The Reproductive Biology and Genetics Group, Room N102A, The West Wing, The Medical School, University of Birmingham, Birmingham, B15 2TJ, UK. E-mail: j.stjohn.1{at}bham.ac.uk
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Abstract |
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Key words: cytoplasmic transfer/embryonic development/ICSI/mtDNA/oocytes
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Introduction |
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Why might CT be advantageous? |
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Along with well-established findings, that mutation to the mitochondrial genome as well as loss of any or all of the mtDNA genes can have severe if not lethal consequences on a cells survival (Wallace, 1993), recent observations show that IVF outcome is significantly related to mtDNA copy number (Reynier et al., 2001
). To this extent, we have previously argued that older women would have fewer intact, non-deleted copies of mtDNA present in their oocytes resulting from mtDNA susceptibility to the ageing process (St John et al., 1997
). It is particularly evident that both embryos and oocytes do have variable levels of mtDNA rearrangements (Brenner et al., 1998
; Barritt et al., 1999
). In this respect, natural selection mechanisms associated with oogenesis, such as atresia, are presumably designed to prevent poor quality oocytes from being ovulated (Jansen and de Boer, 1998
). The evolutionary perspective of atresia and mitochondrial content, as discussed previously (Krakauer and Mira, 1999
, 2000
), suggests that those species producing fewer offspring are more dependent on atresic regulation ensuring that superior oocytes with high quality mitochondrial genomes are selected for fertilization, as in mammals (Perez et al., 2000
). However, superovulation protocols allow poor quality oocytes to be used for fertilization purposes. It is therefore likely that those oocytes will have either depleted mtDNA copy number or deleted mtDNA. Consequently, in those cases of repeated fertilization failure following coitus, the use of superovulation protocols would promote the availability of poorer quality oocytes, perhaps those with reduced numbers of mitochondria present. If such oocytes are proposed for treatment in the infertility clinic then the disparity that exists in copy number from one oocyte to another suggests there is a rational argument for the supplementation of oocytes with donor mitochondria.
MtDNA copy number is dependent on mtDNA transcription and replication, which in turn are reliant on nuclear-encoded transcription factors being translocated to the mitochondria (Clayton, 1998). The timing of the initiation of transcription and replication of the mtDNA genome is critical. These processes are not fully active until after the blastocyst stage (Piko and Taylor, 1987
). Consequently, the genome will be diluted out at each stage of cell division and the availability of ATP diminished, possibly compromising development and implantation when mtDNA copy number is critically low. To this extent, one particular transcription factor, mitochondrial transcription factor A (mtTFA) has been implicated in mtDNA copy number and regulation and associated with mtDNA-depletion disease. Examples of such disease include 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
). Significantly, mtDNA depletion is also associated with benign and malignant trophoblast diseases (Durand et al., 2001
) indicating a relationship with embryo survival. Interestingly, down-regulation of mtTFA through expression of a testis-specific isoform, localized only to the nucleus during later spermatogenesis (Larsson et al., 1996
), could account for the 10-fold reduction in germ cell mtDNA copy number per haploid cell and one copy per mitochondrion (Hecht and Liem, 1984
).
More specifically, expression of mtTFA and other factors associated with the mitochondrial transcription and replication machinery is particularly critical to embryo survival especially through development and differentiation. In the mouse, heterozygous mtTFA knockout embryos have reduced mtDNA copy number and cardiac respiratory chain deficiency. In contrast, homozygous knockout embryos suffer from severe mtDNA depletion and absent OXPHOS. They also proceed through implantation and gastrulation, but survival is limited to embryonic day 10.5 (Larsson et al., 1998; Li et al., 2000
). This clearly stresses the importance of the timing of transcription and replication of mtDNA and the necessity for adequate copy numbers being present at fertilization. The likely outcome for low copy numbers would indicate that the advanced blastocyst may be compromised and the lag between continual dilution of the genome due to the increased numbers of blastomeres and the switching on of embryonic mtDNA transcription and replication could be vital. However, mtTFA activity is highly dependent on interaction with other DNA replication factors such as DNA polymerase
the polymerase specific to mtDNA (Clayton, 1998
)and nuclear respiratory factor (NRF)-1 (Virbasius and Scarpulla, 1994
). The importance of polymerase
is emphasised by its susceptibility to anti-retroviral drugs used to reduce HIV load in HIV positive patients resulting in mtDNA depletion (Nelson et al., 1997
; Dalakas, 2001
; White et al., 2001
). Further, missense mutations to this polymerase facilitate the onset of progressive external ophthalmoplegia (Van Goethem et al., 2001
), a debilitating mitochondrial disease. Putatively, NRF-1 binds to mtTFA in order to facilitate transcription and replication. Disruption to NRF-1 function results in reduced expression of mtTFA and subsequent mtDNA copy number. Interestingly though, embryos generated from NRF knockout mice only survive to embryonic day 3.56.5, noticeably earlier than mtTFA-/- embryos (Huo and Scarpulla, 2001
).
