Human Reproduction, Vol. 15, No. 5, 1149-1154,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes
1 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2 MAR & Molecular Assisted Reproduction and Genetics, Granada, Spain, 3 CIVTE, Centre of Insemination In Vitro and Embryo Transfer, Sevilla, Spain, 4 Centre for Reproductive Medicine, European Hospital, Rome, Italy and 5 Department of Biochemistry and Molecular Biology, University of Granada Faculty of Sciences, Granada, Spain
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Abstract |
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Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.
Key words: human oocyte/membrane fusion/nuclear transfer/oocyte activation/polyethylene glycol
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Membrane fusion techniques were introduced to experimental embryology in the early 1970s when they were used to fuse together early embryonic blastomeres and to study the effects of the resulting tetraploidy on development. The originally designed fusion methods using inactivated Sendai virus (Graham, 1971
) were progressively replaced with less toxic treatments using polyethylene glycol (PEG) (Eglitis, 1980; Spindle, 1981), a chemical fusogen which had previously proven useful for fusing plant protoplasts (Kao and Michayluk, 1974) and for the production of somatic cell hybrids (Pontecorvo, 1975). More recently, cell fusion has been increasingly used for nuclear transfer in mammalian oocytes and its special application – mammalian cloning (Campbell et al., 1996; Wilmut et al., 1997; Wakayama et al., 1998; Wells et al., 1999). In most recent studies, the chemical fusion protocols have been replaced with the more rapid and easily reproducible electrofusion, although a fusion protocol using PEG has been shown to be at least as effective as electrofusion in producing cloned calves by transfer of nuclei from cultured inner cell mass cells (Sims and First, 1993).
Recent experience with human oocytes suggests specific problems with the use of electrofusion in this species. Unlike mouse (Takeuchi et al., 1999) and bovine oocytes (Stice et al., 1996; Wells et al., 1999), human oocytes are almost invariably activated by the electrofusion procedure (Cohen et al., 1998). This represents a serious limitation to the use of standard electrofusion techniques in clinically assisted reproduction treatment protocols aimed at improving embryo quality by nuclear transfer into enucleated donor oocytes (Cohen et al., 1998). In a more remote perspective, this might also be relevant to the research into eventual medical applications of human cloning because recent data show an improvement of cloning efficacy by prolonging the exposure of somatic cell nuclei to metaphase II cytoplasm, apparently by allowing better nuclear reprogramming (Cibelli et al., 1998; Wells et al., 1999).
In this study, we evaluated the ability of a modified PEG treatment protocol to induce fusion between two intact zona-free human oocytes and between enucleated metaphase II oocytes, on the one hand, and metaphase II karyoplasts on the other hand. The effect of the PEG treatment on the cell cycle of oocytes and the subsequent capacity of the reconstituted oocytes to be artificially activated were also examined. Finally, a novel, mechanical technique of inducing cell fusion was tested.
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Materials and methods |
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Source of oocytes
Metaphase II human oocytes were obtained from three different sources. First, oocytes that failed to fertilize after conventional IVF attempts and that were donated for this research by consenting patients were used in individual experiments between 24 and 28 h after in-vitro insemination. Second, oocytes that were shown to be meiotically immature (germinal vesicle or metaphase I stage) after the removal of cumulus and corona cells during preparation for intracytoplasmic sperm injection (ICSI) and thus had to be excluded from clinical use but reached metaphase II during subsequent culture (Tesarik and Mendoza, 1995
). Third, a limited number of fresh metaphase II oocytes were donated by some IVF and ICSI patients in whom high numbers of mature oocytes were recovered and who did not wish embryo cryopreservation. In all cases, oocyte donors and their husbands gave their informed consent for the use of their oocytes in these experiments.
