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Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containing single or multiple nuclei

Published online by Cambridge University Press:  26 September 2008

Nicola J. Winston ,
Martin H. Johnson  and
Peter R. Braude
Nicola J. Winston
Affiliation:
Department of Anatomy, University of Cambridge, University of Cambridge, and Department of Obstetrics and Gynaecology, St Thomas' Hospital, London, UK
Martin H. Johnson*
Affiliation:
Department of Anatomy, University of Cambridge, University of Cambridge, and Department of Obstetrics and Gynaecology, St Thomas' Hospital, London, UK
Peter R. Braude
Affiliation:
Department of Anatomy, University of Cambridge, University of Cambridge, and Department of Obstetrics and Gynaecology, St Thomas' Hospital, London, UK
*
Dr M.H. Johnson, Embryo and Gamete Research Group, Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. Tel: (0223) 333789. Fax: (0223) 333786.
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Summary

The nuclear DNA content of intact, live or fixed, human and mouse oocytes and blastomeres has been measured rapidly and reliably. Chromosomal DNA has been stained with DAPI, the fluorescent emission from which has been measured photocytometrically. In vitro fertilised mouse oocytes and embryos at various stages of development were assessed for their DNA content. The mean values of 1C, 2C and 4C DNA content were clearly different, and it was possible to assign correctly individual values for DNA content to each class with 92%, 61% and 81% confidence respectively. Maintaining the cells as whole mounts allowed other morphological and structural features to be examined. When formation of multiple micronuclei was induced in mouse oocytes by their insemination in the presence of nocodazole, the additive signal from all the micronuclei in one zygote was equivalent to the expected DNA content. Application to early human blastomeres of this photocytometric technique for measurement of the total cellular DNA content revealed that multinucleated blastomeres contained 2C to 4C DNA levels, consistent with a diploid DNA content.

Keywords

Cellular DNA contentEmbryoHumanMouseMultinucleationOocyte
Type
Article
Information
Zygote , Volume 1 , Issue 1 , February 1993 , pp. 17 - 25
Copyright
Copyright © Cambridge University Press 1993

