1 The Institute for Reproductive Medicine and Science of Saint Barnabas, Livingston, New Jersey, USA and 2 Department of Obstetrics and Gynecology, Tel Hashomer, Tel-Aviv, Israel
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Abstract |
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Key words: electrofusion/FISH/nuclear transplantation/spectral karyotyping/translocation
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Introduction |
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Cytoplasmic factors have immediate control over the cell cycle and thus govern the behaviour of the nucleus (Masui, 1996). Nuclear transplantation involving unfertilized sheep and cow eggs bears striking testimony to this. Such experiments were first undertaken in the 1980s with the aim of cloning embryos as a means of breeding livestock (Willadsen, 1986
, 1989
). It was found that viable embryos could be produced by transfer of a nucleus from a cleaving embryo to an enucleated metaphase II (M-II) oocyte, which was collected an estimated 1012 h after completion of the first meiotic division. However, when freshly matured oocytes (collected 26 h after completion of the first meiotic division) were similarly tried, it was found that hardly any of the resulting nuclear transplant `embryos' cleaved during culture. Instead, the transferred nucleus was arrested in a configuration that, at least superficially, looked very similar to an M-II. The explanation was thought to be that while the ooplasm of older oocytes was easily activated, making the transferred nucleus behave like that of a 1-cell embryo, the ooplasm of freshly matured oocytes was not activated by the procedure. In the latter instance, therefore, the composite cell produced by the nuclear transfer behaved, not as a 1-cell embryo, but as an unfertilized egg and, in accordance with this, the transferred nucleus assumed a metaphase configuration. The ability of such nuclear transplant eggs to emit a `second polar body' upon subsequent activation lent support to this explanation (Willadsen, 1992
).
These observations were confirmed with in-vitro matured cattle oocytes, where the inherent precise timing of meiotic events is more easily controlled and exploited (Willadsen, 1973, 1992
). As expected, similar results were obtained when donor nuclei other than those of cleaving embryos were used (Willadsen, 1992
). Similar events were described recently after fusion of mouse oocytes with spermatogenic cells (Sasagawa et al., 1998
). A primary spermatocyte nucleus injected into an oocyte undergoing the first meiotic division will arrange itself into an M-I spindle and emit a `first polar body' and form a (male) M-II at the same time as the oocyte emits its first polar body and reaches M-II. If the oocyte is subsequently activated, a male pronucleus is formed from the spermatocyte-derived M-II after emission of a `second polar body'.
The cytoplasmic factors and mechanisms governing nuclear behaviour appear to be similar in all mammals and, indeed, in all eukaryotes (Kishimoto and Okumura, 1997). In at least some instances, they have been shown to operate across species boundaries (Pavlath et al., 1989
; Willadsen, 1989
; Mendonca et al., 1991
). The use of in-vitro matured bovine oocytes isolated from slaughterhouse material has become a routine procedure. With the above in mind, the present study was undertaken to examine the possibility of using freshly matured cattle oocytes to force transferred human nuclei into metaphase, thereby opening the way for rapid karyotyping of embryo-derived cells for preimplantation diagnosis of structural and numerical chromosome abnormalities. The study was aimed primarily at karyotyping of blastomeres in cases where alternative diagnostic techniques do not exist, are inadequate or have failed.
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Materials and methods |
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In a preclinical study, blastomeres isolated from day 3 and day 4 embryos arrested at the 2- to uneven 8-cell stage were used. The zonae pellucidae were dissolved by brief exposure to Ca2+/Mg2+-free M2 containing 3 mg protease (Protease Streptomyces griseus; Sigma). The blastomeres were separated after brief incubation at 37°C in Ca2+/Mg2+-free M16 medium, using a fine-bore pipette. The media compositions were described by Hogan et al. (1994).
Micromanipulation
With a thin glass needle, a 270° equatorial slit was made in the zona pellucida of the bovine oocyte, centring over the first polar body. The polar body was removed with a fine-bore pipette, and a human blastomere was inserted into the perivitelline space, well clear of the slit in the zona (Figure 1).
