Matured bovine oocytes and zygotes obtained by in vitro maturation and fertilization techniques (IVM and IVF) were cytogenetically prepared by using an improved method for chromosome preparations. The method, which involves use of trypsinizedhypotonic solution plus a vortex-agitated system and very cold two-step fixation process, contributes to weaken the zona pellucida and allows the swelling of oocytes and zygotes which helps the subsequent spreading of the chromosomes. This method permits to obtain preparations of good quality for examining the number and morphology of the chromosomes of oocytes and zygotes for any meiotic and mitotic stage.Se logró optimizar un método rápido para analizar citogenéticamente ovocitos y cigotos de bovino obtenidos mediante técnicas de maduración y fecundación in vitro (MIV y FIV). El método, el cual utiliza una solución hipotónica con tripsina, un sistema de agitación mediante vortex y un sistema doble de fijación en frío, contribuye a eliminar fácilmente la zona pelúcida y a hinchar a los ovocitos y cigotos favoreciendo posteriormente la extensión de los cromosomas. Este método permite obtener preparaciones de muy buena calidad para el análisis tanto del número como de la morfología de los cromosomas de los ovocitos y cigotos en cualquier estadío meiótico y mitótico

Moreno-Millán, M.

Ocaña Quero, J.M.

Ortega Mariscal, M.A.

Pinedo Merlín, M.

Archivos de Zootecnia

Se logró optimizar un método rápido para analizar citogenéticamente ovocitos y cigotos de bovino obtenidos mediante técnicas de maduración y fecundación in vitro (MIV y FIV). El método, el cual utiliza una solución hipotónica con tripsina, un sistema de agitación mediante vortex y un sistema doble de fijación en frío, contribuye a eliminar fácilmente la zona pelúcida y a hinchar a los ovocitos y cigotos favoreciendo posteriormente la extensión de los cromosomas. Este método permite obtener preparaciones de muy buena calidad para el análisis tanto del número como de la morfología de los cromosomas de los ovocitos y cigotos en cualquier estadío meiótico y mitótico

Moreno Millán, Miguel

Ortega Mariscal, M. A.

DIALNET

Un método optimizado para el análisis cromosómico de ovocitos y cigotos obtenidos mediante técnicas de maduración y fecundación in vitro

