El Ensayo Cometa: una técnica para evaluar genotoxicidad en el ADN de oocitos bovinos
DOI:
https://doi.org/10.17533/udea.rccp.324014Keywords:
chromatin, DNA strand break, granulose cells, zona pellucidaAbstract
Resumen
La electroforesis en gel de células individuales o Ensayo Cometa, es frecuentemente usada para medir el daño en el ADN de las células. En la mayoría de estudios, los linfocitos de sangre periférica son utilizados como modelo celular. Sin embargo, en los ˙últimos años, la aplicación de esta técnica se ha extendido a las células germinales, con el fin de evaluar la integridad del ADN espermático, las condiciones de estrés oxidativo en oocitos porcinos y en embriones bovinos; además, la muerte celular en embriones de ratón. Para la realización de la electroforesis de células individuales en un sistema como los oocitos bovinos se hace imprescindible superar dos grandes limitantes en la estructura complejo-cúmulo-oocito (CCOS): una, la degradación de las glicoproteínas de la zona pelúcida y dos, eliminar el nivel de compactación del genoma haploide bovino en metafase II. Dichas limitantes no permiten una migración adecuada del ADN dentro del gel de agarosa. En este estudio se demuestra que el uso de una solución de lisis con proteinasa K durante 3 h y 10 min de corrido electroforético, permite obtener cometas cuantificables de oocitos bovinos madurados in vitro que pueden ser utilizados en estudios genotóxicos en este sistema celular.
Summary
The single cell gel electrophoresis or the comet assay is used to measure the DNA damage of individual cells, for most studies the lymphocytes from peripheral blood are used as cellular model. However, during the last years, the application of this technique has been extended to the germinal cells with the purpose of evaluating the integrity of the spermatic DNA, oxidative stress conditions in boar oocytes and bovine embryos besides cellular death in mouse embryos. Applying the comet assay to a system such as the bovine oocytes it becomes indispensable to overcome two big structural barriers in the cumulus-oocyte-complex (COC): first, the degradation of the glycoproteins in the zona pellucida and second, the elimination of the flatten level of the haploid metaphase II bovine oocyte genome. These obstacles do not allow an appropriate migration of the DNA through the agarose gel. In this study it is demonstrated that the use of a lysis solution with proteinase K during 3 h and 10 min in electrophoretic running allows to obtain quantifiable comets of bovine oocytes matured in vitro, which can be used in genotoxic studies.
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References
Aëarabi SY, Roussel J.D, Chandler JE. Chromosomalanalysis of mammalian oocytes matured in vitro withvarious culture systems. Theriogenology 1997; 48:1173-1183. DOI: https://doi.org/10.1016/S0093-691X(97)00350-6
Beker ARCL, Zeinestra E, Colenbrander B, Bevers MM.Effect of addition of 17‚- estradiol during IVM on bovineoocyte nuclear maturation and subsequent blastocystformation. Theriogenology 2002; 58: 1663-1673. DOI: https://doi.org/10.1016/S0093-691X(02)01082-8
Bond DJ, Chandley AC. Oxford Monographs on MedicalGenetics. No 11. Aneuploidy. Oxford University Press.Oxford. 1983.
Dekel N. Protein phosphorylation in the meiotic cell cycle ofmammalian oocytes. Reviews Reproduction 1996; 1: 82-88. DOI: https://doi.org/10.1530/revreprod/1.2.82
Dusan F, Rejak P, Czikkova S, Ilíkova G, Baran V, Koppel J.Induced cell death of preimplantation mouse embryoscultured in vitro evaluated by comet assay. Theriogenology2003; 60: 691-706. DOI: https://doi.org/10.1016/S0093-691X(03)00087-6
Gedick CM, Even SWB, Collins AR. Single cell gelelectrophoresis applied to the analysis of UV-C damage and itsrepair in human cells. Int. J. Radiat. Biol 1992; 62: 313-320. DOI: https://doi.org/10.1080/09553009214552161
Green MHL, Lowe JE, Harcourt SA, Akinluyi P, Rowe T,et al. UV-C Sensivity of unstimulated and stimulated humanlymphocytes from normal and xeroderma pigmentosumdonors in the comet assay: A potential diagnostic technique.Mutat. Res 1992; 273:137-144. DOI: https://doi.org/10.1016/0921-8777(92)90075-E
Hansmann I, Probeck HD. Detection of nondisjunction inmammals, Environ. Health Perspect. 1979; 31:161.165. DOI: https://doi.org/10.1289/ehp.7931161
Hawk HW, Wall RJ. Improved yields of bovine blastocystsfrom in vitro-produced oocytes. I. Selection oocytes andzygotes. Theriogenology 1994; 41:1571-83. DOI: https://doi.org/10.1016/0093-691X(94)90822-Z
Hendriksen PJM, Viuff D, Greve T, Vos PLAM, Mullaart E, etal. Effect of the origin of bovine oocytes on cell numbers andnumerical chromosome aberration in in vitro embryos collectedat day 3 post-insemination. Theriogenology 2001; 55: 475.
