Ooplasm donation in humans

The need to investigate the transmission of mitochondrial DNA following cytoplasmic transfer

Justin C. St. John

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


    Abstract
 Top
 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
The use of cytoplasmic transfer as an assisted reproductive technique has generated much attention. This arises as donor mitochondria are introduced into the cytoplasm of the recipient oocyte. The consequences are the possible transmission of two mitochondrial (mt)DNA populations to the offspring. This pattern of inheritance is in contrast to the strictly maternal manner in which mtDNA is transmitted following natural fertilization and ICSI. This paper discusses the advantages of using such a technique to enhance embryonic development from poor quality oocytes with respect to the low copy number of mtDNA found in some oocytes following superovulation protocols. However, it also cautions against using such a technique before a clearer understanding of the patterns of inheritance and transmission of mtDNA has been established and suggests that animal models be utilised to do so.

Key words: cytoplasmic transfer/embryonic development/ICSI/mtDNA/oocytes


    Introduction
 Top
 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
As new technologies are introduced to advance treatments offered to those patients seeking assisted reproduction, there has been considerable debate regarding their overall safety (Stromberg et al., 2002Go). Concerns associated with ICSI are related to those genetic disorders previously considered non-transmissible, such as cystic fibrosis (Wong, 1998Go), Y-chromosome microdeletions (Kent-First et al., 1996Go) and possible outcomes arising from the use of gametes carrying chromosomal abnormalities (Emiliani et al., 2000Go; Ron-El et al., 2000Go; Hennebicq et al., 2001Go; Macas et al., 2001Go). Furthermore, associated learning disabilities and possible developmental abnormalities have also been investigated and discussed with outcomes unresolved (Bonduelle et al., 1998Go; Bowen et al., 1998Go). However, more recent data indicate that the use of surgically obtained sperm as opposed to ejaculated sperm results in a greater risk of gestational hypertension and pre-eclampsia (Wang et al., 2002Go). Furthermore, infants conceived using both ICSI and IVF are at twice the risk of a major birth defect than naturally conceived infants (Hansen et al., 2002Go). Concerns related to aberrant patterns of transmission have not just been restricted to chromosomal genetic material, with several investigations conducted into the fate of sperm mitochondrial (mt)DNA (Houshmand et al., 1997Go; Torroni et al., 1998Go). The most stringent of these investigations concluded that the sperm mtDNA was not transmitted to the offspring following ICSI (Danan et al., 1999Go), as with natural fertilization. However, cytoplasmic transfer (CT), the injection of donor oocyte cytoplasm along with a single spermatozoon, does allow transmission of both donor and recipient oocyte mtDNA to the offspring (Brenner et al., 2000Go; Barritt et al., 2001aGo). This leads us to question some of the profound difficulties associated with this kind of treatment and compels us to revisit the issues of mtDNA transmission, the role of mitochondria post-fertilization and, more specifically, nucleo-mitochondrial intercommunication.


    Why might CT be advantageous?
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 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
Infertility resulting from repeated embryonic development failure is a concern for many couples attending infertility clinics. Male factor infertility can, in some circumstances, be overcome by ICSI. However, ICSI is limited in its use for oocytes failing to respond to IVF and more specifically for those failing to activate post-fertilization. Consequently, Cohen and colleagues proposed the supplementation of oocytes with donor cytoplasm for women with repeated embryonic development failure (Cohen et al., 1997Go) arguing that defective ooplasm could account for infertility in some of these women due to ATP depletion, as observed previously (Van Blerkom et al., 1995Go). Oocytes on average possess 100 000 mitochondria with each oocyte mitochondrion containing a single copy of mtDNA. This circular, 16.6 kb genome encodes 13 of the polypeptides of the electron transport chain (Anderson et al., 1981Go) associated with the process of oxidative phosphorylation (OXPHOS), the cell’s most important ATP-generating pathway (Moyes et al., 1998Go).

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 cell’s survival (Wallace, 1993Go), recent observations show that IVF outcome is significantly related to mtDNA copy number (Reynier et al., 2001Go). 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., 1997Go). It is particularly evident that both embryos and oocytes do have variable levels of mtDNA rearrangements (Brenner et al., 1998Go; Barritt et al., 1999Go). 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, 1998Go). The evolutionary perspective of atresia and mitochondrial content, as discussed previously (Krakauer and Mira, 1999Go, 2000Go), 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., 2000Go). 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, 1998Go). 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, 1987Go). 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., 1994Go), familial mtDNA-associated liver disease (Spelbrink et al., 1998Go), fatal childhood myopathy (Larsson et al., 1994Go), skeletal muscle and mitochondrial encephalomyopathy disorders (Siciliano et al., 2000Go), and ocular myopathy, exercise intolerance and muscle wasting (Tessa et al, 2000Go). Significantly, mtDNA depletion is also associated with benign and malignant trophoblast diseases (Durand et al., 2001Go) 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., 1996Go), could account for the 10-fold reduction in germ cell mtDNA copy number per haploid cell and one copy per mitochondrion (Hecht and Liem, 1984Go).

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., 1998Go; Li et al., 2000Go). 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 {gamma}—the polymerase specific to mtDNA (Clayton, 1998Go)—and nuclear respiratory factor (NRF)-1 (Virbasius and Scarpulla, 1994Go). The importance of polymerase {gamma} 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., 1997Go; Dalakas, 2001Go; White et al., 2001Go). Further, missense mutations to this polymerase facilitate the onset of progressive external ophthalmoplegia (Van Goethem et al., 2001Go), 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 3.5–6.5, noticeably earlier than mtTFA-/- embryos (Huo and Scarpulla, 2001Go).

