Human Reproduction, Vol. 17, No. 8, 2165-2173,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Technical approaches to correction of oocyte aneuploidy
Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA
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Abstract |
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BACKGROUND: This study describes the technical approaches used in treatment of age-related oocyte aneuploidy, the efficiency of each step of nuclear transplantation into mouse and human oocytes, and the ability of germinal vesicle (GV) transplantation to restore artificially induced ooplasmic damage. Finally, it examines the possibility of constructing viable female gametes by transferring diploid somatic cell nuclei into enucleated oocytes. METHODS: GV stage mouse oocytes were collected from unstimulated ovaries, and human GV oocytes were donated from consenting patients undergoing ICSI. Stromal (somatic) cells were isolated from uterine biopsies of consenting patients. Mouse cumulus cells were obtained after ovarian stimulation. GV ooplasts prepared by removing nuclei were transplanted either with GV nuclei or with somatic cells by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were inseminated or analysed cytogenetically. Ooplasmic dysfunction was induced by photosensitization with a mitochondria-specific fluorescent dye. RESULTS: GV transplantation had an overall efficiency of 87 and 73% in the mouse and humans respectively. Maturation rates of 95 (mouse) and 64% (human) following reconstitution were comparable with those in control oocytes, as was the incidence of aneuploidy for five chromosome-specific probes after aneuploidy among the reconstituted oocytes. Photosensitization of oocytes significantly reduced the maturation rate to 4.2%, whereas 61.9% of oocytes matured after transfer of photosensitized GV karyoplasts into healthy ooplasts, with 52% of these mature oocytes being successfully fertilized by ICSI. Enucleated immature oocytes receiving mouse cumulus or human endometrial cell nuclei extruded a polar body in >40% of cases. Five out of seven successfully transferred aged human nuclei exhibited the expected number of signals with five chromosome-specific probes suggesting an appropriate chromosome separation in young ooplasm. CONCLUSIONS: Nuclear transplantation itself does not appear to interfere with chromosome segregation and can possibly rescue oocytes with damaged mitochondria. Finally, immature mouse ooplasm supported separation of somatic chromosomes to expected numbers, implying that haploidization may be occurring. The roles of genetic imprinting and fidelity of chromosome segregation are unknown.
Key words: aneuploidy/cell fusion/in-vitro maturation/nuclear transplantation/oocyte micromanipulation
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Introduction |
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The chances of conception decrease inversely with age, more or less reaching a nadir as early as 40 years of age (Tietze, 1957
). The reason resides primarily in the status of the egg and the conceptus rather than in the endometrium (Munné et al., 1995; Dailey et al., 1996), as demonstrated by higher pregnancy rates in older women receiving donor embryos. Coincidentally, the chance of generating chromosomally abnormal fetuses increases from 6.8% for women aged 35–39 years to ~50% in 45 year old women (Hassold and Chiu, 1985).
A clear relationship exists between oocyte ageing and the non-disjunction of bivalent chromosomes during meiosis (Dailey et al., 1996). It has been suggested that such ageing compromises the ability of the meiotic apparatus to direct a balanced chromosome segregation (Battaglia et al., 1996; Volarcik et al., 1998), and this effect has been linked indirectly to a suboptimal perifollicular circulation (Gaulden, 1992; Van Blerkom, 1996; Van Blerkom et al., 1997) that might compromise oocyte mitochondria (Beermann et al., 1988; Van Blerkom, 1994). Indeed, mutations in mitochondrial DNA (mtDNA) have been observed in the oocytes of older women (Shigenaga et al., 1994; Keefe et al., 1995; Barritt et al., 2000). Attempts to improve the chance of achieving a pregnancy in women who are at increased risk for oocyte aneuploidy have involved selection of oocytes and embryos by preimplantation genetic diagnosis (PGD) (Gianaroli et al., 1997; 1999; Munné et al., 1999; Verlinski et al., 1999). Two further logical ways of avoiding oocyte aneuploidy would be cryopreservation of younger mature oocytes (van Uem et al., 1987; Chen, 1988; Porcu et al., 1997) or of the entire ovarian cortex taken at a younger age (Gosden et al., 1994; Newton et al., 1996; Oktay and Gosden, 1996; Oktay et al., 1998).
