Reproductive Genetics Institute, Illinois Masonic Medical Center, 836 W.Wellington, Chicago, IL 60657, USA
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Abstract |
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Key words: cytochalasin/cytokinesis/cytoplast/enucleation/tripronuclear human zygotes
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Introduction |
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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., 1986), 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, 1979
; Hara et al., 1980
; Sakai and Kubota, 1981
; Waksmundzka et al., 1984
), 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.
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Materials and methods |
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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 1A). 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 150180 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 1518 µm outer diameter and flame polished on the DeFronbrune microforge so that the internal diameter of the opening measured 1315 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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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, I) or intermittently through a single slit opening after recovery of the zygote to its initial spherical shape (Figure 1F
). Each pronucleus required 35 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 2472 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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Results |
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Cleavage of the cytoplasts was compared with the cleavage of 238 normally fertilized diploid embryos. At 3032 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 2FI), 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.
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Discussion |
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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, 1975; Hoppe and Illmensee, 1977
; Feng et al., 1996) or multiple (McGrath and Solter, 1983) pronuclei from mouse zygotes. Reported survival rates ranged from 65% (Hoppe and Illmensee, 1977
) 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., 1999
). The cytoskeleton is also known as a thermally sensitive structure and can be disrupted by low temperatures (Pickering et al., 1987, 1988
). This may explain the better survival rates when intracytoplasmic sperm injections in mouse oocytes were performed at temperatures of 1718°C (Kimura and Yanagimachi, 1995
). 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 1977
; Modlinski, 1980
) 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 1012 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 23 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., 1984), 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 nucleicytoplasmic behaviour in human embryos.
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Notes |
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References |
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Balakier, H. and Tarkowski, A.K. (1976) Diploid parthenogenetic mouse embryos produced by heat-shock and cytochalasin B. J. Embryol. Exp. Morphol., 35, 2539.[ISI][Medline]
Boyers, S.P., Diamond, M.P., Lavy, G. et al. (1987) The effect of polyploidy on embryo cleavage after in vitro fertilization in humans. Fertil. Steril., 48, 624627.[ISI][Medline]
Cohen, J., Willadsen, S., Schimmel,T. et al. (1999) Micro manipulation as a clinical tool. In Tronson, A.O. and Gardner, D.K. (eds), Handbook of In Vitro Fertilization. CRC Press, Boca Raton, pp. 265306.
Evsikov, S. and Verlinsky, Y. (1999) Visualization of chromosomes in single human blastomeres. J. Assist. Reprod. Genet., 16, 133137.[ISI][Medline]
Feng,Y.-L. and Gordon, J.W. (1996) Birth of normal mice after removal of the supernumerary male pronucleus from polyspermic zygotes. Hum. Reprod., 11, 341344.[Abstract]
Fryns, J.P., van de Kerckhove, A., Goddeeris, P. and van den Berghe, H. (1977) Unusually long survival in case of full triploidy of maternal origin. Hum. Genet., 38, 147155.[ISI][Medline]
Gordon, J.W., Grunfeld, L., Garrisi, G.J. et al. (1989) Successful microsurgical removal of a pronucleus from tripronuclear human zygotes. Fertil. Steril., 52, 367.[ISI][Medline]
Hara, K., Tydeman, P. and Kirshner, M. (1980) A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. Proc. Natl Acad. Sci. USA, 77, 462466.[Abstract]
Hoppe, P.C. and Illmensee, K. (1977) Microsurgically produced homozygousdiploid uniparental mice. Proc. Natl Acad. Sci. USA, 74, 56575661.[Abstract]
Kimura, Y. and Yanagimachi, R. (1995) Intracytoplasmic sperm injection in the mouse. Biol. Reprod., 52, 709720.[Abstract]
Kola, I., Trounson, A., Dawson, G. et al. (1987) Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol. Reprod., 37, 395401.[Abstract]
Malter, H.E. and Cohen, J. (1989) Embryonic development after microsurgical repair of polyspermic human zygotes. Fertil. Steril., 52, 373379.[ISI][Medline]
McGrath, J. and Solter, D. (1983a) Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell, 37, 179.[ISI]
McGrath, J. and Solter, D. (1983b) Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science, 220, 1300.[ISI][Medline]
Modlinski, J.A. (1975) Haploid mouse embryos obtained by microsurgical removal of one pronucleus. J. Embryol. Exp. Morphol., 33, 897905.[ISI][Medline]
Modlinski, J.A. (1980) Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with cytochalasin B on enucleated eggs. J. Embryol. Exp. Morphol., 60, 153161.[ISI][Medline]
Palermo, G., Munne, S. and Cohen, J. (1994) The human zygote inherits its mitotic potential from the male gamete. Hum. Reprod., 9, 12201225.[Abstract]
Pickering, S.J. and Johnson, M.H. (1987) The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum. Reprod., 2, 207216.[Abstract]
Pickering, S.J., Johnson, M.H., Braude, P.R. and Houliston, E. (1988) Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Hum. Reprod., 3, 978989.[Abstract]
Plachot. M., Mandelbaum, J., Junka, A.-M. et al. (1989) Cytogenetic analysis and developmental capacity of normal and abnormal embryos after IVF. Hum. Reprod., 4, 99103.[Abstract]
Raucher, D. and Sheetz, M.P. (1999) Characteristics of a membrane reservoir buffering membrane tension. Biophys. J., 77, 19922002.
Rawlins, R.G., Binor, Z., Radwanska, E. et al. (1988) Microsurgical enucleation of tripronuclear human zygotes. Fertil. Steril., 50, 266272.[ISI][Medline]
Sakai, M. and Kubota, H.Y. (1981) Cyclic surface changes in non-nucleate egg fragment of Xenopus laevis. Dev. Growth Diff., 23, 4149.[ISI]
Sawai, T. (1979) Cyclic changes in the cortical layer of non-nucleate fragments of the newt's egg. J. Embryol. Exp. Morphol., 51, 183193.[ISI][Medline]
Tarkowski, A.K. (1977) In vitro development of haploid mouse embryos produced by bisection of one cell fertilized eggs. J. Embryol. Exp. Morphol., 38, 187202.[ISI][Medline]
Togo, T., Alderton, J.M., Bi, G.Q. and Steinhardt, R.A. (1999) The mechanism of facilitated cell membrane resealing. Cell Sci., 112, 719731.
Tsunoda, Y., Yasui, T., Nakamura, T. et al. (1986) Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse. J. Exp. Zool., 240, 119125.[ISI][Medline]
Tsunoda, Y., Shioda, M., Onodera, K. et al. (1988) Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation. J. Reprod. Fertil., 82, 173178.[Abstract]
Van Blerkom, J., Henry, G. and Porreco, R. (1984) Preimplantation human embryonic development from polypronuclear eggs after in vitro fertilization. Fertil. Steril., 41, 686696.[ISI][Medline]
Veeck, L. (ed.) (1999) An Atlas of Human Gametes and Conceptuses. Parthenon Publishing Group, New York, pp. 5960.
Verlinsky, Y., Cieslak, J., Ivakhnenko, V. et al. (1996) Polar body diagnosis of common aneuploidies by FISH. J. Assist. Reprod. Genet., 13, 157162.[ISI][Medline]
Waksmundzka, M., Krysiak, E., Karasiewicz, J. et al. (1984) Autonomous cortical activity in mouse eggs controlled by a cytoplasmic clock. J. Embryol. Exp. Morphol., 79, 7796.[ISI][Medline]
Submitted on August 31, 1999; accepted on December 16, 1999.