A microsurgical technique for enucleation of multipronuclear human zygotes

Victor Ivakhnenko1, Jeanine Cieslak and Yury Verlinsky

Reproductive Genetics Institute, Illinois Masonic Medical Center, 836 W.Wellington, Chicago, IL 60657, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tripronuclear human zygotes were used to determine the feasibility of an enucleation procedure without the use of cytoskeleton inhibitors to obtain intact cytoplasts for nuclear transfer research. Of 61 tripronuclear human zygotes manipulated, 100% of the zygotes survived after removal of one pronucleus and 90.1% after complete enucleation, proving the reliability of the proposed microsurgical technique. Morphological changes were observed in the resulting cytoplasts during extended culture. At 36 h post-insemination, 87.5% of cultured cytoplasts showed morphological changes, which included cleavage into two `blastomeres', three `blastomeres' with various degrees of fragmentation, one cell with fragments or gross fragmentation. Comparison with the diploid human zygotes from the same harvests, which had undergone the first cleavage division by this time, showed similar timing in cleavage suggesting autonomous cytoplasmic activity in human zygotes.

Key words: cytochalasin/cytokinesis/cytoplast/enucleation/tripronuclear human zygotes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The microsurgical removal of pronuclei from zygotes has proved to be efficient and valuable for embryological studies in several mammalian species (Modlinski, 1975Go; Hoppe and Illmensee, 1977Go; McGrath and Solter, 1983aGo); however, it has not been investigated sufficiently in humans. First attempts to remove a single pronucleus microsurgically from human triploid zygotes, in order to correct them to diploid status, was reported in 1988 (Rawlins et al., 1988Go). In this study, a micromanipulation technique in conjunction with cytoskeletal relaxants (McGrath and Solter, 1983bGo) was employed; however, survival was poor. Using the same technique a 55% survival rate was achieved (Gordon et al., 1989Go) after removal of a single pronucleus from human triploids in the presence of cytochalasin D. During that same year, a 36% survival rate was reported after pronucleus removal in the absence of cytoskeletal relaxants (Malter and Cohen, 1989Go). In a more recent study, a single pronucleus was removed by penetration of the zygote plasma membrane with a sharp needle (Palermo et al., 1994Go); however, survival rates and information regarding whether or not cytochalasin was used were not reported.

In the present study, we refer to the term of `enucleation' as the removal of all existing pronuclei from abnormally fertilized zygotes, to obtain intact cytoplasts. In an attempt to adopt the modification of the original enucleation technique (Tsunoda et al., 1986Go), we noticed that in the absence of cytoskeletal inhibitors, the plasma membrane of the zygotes is stretchable enough to envelop the pronucleus during the removal without disruption. Based on this observation, changes implemented in the enucleation procedure allowed for repeatedly high survival rates of the manipulated zygotes and cytoplasts, proving that the plasma membrane of the human zygotes has significant reserve to withstand mechanical force. When resulting cytoplasts were cultured in vitro, autonomous activity of the cytoplasm was observed during the first mitotic cell cycle and in some cases beyond 36 h post-insemination. Autonomous activity of the cytoplasm, independent from nucleus activity, was reported for a few animal species (Sawai, 1979Go; Hara et al., 1980Go; Sakai and Kubota, 1981Go; Waksmundzka et al., 1984Go), but no data are available for human embryos. Morphological changes in the human cytoplasts during extended culture, which were similar to nucleated embryos, i.e. the time of cleavage, demonstrate the presence of such activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tripronuclear human zygotes resulting from conventional insemination or intracytoplasmic sperm injection were obtained for micromanipulation from couples undergoing infertility treatment in the IVF programme after signing of informed consent. The use of abnormally fertilized human zygotes for this study was approved by the facilities' Internal Review Board.