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References

Angell, R.R., Aitken, R.J., van Look, P.F.A., Lumbsden, M.A. & Templeton, A.A. (1983). Chromosome abnormalities in human embryos after in vitro fertilisation. Nature 303, 336–8.Google Scholar
Angell, R.R., Sumner, A.T., West, J.D., Thatcher, S.S., Glasier, A.F. & Baird, D.T. (1987). Post-fertilisation polyploidy in human preimplantation embryos fertilised in vitro. Hum. Reprod. 2, 721–7.Google Scholar
Barlow, P.W. & Sherman, M.T. (1972). The biochemistry of differentiation of mouse trophoblast: studies on polyploidy. J. Embryol. Exp. Morphol. 27, 477–505.Google Scholar
Bolton, V.N., Oades, P.J. & Johnson, M.H. (1984). The relationship between cleavage, DNA replication and gene expression in the mouse 2-cell embryo. J. Embryol. Exp. Morphol. 79, 139–63.Google ScholarPubMed
Chatelain, C. & Burstein, S.A. (1984). Fluorescence cytophotometric analysis of megakaryocytic ploidy in culture: studies of normal and thrombocytopenic mice. Blood 64, 1193–9.CrossRefGoogle ScholarPubMed
Chisholm, J.C., Johnson, M.H., Warren, P.D., Fleming, T.P. & Pickering, S.J. (1985). Developmental variability within and between mouse expanding blastocysts and their ICMs. J. Embryol. Exp. Morphol. 86, 311–36.Google ScholarPubMed
Coleman, A.W., Maguire, M. & Coleman, J.R. (1981). Mithramycin and 4′,6-diamidino-2-phenylindole (DAPI) staining for fluorescence microspectrometric measurement of DNA in nuclei, plastids and virus particles. J. Histochem. Cytochem. 29, 959–68.CrossRefGoogle Scholar
Howlett, S.K. (1986 a). The effect of inhibiting DNA replication in the one-cell mouse embryo. Wilhelm Roux Arch. Dev. Biol. 195, 499–505.Google Scholar
Howlett, S.K (1986 b). Control of cell cycle events during meiotic maturation and early cleavage in the mouse. PhD thesis, Cambridge University.Google Scholar
Howlett, S.K. & Bolton, V.N. (1985). Sequence and regulation of morphological and molecular events during the first cell cycle of mouse embryogenesis. J. Embryol. Exp. Morphol. 87, 175–206.Google Scholar
Ikegami, S., Tauchi, T., Ohashi, M., Oguro, M., Nagano, H. & Mano, Y. (1978). Aphidicolin prevents mitotic cell cycle division by interfering with the activity of DNA poly merase-a. Nature 275, 458–60.CrossRefGoogle Scholar
Maro, B., Johnson, M.H., Pickering, S.J. & Flach, G. (1984). Changes in actin distribution during fertilisation of the mouse egg. J. Embryol. Exp. Morphol. 81, 211–37.Google ScholarPubMed
Maro, B., Johnson, M.H., Webb, M. & Flach, G. (1986). Mechanisms of polar body formation in the mouse oocytes: an interaction between chromosomes, the cytoskeleton and the plasma membrane. J. Embryol. Exp. Morphol. 92, 11–32.Google ScholarPubMed
McConnell, J., Pickering, S.J., Johnson, M.H. & Bashford, J. (1990). A technique for quantifying the amount of Macromolecule injected into cells of the early mouse embryo. J. Reprod. Fert. 88, 375–81.CrossRefGoogle ScholarPubMed
Nasr-Esfahani, M.H., Aitken, J.R. & Johnson, M.H. (1990 a). Hydrogen peroxide levels in oocytes and early cleavage stage embryos from blocking and non-blocking strains of mice. Development 109, 501–7.CrossRefGoogle Scholar
Nasr-Esfahani, M., Johnson, M. & Aitken, R.J. (1990 b). The effect of iron and iron chelators on the in vitro block to development of the mouse preimplantation embryo: BAT6, a new medium for improved culture of mouse embryos in vitro. Hum. Reprod. 5, 997–1003.CrossRefGoogle ScholarPubMed
Nurse, P. (1975). Genetic control of cell size at cell division in yeast. Nature 256, 547–51.CrossRefGoogle ScholarPubMed
Pickering, S.J., Braude, P.R., Johnson, M.H., Cant, A. & Currie, J. (1990). Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril. 54, 102–8.CrossRefGoogle ScholarPubMed
Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J. & Gray, J. (1988). Fluorescent in situ hybridisation with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 85, 9138–42.CrossRefGoogle ScholarPubMed
Rao, P.N. & Johnson, R.T. (1974). Regulation of the cell cycle in hybrid cells. In: Control of Proliferation in Animal Cells. Cold Spring Harbor Conference on Cell Proliferation, ed. Clarkson, B. & Baserga, R., pp. 785–800. New York: Cold Spring Harbor Laboratory Press.Google Scholar
Sathananthan, A.H., Wood, C. & Leeton, J. (1982). Ultrastructural evaluation of 8–16 cell human embryos cultured in vitro. Micron 13, 193–203.Google Scholar
Sathananthan, A.H., Trounson, A., Freeman, L. & Brady, T. (1988). The effect of cooling human oocytes on the meiotic spindles and oocyte structure. Hum. Reprod. 3, 968–77.Google Scholar
Smith, R.K.W. & Johnson, M.H. (1985). DNA replication and compaction in the cleaving embryo of the mouse. J. Embryol. Exp. Morphol. 89, 133–48.Google Scholar
Tesarik, J., Kopecny, V., Plachot, M. & Mandelbaum, J. (1987). Ultrastructural and autoradiographic observations on multinucleated blastomeres of human cleaving embryos obtained by IVF. Hum. Reprod. 2, 127–36.CrossRefGoogle Scholar
Trounson, A.O. & Sathananthan, A.H. (1984). The application of electron microscopy in the evaluation of 2–5 cell human embryos cultured in vitro for embryo transfer. J. In Vitro Fertil. Embryo Transf. 3, 153–65.CrossRefGoogle Scholar
Winston, N.J., Braude, P.R., Pickering, S.J., George, M.A., Cant, A., Currie, J. & Johnson, M.H. (1991 a). The incidence of abnormal morphology and nucleocytoplasmic ratios in 2, 3 and 5 day human pre-embryos. Hum. Reprod. 6, 17–24.CrossRefGoogle Scholar
Winston, N.J., Johnson, M.H., Pickering, S.J. & Braude, P.R. (1991 b). Parthenogenetic activation and development of fresh and aged human oocytes. Fertil. Steril. 56, 904–12.Google Scholar