It has been suggested (Zhang et al., 1999) that the transfer of a germinal vesicle (GV) from an aged oocyte into a younger ooplast might form an additional approach to prevention of aneuploidy. In such a case, nuclear transplantation needs to be performed at the GV stage prior to the segregation of chromosomes, the younger cytoplasm providing a healthy spindle and so allowing normal chromosomal segregation during meiosis.
It is clear that nuclear transplantation can be accomplished efficiently in the mouse without affecting oocyte maturation or the incidence of chromosomal abnormalities (Takeuchi et al., 1999a). Therefore, utilizing mouse and spare immature human oocytes, we have evaluated the efficiency of the nuclear transfer approach and its success in terms of oocyte maturation and fertilizability as well as early embryonic development. Some grafted oocytes were subjected to cytogenetic analysis.
In addition, we have evaluated whether germinal vesicle (GV) oocytes with damaged photoirradiated mitochondria could be rescued by nuclear transplantation. In an attempt to overcome the poor quality of oocytes in the ovaries of women aged >40 years, we have explored the possibility that transferred somatic cell nuclei will undergo normal haploidization within young donor enucleated oocytes (ooplasts).
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Materials and methods |
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Oocyte collection
GV oocytes were retrieved by puncturing unstimulated ovarian follicles of 7–11 week old B6D2F1 female mice. Cumulus–corona cells were removed by repeated aspiration through the tip of a hand-drawn pipette. In order to prevent spontaneous germinal vesicle breakdown (GVBD), oocytes were cultured for ~2 h in M199 (Sigma Chemical, St Louis, MO, USA) supplemented with 0.2 mmol/l 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor (Sigma Chemical), until they exhibited a perivitelline space. Immature oocytes also were maintained in these conditions.
Human GV oocytes were obtained from consenting patients undergoing ICSI. The procedures as well as the handling of patient material were performed in accordance with a research protocol approved by the Committee of Human Rights of New York Presbyterian Hospital–Weill Medical College of Cornell University (IRB No. 0198-082). Oocyte retrieval was performed after treatment with a GnRH agonist and gonadotrophins, as described previously (Palermo et al., 1995, 1996a,b). After cumulus–corona cells were removed by enzymatic and/or mechanical treatment, the denuded oocytes were examined under an inverted microscope to assess their condition and maturational stage.
Micromanipulation settings
Micromanipulation was performed in a shallow plastic Petri dish (Model: 1006, Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA) where 5 µl droplets of M2 medium supplemented with the 0.2 mmol/l IBMX were used for mouse oocytes. However, for procedures involving human oocytes, M2 medium was substituted with HTF–HEPES medium supplemented with 0.4% human serum albumin (HSA; Irvine Scientific, Santa Ana, CA, USA). Micromanipulation using a micromanipulator and two microinjectors (Model: IM-6; Narishige, New York/New Jersey Scientific Inc.) was performed on the heated stage (Eastech Laboratory, Centereach, NY, USA) of an inverted microscope (Olympus IX-70, New York/New Jersey Scientific Inc.) equipped with two electrical/hydraulic micromanipulators (Model: MM-188 and MO-109; Narishige, New York/New Jersey Scientific Inc.).
Enucleation, reconstitution and electrofusion of oocytes
Enucleation of mouse and human oocytes was performed as described elsewhere (Takeuchi et al., 1999a, 2001). Briefly, after oocytes were exposed to cytochalasin B (Sigma Chemical), a slit was made in the zona pellucida with a glass microneedle and a GV karyoplast was removed with a micropipette of 20 µm inner diameter (mouse) or 30 µm inner diameter (human) (Figure 1). Subsequently, the GV karyoplast (Figure 2) was inserted into the perivitelline space of another enucleated oocyte to constitute a grafted oocyte (Figure 3).
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Grafted mouse oocytes were washed in fresh medium to remove cytochalasin B, exposed to IBMX to inhibit maturation, and cultured until electrofusion. Using an Electro Cell Manipulator (BTX 200 and 2001; BTX Inc., San Diego, CA, USA), each grafted oocyte was aligned between two micro-electrodes of 100 µm diameter (ECF-100; Tokyo Rikakikai Co. Ltd, Tokyo, Japan), and a single direct pulse was delivered to induce fusion (Takeuchi et al., 1999a
) (Figure 4). Up to four electrical pulses were applied at 30 min intervals, and after washing and culture for 30 min the oocytes were examined (Figure 5). Extrusion of the first polar body (PB) was assessed 12–16 h later in the mouse, and 24 h later in human oocytes.
