1997). One of the major abnormalities associated with NT offspring is the production of abnormal placentae which are thought to be the cause of many fetal deaths (Cibelli et al., 2002). Incorrect reprogramming of imprinted genes has also been cited as one of the major factors which leads to early fetal death. This was highlighted by non-surviving NT calves having incorrect expression of imprinted genes, namely Igf2, Igf2r and H19 in some tissues, whereas surviving adult clones had normal expression at these loci except for Igf2 in muscle (Yang et al., 2005). Furthermore, in mouse NT embryos and offspring irregular patterns of gene expression have been observed most likely as a result of inappropriate reprogramming (Humpherys et al., 2002).
Although the process of NT for human reproductive purposes is ethically unacceptable and is banned in many countries (UK – Human Fertilisation and Embryology Act, 1990; USA – H.R. 534 Office of Legislative Policy and Analysis, 2003; South Korea – see Normile and Mann, 2005), its use as a means of producing histocompatible stem cells for treatment of various diseases is likely to have a significant impact. Cell replacement therapy has great potential for the treatment of certain conditions such as myocardial infarction (Rubart and Field, 2006) and Parkinson’s disease (Taylor and Minger, 2005) and as a replacement for tissue/organ transplantation (Cortesini, 2005). To date, encouraging results have been obtained in animal studies, whereby hESC-derived neural precursors (Wernig et al., 2004) and cardiomyocytes (Laflamme et al., 2005) were successfully transplanted into animal organs. However, considerable characterization and validation of precursor cells derived from hESCs still needs to be undertaken before cell replacement can be used as an effective therapy in humans. Some success at generating NT-derived ESCs has so far been achieved in mouse models (Munsie et al., 2000; Wakayama et al., 2001), and its use as a means of delivering the patient’s own chromosomal DNA with the corrected gene for a single gene disorder has been demonstrated in the mouse (Rideout et al., 2002).
One of the rate-limiting factors in generating autologous ESCs, i.e. stem cells possessing the patient’s own chromosomal and mtDNA compositions is the regulation of the mtDNA composition. In this respect, a considerable amount can be learnt from those offspring derived from NT through either ECNT or SCNT. Here, transmission results in either homoplasmy derived from the recipient oocyte mtDNA or heteroplasmy resulting from transfer of both donor and recipient mtDNA (reviewed in St. John et al., 2004a,b). This is probably because of the random pattern of mtDNA segregation during embryogenesis (reviewed in Shoubridge, 2000). Consequently, it is apparent that no real mechanism is currently understood that could account for the irregular patterns of mtDNA transmission observed. One parallel that can be drawn from both ECNT and SCNT is that the range of donor mtDNA transmitted is relatively similar at 0–57% (Hiendleder et al., 1999) and 0–59% (Takeda et al., 2003), respectively. However, two explanations could exist for those studies not detecting donor mtDNA in live offspring: (i) no donor mtDNA is actually present or (ii) too few tissues have been analysed. Consequently, the true extent of heteroplasmy is difficult to determine. This is perhaps exemplified by the recent studies of pig (Takeda et al., 2006), cow (Takeda et al., 2003) and mouse (Inoue et al., 2004) NT-derived offspring, where varying levels of heteroplasmy has been observed between the tissues. This would indicate that if all tissues are not analysed, then these studies are not truly subjective. Furthermore, investigations should include analysis of the surrogate mother’s mtDNA as leakage through the placenta into fetal blood has been reported in cattle (Hiendleder et al., 2004).
The initial number of donor mtDNA molecules introduced into the recipient oocyte also does not necessarily influence whether donor mtDNA is actually transmitted. Using blastomeres from more advanced embryonic stages in bovine NT has resulted in reduced levels of heteroplasmy being reported (Steinborn et al., 1998). However, this was not a uniform pattern as two offspring derived from 52-cell morula blastomeres contained less donor mtDNA than offspring derived from 92-cell morula blastomeres. A further issue is the stage of embryonic development of the donor cell. A recent analysis of two NT-derived monkey offspring (St. John and Schatten, 2004) demonstrated mtDNA transmission from both the recipient oocyte and the donor cell. In this instance, the early IVF blastomere (8- to 12-cell stage) still contained sperm mtDNA. Consequently, the offspring possessed three different populations of mtDNA. Furthermore, the depletion of donor cell mtDNA to residual levels (<0.02% of its original content) still resulted in the persistence of some mtDNA at the blastocyst stage in sheep NT (Lloyd et al., 2006).
The use of a somatic donor cell with accompanying mitochondria not only raises interesting questions with regard to the differentiated status of nucleus but also of the mitochondria. During development, from the inner cell mass to a fully differentiated cell, mitochondria increase in number to meet the metabolic requirements of that specialized cell. The proliferation of mitochondria further discriminates between the number of mtDNA copies per mitochondrion and cell type, with higher ATP users possessing greater copy numbers (Moyes et al., 1998). Therefore, for SCNT, the mitochondria accompanying the donor cell should also be reprogrammed to the embryonic state, as is the case for the donor nucleus. Failure to do this may result in embryos containing mitochondria with insufficient plasticity to suit a multitude of cell types that they will contribute to. The persistence of donor mitochondria could also influence the metabolic pathways used during early development and explain why improved developmental rates are observed in those embryos cultured in media that enhance anaerobic respiration (Chung et al., 2002). It is further evident that the injection of somatic donor mitochondria into oocytes which are then parthenogenetically activated is detrimental to embryonic development though this is not observed with donor ooplasm (Takeda et al., 2005).
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Interaction between multiple wild-type mitochondrial genomes |
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It has not been clearly demonstrated whether heteroplasmy resulting from two wild-type mtDNA molecules can be detrimental to the offspring and result in mitochondrial disease. This is partly because many post-mortem results have not been published, and the cause of many deaths following NT is not documented. Consequently, we are unsure whether those offspring that survive are the exception to the rule. However, from one report, some common causes of death in sheep NT offspring are similar to symptoms of mitochondrial diseases, including brain, kidney and liver defects (McCreath et al., 2000
) and in a series of other species (Cibelli et al., 2002). Sequence differences due to mtDNA from a ‘foreign’ source can give rise to proteins with altered amino acid sequences. This has been demonstrated in both cattle (Steinborn et al., 2002) and pigs (St. John et al., 2005b) and may result in inadequate interaction between the individual subunits of the ETC. This would result in reduced energy production capacity and symptoms common to mitochondrial disease and influence embryo and fetal survival. The question is whether handmade cloning, the fusion of one or more oocyte cytoplasts with a somatic cell (Vajta et al., 2001), would resolve such issues or complicate them. |
The significance of nucleo–mitochondrial interaction |
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