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Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse oocytes

Published online by Cambridge University Press:  13 November 2000

Hisayo Nakasaka ,
Shuji Yamano ,
Kenji Hinokio ,
Koji Nakagawa ,
Midori Yoshizawa  and
Toshihiro Aono
Hisayo Nakasaka
Affiliation:
Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Shuji Yamano
Affiliation:
Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Kenji Hinokio
Affiliation:
Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Koji Nakagawa
Affiliation:
Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Midori Yoshizawa
Affiliation:
Department of Animal Breeding and Reproduction, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.
Toshihiro Aono
Affiliation:
Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
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Abstract

Freshly ovulated mouse oocytes exposed to 5 mM calcium ionophore A23187 for 5 min and controls (not exposed) were cultured in TYH medium with 10 μg/ml puromycin (the puromycin group) or 2 mM 6-dimethylaminopurine (DMAP; the DMAP group) for 4 h. Among the controls, few oocytes were activated even if they were treated with DMAP or puromycin. In the oocytes exposed to A23187, in contrast, the activation rate, i.e. the rate of oocytes showing at least one pronucleus (PN) after the treatment, was 46.2% (48/104) in the DMAP group and 90.0% (118/131) in the puromycin group. Activation rate in the puromycin group was significantly higher than in the DMAP and control groups (p < 0.0001, respectively). Furthermore, 82.4% (108/131) of the activated oocytes in the puromycin group showed one PN with extrusion of the second polar body (PB). In the puromycin group, the DNA content of the PN of parthenogenones with 1PN2PB was half that of a set of metaphase II chromosomes. Chromosomal analysis was possible in 14 parthenogenones with 1PN2PB in the puromycin group. The parthenogenones possessed a normal set (n = 20) of haploid chromosomes. The combination of A23187 and puromycin proved to be an effective method of producing haploid parthenogenones.

Keywords

Calcium ionophoreHaploidOocyte activationParthenogenonePuromycin
Type
Research Article
Information
Zygote , Volume 8 , Issue 3 , August 2000 , pp. 203 - 208
Copyright
2000 Cambridge University Press

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