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., 2000Go; Van Blerkom et al., 2001Go). 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, 2000Go; Squirrell et al., 2001Go), two events mediated by the microtubule organizing centres (Van Blerkom, 1991Go).


    The necessity for caution
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 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
Whilst the arguments in favour of supplementing poor quality oocytes with mitochondria have some scientific justification, CT does not just introduce this vital organelle into the recipient oocyte. In addition to mitochondria, the unpurified cytoplasm consists of various components such as mRNAs and proteins. CT is still an experimental technique that, while offering treatment to some infertile couples, is equally capable of generating unexpected abnormalities. These include reports of 45,XO syndrome resulting in one spontaneous miscarriage and one selective abortion and one case of pervasive development disorder, diagnosed at 18 months of age (Barritt et al., 2001bGo). However, the available data are from a restricted sample size, which effectively reduces the possibility of predicting the frequency of any serious clinical outcomes. In 1997, shortly after the first publication of CT, these investigators were asked to provide further evidence that this technique did not jeopardize the survival of offspring (St John and Barratt, 1997Go). They were further asked to consider several possible outcomes in terms of biological, social and ethical implications: (i) triparental inheritance—the expected transmission of the mother’s and father’s nuclear DNA and mtDNA from the mother and possibly from the donor oocyte; (ii) the necessity of understanding mitochondrial–mitochondrial and nucleo-mitochondrial interactions; and (iii) the necessity of screening oocytes from potential donors (St John and Barratt, 1997Go). In reply to these issues, the investigators advised that donor mtDNA would not be transmitted (Brenner, 1997Go). However, subsequent reports clearly indicated that both donor and recipient mtDNA are being transmitted to the offspring (Brenner et al., 2000Go; Barritt et al., 2001aGo). This biparental transmission violates the strict maternal patterns of mtDNA transmission that arise post-fertilization in the human (Giles et al., 1980Go).

It is conceivable that the developing embryo regulates transmission of a mother’s mtDNA to the oogonia of her daughter (Poulton, 1995Go). 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., 1996Go) and the ‘bottleneck’ hypothesis (Hauswirth and Laipis, 1982Go) 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., 1997Go) is subsequently amplified clonally and has been used to account for Kearns–Sayre syndrome in a patient showing significant levels of rearranged mtDNA molecules in her oocytes (Marchington et al., 1998Go) and preferential amplification in another (range 0–95%) (Blok et al., 1997Go). Although the degree of restriction is contestable (Jenuth et al., 1996Go), 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, 1998Go). 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., 2001Go) and from interspecific crossings of mice strains (Jenuth et al., 1996Go). These studies suggest that the patterns of transmission are more indicative of random genetic drift.


    What do we know from the manipulation of oocytes and embryos from other species?
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 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
The transmission of mtDNA is not just necessarily refined to those copies present in the non-fertilized oocyte. Indeed, studies on mice oocyte manipulation should have forewarned Cohen and colleagues that their technique would violate this strict mtDNA pattern of transmission. Introduction of foreign mtDNA from different strains of mice results in transmission to the offspring. This technique has been used to generate heteroplasmic offspring to study the segregation of mtDNA following fertilization in a variety of tissues. Indeed, the location at which the foreign mtDNA is placed in the recipient cell can also favour the degree of foreign mtDNA transmission (Merielles and Smith, 1998). Transmission has been observed within ranges of 5–80% (Laipis, 1996Go), 0–30% (Jenuth et al., 1996Go) and 16–100% (Meirelles and Smith, 1997Go). This suggests that post-fertilization, any foreign mtDNA introduced can be transmitted to the offspring. Effectively, CT in humans mimics the early mouse experiments.

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., 1995Go). 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., 1999Go) or at reduced respiratory rates in rat–murine cybrids (Dey et al., 2000Go). 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., 1998Go; Hiendleder et al., 1999Go; Takeda et al., 1999Go). Somatic cell NT can result in the transmission of only recipient mtDNA, for example ‘Dolly the sheep’ (Evans et al., 1999Go) and an interspecific bovine cross (Meirelles et al., 2001Go), and in the transmission of both donor and recipient mtDNA (Steinborn et al., 2000Go). This disparity in transmission is as reflected in human CT outcome (Brenner et al., 2000Go; Barritt et al., 2001aGo). Interestingly, mixed populations of oocytes in cattle have led to variable cytoplasmic backgrounds (Takeda et al., 1999Go) 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., 1997Go).


    Where do we go from here?
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 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
To disband CT completely would not resolve the issue of whether this technique could be feasibly employed for treating female factor infertility. There are sufficient means to devise animal models in order to test the effectiveness of this technique. In this respect, animal models have been generated to investigate outcomes related to ICSI in the mouse and Rhesus macaque, a non-human primate, whilst follow-up studies relating to blastomere-donor NT have been restricted to the mouse though Rhesus macaque offspring do exist. If we are to fully appreciate the outcomes associated with embryo manipulation then extensive investigations are required that incorporate genetic, biochemical and physiological analyses, accompanied by clinical and social behavioural monitoring. This is especially so for CT where considerable debate persists regarding the feasibility of employing such a technique and whether an individual cytoplasmic component alone could act as a fertilization trigger for poor quality oocytes. Consequently, these studies in turn may provide considerable insight into oocyte activation per se, with animal models demonstrating the suitability of these techniques for human therapeutic use.


    References
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 Abstract
 Introduction
 Why might CT be...
 The necessity for caution
 What do we know...
 Where do we go...
 References
 
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