Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells

彗星试验 透明带 卵母细胞 DNA损伤 男科 彗星 遗传毒性 化学 分子生物学 DNA 生物 胚胎 遗传学 毒性 医学 有机化学 天体生物学
作者
Anaïs Berthelot-Ricou,Jeanne Perrin,Carole Di Giorgio,Michel De Méo,Alain Botta,Blandine Courbière
出处
期刊:Fertility and Sterility [Elsevier BV]
卷期号:95 (4): 1452-1457 被引量:35
标识
DOI:10.1016/j.fertnstert.2010.09.016
摘要

To develop and validate an efficient comet assay on mouse oocytes without depellucidation.In vitro experiments using a murine model.Biogenotoxicology research laboratory in Aix-Marseille II University, France.CD1 prepubescent female mice.DNA lesions in oocytes were evaluated by the alkaline comet assay. After oocyte retrieval, we first studied the effect of zona pellucida (ZP) on comet morphology. For this study, we applied the comet assay to mature oocytes with and without ZP after exposure to simulated sunlight irradiation (SSI) compared with negative controls. Next, nondepellucidated mouse oocytes were exposed to three well-known genotoxic agents (SSI, methylmethanesulfonate [MMS], and hydrogen peroxide [H(2)O(2)]) and compared with negative controls. Images of oocytes were analyzed with Komet software.DNA damages were quantified and expressed as olive tail moment (OTM), defined as the product of the tail length and the fraction of total DNA in the tail. OTMχ(2) were calculated from OTM; they corresponded to the degrees of freedom (n) of each OTM distribution obtained from at least 50 oocytes. OTMχ(2) is an indicator of DNA lesions. The test was considered positive and statistically significant when OTMχ(2) increased in oocytes compared with the medium-only control cells.There was no difference in comet aspect between oocyte groups with and without ZP. The three genotoxic agents significantly increased DNA damages as compared with the control groups. The OTMχ(2) values were (mean ± SD): 2.1 ± 0.07, 7.73 ± 0.35, 3.35 ± 0.15, and 12.4 ± 0.51 in control, SSI, MMS, and H(2)O(2) groups, respectively.Comet assay on non depellucidated mouse oocytes is a rapid and easy test. This assay would be useful to assess the genotoxicity on female germ cells of chemicals, drugs, or environmental pollutants and the efficiency of antioxidant molecules.
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