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Cytogenetic analysis
Mouse oocytes that had extruded the first PB were prepared for karyotyping by treatment in a hypotonic solution for 5 min, and then in three different fixatives (Takeuchi et al., 1999a
). Finally, oocytes were stained with Giemsa for visualization of their chromosomes. In the case of human oocytes, a sequential fluorescent in-situ hybridization (FISH) procedure was performed utilizing probes for chromosome 13, 16, 18, 21 and X (Takeuchi et al., 2001).
ICSI of GV-transplanted oocytes
The use of reconstituted mouse oocytes was abandoned because <10% survived the ICSI procedure. Human reconstituted oocytes matured in vitro were subjected to ICSI (Palermo et al., 1995, 1996b) 3–5 h after PB extrusion, and were examined 16–20 h later for the presence of two distinct pronuclei and two PB. To further evaluate their ability to cleave, zygotes were cultured for up to 72 h.
Simulation of ooplasmic damage and rescue attempt by nuclear transplantation
To induce mitochondrial damage, GV oocytes were cultured for ~30 min in M199 supplemented with IBMX containing 500 nmol/l of chloromethyl-X-rosamine (CMXRos, MitoTracker® Red; Molecular Probes, Eugene, OR, USA), washed in M199 supplemented with IBMX, then exposed to UV light for 10 s. Control oocytes were exposed either to CMXRos alone or to photoirradiation alone. All the micromanipulation and electrofusion procedures were carried out as described above, but under dim light. Then, GV nuclei were transferred to irradiated oocytes and to non-irradiated oocytes (controls). This provided material in which (i) only the karyoplast, or (ii) only the ooplast, had been photosensitized.
Successfully reconstituted oocytes were cultured for 14–16 h in M199 to allow nuclear maturation as judged by PB formation. Changes in the morphology and distribution patterns of the mitochondria were identified according to the pattern of fluorescent signals in the photosensitized GV-arrested oocytes. Cytosolic damage was assessed according to dilatation and clustering of mitochondria, characteristic of the initial stage of cell apoptosis (Minamikawa et al., 1999).
Somatic cell haploidization
Cumulus cells were obtained by brief exposure to hyaluronidase (100 IU/ml) of cumulus–oocyte complexes from pregnant mare's serum gonadotrophin/HCG-stimulated B6D2F1 mice. These cells were then cultured for up to 30 days with several passages in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies). Human endometrial cells were obtained from biopsies of consenting patients undergoing endometrial cell co-culture during IVF (IRB No. 1195-099). Stromal and glandular cells were isolated by enzymatic digestion using 0.2% collagenase type II and separated by differential sedimentation (Barmat et al., 1998), then stromal cells were cultured in DMEM medium supplemented with 10% FBS.
As mentioned, mouse ooplasts were prepared by enucleating GV oocytes. The human endometrium stromal cells and mouse cumulus cells (Figure 6) were freed from the culture dish by a standard trypsin–EDTA procedure. One or the other cell type was then inserted subzonally into an enucleated oocyte (Figure 7), and subjected to electrofusion. In reconstituting eggs, the latter was preferred over direct injection because of the fragility of the GV stage ooplasts. The resultant reconstituted oocytes were cultured for 14–16 h until extrusion of the first PB. To assess the distribution of their chromatin, some such oocytes were stained with 0.5 µg/ml Hoechst 33342 solution and evaluated under a fluorescent microscope, while others were fixed beneath a coverslip with methanol/acetic acid (3:1; v/v), and stained with 1% aceto-orcein solution (Figure 8). Other oocytes were processed for karyotyping with Giemsa staining (Figure 9), or were subjected to FISH analysis as described above.
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Data analysis
The Pearson
2-test was utilized for discrete univariate and bivariate data, except where test assumptions were violated, necessitating Fisher's exact test. Statistical significance was stipulated as 0.05 for discrete and continuous analysis. All statistical computations were conducted using the Statistical Analysis System (SAS Institute, Cary, NC, USA). Statistical comparisons were reported only when they reached significance. |
Results |
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Efficiency of nuclear transfer
Of 110 mouse GV oocytes, 96 (87.3%) were successfully enucleated and reconstituted with GV, and of those 91 (94.8%) had reached the metaphase II (MII) stage after 12–16 h in vitro. Moreover, 29 out of 30 (96.7%) tested proved to have a normal karyotype.