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Cell culture and fixation
After electrofusion treatment, the micromanipulated eggs were incubated in IVF-50 medium (Scandinavian IVF Science, Gothenburg, Sweden), and an hour later transferred to IVF-50 medium containing 0.02 µg/ml colcemid (demecolcine; Sigma) and incubated for another 3 to 4 h. Demarcation lines were not always visible (Figure 1). The humanbovine hybrid cells were fixed on slides according to Tarkowski's method (1966) except that 0.02 µl/ml colcemid was added to the hypotonic solution. A typical karyotype of a human blastomere after this procedure is depicted in Figure 2
.
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In two clinical cases, both involving a female carrier of reciprocal translocations, single blastomeres were biopsied from 8-cell stage embryos resulting from fertilized eggs with dubious or no results after polar body analysis. The first case (A) concerned a 37-year-old woman carrier of a 46,XX,t(9;11)(p24;q12) translocation, from whom five mature eggs were obtained and biopsied. One embryo that had yielded unclear FISH signals on first polar body analysis, presumably because the cell was in advanced degeneration, was biopsied again on day 3, and the blastomere was processed using the fusion method described above.
The second case (B) concerned a 31-year-old woman carrier of 46,XX,t(11;16)(q21;q22). Eleven first polar bodies were biopsied. Eight of these either showed that the corresponding oocyte was unbalanced or did not fertilize. Three embryos were biopsied, and the extracted blastomeres were fused with frozenthawed bovine oocytes. The fusion products were fixed as described above.
Fluorescence in-situ hybridization (FISH)
FISH was performed on the human metaphases by using whole chromosome painting probes. For case A, painting probes for chromosomes 9 (labelled in Spectrum Green; Vysis; Chicago, Illinois, USA) and 11 (labelled in Spectrum Orange; Vysis) were used in combination with a telomeric probe for 11q (biotin-labelled; Ning et al., 1996) and a centromeric probe for chromosome 9 (labelled in Spectrum Aqua; Vysis), as described previously (Munné et al., 1998a
,b
). Repeated analysis of the same metaphases was performed using spectral karyotyping (SKY), which allows visualization of all 24 chromosomes. SKY hybridization was performed using the SKYTM kit (Applied Spectral Imaging Inc., Carlsbad, CA, USA) with a conventional FISH regime similar to the one described above and elsewhere (Márquez et al., 1998).
The metaphases were evaluated with an Olympus BX60 fluorescent microscope equipped with a high-pressure mercury lamp for the excitation; a triple-band pass filter to detect Spectrum Aqua/Spectrum Orange/Spectrum Green dyes, another for 4',6-diamidino-2-phenylindole (DAPI) observation, and a third one for fluorescein isothiocyanate/propidium iodide observation. This microscope was also equipped with a SpectraCubeTM SD 200 (Applied Spectral Imaging, Migdal HaEmek, Israel), which combines spectroscopy with imaging and enables the measurement of the visible spectrum for each pixel on the image observed. These metaphases were analysed using the inverted image of the DAPI stain, `display colours' and `spectra-based classification colours' simultaneously. `Display colours' allows all chromosomes to be readily visualized after spectral imaging; while `spectra-based classification colours' describes a chromosome classification algorithm based on spectral measurements at each pixel, permitting resolution of 24 colours (Schröck et al., 1996).
For the second case, painting probes for chromosomes 11 (labelled in Spectrum Orange) and 16 (labelled in Spectrum Green) in combination with a centromeric probe for chromosome 11 (labelled in Spectrum Aqua) were used as described previously (Munné et al., 1998a,b
).
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Results |
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Clinical study
Clinical results are shown in Table I. In case A, the 37-year-old female partner of the couple concerned had a history of habitual abortion and had been diagnosed as having a 46XX,t(9;11)(p24;q12) karyotype. Five of the six recovered oocytes were mature, but their general morphology was poor. The oocytes underwent first polar body biopsy and intracytoplasmic sperm injection. In two oocytes, the chromosomal material of the polar bodies was too condensed to allow analysis. One of these two was the only oocyte that became fertilized normally and developed properly, but its second polar body was degenerated and could not be analysed for the translocation. Blastomere biopsy was therefore carried out on day 3, when the embryo had reached the 8-cell stage. A single blastomere was removed and fused with a frozenthawed bovine M-II oocyte as described above. On the basis of the FISH analysis of the metaphase-transformed blastomere nucleus, the embryo was judged to have a normal karyotype (Figure 3
). Repeat analysis with SKY, although performed after replacement, confirmed the result. The embryo was replaced on day 4, resulting in a XX pregnancy. Amniocentesis performed at 18 weeks confirmed the outcome of the PGD. The baby was delivered at 200 g but suffered from ventricular septal defect (VSD), a congenital anomaly that is not known to be linked with translocations. Repair of VSD at 5 months resulted in a variety of complications. The outcome of this was unknown at the time of writing.
