XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
Lab Anim OnlineFirst, published on December 31, 2014 as doi:10.1177/0023677214567136
l
ab orator y
Short Report
a
n imals
l i m i t e d
A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus–oocyte complexes
Laboratory Animals 0(0) 1–4 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677214567136 la.sagepub.com
`re1 and F Greco1, J Perrin1,2,3, M Auffan2, V Tassistro1, T Orsie 1,4 B Courbiere
Abstract Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original–CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus–oocyte complexes (COC–CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC–CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC–CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC–CA is less timeconsuming and simplifies the protocol of the original–CA, because COC-CA is easier to perform without the washing-out procedure.
Keywords oocyte, cumulus–oocyte complexes, genotoxicity, comet assay, environment
Conventional genotoxicity tests such as micronucleus test, comet assay (CA) or Ames test1–3 are technically difficult to apply to oocytes, and results obtained with somatic cells cannot be extrapolated to gametes.4 In ovaries, follicle and oocyte development is dependent on communication between oocyte and somatic cells of cumulus–oocyte complexes (COC), thanks to paracrine pathways.5 CA is a well-validated technique for assessing low-level DNA damage in individual somatic cells.6 Regarding oocytes, only a few studies have used CA to assess DNA damage in mammalian oocytes.7–15 In a previous work, we adapted CA to denuded mouse oocytes with zona pellucida (ZP).12 However, in vivo, oocytes in ovaries are dependent on COC and are not isolated from follicular cells. Our objective was to adapt the CA for a more physiological approach for studying the genotoxicity of environmental factors on oocytes.
Materials and methods Reagents used were from Sigma Aldrich (Saint Quentin, Fallavier, France), and prepubescent female CD1 mice were from Charles River Laboratory 1
Aix Marseille Universite ´, CNRS, IRD, Avignon Universite ´, IMBE UMR 7263, Marseille, France 2 CEREGE, Aix-Marseille Universite ´, CNRS, UM34, UMR 7330, Aix en Provence, France 3 CECOS – Laboratory of Reproductive Medicine, AP-HM La Conception, Marseille, France 4 Department of Gynaecology, Obstetrics, and Reproduction, AP-HM La Conception, Marseille, France Corresponding author: Blandine Courbiere, MD, PhD, Department of Gynaecology, Obstetrics, and Reproduction, AP-HM La Conception, 13 005 Marseille, France. Emails: [email protected]; [email protected]
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
2
Laboratory Animals 0(0)
(L’Arbresle, France). IRB approval (No. 12-18042012) was obtained from the National Ethics Committee on Animal Experimentation. Metaphase II oocytes were collected after superovulation and mouse euthanasia, as described previously.12
Exposure conditions Two CAs were compared: (i) the original CA (original– CA), as previously described, with denuded oocytes,12 and (ii) the CA on COC (COC–CA), as described below. Two agents inducing DNA damage toward mouse oocytes (already described with the original– CA) were used to validate the COC–CA: hydrogen peroxide (H2O2) and cerium dioxide nanoparticles (CeO2 NPs).16 Three conditions were studied: . Negative control group: denuded oocytes (original– CA)/COC (COC–CA) incubated 2 h in M16 medium (37 C, 5% CO2). . H2O2 positive control group: after incubation over 2 h in M16 medium (37 C, 5% CO2), oocytes were placed in 250 mmol H2O2 solution for 5 min at 4 C. For COC–CA, hyaluronidase was added after incubation with H2O2. . CeO2 NP group: incubation with CeO2 NPs at 100 mg/L for 2 h in M16 medium (37 C, 5% CO2). At least 40 denuded oocytes for the original–CA and 40 COC for the COC–CA were used for each condition; all conditions were carried out in triplicate.
Alkaline CA on COC The original–CA was performed as previously described by Berthelot-Ricou et al.12 For the COC– CA, modifications of the original–CA were performed at the stage where hyaluronidase was used to separate the oocyte from their surrounding follicular cells. For the original–CA, hyaluronidase (10 mg/L over 10 min) was added before exposure. Then, oocytes and follicular cells were washed out to remove hyaluronidase and were incubated separately with the tested agents before the CA. For COC–CA, COC were removed from the oviducts, then incubated immediately with the tested agents. After incubation, hyaluronidase was added to separate the oocyte from the follicular cells, and the CA was performed immediately without the washing-out step.