Consequently, insufficient numbers of mitochondria post-fertilization could compromise embryo development. This is certainly the case for individual blastomeres, which, when deprived of sufficient ATP, undergo apoptosis (Chan et al., 2000; Van Blerkom et al., 2001
). Mitochondria tend to be dynamic during embryo development, providing sufficient energy to accompany cell division. This clustering around the nucleus is in synchrony with each cell division and is followed by dispersal through the cytoplasm post-cell division (Bavister and Squirrell, 2000
; Squirrell et al., 2001
), two events mediated by the microtubule organizing centres (Van Blerkom, 1991
).
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The necessity for caution |
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It is conceivable that the developing embryo regulates transmission of a mothers mtDNA to the oogonia of her daughter (Poulton, 1995). This regulation therefore ensures that transmission is strictly limited to a few molecules present in the oocyte at fertilization. Both random genetic drift (Jenuth et al., 1996
) and the bottleneck hypothesis (Hauswirth and Laipis, 1982
) have been postulated as possible explanations for regulating such segregation to the oocyte. The bottleneck is a restriction event occurring very early during embryogenesis, where a limited number of mitochondria are segregated to those inner cell mass cells destined to become oogonia. This founder population of mtDNA (Marchington et al., 1997
) is subsequently amplified clonally and has been used to account for KearnsSayre syndrome in a patient showing significant levels of rearranged mtDNA molecules in her oocytes (Marchington et al., 1998
) and preferential amplification in another (range 095%) (Blok et al., 1997
). Although the degree of restriction is contestable (Jenuth et al., 1996
), examination of the number of mtDNA copies present at the primordial follicle stage of embryonic development suggests that
10 copies per single cell are present (Jansen and de Boer, 1998
). It is these mtDNA molecules that are transmitted to the next generation, thus restricting transmission, whenever possible, to a homoplasmic wild-type mitochondrion. Other studies limited to the analysis of heteroplasmy and the segregation of mtDNA within primary oocytes reported on a patient with a mtDNA point mutation (Brown et al., 2001
) and from interspecific crossings of mice strains (Jenuth et al., 1996
). These studies suggest that the patterns of transmission are more indicative of random genetic drift.
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What do we know from the manipulation of oocytes and embryos from other species? |
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Little is really understood about the mechanisms associated with transmission. However, studies using cell culture techniques to generate cybrids, a technology that allows enucleated cells to be fused with mtDNA-depleted cells, have shown varying outcomes. For example, it is apparent that the nuclear background of an individual cell type will determine whether wild type or deleted mtDNA molecules are preferentially replicated and transcribed, a clear indication that mtDNA transcription and replication are under the control of the nucleus (Dunbar et al., 1995). Furthermore, cross-species cybrid models show that foreign primate mtDNA will only be amplified in the host cell when its own mtDNA has been eliminated (Moraes et al., 1999
) or at reduced respiratory rates in ratmurine cybrids (Dey et al., 2000
). For those offspring possessing both mtDNA populations, it is important to establish whether donor mtDNA will not out-compete its recipient couterpart.
Transmission of foreign mtDNA is not just restricted to CT. Nuclear transfer (NT) is another advanced technology that can result in transmission of both donor and recipient mtDNA. The fusion of bovine blastomeres to recipient oocytes resulted in transmission of both donor and recipient mtDNA (Steinborn et al., 1998; Hiendleder et al., 1999
; Takeda et al., 1999
). Somatic cell NT can result in the transmission of only recipient mtDNA, for example Dolly the sheep (Evans et al., 1999
) and an interspecific bovine cross (Meirelles et al., 2001
), and in the transmission of both donor and recipient mtDNA (Steinborn et al., 2000
). This disparity in transmission is as reflected in human CT outcome (Brenner et al., 2000
; Barritt et al., 2001a
). Interestingly, mixed populations of oocytes in cattle have led to variable cytoplasmic backgrounds (Takeda et al., 1999
) questioning the true genetic identity of the offspring generated, again a concern for CT. Equally importantly, we are unsure of the consequences of the prevalence of heteroplasmy in offspring as the failure rate in these manipulation processes is disproportionate to the success rate (Wilmut et al., 1997
).
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Where do we go from here? |
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References |
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