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Discussion |
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It is interesting to note that, although the ability of frozenthawed bovine oocytes to develop to term after fertilization has been found to be low (Martino et al., 1996; Izadyar et al., 1998
), such oocytes were quite adequate for the purposes of the present study, provided that they exhibited apparently normal morphology after thawing. This greatly simplifies the logistics of karyotyping by cell fusion.
It is likely that a range of nuclear phases, including developmental arrest, was represented among the blastomeres prior to cell fusion in the preclinical study. Yet at least two-thirds of the fused cells had metaphase sets which could be analysed even though it is possible that the efficiency was reduced due to the poor state of some of the donated material. In addition, the human nuclear material may have been lost during fixation. With more practice, the fixation efficiency should rise. It may also be that some of the cells were anucleate and represented type IV fragmentation (Warner et al., 1998). In any event, it appears that the cell cycle status of the nucleus prior to fusion with the bovine oocyte is of little importance. Also, although blastomeres were the cells investigated in the present study, it is reasonable to expect that the method could be applied to other cell types.
In other preliminary experiments, enucleated freshly ovulated murine, rather than bovine oocytes, were used to provoke metaphase transformation. But both the frequency of metaphase transformation and the quality of the metaphases were very low. Somewhat better results were obtained by using two enucleated mouse oocytes for fusion with each blastomere, instead of one, which suggested that the relatively small ooplasmic volume of the enucleated mouse oocyte and its associated molecular signals were the main reasons for failure. If so, mouse oocytes should work well for karyotyping cells of substantially smaller volume than the blastomeres in the present study.
The method is well suited to determine any type of numerical and structural chromosome abnormality, and will be particularly useful for detecting translocations of paternal origin. Numerical chromosomal abnormalities in preimplantation human embryos are very common (Munné et al., 1995). The molecular techniques of polymerase chain reaction and FISH are powerful diagnostic tools and are widely used today in clinical human genetics and the study of human development (Munné and Cohen, 1998
). However, such diagnostic methods have major drawbacks. Firstly, in interphase cells, only a few chromosomes can be visualized with FISH and, in randomly sampled cells, metaphase chromosomes are rarely available for analysis. Complete metaphase karyotypes can provide much more information and do not require a patient-specific approach. Secondly, only maternally derived translocations can be investigated using painting of metaphase-stage chromosomes of first polar bodies. Transformation of cells into metaphase, as proposed above, has the advantage that paternal as well as maternal translocations can be determined in blastomeres without the development of case-specific probes. Consequently, metaphase transformation by cell fusion would allow the differentiation between normal, balanced and unbalanced karyotypes (Cassel et al., 1997
; Conn et al., 1998; Munné and Cohen, 1998
). The present method for metaphase transformation of single cells by cell fusion can help to overcome these problems by allowing metaphases to be studied with conventional karyotyping.
With the advent of spectral imaging, it is now possible to detect all of the 24 different human chromosomes in polar bodies and blastomeres (Márquez et al., 1998). Although the method could possibly be applied to interphase nuclei, the number of discernible pixels left for analysis would probably be too small to produce a complete chromosome count. Detection of structural chromosomal abnormalities using spectral imaging may only be possible with metaphase chromosomes. PGD involving full karyotyping, rather than analysis of a limited number of chromosomes, might enable more strict embryo selection. This might lead in turn to improvements in implantation and ongoing pregnancy rates. The present method of karyotyping will also be of value in the scientific study of embryonic development, and has a number of other potential fields of applications because of its modest time requirements and apparent effectiveness for analysis of non-dividing cells.
In conclusion, metaphase chromosomes of human preimplantation embryos can now be visualized just a few hours after blastomere biopsy. The method whereby this may be achieved relies on such general principles that its usefulness, in all likelihood, extends to cells other than blastomeres and, indeed, to other species than the human. It is interesting to note that this diagnostic tool is based on nuclear transplantation experiments aimed at the cloning of livestock.
