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Protein modification by phosphorylation during the process of nuclear membrane dissolution in puromycin-treated mouse
1Department of Zoology, College of Natural Sciences, Seoul National University, Korea.
Abstract:
The present study was undertaken to elucidate the mechanism of nuclear membrane dissolution (NMD) in puromycin-treated mouse oocytes. Treatment of germinal vesicle breakdown (GVBD) oocytes with puromycin (50 micrograms/ml) induced chromosome decondensation with formation of a polar body; these are designated nuclear membrane (NM) oocytes. After withdrawal of puromycin, NM oocytes underwent NMD (approximately 70%) during a 12-h culture period. Either dibutyryl cyclic AMP (dbcAMP, 25-100 micrograms/ml) or isobutylmethylxanthine (IBMX, 0.1-1.0 mM) inhibited the process of NMD in a dose-dependent manner, suggesting the involvement of cAMP in the process of NMD. To determine which protein(s) participated in the transition from interphase to metaphase II during NMD, NM oocytes were labeled with [35S]methionine, and one- and two-dimensional gel electrophoresis were performed. Although the synthesis of stage-specific proteins during NMD was not found, two specific proteins of Mr 27,000 and 46,000, which were synthesized at interphase following removal of puromycin, were modified during NMD. Phosphatase treatment and 32PO4-labeling experiments indicated that phosphorylation was responsible for these modifications, which were inhibited by either dbcAMP or IBMX. Therefore, it appears that phosphorylation of specific proteins may play an important role in the transition from interphase to metaphase II.
Insights
Nuclear membrane dissolution (NMD) in mouse oocytes involves specific protein phosphorylation. Cyclic AMP (cAMP) signaling regulates this crucial process for the transition to metaphase II.
Area of Science:
- Reproductive Biology
- Cellular and Molecular Biology
- Developmental Biology
Background:
- Nuclear membrane dissolution (NMD) is a critical event in oocyte maturation.
- Understanding the molecular mechanisms regulating NMD is essential for reproductive biology.
Purpose of the Study:
- To elucidate the mechanism of nuclear membrane dissolution (NMD) in mouse oocytes.
- To identify proteins involved in the transition from interphase to metaphase II during NMD.
Main Methods:
- Puromycin treatment to induce germinal vesicle breakdown (GVBD) and nuclear membrane (NM) oocytes.
- Culture of NM oocytes to observe NMD, with or without cAMP modulators (dbcAMP, IBMX).
- Radioactive labeling ([35S]methionine) and gel electrophoresis (1D and 2D) to analyze protein synthesis and modification.
- Phosphatase treatment and 32PO4 labeling to investigate protein phosphorylation.
Main Results:
- Puromycin-treated oocytes, after drug withdrawal, underwent NMD.
- Cyclic AMP (cAMP) signaling, modulated by dbcAMP and IBMX, inhibited NMD in a dose-dependent manner.
- Two specific proteins (Mr 27,000 and 46,000) were modified during NMD, and this modification was identified as phosphorylation.
- Protein phosphorylation was inhibited by dbcAMP and IBMX, suggesting a link to cAMP signaling.
Conclusions:
- Cyclic AMP (cAMP) signaling plays a regulatory role in nuclear membrane dissolution (NMD).
- Phosphorylation of specific proteins (Mr 27,000 and 46,000) is crucial for the transition from interphase to metaphase II during NMD in mouse oocytes.