Chemicals and culture media
Embryo-tested proteinase from Streptomyces griseus (pronase), 6-dimethylaminopurine (6-DMAP), cell culture-tested lectin from Phaseolus vulgaris (phytohaemagglutinin), PEG (average molecular weight: 1000), cytochalasin B, ionophore A23187, bis benzimide (Hoechst 33342) and dimethylsulphoxide (DMSO) were purchased from Sigma (St Louis, MO, USA). Gamete-100 and IVF-50 media, and embryo-tested mineral oil were from Scandinavian IVF (Gothenborg, Sweden).
Chemical fusion of zona-free oocytes
To dissolve the zona pellucida, oocytes were incubated at 37°C in a solution of pronase (1 mg/ml in PBS) for 2–3 min. After transfer to Gamete-100 medium, remnants of the zona were removed mechanically by repeated aspirations into a finely drawn Pasteur pipette. Pairs of zona-free oocytes were put in contact with each other in sterile watch glasses with IVF-50 medium supplemented with 300 µg/ml phytohaemagglutinin and incubated at 37°C under 5% CO2 in air for 1 h to achieve agglutination. Agglutinated pairs of zona-free oocytes were placed, for 45 s, in a solution of PEG (0.9 g/ml) in Gamete-100 medium, followed by brief sequential washings in 4.5 g/ml PEG, 2.25 g/ml PEG, and three times Gamete-100 medium alone. Pairs of oocytes were then left in Gamete-100 medium for an additional 1 h, during which they were repeatedly observed for signs of fusion.
Chemical fusion of karyoplasts with enucleated oocyte cytoplasts
Zona-intact metaphase II oocytes were first incubated at 37°C with 7.5 µg/ml cytochalasin B in Gamete-100 medium. Pairs of oocytes were then placed in individual drops of Gamete-100 medium under mineral oil for micromanipulation which was performed in an inverted microscope (Nikon or Zeiss) equipped with Hoffman modulation contrast optics, a Narishige micromanipulator set (Tokyo, Japan) and a Fertilaze laser zona-drilling equipment. One of the two oocytes in each drop was immobilized by suction on the holding pipette with the polar body located on the side opposite to the point of fixation, and a hole of ~20 µm was laser-drilled in the polar body area. A blastomere-separation pipette (Humagen Fertility Diagnostics, Charlottesville, VA, USA) was introduced through the drilled hole, and the polar body, together with the adjacent oocyte cytoplasm presumably containing metaphase II chromosomes, was gently aspirated into the pipette (Figure 1A). Care was taken to maintain the integrity of the plasma membrane surrounding the aspirated nucleated part of the oocyte (karyoplast) as well as that surrounding the enucleated rest of the oocyte (cytoplast) during this manipulation. The cytoplasmic bridge connecting the karyoplast with the cytoplast was interrupted by forcing it against the margins of the hole previously drilled in the zona pellucida. Both the karyoplast and the polar body were then expelled from the blastomere-separation pipette near the manipulated oocyte, and the whole procedure was repeated with the other oocyte in the drop. The karyoplast originating from the one oocyte was then inserted into the other oocyte through the previously made hole in the zona pellucida and vice versa (Figures 1B and 1C).
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Each enucleated oocyte cytoplast containing a karyoplast originating from another oocyte in the perivitelline space was then incubated for 1 h (37°C, 5% CO2 in air) in IVF-50 medium containing 300 µg/ml phytohaemagglutinin to obtain agglutination between both entities. Agglutinated karyoplast–cytoplast pairs were induced to fuse by treating them with PEG using the same protocol as for the fusion of whole zona-free oocytes (see previous section).
Mechanical fusion of karyoplasts with enucleated oocyte cytoplasts
Oocyte enucleation in this experiment was done by using two alternative techniques. A fraction of the oocytes were enucleated through a laser-drilled hole in the zona pellucida as described in the previous section, whereas other oocytes were enucleated through a slit previously cut in the zona pellucida by transpiercing it at two adjacent points (~20 µm from each other) with a finely drawn glass microneedle and opening it by rubbing the area between both puncture sites against the holding pipette. This technique was essentially the same as the partial zona dissection originally designed to assist penetration of spermatozoa through the zona pellucida in human assisted reproduction (Malter and Cohen, 1989). In each group, some of karyoplast–cytoplast pairs were incubated for 1 h with 300 µg/ml phytohaemagglutinin (as above), whereas other karyoplast–cytoplast pairs were processed further without this step.