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Notes |
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References |
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Conn, C.M., Harper, J.C., Winston, R.M.L., and Delhanty, J.D.A. (1999) Infertility couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions. Hum. Genet., in press.
Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, USA.
Izadyar, F., Hage, W.J., Colenbrander, B., and Bevers, M.M. (1998) The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation. Mol. Reprod. Dev., 49, 444453.[ISI][Medline]
Kishimoto, T. and Okumura, E. (1997) In-vivo regulation of the entry into M-phase: initial activation and nuclear translocation of cyclin B/Cdc2. Prog. Cell Cycle Res., 3, 241249.[Medline]
Márquez, C., Cohen, J., and Munné, S. (1999) Chromosome identification on human oocytes and polar bodies by spectral karyotyping. Cytogenet. Cell Genet., in press.
Martino, A., Pollard, J.W. and Leibo, S.P. (1996) Effect of chilling bovine oocytes on their developmental competence. Mol. Reprod. Dev., 45, 503512.[ISI][Medline]
Masui, Y. (1996) A quest for cytoplasmic factors that control the cell cycle. Prog. Cell Cycle Res., 2, 113.[Medline]
Mendonca, M.S., Antoniono, R.J., Latham, K.M. et al. (1991) Characterization of intestinal alkaline phosphatase expression and the tumorigenic potential of gamma irradiated HeLaxfibroblast cell hybrids. Cancer Res., 51, 44554462.[Abstract]
Munné, S. and Cohen, J. (1998) Chromosome abnormalities in human embryos. Hum. Reprod. Update 4, 842855.
Munné, S., Alikani, M., Tomkin, G. et al. (1995) Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil. Steril., 64, 382391.[ISI][Medline]
Munné, S., Scott, R., Sable, D., and Cohen, J. (1998a) First pregnancies after pre-conception diagnosis of translocations of maternal origin. Fertil. Steril., 69, 675681.[ISI][Medline]
Munné, S., Morrison, L., Fung, J. et al. (1998b) Spontaneous abortions are reduced after pre-conception diagnosis of translocations. J. Assist. Reprod. Genet., in press.
Ning, Y. et al. (1996) A complete set of human telomere probes and their clinical implication. Nature Genet., 14, 8689.[ISI][Medline]
Pavlath, G.K., Chiu, C.P. and Blau, H.M. (1989) In-vivo aging of human fibroblasts does not alter nuclear plasticity in heterokaryons. Somat. Cell Mol. Genet., 15, 191202.[ISI][Medline]
Santaló, J., Veiga, A., Calafell, J.M. et al. (1995) Evaluation of cytogenetic analysis for clinical preimplantation diagnosis. Fertil. Steril., 64, 4450.[ISI][Medline]
Sasagawa, I., Kuretake, S., Eppig, J.J. and Yanagimachi, R. (1998) Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm. Biol. Reprod., 58, 248254.[Abstract]
Schröck, E., du Manoir, S., Veldman, T. et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science, 273, 494497.[Abstract]
Tarkowski, A.K. (1966) An air drying method for chromosome preparation from mouse eggs. Cytogenet., 5, 394400.[ISI]
Warner, C.M., Cao, W., Exley, G.E. et al. (1998) Genetic regulation of egg and embryo survival. Hum. Reprod., 13 (suppl.), in press.
Whittingham, D.G. (1971) Culture of mouse ova. J. Reprod. Fertil., 14 (suppl.), 721.
Willadsen, S.M. (1973) Bovine eggs in vivo and in vitro. Thesis Institut for Husdyrenes Reproduktion. Royal Veterinary and Agricultural School. Copenhagen, Denmark.
Willadsen, S.M. (1986) Nuclear transplantation in sheep embryos. Nature, 320, 6365.[ISI][Medline]
Willadsen, S.M. (1989) Cloning of sheep and cow embryos. Genome, 31, 956962.[ISI][Medline]
Willadsen, S.M. (1992) Observations on the behavior of foreign nuclei introduced into in vitro matured oocytes. In Seidel, G.E. (ed.), Proceedings of a Symposium on Cloning of Mammals by Nuclear Transplantation. Colorado State University, Fort Collins, USA.
Submitted on July 3, 1998; accepted on November 5, 1995.