Main outcome measures and statistical analysis Examination was performed after propidium iodide staining using a fluorescence microscope (X200, Olympus BX-60; Olympus, Rungis, France). Pictures were obtained with Visilog software version 6.7 (Noesis SA, Saint Aubin, France). DNA damage was assessed with olive tail moment (OTM) analysed by Komet software (version 5.5; Andor Bioimaging, Nottingham, UK). OTM was defined as the product of the tail length by DNA% contained in the tail of the comet.1 An analysis of variance (ANOVA) analysis was performed followed by Fisher LSD post hoc test
Figure 1. Genotoxicity assessment with the original comet assay (a) and the comet assay performed on cumulus–oocyte complexes (b). *P 0.05. OTM: olive tail moment; CeO2 NP: cerium dioxide nanoparticles.
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
Greco et al.
3
Figure 2. Images obtained with comet assay on cumulus–oocyte complexes for negative control group (a) and positive control group (b).
Table 1. Summary of different studies using comet assay on mammalian oocytes.
Authors Chan et al. 2000
13
Jebelli et al. 200114
Men et al. 20038
Wang et al. 200615
Huang et al. 20087
Stachowiak et al. 200911
Sharma et al. 201010
Novotna et al. 20109
Berthelot-Ricou et al. 201112
No. of studied oocyte and main outcome measure for DNA damage
Objective of comet assay
Animals
To develop a comet assay in thawed cryopreserved oocyte To assess impact of luteal phase sera from ART patients on oocyte To examine the effect of three cryopreservation vitrification protocols on the integrity oocyte DNA To assess effect of gonadotropin stimulation on oocyte
Cryopreserved hamster oocyte ZPþ Hamster oocyte ZPþ
2 oocytes/slide Pixel intensity
Bovine oocyte ZP–
142 oocytes/condition Length of comet tail
Mice oocyte ZP–
To evaluate the effect of vitrification on in vitro-matured oocytes To test three vitrification protocols on oocyte
Mice oocyte ZP–
32 oocytes/condition No. of damaged oocyte and length of comet tail 50 oocytes/condition Length of comet tail
To investigate the impact of two cryodevices for oocyte vitrification To test effect of different activation modes on oocyte maturated in vitro To develop a easy and reproductible comet assay on oocyte
Bovine oocyte ZP–
Bovine oocyte ZP–
2 oocytes/slide Pixel intensity
30 oocytes/condition Semi-quantitative scale based on length and intensity of comet tail 220 oocytes/condition Length of DNA migrated
Porcine oocyte ZP–
32 oocytes/condition DNA% in the tail
Mice oocyte ZPþ
120 oocytes/condition OTM
ZP–: oocyte without zona pellucida; ZPþ: oocyte with zona pellucida; ART: assisted reproduction technique; OTM: olive tail moment.
using StatviewÕ 5.1 for Windows (Abacus Concepts, Berkeley, CA, USA). For each condition, results were expressed as mean OTM values SEM. Results were considered significant at P < 0.05.
Results An average of 100 oocytes were analysed by condition. With the original–CA (Figure 1a), significant DNA
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
4
Laboratory Animals 0(0)
damage was observed following H2O2 (OTM ¼ 38 1.3) and CeO2 NP (OTM ¼ 11.6 0.8) exposures compared with the negative control (OTM ¼ 2.9 0.2). With COC–CA (Figure 1b), significant DNA damage was observed following H2O2 (OTM ¼ 36 1.3) and CeO2 NP (OTM ¼ 20 1.6) exposures compared with the negative control (OTM ¼ 5.4 0.6). Figure 2 shows that comet pictures obtained with COC–CA looked the same as those obtained with the original–CA.
