Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay.
The occurrence of apoptosis in mouse preimplantation embryos was analyzed using DNA staining (Hoechst 33342, PI) for the visualization of nuclear changes and by the comet assay, a single-cell gel electrophoresis assay, modified for the analysis of blastocysts. Mouse preimplantation embryos isolated 56 h after superovulation were cultured in vitro for 64 h. Apoptosis was induced by treatment with camptothecin and actinomycin D during the first 15 h of culture. After culture in vitro, a number of embryos were stained and analyzed using morphological criteria. The remaining embryos were examined using the comet assay for the detection of DNA fragmentation. The proportion of damaged embryos in experimental groups, in comparison to controls, was dependent on the dose of apoptosis inductor. At high doses (camptothecin, microg/ml and actinomycin D, 0.05 microg/ml) over 90% (chi-square test, P<0.001) of embryos had apoptotic comets, at medium doses (camptothecin, 0.01 microg/ml and actinomycin D, 0.005 microg/ml) comets appeared only in 30-70% of embryos (camptothecin, P<0.01 and actinomycin D, P<0.001). At low doses (camptothecin, 0.001 microg/ml and actinomycin D, 0.0005 microg/ml) the increase in damaged embryos was not statistically significant. Hoechst/PI staining showed a higher percentage of damaged blastomeres at high doses. Morphological changes correlated with the outcome of the comet assay. Our results show that comet assay is an appropriate method for studying apoptosis in preimplantation embryos, and it appears to be more sensitive than the classically used morphological analyses.
Fabian
Dusan
D
Institute of Animal Physiology, Slovak Academy of Sciences, Soltésovej 4, 040 01 Kosice, Slovak Republic.
Rehák
Pavol
P
Czikková
Sona
S
Il'ková
Gabika
G
Baran
Vladimír
V
Koppel
Juraj
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001562:Benzimidazoles; D001755:Blastocyst / Q000737:chemistry / Q000166:cytology; D002166:Camptothecin / Q000494:pharmacology; D020552:Comet Assay; D046508:Culture Techniques; D004247:DNA / Q000032:analysis; D003609:Dactinomycin / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology*; D047108:Embryonic Development; D005260:Female; D005456:Fluorescent Dyes; D051379:Mice; D008813:Mice, Inbred ICR; D011247:Pregnancy; D011419:Propidium
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D003609:Dactinomycin; D011419:Propidium; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride; D002166:Camptothecin