A reliable technique of nuclear transplantation for immature mammalian oocytes

Takumi Takeuchi, Berrin Ergün, Tian Hua Huang, Zev Rosenwaks and Gianpiero D. Palermo1

The Center for Reproductive Medicine and Infertility, The New York Hospital–Cornell Medical Center, 505 East 70th Street, HT-336, New York, NY 10021, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transplanting a germinal vesicle (GV) to another enucleated oocyte provides a possible way to avoid age-related aneuploidy in metaphase II (MII) oocytes from older women. This study was conducted to examine the efficiency of each step of nuclear transplantation as reflected in the survival and maturation capacity of immature mouse oocytes subjected to this procedure. GV stage oocytes were retrieved from unstimulated ovaries. A GV removed with a small amount of cytoplasm (karyoplast) was transferred subzonally into a previously enucleated oocyte, which was then exposed to direct current to promote fusion. Such reconstituted oocytes were placed in culture to allow maturation, and some that had extruded a first polar body were fixed and processed for chromosome analysis. Each step of nuclear transplantation – survival, enucleation, grafting, and reconstitution – was successful in >90%, with the overall efficiency of reconstitution being 80%. The observation of normal karyotypes confirmed that the procedure did not increase chromosomal aneuploidy. An electrolytic medium, revealed to be superior for the reconstitution procedure, also allowed haploidization of the transplanted nucleus. These findings suggest that this technique can be applied to study the effects of a `younger' woman's ooplasm on the disjunction of an `older' woman's chromosomes during meiosis I.

Key words: aneuploidy/cell fusion/in-vitro maturation/nuclear transplantation/oocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Advancing maternal age presents a clear inverse relationship with fertility, the decline in which is particularly evident by age 40 years (Tietze, 1957Go), and appears to be primarily related to an increased incidence of oocyte aneuploidy (Dailey et al., 1996Go). Women of virtually any age can become pregnant by replacing embryos derived from young donor oocytes. The risk of conceiving an aneuploid fetus during in-vitro fertilization (IVF) increases from 6.8% for women 35–39 years old to ~50% in women 45 or older (Hassold and Chiu, 1985Go). The finding that 37.2% of morphologically normal 8-cell embryos in the 40–45 year maternal age group expressed chromosomal aberrations (Munné et al., 1995Go) shows that aneuploidy impairs embryo implantation.

A clear relationship exists between ageing and abnormality of the chromosome/chromatid segregation, due to the non-disjunction of bivalents during meiosis (Dailey et al., 1996Go). The meiotic spindle is generated by the ooplasm and it has been suggested that ageing of its organelles has an effect on the spindle's ability to undergo a balanced chromosome segregation/haploidization (Muggleton-Harris et al., 1982Go; Pratt and Muggleton-Harris, 1988Go; Battaglia et al., 1996Go). Why aneuploid oocytes are more common in aged women is still unknown, but it has been proposed that oxidative stress acting on primary oocytes and/or surrounding ovarian cells may be a major factor (Tarín, 1995Go; Tarín et al., 1996Go; Van Blerkom et al., 1997Go). Other recent studies have demonstrated that the potential fertility of ovarian tissue can be maintained by cryopreservation of ovarian cortices (Gosden et al., 1994Go; Newton et al., 1996Go; Oktay and Gosden, 1996Go). This approach suggests that through ovarian tissue banking, it should be possible to preserve fertility in patients undergoing chemotherapy, and in women that opt to postpone their childbearing (Oktay et al., 1998Go).

The problem remains for women that have already entered the `downward slope' in regard to their ability to reproduce. Cytoplasmic transfusion into eggs or zygotes of non-human mammals has been shown to improve their developmental potential (Flood et al., 1990Go; Levron et al., 1996Go), and the transfer of cytosol from fertile donor oocytes has resulted in a successful delivery (Cohen et al., 1997Go, 1998aGo). However, since this procedure was performed on mature oocytes, it cannot change any chromosomal imbalance that may appear during meiosis. This can be overcome only by transfer of the nucleus of a germinal vesicle (GV) stage oocyte to an enucleated immature oocyte from a younger woman (Zhang et al., 1997Go).