Hlinka D, Dudas M, Herman M, Kalina I. Experimentalattempts to extend the current preimplantation geneticdiagnosis with individual karyotypization of humanblastomeres. Reprod. Nut. Dev 2000; 41:91-106. DOI: https://doi.org/10.1051/rnd:2001115
Huges CM, Lewis SE, Mckelvey-Martin VJ, Thompson W.The effects of antioxidant supplementation during percollpreparation on human sperm DNA integrity. Hum Rep 1998;15: 1240-1247. DOI: https://doi.org/10.1093/humrep/13.5.1240
Katoh MA, Cain CT, Hughes LA, Foxworth LB, Bishop JB,Generoso WM. (1990). Female-specific dominant lethaleffect in mice. Mut Res 1990; 230: 205-217. DOI: https://doi.org/10.1016/0027-5107(90)90058-C
Kohn KW, Erickson LC, Ewig RG, Friedman CA.Fractionation of DNA from mammalian cells by alkalineelution. Biochem 1976; 15: 4629ñ4637. DOI: https://doi.org/10.1021/bi00666a013
Ledan E, Polanski Z, Terret ME, Maro B. Meiotic maturation ofthe mouse oocyte requires an equilibrium between cyclin Bsynthesis and degradation. Develop Biology 2001; 232: 400-413. DOI: https://doi.org/10.1006/dbio.2001.0188
Leibfried ML, Critser ES, Parrish JL, Fist NL. In vitromaturation and fertilization of bovine oocytes.Theriogenology 1989; 31: 6174-6180. DOI: https://doi.org/10.1016/0093-691X(89)90564-5
Lett JT, Klucis ES, Sun C. On the size of the DNA in themammalian chromosome. Biophys. J. 1970; 10: 277ñ292. DOI: https://doi.org/10.1016/S0006-3495(70)86300-7
Lipetz PD, Brash DE, Joseph LB Jewett HD, Lisle DR, etal. Determination of DNA super helicity and extremely lowlevel of DNA strand breaks in low numbers of non radiolabeled cells by 40,60-diamidino-2-phenylindol fluorescencein nucleoid gradients, Anal. Biochem 1982; 121: 330ñ348. DOI: https://doi.org/10.1016/0003-2697(82)90491-2
Mailhes JB, Young D, London SN. Cytogenetic effects ofcaffeine during in vivo mouse oocyte maturation. Mut Res1996; 11:395 ñ399 DOI: https://doi.org/10.1093/mutage/11.4.395
Ostling O, Johanson KJ. Microelectrophoretic study ofradiation induced DNA damages in individual mammaliancells. Biochem. Biophys. Res. Common 1984; 123: 291-298. DOI: https://doi.org/10.1016/0006-291X(84)90411-X
Pope CE, Mcrae MA, Plair BL, Keller GL, Dresser BL. In-vitro and in- vivo developmental of embryos produced by invitro maturation and in -vitro fertilization of cat oocytes.Journal of Reproduction and Fertility 1997; 51†:69-82.
Rydberg R, Johanson KJ. Estimation of DNA strand breaksin single mammalian cells. In: Hanwalt PC, Friedberg ECeditors. DNA Repair Mechanisms. New York : AcademicPress; 1978 p. 465ñ468. DOI: https://doi.org/10.1016/B978-0-12-322650-1.50090-4
Shimada T, Watanabe T, Endo A. Potential mutagenicity ofcadmium in mamalian oocytes. Mut. Res 1976; 40: 389-396. DOI: https://doi.org/10.1016/0165-1218(76)90038-0
Singh NP, Mccoy MT, Tice RR, Schneider EL. A simpletechnique for quantitation of low levels of DNA damage inindividual cells. Expl Cell Res 1988; 175:184-191. DOI: https://doi.org/10.1016/0014-4827(88)90265-0
Singh NP, Tice RR, Stephens RE, Schneider EL. A microgelelectrophoresis technique for the direct quantitation of DNAdamage and repair in individual fibroblasts cultured onmicroscope slides. Mutat Res 1991; 252: 289-296. DOI: https://doi.org/10.1016/0165-1161(91)90008-V
Singh NP, Stephens RE, Schneider LE. Modifications ofalkaline microgel electrophoresis for sensitive detection ofDNA damage, Int. J. Radiat. Biol. 1994; 66: 23ñ28. DOI: https://doi.org/10.1080/09553009414550911
Singh NP, Graham MM, Singh V, Khan A. Induction of DNAsingle strand breaks in human lymphocytes by low doses ofGamma Rays, Int. J. Radiat. Biol 1995; 68: 563ñ570. DOI: https://doi.org/10.1080/09553009514551551
Singh NP. Microgels for estimation of DNA strand breaks,DNA protein crosslinkís and apoptosis. Mutat Res 2000;455:111ñ127. DOI: https://doi.org/10.1016/S0027-5107(00)00075-0
Sudman PD, Rutledge JC, Bishop JB, Generoso WM.Bleomycin: female-specific dominant lethal effects in mice.Mut Res 1992; 296:143-156. DOI: https://doi.org/10.1016/0165-1110(92)90038-B
Takahashi M, Keicho K, Takahashi H, Ogawa H, Schultz M,Okano A. Effect of oxidative stress on development andDNA damage in in vitro cultured bovine embryos by cometassay. Theriogenology 2000; 54:137-145. DOI: https://doi.org/10.1016/S0093-691X(00)00332-0
Tatemoto H, Sakurai N, Muto N. Protection of porcineoocytes against apoptotic cell death caused by oxidativestress during in vitro maturation: role of cumulus cells. BiolRep 2000; 63: 805-810. DOI: https://doi.org/10.1095/biolreprod63.3.805
Tice R, Chaillet J, Schneider EL. Demonstration ofspontaneous sister chromatid exchanges in vivo. Exp. Cell.Res 1976; 102: 426-429. DOI: https://doi.org/10.1016/0014-4827(76)90062-8
Yuan Z, Mailhes JB. Aneuploidy determination in C-bandedmouse metaphase II oocytes following cyclophosphamidetreatment in vivo. Mut Res 1987; 179: 209-214. DOI: https://doi.org/10.1016/0027-5107(87)90311-3
Wassarman PM, Albertini DF. The mammalian ovum. In:Knobil E, Neill JD, editors. The Physiology of Reproduction.2nd ed. New York: Raven Press; 1994. p. 79-115.
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