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Evaluation of a simple method for measuring the cellular DNA content of mouse oocytes and embryos, human fibroblasts and parthenogenetically activated human oocytes using a computerised image analysis system(Seescan)

Published online by Cambridge University Press:  26 September 2008

Alison S. Taylor  and
Peter R. Braude
Alison S. Taylor*
Affiliation:
Assisted Conception Unit, Guy's and St Thomas' Hospital Trust, London, UK.
Peter R. Braude
Affiliation:
Assisted Conception Unit, Guy's and St Thomas' Hospital Trust, London, UK.
*
A.S. Taylor, Assisted conception Unit, UMDS department of Obstetrics and Gynaecology, Guy's and St Thomas' Hospital Trust, Lambeth palace Road, London SE1 7EH, UK. Telephone: 071-928 8105. Fax: 071-620 1227.
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Summary

We report the use of a simple, reproducible, photocytometric method for measuring nuclear DNA content of DAPI-stained cells, using a computerised image analysis system: Seescan. As this technique is non-destructive and uses very short exposure to ultraviolet light, it can be used for either fixed or vital material. After correcting for any background cytoplasmic staining, the intensity of nuclear stain was measured by the Seescan and compared with that of control cells of known ploidy. Fixed material was found to stain more intensely than live material initially, but demonstrated a rapid loss of nuclear intensity over the first 90 min following removal from DAPI, after which the level plateaued. In contrast, live cells showed no change in nuclear intensity with time. The system was validated by measuring the DNA content of carefully timed mouse blastomeres, human fetal lung fibroblasts and parthenogenetically activated human oocytes. The results obtained were appropriate for the developmental stage or phenotypic appearance of each of the cell types measured.

Keywords

Cellular DNA contentComputerised image analysisDAPIHuman fibroblasts and parthenogenetically activated oocytesMouse embryos
Type
Article
Information
Zygote , Volume 3 , Issue 1 , February 1995 , pp. 85 - 94
Copyright
Copyright © Cambridge University Press 1995

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References

Angell, R.R., Sumner, A.T..,West, J.D. et al. ., (1987). Post-fertilisation polyploidy in human preimplantation embryos fertilised in-vitro. Hum. Reprod. 2, 721–2.CrossRefGoogle Scholar
Artley, J., Braude, P. &Johnson, M.. (1992). Gene activity and cleavage arrest in human pre-embryos. Hum. Reprod. 7, 1014–21.CrossRefGoogle ScholarPubMed
Balakier, H..,Maclusky, N.J. &Casper, R.F.. (1993), Characterization of the first cell cycle in human zygotes: implications for cryopreservation, Fertil. Steril. 59, 359–65.CrossRefGoogle 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. et al. ., (1985), Developmental variability within and and between mouse expanding blastocysts and their ICMs, J. Embryol Exp. Morphol. 86, 311–36.Google ScholarPubMed
Coleman, A.W. &Goff, L.J.. (1985). Applications of fluorochromes to pollen biology. I. Mithramycin and 4',6-diamidino-2-phenylindole(DAPI) as vital stains for quantitation of nuclear DNA. Stain Technol. 60, 145–54.CrossRefGoogle Scholar
Coleman, A.W., Maguire, M.J. &Coleman, J.R.. (1981). Mithramycin- and 4'-6'-Diamidino-2-phenylindole [DAPI]-DNA staining for fluoroscence microspectrophotometric meassurement of DNA in nuclei, plastids, and virus particles. J. Histochem. Cytochem. 29, 959–68.CrossRefGoogle Scholar
Frindel, E., Tubiana, M.. (1971). Radiobiology and the cell cycle. The cell Cycle and Cancer, ed. Baserg, et al. , pp. New York: Marcel Dekker, 391–447.Google Scholar
Fulka, J., Bradshaw, J., Jung, T. et al. . (1993). UV irradiation of metaphase II bovine oocytes (abstract).In Annual Conference of the Society for Study of Fertility, Cambridge, UK, p. 29. Dorchester, Dorset: Henry Ling, Doret press.Google Scholar
Gentry, W.L., Smith, A.L., Buster, J.E. et al. . (1989). Vital staining of murine blastocysts: stairns without decreasing viability, wheres DAPI impairs, embryogenesis(abstract).Google Scholar
Hardy, K., Winston, R.M.L. &Handyside, A.H.. (1991). Nuclear abnormalities and development arrest in human preimplantation embryos in vitro. Hum Reprod. 6, 152.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 ScholarPubMed
Maltzman, W. &Czyzyk, L.. (1984), UV irradiation stimulates levels of p53 cellular tumour antigen in nontransformed mouse cells. Mol. Cell Bio., 4, 1689–94.Google Scholar
Muggleton-Harris, A.L. &Aroian, M.A.. (1982). Replicative potential of individual cell hybrids derived from young and old donor human skin fibroblasts. Somatic Cell Genet. 8, 41–50.CrossRefGoogle Scholar
Munné, S., Lee, A., Rosenwaks, Z. et al. . (1993). Diagnosis of major chromosome aneuploides in human preimplantation embryos. Hum. Reprod. 8, 2185–91.CrossRefGoogle Scholar
Nasr-Esfahani, M., Johnson, M. &Aitken, R.J.. (1990). The effect of iron and iron chelators on the in vitro block to cevelopment of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod. 5, 997–1003.CrossRefGoogle Scholar
Otto, F., Tsou, K.C.. (1985). A comparative study of DAPI, DIPI, and Hoechst 33258 and 33342 as chromosomal DNA stains. Stain Technol. 60, 7–11.CrossRefGoogle ScholarPubMed
Smith, R. &Johnson, M.H.. (1985). DNA replication and compaction in the cleaving embryo of the mouse. J. Embryol. Exp. Morphol. 89, 133–48.Google ScholarPubMed
Taylor, A.S., Braude, P.R.. (1994). The early development and DNA content of activated human oocytes and parthenogenetic embryos. Hum Reprod. 9, 2389–97.CrossRefGoogle Scholar
Winston, N.J., Braude, P.R., Pickering, S.J. et al. . (1991). The incidence of abnormal morphology and nucleo-cytoplasmic ratios in 2, 3 and 5 day human pre-embryos. Hum Reprod. 6, 17–24.CrossRefGoogle Scholar
Winston, N.J., Johnson, M.H. &Braude, P.R.. (1993). Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containting single or multiple nuclei. Zygote. 1, 17–25.CrossRefGoogle ScholarPubMed
Woods, M.J., Trowbridg, E.A.. (1990). Megakaryocyte ploidy in thrombocytosis: improved microdensitometric measurements with a new image analysis system. Eur J Haematol. 44, 220–6.CrossRefGoogle ScholarPubMed