In this study we have evaluated the efficacy of nuclear transplantation using mouse primary oocytes – as reflected in their subsequent survival, nuclear-cytoplasmic reconstitution, and nuclear maturation – with a focus on the optimal conditions for electrofusion, on the influence of karyoplast nuclear/cytoplasmic ratios and the relative ability of GV- and metaphase II (MII)-derived cytoplasts to support nuclear maturation. Finally, some oocytes were karyotyped in order to assess possible genetic consequences of this procedure.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
B6D2F1 female mice were purchased and housed in a temperature- and light-controlled room on a 12 h light:12 h dark photoperiod and were provided with food and water ad libitum. The experimental protocol (No. 9707–415A) was fully approved by the Institutional Animal Care and Use Committee of Cornell University Medical College.

Oocyte collection
Immature oocytes at the GV stage (GV) were retrieved by puncturing follicles of unstimulated ovaries dissected from B6D2F1 female mice 7–11 weeks old. Cumulus–corona cells were removed mechanically by repeated aspiration through the tip of a hand-drawn pipette. In order to prevent spontaneous germinal vesicle breakdown (GVBD), oocytes were cultured in medium (M199) supplemented with a phosphodiesterase inhibitor (0.2 mM 3-isobutyl-1-methylxanthine; Sigma, St Louis, MO, USA). Immature oocytes were kept in these conditions for ~2 h until they exhibited a perivitelline space.

Mature oocytes with first polar body extruded (MII stage) were collected 15 h after a luteinizing hormone (LH) surge induced by human chorionic gonadotrophin (HCG, 10 IU i.p.; Sigma) administration to female mice where pregnant mare serum gonadotrophin was administered (10 IU i.p.; Sigma) 48 h earlier. The cumulus oophorus was removed by brief exposure to 100 IU/ml of hyaluronidase (Type VIII; Sigma) and completed with a glass pipette.

Enucleation technique
Micromanipulation was performed in a shallow plastic Petri dish (model: 1006; Falcon Becton Dickinson Labware, Franklin Lakes, NJ, USA) in 5 µl droplets of M2 medium supplemented with 3 mg/ml of bovine serum albumin (BSA; Sigma). All tools were made from borosilicate glass (Drummond Scientific, Broomall, PA, USA) drawn on a horizontal micropuller (model: 753; Campden Instruments Ltd, Loughborough, UK) and calibrated, cut, and fire-polished on a microforge (model: MF-9/900; Narishige, New York/New Jersey Scientific Inc., Middlebush, NJ, USA). Tools were controlled by two microinjectors (model: IM-6; Narishige, New York/New Jersey Scientific Inc.). Micromanipulation procedures were carried out on a heated stage (Eastech Laboratory, Centereach, NY, USA) placed on 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.).

The zona pellucida was penetrated by pressing a glass microneedle tangentially into the perivitelline space against the holding pipette. Prior to enucleation, oocytes were exposed for at least 15 min to M2 medium containing 25 µg/ml of cytochalasin B (CCB; Sigma) and 3 mg/ml BSA. The GV nucleus surrounded by a small amount of cytoplasm (GV karyoplast) was removed by a micropipette with a 20 µm inner diameter (Figure 1Go). In earlier reports, GV enucleation was performed rather `indirectly' (Sun and Moor, 1991Go; Meng et al., 1996Go), by increasing the pressure inside a holding pipette to expel a GV karyoplast through a slit made in the zona. In the present study, the enucleation was performed `directly' with a calibrated enucleation pipette for speed and to be able to determine the size of the karyoplast. Thereafter, the isolated GV karyoplast was inserted with the same tool into the perivitelline space of another previously enucleated oocyte at either the GV (GV cytoplast) or MII stage (MII cytoplast). A `grafted oocyte' constitutes a manipulated oocyte in which an isolated karyoplast and an isolated cytoplast are still distinct entities.



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Figure 1. Removal of the nucleus surrounded by a small amount of ooplasm from a mouse germinal vesicle oocyte.

 
Enucleation of oocytes was performed by removing the metaphase II spindle with a small amount of ooplasm, and the first polar body (PB) (Wakayama et al., 1998Go). In the mouse, the spindle is identifiable as a translucent region. Similar enucleation of the MII spindle from mature oocytes required a lower amount of CCB (7.5 µg/ml) for the same length of time.

