AN IMPROVED METHOD FOR CHROMOSOME PREPARATIONS FROM BOVINE OOCYTES AND ZYGOTES OBTAINED BY IN VITRO MATURATION AND FERTILIZATION TECHNIQUES (UN MÉTODO OPTIMIZADO PARA EL ANÁLISIS CROMOSÓMICO DE OVOCITOS Y CIGOTOS OBTENIDOS MEDIANTE TÉCNICAS DE MADURACIÓN Y FECUNDACIÓN IN VITRO)

Ocaña-Quero J.M.

Description

Abstract
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.
Resumen
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.

Sujets

• Cytogenetics
• Bovinae
• Oocyte
• Zygote
• In vitro fertilisation
• Citogenética
• Oocito
• Cigoto
• Fecundación in vitro

Informations

• Publié par: erevistas
• Publié le: 01 janvier 1998
• Nombre de lectures: 41

Extrait

AN IMPROVED METHOD FOR CHROMOSOME PREPARATIONS
FROM BOVINE OOCYTES AND ZYGOTES OBTAINED BY IN
VITRO MATURATION AND FERTILIZATION TECHNIQUES
UN MÉTODO OPTIMIZADO PARA EL ANÁLISIS CROMOSÓMICO DE OVOCITOS Y
CIGOTOS OBTENIDOS MEDIANTE TÉCNICAS DE MADURACIÓN Y
FECUNDACIÓN IN VITRO
*1 1 2 3Ocaña Quero, J.M. , M. Moreno Millán , M. Pinedo Merlín y M. Ortega Mariscal
1Departamento de Genética. Laboratorio de Citogenética. Facultad de Veterinaria. Universidad de
Córdoba. Avda. Medina Azahara 9. 14005 Córdoba. España.
2Departamento de Patología Animal. Facultad de Veterinaria. Universidad de Córdoba. Avda. Medina
Azahara 9. 14005 Córdoba. España.
3Departamento de Bromatología y Tecnología de los Alimentos. Edificio C 1. Rabanales. 14071 Córdoba.
España.
ADDITIONAL KEYWORDS PALABRAS CLAVE ADICIONALES
Cytogenetic. Bovine. Oocyte. Zygote. In vitro Citogenética. Vacuno. Ovocito. Cigoto. Fecun
fertilization. dación in vitro.
SUMMARY
Matured bovine oocytes and zygotes obtained morphology of the chromosomes of oocytes and
by in vitro maturation and fertilization techniques zygotes for any meiotic and mitotic stage.
(IVM and IVF) were cytogenetically prepared by
using an improved method for chromosome
preparations. RESUMEN
The method, which involves use of trypsinized
hypotonic solution plus a vortex agitated system Se logró optimizar un método rápido para
and very cold two step fixation process, analizar citogenéticamente ovocitos y cigotos de
contributes to weaken the zona pellucida and bovino obtenidos mediante técnicas de madura
allows the swelling of oocytes and zygotes which ción y fecundación in vitro (MIV y FIV).
helps the subsequent spreading of the chromoso El método, el cual utiliza una solución
mes. This method permits to obtain preparations hipotónica con tripsina, un sistema de agitación
of good quality for examining the number and 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*Correspondencia: Ocaña Quero.
E Mail: [e-mail]. favoreciendo posteriormente la extensión de los
Arch. Zootec. 47: 669 675. 1998.OCAÑA QUERO ET AL.
cromosomas. Este método permite obtener pre for the demonstration of chromosomes
paraciones de muy buena calidad para el análisis of bovine oocytes and zygotes obtained
tanto del número como de la morfología de los by in vitro maturation and fertilization
cromosomas de los ovocitos y cigotos en cual techniques, which may be called a
quier estadío meiótico y mitótico. trypsinized hypotonic and double
fixation method, and is believed to
have advantages over the previous one
INTRODUCTION in that it constantly prepares clear and
well spread metaphase plates.
The increasing interest in repro
ductive cytogenetics that used various
embryo manipulation procedures calls MATERIALS AND METHODS
for a reliable, simple and constant
method for chromosomal analysis of PRODUCTION OF IN VITRO-MATURED
early embryos of domestic mammals. BOVINE OOCYTES AND ZYGOTES
The first effective air drying method Bovine matured oocytes and zygo
for making chromosome preparations tes were produced by following an
from pre implantation mouse embryos IVM IVF technique. Briefly, cumulus
was devised by Tarkowski (1966). oocyte complexes (COC) were obtai
Although widely used, not only with ned by aspirating immature follicles
mouse but also with other mammalian (<7 mm) from ovaries collected at
species, it requires considerable skill slaughter. Selected COC were cultured
to obtain high quality preparations and for maturation in 1 ml droplets of TCM
is particularly difficult to use on zona 199 medium supplemented with 1 IU/
free embryos or isolated blastomeres, ml PMSG and 0.5 IU/ml HCG accor
which are difficult to handle by this ding to the procedure described by
method. Several investigators have Ocaña et al. (1994). After 24 h of
modified the method of Tarkowski for culture, the expanded COC were
chromosome preparations of mouse inseminated with spermatozoa selected
embryos (Garside and Hillman, 1985) by a modified swim up through Percoll
and other mammalian species such as gradient system (Utsumi et al. , 1991).