Discussion CA is usually described with regard to denuded oocytes without ZP (Table 1). CA on COC offers an interesting tool for testing the genotoxicity of environmental agents towards germinal cells. This method gets closer to in vivo conditions of exposure. In addition, the COC–CA is less time-consuming and simplifies the protocol of the original–CA, because COC-CA is easier to perform without the washing-out procedure. A further development would be to perform COC–CA after in vivo exposure of mice. Declaration of competing interests FG was awarded a scholarship from the ‘Foundation, health, sport and development’, Aix Marseille University for a master’s degree. JP, MA, TO, and BC have nothing to disclose.
Funding This work is a contribution to the LABEX SERENADE (No. ANR-11-LABX-0064) funded by the French Government program of the French National Research Agency (ANR) through the A*Midex project (No. ANR11-IDEX-0001-02).
References 1. Tice RR, Agurell E, Anderson D, et al. Single cell gel/ comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000; 35: 206–221. 2. Ames BN, Mccann J and Yamasaki E. Methods for detecting carcinogens and mutagens with the salmonella/mammalian-microsome mutagenicity test. Mutat Res 1975; 31: 347–364. 3. Burlinson B, Tice RR, Speit G, et al. Fourth International Workgroup on Genotoxicity Testing: results of the in vivo comet assay workgroup. Mutat Res 2007; 627: 31–35. 4. Aye M, Di Giorgio C, De Meo M, Botta A, Perrin J and Courbie`re B. Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification: dimethyl sulfoxide, ethylene glycol and propylene glycol. Food Chem Toxicol 2010; 48: 1905–1912.
5. Sutton ML, Gilchrist RB and Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus–oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update 2003; 9: 35–48. 6. Singh NP, McCoy MT, Tice RR and Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184–191. 7. Huang JYJ, Chen HY, Park JYS, Tan SL and Chian R-C. Comparison of spindle and chromosome configuration in in vitro- and in vivo-matured mouse oocytes after vitrification. Fertil Steril 2008; 90: 1424–1432. 8. Men H, Monson RL, Parrish JJ and Rutledge JJ. Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation. Mol Reprod Dev 2003; 64: 245–250. 9. Novotna´ B, Petr J, Sedmı´ kova´ M, Kratochvilova´ J and Jı´ lek F. Effect of different activation modes on DNA integrity of porcine M II oocytes matured in vitro. Zygote Camb Engl 2010; 18: 81–87. 10. Sharma GT, Dubey PK and Chandra V. Morphological changes, DNA damage and developmental competence of in vitro matured, vitrified-thawed buffalo (Bubalus bubalis) oocytes: a comparative study of two cryoprotectants and two cryodevices. Cryobiology 2010; 60: 315–321. 11. Stachowiak EM, Papis K, Kruszewski M, Iwanenko T, Bartomiejczyk T and Modlinski JA. Comparison of the level(s) of DNA damage using comet assay in bovine oocytes subjected to selected vitrification methods. Reprod Domest Anim Zuchthyg 2009; 44: 653–658. 12. Berthelot-Ricou A, Perrin J, Di Giorgio C, De Meo M, Botta A and Courbiere B. Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells. Fertil Steril 2011; 95: 1452–1457. 13. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chana SR and King A. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001; 75: 186–192. 14. Jebelli B, Chan PJ, Corselli J, Patton WC and King A. Clinical assisted reproduction: oocyte comet assay of luteal phase sera from nonpregnant patients after assisted reproductive procedures. J Assist Reprod Genet 2001; 18: 421–425. 15. Wang Y, Ock S-A and Chian R-C. Effect of gonadotrophin stimulation on mouse oocyte quality and subsequent embryonic development in vitro. Reprod Biomed Online 2006; 12: 304–314. 16. Courbiere B, Auffan M, Rollais R, et al. Ultrastructural interactions and genotoxicity assay of cerium dioxide nanoparticles on mouse oocytes. Int J Mol Sci 2013; 14: 21613–21628.