Tool construction and micromanipulation setup
Three tools were required for the enucleation procedure: a holding pipette, a partial zona dissection needle and an enucleation pipette (Figure 1AGo). Microtools were fabricated from glass capillary tubes (Drummond Scientific Co., Broomall, PA, USA, 30 µl) with a 1.0 mm outer diameter. Holding pipettes were hand-pulled, then broken at 150–180 µm outer diameter and flame polished on the DeFronbrune microforge (Technical Products International, St Louis, MO, USA) so that the internal diameter of the opening measured 15 µm. Two pipettes were pulled on a Flaming Brown micropipette puller (Sutter Instruments, San Rafael, CA, USA) to obtain fine, sharp needles. To prepare the enucleation pipette, a needle was broken at a 15–18 µm outer diameter and flame polished on the DeFronbrune microforge so that the internal diameter of the opening measured 13–15 µm. To accommodate the micromanipulation setup, both the enucleation pipette and the microneedle were bent to a 35° angle. All micromanipulations were carried out at room temperature in 10 µl drops of human tubal fluid (HTF) medium supplemented with 10% Plasmanate (Bayer Biological, West Haven, CT, USA) under oil (Squibb-Bristol Myers, Eatontown, NJ, USA) on an inverted microscope (Diaphot, Nikon, Garden City, NY, USA) fitted with two hydraulic micromanipulators (Narishige, Tokyo, Japan), and a micrometer-controlled, 100-µl syringe (Hamilton #1710, Fisher, IL, USA). Teflon tubing was used for all hydraulic systems, filled with light paraffin oil, while the microtools were filled with highly viscous silicone oil (dimethylpolysiloxane, 12 500 centistokes, Sigma, St Louis, MO, USA) for fine control during aspiration.



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Figure 1. Enucleation procedure. Original magnification x200. (A) Tripronuclear zygote and three-tool setup. (B) Invagination of the pronucleus created by the flow of the medium from the micropipette. (C) Initial aspiration of the pronucleus into the micropipette. (D) Thread-like membrane connection between the zygote and the pronucleus. (E) Distortion of the zygote immediately after pronucleus removal. (F) Recovery of the zygote to its spherical shape after the first pronucleus was removed. (G) Removal of the second pronucleus through the second opening in the zona pellucida. (H) Pronucleus removal through three separate openings; distortion of the cytoplast immediately following. (I) Pronuclei after removal.

 
Micromanipulation
Prior to enucleation, both first and second polar bodies were removed (Verlinsky et al., 1996Go) to eliminate interference during examination of cytoplasts for complete removal of nuclear material. Afterwards, the zygote was rotated so that the slit opening created in the zona pellucida for polar body removal was in focus at the 3 o'clock position and the zygote was held firmly by suction from the holding pipette. At this point, each individual pronucleus could be visualized clearly using the microscope fine focus adjustment. The enucleation pipette was back-filled with a substantial amount of culture medium and passed through the slit opening. The pipette was brought up to the plasma membrane and the target pronucleus was selected. Pressure on the plasma membrane and cytoplasm was applied by gentle thrust of the enucleation pipette with simultaneous expulsion of the medium from the pipette. This forced cytoplasm between the pronucleus and the plasma membrane to move away, allowing complete contact between the pronuclear membrane and the plasma membrane. At this point, because of the elasticity of both membranes, the flow of the medium created an invagination in the pronucleus (Figure 1BGo). As soon as this invagination was seen, indicating access to the pronucleus, gentle suction (Figure 1CGo) was applied until the entire pronucleus (nuclear organizers and distal pronuclear membrane) was aspirated into the enucleation pipette. Minimal suction was maintained in the enucleation pipette while the pipette was slowly withdrawn from zygote. The pronucleus remained connected to the zygote by a very thin, stretchable thread (Figure 1DGo) until complete withdrawal of the pipette forced the plasma membrane, enveloping the pronucleus, to pinch off. Morphological distortion of the zygotes occurred during the enucleation procedure, similar to what is often seen with oocytes during intracytoplasmic sperm injection (Figure 1EGo).

Subsequent removal of second and third pronuclei was performed in three different ways; sequentially one after another through a single slit opening, through three separate slit openings (Figure 1G, H, IGo) or intermittently through a single slit opening after recovery of the zygote to its initial spherical shape (Figure 1FGo). Each pronucleus required 3–5 min for removal; however, the time of complete enucleation was dependent on the chosen strategy. Shortly after transfer to the original culture dishes, cytoplasmic integrity of the zygotes and cytoplasts was assessed for leakage and localized darkening.