In order to define the influence of the cytoplasm surrounding the enucleated GV, a preliminary study on spontaneous nuclear maturation was performed using isolated karyoplasts of different sizes. Karyoplasts first classified according to their diameter (small: <30 µm; medium: 30–40 µm; large: >40 µm) (Figure 2Go) were then cultured individually and observed at 3, 12 and 24 h intervals for GVBD and PB extrusion.



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Figure 2. Isolated mouse karyoplasts of different sizes.

 
After insertion of the karyoplast, grafted oocytes were washed in fresh medium to remove CCB, and then cultured in M199 medium containing 25 µg/ml gentamicine, 0.22 mM pyruvic acid, and 3 mg/ml BSA for at least 15 min prior to electrofusion.

Electrofusion
An Electro Cell Manipulator (BTX 200 and 2001, BTX Inc., San Diego, CA, USA) was used. Each grafted oocyte was aligned with a micromanipulator between two micro-electrodes of 100 µm diameter (ECF-100 Tokyo Rikakikai Co. Ltd, Tokyo, Japan) perpendicular to their axes (Figure 3Go). To induce fusion, a single or double 1.0 kV/cm direct current (DC) fusion pulse(s) was delivered for 50–99 µs in a non-electrolyte or electrolyte medium. In the non-electrolyte medium, pulses of alternating dielectrophoretic current (AC) (100–200 V/cm, 5–30 s) were applied to ensure appropriate contact between karyoplast and cytoplast. Up to four electrical pulses were applied in total, each at 30 min intervals. Then, after washing and culture for 30 min, fused oocytes were examined to confirm cell survival and fusion (Figure 4Go).



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Figure 3. A grafted oocyte (couplet of a karyoplast and cytoplast) aligned between two microelectrodes.

 


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Figure 4. Fusing grafted oocytes 30 min after electrofusion.

 
Oocytes were randomly assigned to three non-electrolytic and one electrolytic medium (M2 medium). Non-electrolytic media were: (i) 0.3 M concentration of mannitol supplemented with 0.1 mM CaCl2, 0.05 mM MgCl2 and 1 mg/ml BSA; (ii) 0.25 M concentration of sucrose; and (iii) Zimmermann's cell fusion medium [0.28 M sucrose, 0.5 mM Mg (C2H3O2)2H2O, 0.1 mM Ca(C2H3O2)2, 0.1 mM K2HPO4, 0.1 mM glutathione, 0.01 mg/ml BSA] (Wolfe and Kraemer, 1992Go). Each non-electrolytic medium had an osmolarity and pH of ~280 mOsm/l and a pH of ~7.3.

In-vitro maturation of reconstituted oocytes
Oocytes reconstituted by the electrofusion were cultured and then examined at 12 to 16 h after fusion treatment to evaluate nuclear maturation as evidenced by GVBD and extrusion of the first PB.

Cytogenetic evaluation of reconstituted in-vitro matured oocytes
Oocytes that had extruded the first PB were prepared for chromosome analysis by gradual fixation (Kamiguchi and Mikamo, 1986Go; Kamiguchi et al., 1993Go). Briefly, oocytes were treated in a hypotonic solution (0.068 M potassium chloride with 1 mg/ml BSA) for 5 min, and swollen oocytes were plunged in fixative I (methanol:acetic acid:distilled water, 5:1:3, v/v/v) for 5 min. Then the oocyte was placed on a clean slide and covered with fixative II (methanol:acetic acid, 3:1, v/v). The slide was kept in a Coplin jar containing fixative II for at least 5 min, then dipped into fixative III (methanol:acetic acid:distilled water, 3:3:1, v/v/v) for 1 min and dried in moist warm air. Fixed oocytes were stained with Giemsa to score chromosomes.

Data analysis
The customary Pearson {chi}2-test was utilized for discrete univariate and bivariate data, except where test assumptions were violated, necessitating the Fisher 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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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reconstitution of oocytes after GV transplantation
The results presented here were obtained from a set of three experiments performed successfully only after 53 preliminary attempts in which only 3.1% of 748 oocytes from 60 mice were reconstituted successfully. Of those 23 restored oocytes incubated in human tubal fluid (HTF) supplemented with 0.3% human serum albumin, only five (21.7%) extruded the first polar body. At this point, the electrofusion medium was changed and the medium 199 culture step was added. Subsequent to this, at each step >90% of surviving oocytes were successfully enucleated, grafted, and fused (Table IGo). The overall efficiency of the technique from the enucleated immature oocyte to the reconstituted oocyte with an extruded PB was 80%.