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
(LAN)
[1–4] [PREPRINTER stage]
Lab Anim OnlineFirst, published on December 31, 2014 as doi:10.1177/0023677214567136
l
ab orator y
Short Report
a
n imals
l i m i t e d
A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus–oocyte complexes
Laboratory Animals 0(0) 1–4 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677214567136 la.sagepub.com
`re1 and F Greco1, J Perrin1,2,3, M Auffan2, V Tassistro1, T Orsie 1,4 B Courbiere
Abstract Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original–CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus–oocyte complexes (COC–CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC–CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC–CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC–CA is less timeconsuming and simplifies the protocol of the original–CA, because COC-CA is easier to perform without the washing-out procedure.
Keywords oocyte, cumulus–oocyte complexes, genotoxicity, comet assay, environment
Conventional genotoxicity tests such as micronucleus test, comet assay (CA) or Ames test1–3 are technically difficult to apply to oocytes, and results obtained with somatic cells cannot be extrapolated to gametes.4 In ovaries, follicle and oocyte development is dependent on communication between oocyte and somatic cells of cumulus–oocyte complexes (COC), thanks to paracrine pathways.5 CA is a well-validated technique for assessing low-level DNA damage in individual somatic cells.6 Regarding oocytes, only a few studies have used CA to assess DNA damage in mammalian oocytes.7–15 In a previous work, we adapted CA to denuded mouse oocytes with zona pellucida (ZP).12 However, in vivo, oocytes in ovaries are dependent on COC and are not isolated from follicular cells. Our objective was to adapt the CA for a more physiological approach for studying the genotoxicity of environmental factors on oocytes.
Materials and methods Reagents used were from Sigma Aldrich (Saint Quentin, Fallavier, France), and prepubescent female CD1 mice were from Charles River Laboratory 1
Aix Marseille Universite ´, CNRS, IRD, Avignon Universite ´, IMBE UMR 7263, Marseille, France 2 CEREGE, Aix-Marseille Universite ´, CNRS, UM34, UMR 7330, Aix en Provence, France 3 CECOS – Laboratory of Reproductive Medicine, AP-HM La Conception, Marseille, France 4 Department of Gynaecology, Obstetrics, and Reproduction, AP-HM La Conception, Marseille, France Corresponding author: Blandine Courbiere, MD, PhD, Department of Gynaecology, Obstetrics, and Reproduction, AP-HM La Conception, 13 005 Marseille, France. Emails: [email protected]; [email protected]
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
2
Laboratory Animals 0(0)
(L’Arbresle, France). IRB approval (No. 12-18042012) was obtained from the National Ethics Committee on Animal Experimentation. Metaphase II oocytes were collected after superovulation and mouse euthanasia, as described previously.12
Exposure conditions Two CAs were compared: (i) the original CA (original– CA), as previously described, with denuded oocytes,12 and (ii) the CA on COC (COC–CA), as described below. Two agents inducing DNA damage toward mouse oocytes (already described with the original– CA) were used to validate the COC–CA: hydrogen peroxide (H2O2) and cerium dioxide nanoparticles (CeO2 NPs).16 Three conditions were studied: . Negative control group: denuded oocytes (original– CA)/COC (COC–CA) incubated 2 h in M16 medium (37 C, 5% CO2). . H2O2 positive control group: after incubation over 2 h in M16 medium (37 C, 5% CO2), oocytes were placed in 250 mmol H2O2 solution for 5 min at 4 C. For COC–CA, hyaluronidase was added after incubation with H2O2. . CeO2 NP group: incubation with CeO2 NPs at 100 mg/L for 2 h in M16 medium (37 C, 5% CO2). At least 40 denuded oocytes for the original–CA and 40 COC for the COC–CA were used for each condition; all conditions were carried out in triplicate.
Alkaline CA on COC The original–CA was performed as previously described by Berthelot-Ricou et al.12 For the COC– CA, modifications of the original–CA were performed at the stage where hyaluronidase was used to separate the oocyte from their surrounding follicular cells. For the original–CA, hyaluronidase (10 mg/L over 10 min) was added before exposure. Then, oocytes and follicular cells were washed out to remove hyaluronidase and were incubated separately with the tested agents before the CA. For COC–CA, COC were removed from the oviducts, then incubated immediately with the tested agents. After incubation, hyaluronidase was added to separate the oocyte from the follicular cells, and the CA was performed immediately without the washing-out step.