Secretaría de Estado de Cultura

Repositorio Institucional de la Universidad de Córdoba

AN IMPROVED METHOD FOR CHROMOSOME PREPARATIONSFROM BOVINE OOCYTES AND ZYGOTES OBTAINED BY INVITRO MATURATION AND FERTILIZATION TECHNIQUESUN MÉTODO OPTIMIZADO PARA EL ANÁLISIS CROMOSÓMICO DE OVOCITOS YCIGOTOS OBTENIDOS MEDIANTE TÉCNICAS DE MADURACIÓN YFECUNDACIÓN IN VITROOcaña-Quero, J.M.*1, M. Moreno-Millán1, M. Pinedo-Merlín2 y M. Ortega Mariscal31Departamento de Genética. Laboratorio de Citogenética. Facultad de Veterinaria. Universidad deCórdoba. Avda. Medina Azahara 9. 14005 Córdoba. España.2Departamento de Patología Animal. Facultad de Veterinaria. Universidad de Córdoba. Avda. MedinaAzahara 9. 14005 Córdoba. España.3Departamento de Bromatología y Tecnología de los Alimentos. Edificio C-1. Rabanales. 14071 Córdoba.España.*Correspondencia: Ocaña-Quero. E-Mail: Ge2ocquj@uco.es.ADDITIONAL KEYWORDSCytogenetic. Bovine. Oocyte. Zygote. In vitrofertilization.PALABRAS CLAVE ADICIONALESCitogenética. Vacuno. Ovocito. Cigoto. Fecun-dación in vitro.SUMMARYMatured bovine oocytes and zygotes obtainedby in vitro maturation and fertilization techniques(IVM and IVF) were cytogenetically prepared byusing an improved method for chromosomepreparations.The method, which involves use of trypsinized-hypotonic solution plus a vortex-agitated systemand very cold two-step fixation process,contributes to weaken the zona pellucida andallows the swelling of oocytes and zygotes whichhelps the subsequent spreading of the chromoso-mes. This method permits to obtain preparationsof good quality for examining the number andArch. Zootec. 47: 669-675. 1998.morphology of the chromosomes of oocytes andzygotes for any meiotic and mitotic stage.RESUMENSe logró optimizar un método rápido paraanalizar citogenéticamente ovocitos y cigotos debovino obtenidos mediante técnicas de madura-ción y fecundación in vitro (MIV y FIV).El método, el cual utiliza una soluciónhipotónica con tripsina, un sistema de agitaciónmediante vortex y un sistema doble de fijación enfrío, contribuye a eliminar fácilmente la zonapelúcida y a hinchar a los ovocitos y cigotosfavoreciendo posteriormente la extensión de losArchivos de zootecnia vol. 47, núm. 180, p. 670.OCAÑA-QUERO ET AL.cromosomas. Este método permite obtener pre-paraciones de muy buena calidad para el análisistanto del número como de la morfología de loscromosomas de los ovocitos y cigotos en cual-quier estadío meiótico y mitótico.INTRODUCTIONThe increasing interest in repro-ductive cytogenetics that used variousembryo manipulation procedures callsfor a reliable, simple and constantmethod for chromosomal analysis ofearly embryos of domestic mammals.The first effective air drying methodfor making chromosome preparationsfrom pre-implantation mouse embryoswas devised by Tarkowski (1966).Although widely used, not only withmouse but also with other mammalianspecies, it requires considerable skillto obtain high quality preparations andis particularly difficult to use on zona-free embryos or isolated blastomeres,which are difficult to handle by thismethod. Several investigators havemodified the method of Tarkowski forchromosome preparations of mouseembryos (Garside and Hillman, 1985)and other mammalian species such aspig (McFeely, 1966), and bovine (Kinget al., 1979). Earlier modifications,which made possible a higher rate ofanalyzable metaphases, concernedprimary the improvement of thefixation process (Kamiguchi et al.,1976). However, some considerabletechnical difficulties, especially incases of pre-implantation embryos,have been encountered with all thesemethods.In this study, we would like toprovide a simplified, reliable methodfor the demonstration of chromosomesof bovine oocytes and zygotes obtainedby in vitro maturation and fertilizationtechniques, which may be called atrypsinized-hypotonic and doublefixation method, and is believed tohave advantages over the previous onein that it constantly prepares clear andwell-spread metaphase plates.MATERIALS AND METHODSPRODUCTION OF IN VITRO-MATUREDBOVINE OOCYTES AND ZYGOTESBovine matured oocytes and zygo-tes were produced by following anIVM-IVF technique. Briefly, cumulus-oocyte complexes (COC) were obtai-ned by aspirating  immature follicles(<7 mm) from ovaries collected atslaughter. Selected COC were culturedfor maturation in 1 ml droplets of TCM-199 