Cytoplasts culture and microscopic observations
Cytoplasts were cultured in standard IVF culture conditions (37°C, 5% CO2 95% humidity) in 50 µl drops of HTF medium supplemented with Plasmanate 10% (Bayer Biological). After enucleation, some of the cytoplasts were chosen at random and stained with a fluorescent DNA stain (Hoechst 33342, 5 µg/ml; Sigma) and examined under UV light to insure complete removal of all nuclear material. Afterwards, the stained cytoplasts were excluded from further observations. The remaining cytoplasts were left in culture and observed every 4 h for the first 12 h after enucleation, followed by examination every 12 h for the next 24–72 h. In parallel, non-manipulated diploid zygotes from the same harvests were observed in 12 h intervals. The time of first cleavage division in both nucleate and anucleate zygotes, as well as their morphological appearance were recorded and compared. All cytoplasts were stained and examined under UV light once any morphological changes had ceased.


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 Materials and methods
 Results
 Discussion
 References
 
Survival rates
Table IGo contains survival rates of the manipulated zygotes after removal of the first, second and third pronucleus. A total of 61 zygotes were manipulated; all 61 (100%) survived after removal of the first pronucleus, 60 (98.3%) survived removal of the second pronucleus and 55 (90.1%) cytoplasts remained intact after the removal of the third pronucleus. Partial removal of the second pronucleus occurred in two zygotes and of the third pronucleus in one zygote. After enucleation, seven out of a total of 55 intact cytoplasts were stained and examined under UV light. A blue fluorescence usually seen when nuclear material is present was not seen, indicating all nuclear DNA was completely removed from the zygotes.


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Table I. Cytoplast survival rates after enucleation
 
Cytoplast cleavage
Microscopical examinations of 48 intact cytoplasts were performed and morphological changes are summarized in Table IIGo.


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Table II. Morphological changes of 48 cytoplasts during 36 h postinsemination
 
Morphological changes of cytoplasts were categorized as spherical, in which the cytoplast recovered its original round shape: no other visible changes were observed (Figure 2AGo); deformation of the cytoplast due to an undulation of the surface; one cell with fragmentation: several cytoplasmic fragments appeared beside the main body of the cytoplast (Figure 2BGo); immediate fragmentation of the entire cytoplast: multiple cytoplasmic fragments (Figure 2CGo); two cells, when two equal size `blastomeres' with minimal cytoplasmic fragments present (Figure 2DGo); three cells, when three equal size `blastomeres' with minimal cytoplasmic fragments present (Figure 2EGo).



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Figure 2. Cytoplast cleavage. Original magnification x200. (A) Spherical appearance of the cytoplast after recovery. (B) One cell with fragmentation; several cytoplasmic fragments appeared beside the main body of the cytoplast. (C) Immediate fragmentation of the entire cytoplast; multiple cytoplasmic fragments. (D) Two, equal size `blastomeres' with minimal cytoplasmic fragments present. (E) Three, equal size `blastomeres' with minimal cytoplasmic fragments present. (F) Four, equal size `blastomeres' after second cleavage. (G)–(I) Six or more `blastomeres' after second and third cleavage.

 
The first morphological changes were observed after enucleation at 24 h post-insemination. Seven of 48 (14.6%) of the originally spherical cytoplasts exhibited surface deformations, two (4.2%) formed a `one-cell with fragmentation', two (4.2%) underwent immediate complete fragmentation and one (2.1%) cytoplast cleaved into two equal `blastomeres'. Thirty-six (75%) of the cytoplasts showed no activity at this time. Wave-like deformations in the surface of the cytoplast varied in intensity and duration for a period of time and in two cytoplasts led to manifold furrows and immediate fragmentation of the cytoplasts. During the next 12 h, 87.5% of cytoplasts showed similar activity and at the end of hour 36, 22 (45.8%) cleaved into two `blastomeres' and four (8.4%) into three `blastomeres' with various degrees of fragmentation, 11 (22.9%) cleaved into one large cell with fragments and five (10.4%) were grossly fragmented. Only six (12.5%) of the cytoplasts remained unchanged.

