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Updated: Aug 12, 2026
Mouse Oocyte Microinjection, Maturation and Ploidy Assessment
Published on: July 23, 2011
Effective activation method with A23187 and puromycin to produce haploid parthenogenones from freshly ovulated mouse
H Nakasaka1, S Yamano, K Hinokio
1University of Tokushima and Utsunomiya University, Japan. nakasaka@clin.med.tokushima-u.ac.jp
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 microg/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.
Insights
Calcium ionophore A23187 combined with puromycin effectively activates mouse oocytes, producing viable haploid parthenogenones with a high success rate.
Area of Science:
- Reproductive Biology
- Developmental Biology
- Cell Biology
Background:
- Oocyte activation is crucial for initiating embryonic development.
- Chemical and physical stimuli are used to induce oocyte activation in vitro.
- Parthenogenesis offers a model for studying early development and potential applications in assisted reproduction.
Purpose of the Study:
- To evaluate the efficacy of calcium ionophore A23187 and puromycin in inducing mouse oocyte activation.
- To determine the rate of successful parthenogenesis and the ploidy of the resulting embryos.
- To establish an effective method for producing haploid mouse parthenogenones.
Main Methods:
- Freshly ovulated mouse oocytes were treated with calcium ionophore A23187.
- Treated and control oocytes were cultured in medium containing either puromycin or 6-dimethylaminopurine (DMAP).
- Oocyte activation, pronucleus (PN) formation, second polar body (PB) extrusion, and chromosomal analysis were assessed.
Main Results:
- The combination of A23187 and puromycin resulted in a significantly higher oocyte activation rate (90.0%) compared to DMAP (46.2%) and controls.
- A high proportion (82.4%) of activated oocytes in the puromycin group exhibited one PN and extruded the second PB.
- Parthenogenones produced using A23187 and puromycin possessed a normal haploid set of chromosomes (n=20).
Conclusions:
- The combined treatment of calcium ionophore A23187 and puromycin is a highly effective method for inducing parthenogenesis in mouse oocytes.
- This method reliably produces haploid parthenogenones with normal chromosome complements.
- This approach provides a valuable tool for research in developmental biology and assisted reproductive technologies.