medium supplemented with 1 IU/ml PMSG and 0.5 IU/ml HCG accor-ding to the procedure described byOcaña et al. (1994). After 24 h ofculture, the expanded COC wereinseminated with spermatozoa selectedby a modified swim-up through Percollgradient system (Utsumi et al., 1991).Briefly, 2 ml of 45 p.100 Percollsolution was placed on 2 ml of 90p.100 Percoll in a 10-ml test tube. Four0.5 ml-straw of frozen semen wasthawed in 37ºC water, and 2 ml ofsemen were deposited on the upperlayer of the percoll gradient solution.The semen was centrifuged for 15 minat 700 g and the sedimented sper-matozoa displaying good motility inthe bottom of tube were resuspendedin 1 ml of H-TALP medium containing0.6 p.100 BSA (Sigma), and 100 µg/Archivos de zootecnia vol. 47, núm. 180, p. 671.A METHOD FOR CHROMOSOME PREPARATIONSml heparin (Sigma) and were incubatedfor 15 min in a CO2 incubator forcapacitation. 300 µl of the capacitatedsperm suspension were introduced into1 ml of freshly prepared fertilizationmedium (TCM-199 supplemented with10 p.100 FCS), containing 20-40matured oocytes at a concentration of1-2x106 total spermatozoa/ml andcultured for 24 h at 39ºC under 5 p.100CO2 in air.TECHNIQUE FOR CHROMOSOMES PREPA-RATION OF OOCYTES AND ZYGOTESTrypsin and hypotonic treatment.At the end of the culture period formaturation and/or fertilization, matu-red oocytes or zygotes (a total of 480oocytes and 210 embryos wereprocessed using the control method,while 500 oocytes and 200 embryoswere processed using the two-stepmethod) were transferred to 3-mlconical tubes and vortex-agitated for2-5 min in trisodium citrate (0.88p.100) and trypsin (0.02 p.100)hypotonic solution which was pre-viously prepared and stored at 39ºCuntil required for hypotonic treatmentof the oocytes/zygotes. After a slightagitation to remove the cumulusoophorus cells and weak the zonapellucida, denuded oocytes and zygoteswere placed into culture plate contai-ning 2 ml of the same hypotonicsolution without trypsin for 30 min at39ºC.Fixation. The oocytes were placedinto culture plate containing 1 ml of avery cold-initial fixing solution of 1:1methanol:acetic acid for 5 min. Thisfixing solution was prepared on theday of use and maintained in thefreezing compartment of a refrigeratorat -20ºC until required for fixing ofoocytes/zygotes. Then, oocytes andzygotes were fixed in a second fixingsolution of 3:1 methanol:acetic acidfor 24 h. The second fixation time canbe variable between 2 to 24 h withoutaffecting to fixation of the cells.Spreading. Finally, the oocyteswere mounted on slides, stained with 5p.100 Giemsa onto Soremsen buffersolution (Ph=6,8) and examined withthe light microscope at 1500 xmagnification for evaluation of meioticstage.CRITERIA FOR MATURATION ANDFERTILIZATIONOocytes were morphologicallyevaluated for stage of maturation afterculture. The meiotic progress ofoocytes was classified as follows: (1)Germinal vesicle stage: an intact nu-clear membrane with the chromatinmeiotically inactive in which thechromatin is lightly condense; (2)Metaphase I: the nuclear membranebroken and a chromatin patterncharacteristic of an oocyte resumingmeiosis; (3) Metaphase II: a polar bodypresent within the perivitelline spacewith maternal chromatin complementidentified in the oocyte and (4)Degeneration: oocyte showing obviousdegenerative changes such as vacuo-lated or fragmented cytoplasm orscattered chromatin complement. Forfertilization stage, oocytes wereclassified as follows: (1) oocyteswithout both male and female pro-nuclei were judged as unfertilized; (2)oocytes with both male and femalepronuclei and with residual sperm-tailwere defined as normally fertilized;(3) oocytes with more than twoArchivos de zootecnia vol. 47, núm. 180, p. 672.OCAÑA-QUERO ET AL.Table I. Percentages of oocytes and embryos karyotyped using different methods. (Porcentajesde ovocitos y embriones cariotipados usando diferentes métodos).Method No of trials No of processed cells No of karyotyped cells ( p.100)Oocytes Embryos Oocytes EmbryosControl 5 480 210 300 (62.5)a 125 (59.5)aTwo-step 5 500 200 434 (86.8)b 156 (78)ba,bValues with different superscripts within each column are statistically different (p< 0.05). Chi-squaretest.Table II. Method for karyotyping bovine oocytes and zygotes developing from in vitromaturation and fertilization techniques.