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A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus

Published online by Cambridge University Press:  17 May 2001

Koji Nakagawa ,
Shuji Yamano ,
Hisayo Nakasaka ,
Kenji Hinokio ,
Midori Yoshizawa  and
Toshihiro Aono
Koji Nakagawa
Affiliation:
Department of Obstetrics and Gynaecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Shuji Yamano
Affiliation:
Department of Obstetrics and Gynaecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Hisayo Nakasaka
Affiliation:
Department of Obstetrics and Gynaecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
Kenji Hinokio
Affiliation:
Department of Obstetrics and Gynaecology, 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 Gynaecology, School of Medicine, The University of Tokushima, Tokushima, Japan.
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Abstract

Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 μM A23187 for 5 min were treated with 10 μg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.

Keywords

Calcium ionophore A23187Haploid pronucleusHuman parthenogenonesOocyte activationPuromycin
Type
Research Article
Information
Zygote , Volume 9 , Issue 1 , February 2001 , pp. 83 - 88
Copyright
2001 Cambridge University Press

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