The karyoplast–cytoplast pairs were immobilized by suction on the holding pipette with the larger part (enucleated oocyte cytoplast) oriented towards the holding pipette so as to visualize the hole previously drilled in the zona pellucida on the opposite side (Figure 2). An ICSI micropipette (Humagen) was then introduced through the hole in the zona pellucida, first into the karyoplast and then further into the cytoplast. The path of the micropipette was chosen to avoid the central region of the karyoplast where chromosomes were mostly located (Figure 2), as determined by independent experiments in which karyoplasts were stained with Hoechst 33342 (Z.P.Nagy et al., unpublished data). When the tip of the ICSI micropipette was positioned deep in the cytoplast, ~3 pl of cytoplasm was aspirated in a similar way as for the standard ICSI procedure using vigorous cytoplasmic aspiration (Tesarik and Sousa, 1995), and the micropipette was moved back to the karyoplast where the aspirated cytoplasm was released. Thereafter, the same volume of cytoplasm was aspirated from an adjacent karyoplast region, the micropipette was pushed forward to the cytoplast again, and the aspirated cytoplasm was released there. The micropipette was then withdrawn, and the procedure was repeated with another karyoplast–cytoplast pair. After this manipulation, karyoplast–cytoplast pairs were left in Gamete-100 medium for an additional 1 h, during which time they were repeatedly inspected for signs of fusion.
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Activation of reconstituted oocytes
Reconstituted oocytes were kept in IVF-50 medium equilibrated with 5% CO2 in air for 4 h after the detection of the first signs of fusion. They were then exposed for 10 min at 37°C to a solution of 10 µmol/l ionophore A23187 in Gamete-100 medium (prepared from a 2 mmol/l stock solution in dimethylsulphoxide), washed three times in fresh Gamete-100 medium and incubated for 3 h in IVF-50 medium supplemented with 2 mmol/l 6-DMAP (37°C, 5% CO2 in air). Control oocytes were treated with the corresponding concentration of dimethylsulphoxide in culture medium alone and were not incubated with 6-DMAP. Oocytes were inspected several times between 10 h and 24 h after ionophore treatment for signs of activation (development of pronuclei).
Statistics
Percentages of fused and activated oocytes within different treatment and control groups were compared by 
2 and Kruskal–Wallis tests.
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Results |
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Efficiency of the chemical and mechanical fusion techniques
When pairs of whole zona-free oocytes, previously agglutinated with phytohaemagglutinin (Figure 3A
) were treated with PEG, the first signs of fusion usually appeared between 10 and 20 min after treatment. At the origin, they could be recognized as focal losses of the intactness of the plasma membrane of both oocytes at the site of attachment. Soon thereafter, the zone of tight contact between the oocytes began to enlarge progressively (Figures 3B and 3C), so that the original outline of the two oocytes was lost, and the oocyte pair attained an oval form (Figure 3D). The long and the short diameters of the resulting fusion product then approached each other until the cell attained the definitive round form. This process was usually completed within the first hour after the exposure of oocyte pairs to PEG. The efficiency of fusion between two whole oocytes was >50% in this system (Table I).
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When the chemical fusion technique was applied to enucleated oocyte cytoplasts paired with karyoplasts, a similar sequence and timing of events was observed (Figure 4
). After the appearance of the first signs of fusion (Figure 4A), the contact area between the fusing cells showed a stepwise increase (Figures 4B and 4C) until the karyoplast became entirely incorporated into the larger cytoplast (Figure 4D).
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The efficiency of the mechanical fusion technique was dependent on the preincubation of karyoplast–cytoplast pairs with phytohaemagglutinin. Without this preincubation, the manipulation led to immediate degeneration of nine karyoplast–cytoplast pairs out of 10 treated. In contrast, when used after previous tight agglutination between the karyoplast and the cytoplast, the mechanical technique gave a similar yield (>80%) of surviving fusion products as compared to the chemical technique (Table I
).
