Comet assay on one- and two-cell mouse embryos
Introduction
Numerous animal studies have shown that DNA damage transmitted via germ cells leads to a broad spectrum of abnormal reproductive outcomes, including pregnancy loss, birth defects, offspring with chromosomal abnormalities and various genetic diseases (McFadden and Friedman, 1997; Marchetti and Wyrobek, 2005). Understanding the mechanism of induction and the embryonic consequences of transmissible DNA damage is important for assessing the genetic risk associated with exposure of germ cells to genotoxic agents. Among standard methods used for assessing transgenerational transmission of DNA damage are the dominant lethal test, the heritable translocation and specific locus tests (Yauk et al., 2015). However, there is still a need for the development of new, more sensitive and higher throughput techniques in this field.
Comet assay, a technique for highly sensitive detection of DNA damage at the level of an individual eukaryotic cell, is now widely adopted for various types of cells (Koppen et al., 2017). In particular, the assay has been used to assess DNA damage in male germ cells and oocytes (Baumgartner et al., 2009; Speit et al., 2009; Berthelot-Ricou et al., 2011; Einaudi et al., 2014).
The application of comet assay to assess DNA damage in animal embryos has been described in only a few studies in which heterogeneous protocols and species have been used (Takahashi et al., 1999, 2000; Harrouk et al., 2000; Uno et al., 2000; Fabian et al., 2003; Müller et al., 1996; Sturmey et al., 2009). A survey of the literature carried out in the paper (Rolland et al., 2017) showed that there is no standardized comet assay protocol for embryos. The authors optimized the comet assay protocol for thawed embryos. The aim of our study was to employ the comet assay to detect DNA damage in one and two-cell mouse embryos obtained by natural mating, including DNA damage induced by in vivo exposure of maternal germ cells to genotoxic agents.
Section snippets
Animals
The experiments were performed on totally 160 female and 80 male F1 CBA x C57BL/6 mice (20–22 g) obtained from the central animal facility of the Zakusov Research Institute of Pharmacology. The mice were kept under controlled conditions at the room temperature of 22 ± 2 °C and at relative humidity of 55 ± 10 % with an automatically controlled 12 h light/dark cycle. Standard laboratory animal feed (MEST Ltd, Russia) and water were provided ad libitum. Animals were quarantined for at least one
In vitro experiments
Since the main limitation of embryo research lies in the small number of available samples, one of the objectives of the study was to minimize the loss of embryos on the slides. When the protocol with two layers of agarose was used, a significant loss of embryos was observed: the overall recovery rate was 39.2 ± 8.8 %. It is worth noting that no detachment and loss of gels or their parts was observed. Spreading of agarose with embryos using a pipette tip (second layer) and applying the third
Discussion
The comet assay has been developed initially for large numbers of somatic cells and the use of this technique for embryos has met with difficulty due to a limited number of cells available for analysis. So, one of the main issues is to reduce the loss of embryos during slide preparation. All of the published comet assay studies on embryos have used two layers of agarose and only in two studies a protocol with three layers has been used (Takahashi et al., 1999, 2000; Harrouk et al., 2000; Uno et
Conclusion
In this study, we used the comet assay to measure DNA damage in one- and two-cell mouse embryos obtained by natural mating. We confirmed previous data that using a protocol with three layers of agarose reduces the embryo loss and enables the study of a small number of embryos. A significantly lower basal DNA damage level was found in embryos with removed ZP compared to embryos with intact ZP. The in vivo data show that the DNA damage induced in maternal germ cells persists, which can be
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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