A total of 287 immature human oocytes were obtained from 144 patients of 34.6 ± 4 years. As controls, 80 of these were simply cultured in HTF–HSA medium. Due to the limited availability of human material, in most cases enucleated GV were exchanged between sibling oocytes. The results of GV transplantation in 207 oocytes are shown in Table I.
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In the case of human oocytes, 72.9% of 207 were successfully reconstituted with a GV and 64.2% of these reached MII in culture. Among 80 control oocytes, 63.8% were able to extrude a first PB with 23.8% arrested at GV stage and 12.5% at metaphase I.
To identify any deleterious effect of nuclear transplantation, a total of 16 GV-manipulated and 17 control oocytes were analysed by FISH for 13, 16, 18, 21 and X chromosomes. Similar normal complements were observed in 11 out of 14 (78.6%) manipulated oocytes (maternal age 31.6 ± 3 years) and in 11 out of 15 (73.3%) control oocytes.
In order to assess the contribution of the cytoplasmic replacement, 30 GV karyoplasts generated from 15 `aged' (37–43 years) and from 15 `young' (28–32 years) oocytes were reciprocally transplanted into cytoplasts of the opposite age. A total of 24 out of 30 oocytes (80.0%) successfully reconstituted and 19 (63.3%) extruded a first PB. Among seven oocytes where an aged karyoplast (40.0 ± 2 years) had been inserted into a young ooplast (29.8 ± 2 years) five displayed a normal karyotype, whereas nine of 12 young karyoplasts (31.4 ± 2 years) maturing in older ooplasm (38.1 ± 3 years) were abnormal.
Fertilization and early development of human oocytes after ICSI
The survival and fertilization of reconstituted (n = 32; 33.2 ± 4 years) and control (n = 34; 34.1 ± 4 years) MII oocytes are shown in Table II. After three days, 16 of 17 normally fertilized, GV-transplanted oocytes had cleaved, with a mean (± SD) blastomere number and anucleate fragment frequency of 5.0 ± 2 and 24.1 ± 15 respectively. When 22 zygotes generated from non-manipulated oocytes were incubated for the same incubation period, 16 (72.7%) underwent cleavage. The average number of blastomeres and rate of fragmentation at day 3 was 4.8 ± 2 and 35.0 ± 26% respectively. Thus, embryonic development of the manipulated oocytes was comparable to that in the controls.
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`Rescue' by nuclear transfer
Exposure to CMXRos alone or to simple irradiation alone did not affect in-vitro maturation rates, whereas exposure to both CMXRos + photoirradiation (`photosensitization') significantly inhibited maturation of the oocytes (P < 0.001) (Table III
), a majority of which displayed membrane blebbing, degeneration, and altered mitochondria typical of the initial stages of apoptosis.
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Oocytes reconstituted with non-photosensitized GV achieved a reconstitution rate of 84% and a maturation rate of 97%. By contrast, oocytes reconstituted from an irradiated nucleus or from an irradiated ooplast showed extensive cell lysis after electrofusion (P < 0.01) (Table IV
). Moreover, 62% of oocytes successfully reconstituted from a photosensitized karyoplast and intact ooplast underwent maturation, as reflected in extrusion of the first polar body, compared to 21% in oocytes reconstituted with photosensitized ooplasts, and to 4% in photosensitized non-manipulated oocytes (Table III
).
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Somatic cell haploidization
The rates of cell survival, reconstitution and subsequent PB extrusion using human endometrial or mouse cumulus cells in intact GV enucleated mouse oocytes, are shown in Table V
. After up to 16 h in culture, oocytes showed metaphase chromosomes in the ooplasm, and in the corresponding PB. Overall, 60% (n = 6) developed as reconstituted oocytes with an extruded PB after endometrial cell fusion, and 42.8% (n = 15) after mouse cumulus cell fusion. Of five oocytes that had extruded PB and were analysed cytogenetically, four revealed 20 chromosomes as expected for a haploid complement.
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Finally, in analysis of the behaviour of transplanted somatic cell nuclei, 77 out of 78 mouse GV oocytes were successfully enucleated and grafted with a single cumulus cell, 56 being reconstituted with somatic cell nuclei after fusion. After 14–16 h of culture, 51% had extruded a PB, and 13 oocytes at MII with extruded PB proved to be analysable; three (23.1%) had diploid metaphase chromosomes, five (38.5%) were abnormal (two hyperhaploid and three chromatid breakage), and five (38.5%) revealed 20 chromosomes as expected for a normal haploid constitution.