Interestingly, most of the karyoplast–cytoplast pairs that came from the group of oocytes whose zona pellucida had been opened by laser-drilling and that either did not survive after the manipulation or failed to fuse showed a partial or total escape of the karyoplast through the previously drilled hole in the zona. This condition was prevented by using the mechanical technique of zona pellucida opening instead of laser-drilling. The survival and fusion efficiency also tended to be higher in this group (Table I), but this difference was not statistically significant.
Spontaneous and induced activation of reconstituted oocytes
Out of 15 tetraploid cells resulting from PEG-induced fusion of zona-free oocytes that were not treated for artificial oocyte activation, only one became spontaneously activated during subsequent 24 h of culture. On the other hand, 12 of 14 fused oocytes (86%) treated with ionophore A23187 and 6-DMAP showed signs of activation during subsequent 24 h. The spontaneous activation of oocytes resulting from fusion of enucleated oocyte cytoplasts with karyoplasts was also a rare event, irrespective of the fusion technique used. However, the reconstituted oocytes were easily activated by the combined treatment with ionophore A23187 and 6-DMAP (Table II). No difference in the ability to undergo induced activation was detected between reconstituted oocytes resulting from the two fusion techniques (P > 0.05).
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Discussion |
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The present data show that the application to mature human oocytes of a chemical fusion protocol based on a short exposure to PEG of pairs of karyoplasts and enucleated oocyte cytoplasts, previously agglutinated by phytohaemagglutinin, leads to a high yield of fused cells without causing oocyte activation. These high yields are in agreement with earlier studies using PEG to fuse mouse embryo blastomeres (Eglitis, 1980
; Spindle, 1981) and mouse oocytes at the germinal vesicle (Fulka Jr et al., 1995) or metaphase I stage (Fulka Jr et al., 1997). Moreover, this technique appears to be essentially free of harmful effects on fused cells because fused mouse blastomeres easily develop into tetraploid blastocysts (Spindle, 1981) and reconstructed embryos resulting from PEG-mediated fusion between enucleated bovine oocytes and karyoplasts derived from inner cell mass cells develop to term after transfer to recipient cows (Sims and First, 1993). The slightly lower yield of this technique when applied to pairs of whole zona-free oocytes was mainly due to a lower cell survival, which is likely to be related to the lack of the protective function of the zona pellucida or to the toxic effect of protease treatment used for zona removal. The unusually large size of the resulting fusion products may also have a negative impact on its survival. The results with the fusion of whole zona-free oocytes may also be influenced by the fact that most of these experiments were performed at the beginning of this study, in order to test the efficacy of the fusion protocol, whereas most of the experiments with karyoplast–cytoplast fusion were performed later, when more skill had been attained. Surprisingly, a very encouraging yield of fusion was also obtained with the mechanical fusion technique that, to our knowledge, had never been experienced before. The success of this technique was largely dependent on the use of phytohaemagglutinin to tighten the contact between the oocyte cytoplast and the adjacent karyoplast prior to the manipulation. In this condition, the ICSI needle can open the way to free passage of cytoplasmic components between both entities through a zone that is sealed from the extracellular milieu by means of the surrounding agglutinated membrane portions. This can be expected to prevent massive fluxes of ions and other molecules between the extracellular and intracellular compartments along the existing concentration gradients. Furthermore, the agglutinated membranes can establish a very intimate physical contact so that the mechanically disrupted bilayers of adjacent membranes fuse together instead of fusing with other portions of the same disrupted membrane. If this occurs, there are not enough blunt ends of the disrupted membranes available for resealing the puncture canal, and the communication between the karyoplast and the cytoplast thus becomes irreversible. Most of the karyoplast–cytoplast pairs that failed to fuse or degenerated after the manipulation showed a partial or total protrusion of the karyoplast through the hole previously laser-drilled in the zona pellucida. This could be efficiently prevented by using the mechanical zona-opening technique rather than the laser drilling. Because, unlike the laser, the mechanical zona-opening technique produces elastic deformation of the ajacent zona regions, the same elastic forces may tend to close the hole after manipulation and thus limit the risk of escape.
