Comet assay and early apoptosis

Choucroun, P; Gillet, Daniel; Dorange, Germaine; Sawicki, B; Dewitte, J.-D.

doi:10.1016/s0027-5107(01)00123-3

Cited by 145 publications

(75 citation statements)

References 12 publications

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“…The comet assay has been used as a biomarker of oxidative stress-mediated DNA strand breaks in individual cells (Singh et al, 1988;Visvardis et al, 1997;Choucroun et al, 2001) and embryos (Takahashi et al, 2000). In fact, embryo development in vitro under ordinary culture systems is also compromised by the toxic effects of atmospheric oxygen concentration which is approximately 20% (Takahashi et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

“…Comet assay can also be used to detect fragments of DNA that occur in apoptosis (Choucroun et al, 2001) and it has been well documented that in vitro-produced embryos do undergo apoptosis (Knijn et al, 2003). The effect of melatonin on the regulation of cellular apoptosis processes has also been documented (Sainz et al, 2003).…”

Section: Resultsmentioning

confidence: 99%

“…In spite of this method being frequently used to evaluate the genotoxicity of test substances there is evidence that comet pictures might be associated with apoptotic nuclei (Choucroun et al, 2001). Additionally, a significant relationship between supplementation of maturation medium and apoptotic index of resulting embryos has been reported (Warzych et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

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Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Takada

Martins

Mingoti

et al. 2010

Sci. agric. (Piracicaba, Braz.)

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Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum -FCS) at 39°C and 5% CO 2 in air. After 24 hours of culture in MM with 0.5 μg mL -1 FSH and 5.0 μg mL -1 LH; 10 -9 M MEL) or 10 -9 M MEL, 0.5 μg mL -1 FSH and 5.0 μg mL -1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10 -9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM. Key words: comet assay, embryos, gonadotropins, in vitro fertilization, melatonin Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos RESUMO: Melatonin (MEL) atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas) nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro. Complexos cumulus-ovócitos foram aspirados de ovários provenientes de matadouros e cultivados em meio de maturação (Meio TCM-199 suplementado com 10% de soro fetal bovino) a 39°C e 5% CO 2 em ar. Após 24 horas de cultivo em meio de maturação com 0,5 μg mL -1 de FSH e 5,0 μg mL -1 de LH; 10 -9 M de MEL ou 10 -9 M de MEL, 0,5 μg mL -1 de FSH e 5,0 μg mL -1 de LH, os ovócitos foram corados com Hoechst 33342 para avaliação da taxa de maturação nuclear. A fragmentação do DNA dos blastocistos foi avaliada pela técnica do Cometa. Não houve efeito da MEL adicionada ao meio de maturação, com ou sem gonadotrofinas, nas taxas de maturação nuclear, clivagem e blastocistos. Os percentuais variaram entre 88% a 90%, 85% a 88% e 42% a 46%, respectivamente. O grau de fragmentação do DNA também não foi afetado pela adição de MEL ao meio de matur...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Takada

Martins

Mingoti

et al. 2010

Sci. agric. (Piracicaba, Braz.)

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“…2D). To avoid comets originating from apoptotic cells that could perturb the results (Choucroun et al, 2001), we analysed cells from 1-day-old embryoid bodies because apoptosis was not detectable at this time point (Fig. 1C,D).…”

Section: Inhibition Of Kdm6 Protein Activity During Esc Differentiatimentioning

confidence: 99%

Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage

Hofstetter¹,

Kampka²,

Huppertz³

et al. 2016

Development

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Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2-and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.

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“…Therefore, this assay seems to detect cell death earlier than the morphologic assay. Studies showing early detection of cell death by comet assay are few (17,21), and further investigations are required to completely establish this application.…”

Section: Discussionmentioning

confidence: 99%

Single‐cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death

Chandna

2004

Cytometry Pt A

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Background: Single-cell gel electrophoresis, or the comet assay, a technique widely used for DNA damage analysis, has been used recently for detecting DNA fragmentation in cells undergoing apoptosis. However, the number of variants of this assay used thus far primarily detected the late stages of DNA fragmentation. Therefore, monitoring the progression of DNA fragmentation, which could greatly improve the analysis of cell death induction and progression at the single-cell level, has not been possible with this assay. Methods: In the present study, a modification of the original neutral comet assay developed by Ostling and Johanson (Biochem Biophys Res Commun 123:291-298, 1984) was used to detect various stages of DNA fragmentation. This assay involves cell lysis with anionic detergents at nearly neutral pH (9.5) and does not include high salt concentration, unlike most other published methods. BMG-1 human glioma cells were induced to undergo programmed cell death by treating with a large dose (100 M) of etoposide, and comets were prepared after different durations (1-24 h) of treatment. Results: In contrast to results of previously published studies, comets with different shapes reflecting progressive stages of DNA fragmentation were observed. Of these, six distinct shapes were identified and divided into

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Comet assay and early apoptosis

Cited by 145 publications

References 12 publications

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage

Single‐cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death

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