pig (McFeely, 1966), and bovine (KingBriefly, 2 ml of 45 p.100 Percoll
et al., 1979). Earlier modifications, solution was placed on 2 ml of 90
which made possible a higher rate of p.100 Percoll in a 10 ml test tube. Four
analyzable metaphases, concerned 0.5 ml straw of frozen semen was
primary the improvement of the thawed in 37ºC water, and 2 ml of
fixation process (Kamiguchi et al., semen were deposited on the upper
1976). However, some considerable layer of the percoll gradient solution.
technical difficulties, especially in The semen was centrifuged for 15 min
cases of pre implantation embryos, at 700 g and the sedimented sper
have been encountered with all these matozoa displaying good motility in
methods. the bottom of tube were resuspended
In this study, we would like to in 1 ml of H TALP medium containing
provide a simplified, reliable method 0.6 p.100 BSA (Sigma), and 100 μg/
Archivos de zootecnia vol. 47, núm. 180, p. 670.A METHOD FOR CHROMOSOME PREPARATIONS
ml heparin (Sigma) and were incubatedat 20ºC until required for fixing of
for 15 min in a CO incubator for oocytes/zygotes. Then, oocytes and
2
capacitation. 300 μl of the capacitated zygotes were fixed in a second fixing
sperm suspension were introduced into solution of 3:1 methanol:acetic acid
1 ml of freshly prepared fertilization for 24 h. The second fixation time can
medium (TCM 199 supplemented with be variable between 2 to 24 h without
10 p.100 FCS), containing 20 40 affecting to fixation of the cells.
matured oocytes at a concentration of Spreading. Finally, the oocytes
1 2x106 total spermatozoa/ml and were mounted on slides, stained with 5
cultured for 24 h at 39ºC under 5 p.100 p.100 Giemsa onto Soremsen buffer
CO in air. solution (Ph=6,8) and examined with
2
the light microscope at 1500 x
TECHNIQUE FOR CHROMOSOMES PREPA- magnification for evaluation of meiotic
RATION OF OOCYTES AND ZYGOTES stage.
Trypsin and hypotonic treatment.
At the end of the culture period for CRITERIA FOR MATURATION AND
maturation and/or fertilization, matu FERTILIZATION
red oocytes or zygotes (a total of 480 Oocytes were morphologically
oocytes and 210 embryos were evaluated for stage of maturation after
processed using the control method, culture. The meiotic progress of
while 500 oocytes and 200 embryos oocytes was classified as follows: (1)
were processed using the two step Germinal vesicle stage: an intact nu
method) were transferred to 3 ml clear membrane with the chromatin
conical tubes and vortex agitated for meiotically inactive in which the
2 5 min in trisodium citrate (0.88 chromatin is lightly condense; (2)
Metaphase I: the nuclear membranep.100) and trypsin (0.02 p.100)
broken and a chromatin patternhypotonic solution which was pre
characteristic of an oocyte resumingviously prepared and stored at 39ºC
meiosis; (3) Metaphase II: a polar bodyuntil required for hypotonic treatment
present within the perivitelline spaceof the oocytes/zygotes. After a slight
agitation to remove the cumulus with maternal chromatin complement
oophorus cells and weak the zona identified in the oocyte and (4)
pellucida, denuded oocytes and zygotes Degeneration: oocyte showing obvious
were placed into culture plate contai degenerative changes such as vacuo
ning 2 ml of the same hypotonic lated or fragmented cytoplasm or
scattered chromatin complement. Forsolution without trypsin for 30 min at
fertilization stage, oocytes were39ºC.
classified as follows: (1) oocytesFixation. The oocytes were placed
without both male and female pro into culture plate containing 1 ml of a
nuclei were judged as unfertilized; (2)very cold initial fixing solution of 1:1
methanol:acetic acid for 5 min. This oocytes with both male and female
fixing solution was prepared on the pronuclei and with residual sperm tail
day of use and maintained in the were defined as normally fertilized;
freezing compartment of a refrigerator (3) oocytes with more than two
Archivos de zootecnia vol. 47, núm. 180, p. 671.OCAÑA QUERO ET AL.
Table I. Percentages of oocytes and embryos karyotyped using different methods. (Porcentajes
de ovocitos y embriones cariotipados usando diferentes métodos).
Method No of trials No of processed cells No of karyotyped cells ( p.100)
Oocytes Embryos Oocytes Embryos
a aControl 5 480 210 300 (62.5) 125 (59.5)
b bTwo step 5 500 200 434 (86.8) 156 (78)
a,bValues with different superscripts within each column are statistically different (p< 0.05). Chi square
test.
pronuclei and decondensed sperm RESULTS AND DISCUSSION
heads were considered to be polys
permic; (4) oocytes with a cytoplasm The percentajes of oocytes and
vacuolated or fragmented were consi zygotes karyoryped (86.8 and 78 p.100
dered to be degenerated; (5) fertilized respectively, table I) using the two
oocytes with a diploid chrom