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Table I. Individual steps in the nuclear transplantation process of immature mouse oocytes
 
Effect of karyoplast size on nuclear maturation
In order to quantify the influence of cytoplasmic volume on spontaneous nuclear maturation, isolated karyoplasts of three different sizes were maintained in culture and observed for up to 24 h. GVBD was never higher than 5% at 3 h regardless of karyoplast size, but at 12 h only ~50% receiving medium size karyoplasts were at the GVBD stage compared to 100% for the large size karyoplasts (Table IIGo). These proportions had not changed after a further 24 h in culture. In all karyoplasts, nuclear maturation proceeded no further than GVBD without any PB extrusion.


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Table II. Effect of cytoplasmic volume on maturation of isolated germinal vesicle karyoplasts
 
Effect of media on oocyte reconstitution
When transplanted karyoplasts were exposed to one of three non-electrolyte media or to an electrolyte medium, as shown in Table IIIGo, the electrolyte medium was far superior as a supportive environment for electrofusion (P = 0.0001).


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Table III. Effect of media on reconstitution rate of mouse immature oocytes
 
The effect of cytoplast cell cycle stage on nuclear maturation
Ninety-three percent of 57 GV oocytes and all of the 27 MII oocytes were enucleated successfully. A GV was then introduced into each of 26 cytoplasts derived from GV oocytes, and into each of 27 MII oocytes. Interestingly, none of the 22 MII cytoplasts reconstituted successfully supported any final maturation of the transplanted GV. Of these, seven (31.8%) remained arrested at the GV stage and the other 15 underwent GVBD but never extruded their first polar bodies, even after attempts at activation by an electrical pulse (Table IVGo).


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Table IV. Ability of cytoplasts at different cell cycle stages to induce nuclear maturation
 
Cytogenetics of the reconstituted/matured oocytes
Of the 16 mature oocytes karyotyped successfully, 15 were normal (Figure 5Go), and one was hypohaploid for chromosome 19.



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Figure 5. A normal haploid set of chromosomes obtained from a manipulated oocyte after extrusion of the first polar body.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, where a GV was transplanted with an overall efficiency of 80%, four key points became apparent. The adoption of an electrolytic medium increased the fusion rate significantly, and recipient cytoplasts prepared from GV stages supported nuclear maturation whereas those from mature oocytes did not. In addition, the GV needs to be surrounded by the smallest amount of cytoplasm possible during its isolation, to optimize the benefits of the donor cytoplasm. Finally, the rate of first PB extrusion was markedly enhanced by the adoption of a specific medium for in-vitro maturation.

Although the technique of nuclear transplantation described here is feasible and can be performed efficiently, it proved critical to define the size of the karyoplasts and the media used for cell fusion, as well as the electrofusion settings. Cytoplasts prepared from primary oocytes were characterized by poor fusion rates, mature oocytes being more fusogenic because they are more elastic and so contact between the karyoplast and cytoplast was easier to establish. Cell fusion of immature oocytes was optimized by the adoption of an electrolyte medium. In addition, application of the dielectrophoretic AC alignment pulse prior to induction of the DC fusion pulse greatly improved the rate of cell fusion (Zimmermann et al., 1984Go). The electrical alignment is generally performed for a large number of cells in an electrofusion chamber filled with a low-conductive, non-electrolyte solution made up of isotonic concentrations of sugars (Zimmermann et al., 1984Go), and is required to orient the axis along the adjacent cell membranes of the constructs perpendicular to the electric current vector. When an AC pulse passes through a conductive electrolyte medium, localized heating occurs with consequent cell distress. Since nuclear transfer experiments usually deal with a small number of cells, it does not require the formation of pearl chains of cells, and the alignment can be performed manually cell by cell, making the use of non-electrolyte media redundant. Several authors (Kubiak and Tarkowski, 1985; Tsunoda et al., 1987Go) reported a satisfactory fusion rate when DC fusion pulses were applied to 2-cell mouse embryos suspended in either 0.3 M mannitol or PBS without previous application of AC pulses. In these reports, however, cells were manually aligned, eliminating the need for an AC current. In view of the advantage suggested in placing the constructs in a physiologically balanced fusion medium (Rickords and White, 1992Go), we adopted manual alignment to avoid their exposure to the stress of AC. No detrimental effects of M2 as a fusion medium were observed on cell survival and subsequent nuclear maturation of the restored oocytes.