Main outcome measures and statistical analysis Examination was performed after propidium iodide staining using a fluorescence microscope (X200, Olympus BX-60; Olympus, Rungis, France). Pictures were obtained with Visilog software version 6.7 (Noesis SA, Saint Aubin, France). DNA damage was assessed with olive tail moment (OTM) analysed by Komet software (version 5.5; Andor Bioimaging, Nottingham, UK). OTM was defined as the product of the tail length by DNA% contained in the tail of the comet.1 An analysis of variance (ANOVA) analysis was performed followed by Fisher LSD post hoc test
Figure 1. Genotoxicity assessment with the original comet assay (a) and the comet assay performed on cumulus–oocyte complexes (b). *P 0.05. OTM: olive tail moment; CeO2 NP: cerium dioxide nanoparticles.
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
Greco et al.
3
Figure 2. Images obtained with comet assay on cumulus–oocyte complexes for negative control group (a) and positive control group (b).
Table 1. Summary of different studies using comet assay on mammalian oocytes.
Authors Chan et al. 2000
13
Jebelli et al. 200114
Men et al. 20038
Wang et al. 200615
Huang et al. 20087
Stachowiak et al. 200911
Sharma et al. 201010
Novotna et al. 20109
Berthelot-Ricou et al. 201112
No. of studied oocyte and main outcome measure for DNA damage
Objective of comet assay
Animals
To develop a comet assay in thawed cryopreserved oocyte To assess impact of luteal phase sera from ART patients on oocyte To examine the effect of three cryopreservation vitrification protocols on the integrity oocyte DNA To assess effect of gonadotropin stimulation on oocyte
Cryopreserved hamster oocyte ZPþ Hamster oocyte ZPþ
2 oocytes/slide Pixel intensity
Bovine oocyte ZP–
142 oocytes/condition Length of comet tail
Mice oocyte ZP–
To evaluate the effect of vitrification on in vitro-matured oocytes To test three vitrification protocols on oocyte
Mice oocyte ZP–
32 oocytes/condition No. of damaged oocyte and length of comet tail 50 oocytes/condition Length of comet tail
To investigate the impact of two cryodevices for oocyte vitrification To test effect of different activation modes on oocyte maturated in vitro To develop a easy and reproductible comet assay on oocyte
Bovine oocyte ZP–
Bovine oocyte ZP–
2 oocytes/slide Pixel intensity
30 oocytes/condition Semi-quantitative scale based on length and intensity of comet tail 220 oocytes/condition Length of DNA migrated
Porcine oocyte ZP–
32 oocytes/condition DNA% in the tail
Mice oocyte ZPþ
120 oocytes/condition OTM
ZP–: oocyte without zona pellucida; ZPþ: oocyte with zona pellucida; ART: assisted reproduction technique; OTM: olive tail moment.
using StatviewÕ 5.1 for Windows (Abacus Concepts, Berkeley, CA, USA). For each condition, results were expressed as mean OTM values SEM. Results were considered significant at P < 0.05.
Results An average of 100 oocytes were analysed by condition. With the original–CA (Figure 1a), significant DNA
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
XML Template (2014) [30.12.2014–2:15pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140051/APPFile/SG-LANJ140051.3d
(LAN)
[1–4] [PREPRINTER stage]
4
Laboratory Animals 0(0)
damage was observed following H2O2 (OTM ¼ 38 1.3) and CeO2 NP (OTM ¼ 11.6 0.8) exposures compared with the negative control (OTM ¼ 2.9 0.2). With COC–CA (Figure 1b), significant DNA damage was observed following H2O2 (OTM ¼ 36 1.3) and CeO2 NP (OTM ¼ 20 1.6) exposures compared with the negative control (OTM ¼ 5.4 0.6). Figure 2 shows that comet pictures obtained with COC–CA looked the same as those obtained with the original–CA.
