Assessment of DNA damage in individual hamster embryos by comet assay.
DNA damage induced by either light exposure or oxidative stress likely contributes to the compromised development in vitro of cultured preimplantation embryos. Using the comet assay, which entails microgel electrophoresis that can readily detect single-strand breaks in DNA, a significant increase in DNA damage was detected in individual one-cell hamster embryos that were treated with either ultraviolet light or hydrogen peroxide. In addition, an increase in DNA damage also was observed following exposure of one-cell embryos to visible light. When the embryos were placed in drops of culture medium that were covered with mineral oil and the dishes then placed in a portable incubator containing 5% CO(2) in air at 37 degrees C, visible and UV light irradiation for 30 min still induced extensive DNA damage when compared to control embryos that were kept in the dark. In contrast, infrared irradiation did not induce an increase in DNA damage. DNA damage also was measured in individual one- and two-cell stage embryos developed in vivo or in vitro. The extent of DNA damage in the cultured embryos was significantly greater than in embryos that developed in vivo. These results highlight the usefulness of the comet assay to assess DNA damage in individual preimplantation embryos and how the assay can be used to monitor culture conditions in vitro.
Takahashi
M
M
Department of Animal Reproduction, National Institute of Animal Industry, Ibaraki, Japan. masashi@niai.affrc.go.jp
Saka
N
N
Takahashi
H
H
Kanai
Y
Y
Schultz
R M
RM
Okano
A
A
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D004249:DNA Damage; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004622:Embryo, Mammalian / Q000187:drug effects / Q000528:radiation effects*; D005314:Embryonic and Fetal Development / Q000528:radiation effects; D004996:Ethidium; D006861:Hydrogen Peroxide / Q000633:toxicity; D007259:Infrared Rays / Q000009:adverse effects; D008027:Light / Q000009:adverse effects; D008647:Mesocricetus / Q000196:embryology*; D009152:Mutagenicity Tests; D018384:Oxidative Stress; D014466:Ultraviolet Rays / Q000009:adverse effects
D006861:Hydrogen Peroxide; D004996:Ethidium