Neither the chemical nor the mechanical fusion technique caused oocyte activation. This was not surprising for the mechanical technique, because this manipulation is basically the same as that to which the oocyte is subjected during standard ICSI and which is known to be an insufficient stimulus for human oocyte activation (Tesarik and Testart, 1994; Tesarik et al., 1994). The PEG-mediated fusion is also insufficient to induce oocyte activation in bovines (Sims and First, 1993), but this also holds true for electrofusion in this species (Wells et al., 1999). In contrast, most human oocytes become activated by the electrofusion procedure (Cohen et al., 1998). This finding was in agreement with the results of a small series of experiments, performed in our laboratory, in which the same electrofusion method (Cohen et al., 1998) was used. This series involved 10 karyoplast–cytoplast pairs, derived from metaphase II human oocytes, of which seven fused. However, all of the successfully fused karyoplast–cytoplast pairs became spontaneously activated during subsequent 24 h of in-vitro culture (unpublished data). The lack of spontaneous oocyte activation following PEG-induced fusion, observed in this study, thus suggests that both treatments, though similarly efficient in promoting cell fusion, produce different side-effects that condition their different oocyte-activating ability. This may be related to the fact that electrofusion produces an electroporation effect leading to a forced entry of medium components through the plasma membrane. Because the presence of Ca2+ and Mg2+ ions in the fusion medium is required for optimal fusion efficiency, this treatment can alter the ion composition of the cytosol to promote oocyte activation. On the other hand, PEG may be less effective in creating plasma membrane pores and thus keep the intracellular milieu free of ionic changes leading to premature inactivation of metaphase promoting factor (MPF) resulting in oocyte activation. The fertilization ability and post-fertilization developmental potential of reconstituted oocytes were not addressed in this study because the formation of human embryos for research purposes is banned by law in two of the countries in which this work was performed (France and Spain) and is subject to strict regulation in the third (Italy). In fact, approval for experimental fertilization of reconstituted oocytes had not been obtained from the competent ethical authority at the time at which this study was being performed. This issue is dealt with in a separate study (Z.P.Nagy et al., unpublished data).
In practical terms, the two fusion techniques described in this study may prove useful in experiments in which a prolonged exposure of donor nuclei to MPF within the recipient cytoplast is required for adequate nuclear reprogramming. This seems to be particularly important when karyoplasts originating from non-quiescent cells are used (Cibelli et al., 1998), but it is also likely to improve embryo development in cases in which quiescent nuclei are used (Wells et al., 1999). However, the practical use of these techniques with human oocytes cannot be recommended for the time being and will require the achievement of a broad consensus as to the ethical aspects of human cloning, the definition of clear medical indications and the application of protocols inducing an early differentiation of embryonic cells into committed cell lines required for therapy and thus avoiding the destruction of potentially viable embryos.
On the other hand, the data obtained in this study can be applied now for the development of treatment methods whereby metaphase II chromosomes from women with repeated failures of embryo development due to defective oocyte cytoplasm are transferred to enucleated donor oocytes. Compared to standard oocyte donation, this approach will enable a nearly full genetic contribution (with the exception of female extranuclear genes) from both parents to the future embryo and is likely to be more flexible and efficient than oocyte cytoplasmic transfer recently used in this indication (Cohen et al., 1997). Because ICSI will probably be the optimal way of fertilizing the reconstructed oocytes, the mechanical fusion method described in this study may have the advantage of simultaneously operating the fusion between the karyoplast and the enucleated oocyte cytoplast and the introduction of the fertilizing spermatozoon by a single, relatively simple action. The efficiency of this technique is currently being tested in our laboratories.
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Notes |
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6 To whom correspondence should be addressed at: Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France
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References |
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Submitted on October 8, 1999; accepted on January 25, 2000.
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