(Método para cariotipar ovocitos y cigotos de bovinoobtenidos mediante técnicas de maduración y fecundación in vitro).Time Procedure Purpose2-5 min Trypsin (0.02 p.100) plus hypotonic Disggregate zona pellucidatreatment (0.88 p.100 Na-citrate) swell cellsplus vortex-agitated system disperse chromosomes30 min Hypotonic treatment Swell cells and(0.88 p.100 Na-citrate) disperse chromosomes5 min Add fixative (methanol:acetic acid 1:1) Fix and disperse cells;dissolve zona pellucida24 h Add Carnoys fixative Fixation of cells and(methanol:acetic acid 3:1) attachment to slide.Drop cell suspension Attach cells to slideonto cold, wet slides13-30 min Stain (Giemsa 5 p.100 ontoSorensen buffer solution,Ph=6,8)20 min Microscopic examination (1500 x)Total time: 26 h (including an approximation of time required for microscopic examination).pronuclei and decondensed spermheads were considered to be polys-permic; (4) oocytes with a cytoplasmvacuolated or fragmented were consi-dered to be degenerated; (5) fertilizedoocytes with a diploid chromosomesgroup were considered to be zygotes.RESULTS AND DISCUSSIONThe percentajes of oocytes andzygotes karyoryped (86.8 and 78 p.100respectively, table I) using the two-step method were significantly higher(p<0.05) than those observed in theArchivos de zootecnia vol. 47, núm. 180, p. 673.A METHOD FOR CHROMOSOME PREPARATIONSFigure 1. Metaphase plate from an in vitromatured oocyte showing a haploid chro-mosome complement (n=30). The arrowindicates the X chromosome. (Metafase de unovocito madurado in vitro mostrando una dota-ción cromosómica haploide. La flecha indica elcromosoma X).blastomeres and zygotes differedamong authors. Kamiguchi et al.(1976) used 30 p.100 calf serum intheir procedure, Tarkowski (1966)used 1 p.100 sodium citrate, Popescuet al. (1982) and Long (1977) usedKCl. However we used a combinationof trypsin plus sodium citrate, accom-pained with vortex-agitated systemduring short-term treatment. Inaddition, several authors have emplo-yed some enzyme type, such as actinase(Iwasaki et al., 1988), trypsin (Hare etal., 1976; Yoshizawa et al., 1990),pronase (Angell et al., 1983) to weakenthe zona pellucida of the zygotes andembryos. However, they used theenzyme treatment prior to hypotonictreatment. Our method has the advan-tage, with regard to other methods,that enzyme and hypotonic treatmentare only realized at one-step treatment.The hypotonic solution action is veryintensive due to the fact that trypsinquickly weakens the zona pellucida,control method (62.5 and 59.5 p.100respectively).When the oocytes and zygotes wereplaced into a trypsinized-hypotonicsolution of sodium citrate plus trypsin,the cell body was considerably swollen,to fill up the periviteline space, and theswollen cell body began to escape fromthe zona pellucida. This escape wasachieved through a slit made in thezona pellucida which did not rupturethe cytoplasmic membrane, but didusually cause loss of nuclear material.In table II is shown the developmentof the method and the employed totaltime for displaying chromosomes, ofbovine oocytes and zygotes which wereobtained by in vitro maturation andfertilization techniques.The development of a trypsinized-hypotonic treatment for chromosomeanalysis of oocytes and zygotesobtained by in vitro maturation andfertilization techniques was the mostimportant outcome of this study. Hsu(1952) and Hsu and Pomerat (1953)reported the importance of the hypo-tonic treatment as a means of producinga good spreading of chromosomes fromvertebrate cells. Holmquist and Motara(1987) reported that in a hypotonicenvironment, cell actively absorbswater to equilibrate its salt balance.The mechanism by which hypotonicsalt solution exerts its influence on thedividing cells to spread their chromo-somes has been explained by itseffectiveness in desintegrating themitotic spindles (Hsu and Pomerat,1953), or loosening the interchro-mosomal connections (Klasterska andRamel, 1978). The composition of thehypotonic solution for chromosomepreparations of embryos, isolatedArchivos de zootecnia vol. 47, núm. 180, p. 674.OCAÑA-QUERO ET AL.Figure 2. Metaphase plate from a zygoteshowing a diploid chromosome complement(2n=60). The arrows indicate the Xchromosomes. (Metafase de un cigoto mos-trando una dotación cromosómica diploide. Lasflechas indican los cromosomas X).Figure 3. Metaphase plate from a zygoteshowing a hypodiploid chromosomecomplement (2n=53). The arrows indicatethe X chromosomes. (Metafase de un cigotomostrando una dotación cromosómica hipodi-ploide. Las flechas indican los cromosomas X).REFERENCESAngell, R.R., R.J. Aitken, P.F.A. Van Look, M.A.Lumsden and A.A. Templeton. 