Cleavage of the cytoplasts was compared with the cleavage of 238 normally fertilized diploid embryos. At 30–32 h post-insemination, 88.2% of diploid embryos divided into two, three, and four cells compared with 87.5% of cytoplasts which showed cytoplasmic activity of the previously described cleavage patterns. In all, 11.8% diploids and 12.5% of cytoplasts remained unchanged.

Further observations during the next 2 days showed that from 42 cleaved cytoplasts, 13 (30.9%) proceeded to the second and six (14.3%) to the third cleavage (Figure 2F–IGo), while the remaining 23 (54.8%) ceased cleavage or became grossly fragmented. The time of the second and third cleavage of cytoplasts was similar to the time of cleavage of diploid embryos from the same harvests.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
There is extensive experience in enucleation and nuclear transplantation in animal research; however, experience with human oocytes and embryos is difficult to obtain because of the limited numbers available for study. By theoretical calculations (Feng and Gordon, 1996Go) a considerable number of abnormally fertilized human oocytes exist in human IVF programmes. Certainly, with patient consent, it can be a source of a valuable material for diagnostic (Cohen et al., 1999Go; Evsikov and Verlinsky, 1999Go) and therapeutic purposes (Rawlins et al., 1988Go; Gordon et al., 1989Go; Malter et al., 1989; Palermo et al., 1994Go; Veeck, 1999Go) as well as for obtaining information about nuclear and cytoplasmic roles in embryo development (Palermo et al., 1994Go). It was also demonstrated that despite some disadvantages (Boyers et al., 1987Go; Kola et al., 1987Go), developmental potential as well as physiology of tripronuclear human zygotes are comparable to diploid human zygotes (Fryns, et al., 1977Go; Van Blerkom et al., 1984Go; Plachot, et al., 1989; Balakier, 1993Go).

Since enucleation of abnormally fertilized human zygotes is an integral part in all these procedures and high survival rates of manipulated zygotes is important, we investigated and developed an efficient pronucleus removal technique. Previously described enucleation techniques can be divided into microsurgical removal of single (Modlinski, 1975Go; Hoppe and Illmensee, 1977Go; Feng et al., 1996) or multiple (McGrath and Solter, 1983) pronuclei from mouse zygotes. Reported survival rates ranged from 65% (Hoppe and Illmensee, 1977Go) to 97% (McGrath and Solter, 1983) depending on whether or not the plasma membrane of the zygote was penetrated. Attempts at enucleation of human triploid zygotes concentrated mostly on single pronucleus removal, resulting in inadequate survival rates. All authors acknowledged that the sensitivity of the zygotes to micromanipulation is a factor responsible for different survival rates and that mechanical damage of the plasma membrane can be overcome by the use of the cytoskeletal relaxant cytochalasin. Based on these observations, cytochalasin has been used effectively in the majority of reported nuclear transplantation studies in animal oocytes and zygotes. Increased resistance of the eggs to damage by mechanical force in the presence of the cytochalasin emphasizes the importance of the cytoskeleton as an initial obstacle to the egg survival during micromanipulation, since cytochalasin has no direct effect on the plasma membrane. Disruption of the cytoskeleton by the depolymerization of actin filaments probably exerts secondary effects on the plasma membrane, including decrease in surface tension and subsequent increase in membrane reservoir; it also facilitates resealing, as was shown on somatic cells (Raucher et al., 1999; Togo et al., 1999Go). The cytoskeleton is also known as a thermally sensitive structure and can be disrupted by low temperatures (Pickering et al., 1987, 1988Go). This may explain the better survival rates when intracytoplasmic sperm injections in mouse oocytes were performed at temperatures of 17–18°C (Kimura and Yanagimachi, 1995Go). However, we did not notice any difference in performance or survival, whether enucleation was done at room temperature (22°C), which is probably insufficient for the significant disruption of the cytoskeleton or when the temperature of the microscope stage was adjusted to 37°C. In spite of better survival rates, intervention in cytoskeletal organization of the eggs by exposure to cytochalasin or cooling can cause fragmentation, delayed cleavage, early developmental arrest (Balakier et al., 1976; Hoppe and Illmensee 1977Go; Modlinski, 1980Go) or instability of the meiotic spindle (Pickering et al., 1987). It is difficult to estimate the significance of the reported negative effects, since live animals were born from oocytes and embryos that had been exposed to such conditions during micromanipulations.