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Discussion |
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Our results demonstrate that GV nuclear transplantation of immature human oocytes can be accomplished with an efficiency of >80% for each step (Table I
), their final in-vitro maturation rate of 64% being similar to that of non-manipulated GV oocytes. Moreover, oocytes reconstituted by nuclear transplantation clearly can be fertilized normally by ICSI. Thus, the rates of oocyte survival, fertilization and early development do not differ from those in non-manipulated, in-vitro matured oocytes. Since all the oocytes used were exposed to HCG in vivo before collection, and were denuded of corona cells prior to GVBD, these were not isolated and cultured in an ideal setting for maturation (Janssenswillen et al., 1995; Goud et al., 1998). In fact, this is an imperfect technique, and only a few deliveries from such immature oocytes have been reported so far (Nagy et al., 1996; Edirisinghe et al., 1997).
Nonetheless, on the basis of our results, this model can be used to study the relationship between cytoplasmic ageing and oocyte aneuploidy. Substitution of an old cytoplasm with a younger one appears promising as a way of reducing the incidence of aneuploidy due to non-disjunction in older women. However, no one oocyte stage has all the ideal attributes for this. MII cytoplasts are more fusogenic, more easily manipulated, and have greater potential for development, but they cannot bring about maturation of the transplanted GV. On the other hand, those prepared from GV oocytes do support GV maturation, but they are less fusogenic and may be functionally impaired by coronal cell removal (Takeuchi et al., 1999a). Another aspect that needs to be evaluated further is the role of residual maternal mtDNA and its eventual influence on the constructed genome (Keefe et al., 1995; Brenner et al., 1998; Van Blerkom et al., 1998).
The incidence of aneuploidy appears to be 25–35% in human oocytes maturing in vivo (Plachot et al., 1988; Pellestor, 1991; Van Blerkom, 1994), and was reported as 25% also among cumulus-enclosed immature oocytes cultured in vitro in a specific medium supplemented with gonadotrophins (Park et al., 1997). Our preliminary cytogenetic evaluations suggest that nuclear transplantation itself does not increase the incidence of aneuploidy, and that younger ooplasm can support normal meiotic division of older GV (Zhang et al., 1999), but conversely, older ooplasm tends to induce an abnormal segregation of meiotic chromosomes.
In summary, our findings support the current view that ooplasmic factors finally ordain the balanced segregation of bivalent chromosomes during anaphase of meiosis I. An explanation for defective segregation may lie in reactive oxygen species (ROS) damage to mitochondria with consequent reduction in ATP levels (Tarín, 1995; Tarín et al., 1998). As another possibility, effects on the integrity of the meiotic spindle of maturing oocytes may be attributed to an age-related follicular hypoxia and so to a lower intracellular pH coming from an impaired perifollicular microcirculation (Gaulden, 1992; Van Blerkom et al., 1997). Although our inter-patient cross-transplantation was too limited to draw definite conclusions, this technique did result in a reconstitution of hybrid immature oocytes at a rate of 73%, with ~64% maturing normally.
In attempting to mimic ooplasmic `incompetence', we have developed a novel strategy involving mitochondrial photosensitization using CMXRos, which consistently inhibits the maturation of immature mammalian oocytes. One group (Minamikawa et al., 1999) found that CMXRos photosensitizes mitochondria in living cells within 20 min, inducing their swelling due to an altered permeability and electrical depolarization of the mitochondrial inner membrane, and eventually leading to apoptosis (Salet and Moreno, 1990). Generally, photosensitization involves absorption of light by the chromophore after its uptake into the cell, such that the energy due to excitation of outer shell electrons is transferred through molecular activation generating ROS (Foote, 1968). Thus, ROS may damage mitochondrial DNA, proteins, and lipids resulting, with time, in fewer functional mitochondria and decreased ATP levels (Tarín, 1995).
Recently, Tarín et al. (1996) have postulated that induction of age-related aneuploidy during oocyte development involves oxidative stress, and they have demonstrated that antioxidant therapy can prevent associated abnormalities in oocyte chromosomal distribution and segregation (Tarín et al., 1998). However, any ability of antioxidant therapy to do this is only prophylactic, as is the counteracting effect of antioxidants on thiol oxidation-induced apoptosis in mouse 1-cell zygotes (Liu et al., 1999). Thus while oocyte damage induced by oxidants is preventable by coincident antioxidant therapy, once the apoptosis is initiated it seems to be impossible to reverse.