1983.Chromosome abnormalities in humanembryos after in vitro fertilization. Nature,303: 336-338.Fujimoto, S., T.J. Passantino, I. Koenczoel andS.J. Segal. 1975. A simplified method forchromosome preparations of rat pre-implantation embryos. Cytologia, 40: 469-475.thus permiting the penetration of thehypotonic solution into the cell, andthe subsequent breakage of theinterchromosomal connections anddispers of the chromosomes (Figures1, 2, 3). All these mechanisms werefavoured by the vortex-agitated systemwhich facilitated the trypsin-digestiveaction. In our technique, the trypsinconcentration has to be exactlycontrolled at 0.02 p.100 and theexposure time at 5 min as well. Trypsinconcentrations or exposure time higherthan 0.02 p.100 or 5 min respectivelyproduced an intensive digestion of thezona pellucida determining the loss ofboth cytoplasmic and nuclear mate-rial.The two-step fixing process used inour study is similar to the doublefixation method devised by Fujimotoet al. (1975) and Yoshizawa et al.(1990). Our two-step process has theadvantage over the one-step method offixing slides in that the prefixedoocytes or zygotes were not washedaway, and so not lost, during the secondfixing.Archivos de zootecnia vol. 47, núm. 180, p. 675.A METHOD FOR CHROMOSOME PREPARATIONSGarside, W. and N. Hillman. 1985. A method forkaryotyping mouse blastocysts embryosdeveloping from in vivo and in vitro fertilizedeggs. Experientia, 41: 1183-1184.Hare, W.C.D., D. Mitchell, K.J. Betteridge, M.D.Eaglesome and G.C.B. Randall. 1976. Sexingtwo-weel old bovine embryos by chromosomalanalysis prior to surgical transfer:preliminarymethods and results. Theriogenology, 5: 243-253.Holmquist, G.T. and M.A. Motara. 1987. Themagic of cytogenetic technology. In: Obe, G.and Basler, A. (eds). Cytogenetics, Springer-Verlag, Berlin, pp: 123-130.Hsu, T.C. and C.M. Pomerat. 1953. Mammalianchromosome in vitro. II. A method forspreading the chromosomes of cells in tissueculture. J. Hered., 44: 23-29.Hsu, T.C. 1952. Mammalian Chromosomes invitro. I. The karyotype of man. J. Hered., 43:167-172.Iwasaki, S., Y. Shioya, H. Masuda, A. Hanadaand T. Nakahara. 1988. Sex ratio of earlyembryos fertilized in vitro with spermatozoaseparated by percoll. Theriogenology, 30:1191-1198.Kamiguchi, Y., K. Funaki and K. Mikano. 1976. Anew technique for chromosome study of murineoocytes. Proc. Japan Acad., 52: 316-319.King, W.A., T. Linares, I. Gustavsson and A.Bane. 1979. A method for preparation ofchromosome from bovine zygotes andblastocysts. Veterinary Science Commu-nication, 3: 51-56.Klasterska, I. and C. Ramel. 1978. The hypotonicpretreatment in mammalian cytology:itsfunction and effect on the aspect of meioticchromosomes. Hereditas, 90: 21-29.Long, S.E. 1977. Cytogenetic examination ofpre-implantation blastocysts of ewes matedto rams heterozygous for the Massey I (t1)translocation. Cytogenet. Cell Genet., 18:82-89.McFeely, R.A. 1966. A direct method for thedisplay of chromosomes from early pigembryos. J. Reprod. Fert., 11: 161-163.Ocaña-Quero, J.M., M. Moreno-Millán, M. Valera-Córdoba and A. Rodero-Franganillo. 1994.The influence of different types of mediasupplement on the meiotic maturation ofbovine oocytes in vitro. Theriogenology 41:405-411.Popescu, C.P., J. Boscher and E.P. Cribiu. 1982.Collagen substrates for cytogenetic studiesof domestic animal embryos. The Journal ofHeredity, 73: 147-148.Tarkowski, A.K. 1966. An air-drying method forchromosome preparations from mouse eggs.Cytogenetics, 5: 394-400.Utsumi, K., H. Kato and A. Iritani. 1991. Full-termdevelopment of bovine follicular oocytesmatured in culture and fertilized in vitro.Theriogenology, 35: 695-699.Yoshizawa, M., S. Nakamoto, Y. Tsunoda and T.Muramatsu. 1990. A short-term hypotonictreatment for chromosome preparation ofintact and zona penetrated mouse embryos.Theriogenology, 33: 789-797.Recibido: 3-6-97. Aceptado: 18-6-98.

Un método optimizado para el análisis cromosómico de ovocitos y cigotos obtenidos mediante técnicas de maduración y fecundación in vitro

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