Discussion CA is usually described with regard to denuded oocytes without ZP (Table 1). CA on COC offers an interesting tool for testing the genotoxicity of environmental agents towards germinal cells. This method gets closer to in vivo conditions of exposure. In addition, the COC–CA is less time-consuming and simplifies the protocol of the original–CA, because COC-CA is easier to perform without the washing-out procedure. A further development would be to perform COC–CA after in vivo exposure of mice. Declaration of competing interests FG was awarded a scholarship from the ‘Foundation, health, sport and development’, Aix Marseille University for a master’s degree. JP, MA, TO, and BC have nothing to disclose.
Funding This work is a contribution to the LABEX SERENADE (No. ANR-11-LABX-0064) funded by the French Government program of the French National Research Agency (ANR) through the A*Midex project (No. ANR11-IDEX-0001-02).
References 1. Tice RR, Agurell E, Anderson D, et al. Single cell gel/ comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000; 35: 206–221. 2. Ames BN, Mccann J and Yamasaki E. Methods for detecting carcinogens and mutagens with the salmonella/mammalian-microsome mutagenicity test. Mutat Res 1975; 31: 347–364. 3. Burlinson B, Tice RR, Speit G, et al. Fourth International Workgroup on Genotoxicity Testing: results of the in vivo comet assay workgroup. Mutat Res 2007; 627: 31–35. 4. Aye M, Di Giorgio C, De Meo M, Botta A, Perrin J and Courbie`re B. Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification: dimethyl sulfoxide, ethylene glycol and propylene glycol. Food Chem Toxicol 2010; 48: 1905–1912.
5. Sutton ML, Gilchrist RB and Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus–oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update 2003; 9: 35–48. 6. Singh NP, McCoy MT, Tice RR and Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184–191. 7. Huang JYJ, Chen HY, Park JYS, Tan SL and Chian R-C. Comparison of spindle and chromosome configuration in in vitro- and in vivo-matured mouse oocytes after vitrification. Fertil Steril 2008; 90: 1424–1432. 8. Men H, Monson RL, Parrish JJ and Rutledge JJ. Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation. Mol Reprod Dev 2003; 64: 245–250. 9. Novotna´ B, Petr J, Sedmı´ kova´ M, Kratochvilova´ J and Jı´ lek F. Effect of different activation modes on DNA integrity of porcine M II oocytes matured in vitro. Zygote Camb Engl 2010; 18: 81–87. 10. Sharma GT, Dubey PK and Chandra V. Morphological changes, DNA damage and developmental competence of in vitro matured, vitrified-thawed buffalo (Bubalus bubalis) oocytes: a comparative study of two cryoprotectants and two cryodevices. Cryobiology 2010; 60: 315–321. 11. Stachowiak EM, Papis K, Kruszewski M, Iwanenko T, Bartomiejczyk T and Modlinski JA. Comparison of the level(s) of DNA damage using comet assay in bovine oocytes subjected to selected vitrification methods. Reprod Domest Anim Zuchthyg 2009; 44: 653–658. 12. Berthelot-Ricou A, Perrin J, Di Giorgio C, De Meo M, Botta A and Courbiere B. Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells. Fertil Steril 2011; 95: 1452–1457. 13. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chana SR and King A. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001; 75: 186–192. 14. Jebelli B, Chan PJ, Corselli J, Patton WC and King A. Clinical assisted reproduction: oocyte comet assay of luteal phase sera from nonpregnant patients after assisted reproductive procedures. J Assist Reprod Genet 2001; 18: 421–425. 15. Wang Y, Ock S-A and Chian R-C. Effect of gonadotrophin stimulation on mouse oocyte quality and subsequent embryonic development in vitro. Reprod Biomed Online 2006; 12: 304–314. 16. Courbiere B, Auffan M, Rollais R, et al. Ultrastructural interactions and genotoxicity assay of cerium dioxide nanoparticles on mouse oocytes. Int J Mol Sci 2013; 14: 21613–21628.
Downloaded from lan.sagepub.com at UZH Hauptbibliothek / Zentralbibliothek Zürich on January 6, 2015