For human oocytes and embryos, no extensive data on the use of cytochalasin for micromanipulations are available. We have observed fragmentation of triploid human zygotes and cytoplast cleavage delay for up to 10–12 h after exposure to cytochalasin D (1 µg/ml) during micromanipulation; we therefore had good reason to avoid its use. It is questionable whether cytochalasin should be used during micromanipulation on human eggs. Large size, cytoplasm clarity, and significant elasticity make them different from other species, e.g. mouse, bovine or porcine, and favourable for manipulation.

Enucleation without disruption of the cytoskeleton by cytochalasin or cooling was possible when the plasma membrane was pushed in with the help of a flame-polished blunt micropipette to the point of contact with the pronuclear membrane, mechanically moving the cytoplasm with the cytoskeleton. Factors determining the success of this technique, are the natural elasticity of the plasma membrane and construction of the enucleating pipette, similar to the shape of a holding pipette. The pipette's internal narrowing at the tip allows the membrane-bound pronucleus to pass through and expand in the larger part of the enucleation pipette, providing sufficient grip for the extraction. All zygotes remained intact after removal of the first pronucleus and despite each additional pronucleus removed, the survival rate decreased only slightly. Whether pronuclei were removed one at a time, through the same opening, or through separate openings in the zona pellucida, or intermittently after a short incubation period when the zygote was allowed to recover to its original spherical shape, an insignificant difference in survival rates was seen. The extent of zygote distortion can create difficulties in proper orientation and visualization of remaining pronuclei, which may explain the slight difference seen. Survival was better when pronuclei were removed intermittently through the same slit opening in the zona pellucida after the zygote regained its original shape. Although the whole pronucleus was usually aspirated, in a few cases only part of the pronucleus was removed and the retained portion was removed later in the same manner. Slow aspiration of the pronucleus and slow withdrawal of the pipette helped to avoid this from occurring again.

Since minimal or no cytoplasm was lost during enucleation, it was assumed that obtained cytoplasts were intact and retained qualities of the originally nucleated zygotes. Supporting this assumption, systematic microscopic observations showed that intact cytoplasts had obvious cleavage potential. From 48 initial cytoplasts, 15 (31.2%) underwent at least two symmetrical divisions and six (12.5%) underwent three. Whereas 27 (56.3%) arrested, they still showed some degree of cytoplasmic activity. Overall, the rate of cleavage was 2–3 h behind the development of diploid zygotes. However, in many instances, when compared to the cleavage rate of the sibling diploids, no significant difference was observed. Similar to a study describing autonomous cortical activity in bisected mouse eggs (Waksmundzka et al., 1984Go), we observed the same phenomenon in enucleated human zygotes, represented by cytoplast surface undulations, furrowing, symmetrical cleavage or fragmentation. It is of interest that the resulting cytoplasts continued to develop during the second and third days after enucleation and showed cyclic patterns of cleavage similar to diploid embryos, with the exception of failure to continue beyond the 8-cell stage. This indicates the presence of a mechanism of cytokinesis in human zygotes which continues to function in the absence of nuclei similar to that found in other animal species. The high success rate of the enucleation procedure demonstrates that the plasma membrane of the human zygote has significant reserve against stretching and that the stiffness in the presence of an intact cytoskeleton is solely responsible for the fragility, which can be overcome by a simple mechanical approach. The proposed enucleation technique can become a useful tool in the use of abnormally fertilized human oocytes for diagnostic and therapeutic purposes as well for studying nuclei–cytoplasmic behaviour in human embryos.


    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 31, 1999; accepted on December 16, 1999.