Although the potential for maturation was not compromised when GV were transferred to non-photosensitized oocytes (Takeuchi et al., 1999a), meiosis resumption was significantly reduced in reconstituted oocytes generated from photosensitized ooplasts. The ability of nuclear transplantation to `rescue' in this case is only partial, perhaps because some damaged mitochondria are introduced with the photosensitized karyoplast. Thus, it is possible that the outcome can be negative, even where only a small proportion of the oocyte mitochondria are damaged.
In conclusion, although these avenues need to be explored further, we have been able to simulate oocyte damage in the ovary of ageing patients by photosensitization, thus providing a model which permits assessment of the potential for nuclear transplantation to `rescue' oocytes in such cases. However, before we apply these techniques to the clinic, critical aspects such as the role of mtDNA in development of the resulting embryos (Tsai et al., 1999), and the availability and viability of donor ooplasm, need to be pursued further.
Recent reports suggesting haploidization of cumulus cells and fibroblasts (Lacham-Kaplan et al., 2001; Tesarik et al., 2001), suggest that this ability is retained by the cytoplast of an enucleated MII oocyte. In contrast to GV ooplasm, mature oocytes require activating stimuli by spermatozoa or artificial activation to accomplish haploidization of transplanted somatic nuclei, following which fertilization can occur with development as far as the blastocyst stage (Lacham-Kaplan et al., 2001; Tesarik et al., 2001). The present study supports notions that a GV ooplast can stimulate a somatic interphase nucleus to undergo a reductive segregation to normal chromosome numbers (Kubelka and Moor, 1997). Mouse GV oocytes reconstituted with somatic cells displayed a lower rate of maturation compared to the 92% obtained after reconstitution with GV (Takeuchi et al., 1999a,b; Tsai et al., 2000). However, in successful cases the timing of PB extrusion during culture was similar to that in the untreated GV oocyte population.
In the present case, where the embryonic nuclear genome is created as a blend of the male gamete genome and of a presumably haploidized somatic cell genome, one major concern is the risk of imprinting abnormalities (Trounson, 2001). In somatic cells, some alleles are imprinted either maternally or paternally. Moreover, the imprinted status of the somatic cell nucleus may be subjected to trans-modification of germ cell cytoplasm, as demonstrated in germ–somatic hybrids (Surani, 1999), or may be affected by epigenetic events in the early embryo (Reik et al., 1993). The risk in the present case may be less than in conventional cloning because at least one allele of each chromosome originates directly from a gamete. Monoallelic expression of imprinted genes is maintained by epigenetic regulatory mechanisms such as DNA methylation and histone acetylation (Monk et al., 1987; Surani et al., 1998). Gametes presumably need a full set of imprinted genes, that is a complete maternal or paternal epigender, for normal offspring. The quality of the maternal and paternal genomes is determined by the epigenetic marks on its imprinted alleles, but the extent to which the genome's epigender is reflected in a gene expression pattern or a developmental phenotype depends on the cellular environment (Spielman et al., 2001). Both the epigender of the somatic nucleus and the status of the recipient cell would affect the outcome of the manufactured oocyte. Therefore the effect of artificial gamete production on imprinting is still unknown.
Although the overall efficiency of chromosome segregation leading to the expected numbers for haploidization appears to be limited at present, its success will be measured by the ability to produce normal haploid oocytes and healthy progeny and whether this progeny is normal. Nonetheless, we conclude that nuclear transplantation does offer the prospect of creating reconstituted oocytes, for studies on regulation of chromosome segregation and nuclear cytoplasmic interactions during gametogenesis.
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Acknowledgements |
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We thank all ICSI patients that generously donated their immature oocytes for the completion of this work. We are very appreciative to all clinicians and scientists of Cornell Institute for Reproductive Medicine for assistance with this study. We are most grateful to Professor J.Michael Bedford for his critical scientific advice and to Ms Queenie V.Neri for editing the manuscript.
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1 To whom correspondence should be addressed. E-mail: gdpalerm{at}med.cornell.edu
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Submitted on August 8, 2001; resubmitted on December 13, 2001; accepted on March 7, 2002.
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