The observation of a direct positive relationship between the amount of perinuclear residual cytoplasm present in an isolated karyoplast and its effect on the nuclear maturation rate (Fulka et al., 1998Go) has drawn attention to the nuclear:cytoplasmic ratio, which often has been overlooked in nuclear transplantation experiments. In this present study, moreover, GV karyoplasts with a very thin rim of cytoplasm seldom underwent GVBD, and no karyoplasts bearing less than one-third of the normal amounts of cytoplasmic volume extruded a PB. A possible explanation may be seen in the analogy with incompetent oocytes that acquire the ability to undergo GVBD only after their volume increases to at least 80% of the fully grown oocyte (Wassarman, 1988Go). An insufficient complement of cell cycle proteins in incompetent oocytes might be responsible for this inability (Motlik and Kubelka, 1990Go). While a total replacement with host ooplasm would be ideal and might be achieved by injecting nuclear material into the recipient ooplast, this is still not possible due to the large size of the nucleus. Therefore, during enucleation, the smallest amount of surrounding cytoplasm should be removed, in order to maximize the positive effect of the host ooplasm on subsequent nuclear maturation.

MII oocytes seem in many respects ideal for cytoplast preparation because of their fusogenicity, their ability to support future development, and the ease with which their cumulus can be removed (Zhang et al., 1995Go; Cecconi et al., 1996Go). On the other hand, they cannot promote GVBD and extrusion of the first PB. In that last regard, several studies have identified cytoplasmic factors such as maturation promoting factor (MPF) as responsible for inducing nuclear envelope breakdown and chromosome condensation (Masui and Markert, 1971Go; Meyerhof and Masui, 1979Go; Muggleton-Harris et al., 1982Go; Kishimoto, 1986Go; Muggleton-Harris et al., 1988). MPF activity appears shortly before GVBD, maintains a high level during MI, decreases prior to extrusion of the first PB, to rise again throughout the MII stage (Campbell et al., 1996Go; Fulka et al., 1992Go, 1996Go). When MII cytoplasts are used, the same electric current that induces fusion may activate the oocyte as well (Campbell et al., 1996Go; Kono, 1997Go). In turn, the activation induces a reduction of MPF, by degradation of cyclin and a tapering off of the cytostatic factor (CSF) activity, allowing arrested MII oocytes to undergo cleavage division (Whitaker, 1996Go). In this study, we have applied electrical pulses to induce cell fusion, GVBD and eventual extrusion of the first PB, but these may have caused activation as well. Therefore, although oocytes derived from MII cytoplasts were not able to induce haploidization, they were able in 16% of the cases (3/22) to undergo first cleavage division. On the other hand, because immature oocytes are insensitive to electrical stimulation, the MPF activity is low throughout the fusion step and beyond (Kubelka and Moor, 1997Go).

To conclude, nuclear transplantation can be efficiently performed and apparently does not increase chromosomal errors. Thus, this 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 in oocytes from older women. However, no one oocyte stage has all the ideal attributes for this. Though it is more fusogenic, is more easily manipulated, and has greater potential for development, the MII cytoplast cannot bring about maturation of the transplanted GV. Although cytoplasts prepared from GV oocytes support GV maturation, they are less fusogenic and may be functionally impaired by the attendant cumulus coronal cell removal. In spite of the fact that this impairment may be reversed by the addition of mature cytoplasm (Flood et al., 1990Go; Cohen et al., 1998aGo,bGo), this remains a limiting factor. Another aspect that needs to be evaluated further is the role of maternal mitochondrial DNA and its eventual influence on the original genome (Keefe et al., 1995Go; Houshmand et al., 1997Go; Brenner et al., 1998Go; Van Blerkom et al., 1998Go).


    Acknowledgments
 
We thank Prof. J.Michael Bedford for his critical review of the manuscript and Queenie Neri for editing the manuscript.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 28, 1998; accepted on January 18, 1999.