Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containing single or multiple nuclei.
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.
Winston
N J
NJ
Department of Anatomy, University of Cambridge, UK.
Johnson
M H
MH
Braude
P R
PR
D000818:Animals; D001755:Blastocyst / Q000737:chemistry; D001757:Blastomeres / Q000737:chemistry*; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D004171:Diploidy; D005260:Female; D005307:Fertilization in Vitro; D005456:Fluorescent Dyes; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007211:Indoles; D051379:Mice; D015739:Nocodazole / Q000494:pharmacology; D009865:Oocytes / Q000737:chemistry* / Q000187:drug effects; D015053:Zygote / Q000737:chemistry
D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA; D015739:Nocodazole