Demecolcine-induced enucleation of sheep meiotically maturing oocytes.
Demecolcine-induced enucleation of sheep meiotically maturing oocytes.
复制标题
德美考辛诱导绵羊减数分裂成熟卵母细胞的去核。
DOI：
10.1051/rnd:2006002
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Yongfu Chen
中科院分区：
文献类型：
--
作者：
J. Hou;T. Lei;Lei Liu;X. Cui;X. An;Yongfu Chen
本研究的目的是研究 demecolcine（一种微管破坏试剂）对绵羊减数分裂成熟卵母细胞诱导去核 (IE) 的可能影响。使用抗微管蛋白抗体的免疫荧光染色来检查卵母细胞的纺锤体状态。当具有完整生发囊泡（GV）的卵母细胞在含有不同浓度的德美考辛（0.01～0.4μg.mL-1）的培养基中培养20～22小时时，纺锤体微管组织和第一极体（PB1）的挤出受到德美考辛的剂量依赖性抑制。 IE率最高(58.1%)来自0.04μg.mL-1德美可辛处理。生泡破裂 (GVBD) 或中期 (M) 后应用 Demecolcine 处理产生的 PB1 挤出率和 IE 效率与成熟开始时应用的处理相似。免疫荧光分析表明，非纺锤体微管和纺锤体微管均被 demecolcine 显着破坏。德美秋碱和放线菌酮 (CHX) 或 6-二甲氨基嘌呤 (6-DMAP) 联合治疗导致单一原核形成，而不是 PB1 挤出。当将经过德美考辛处理的卵母细胞转移到不含德美考辛的培养基中时，挤出PB1的能力很快恢复，并且在这种处理后获得了72.1%的IE率。这些结果表明，demecolcine 可用作诱导绵羊减数分裂成熟卵母细胞去核的潜在试剂，并可极大地促进核移植研究。
The objective of this study was to investigate the possible effect of demecolcine, a microtubule-disrupting reagent, on induced enucleation (IE) of sheep meiotically maturing oocytes. Immunofluorescent staining with anti-tubulin antibodies was used to examine the spindle status of the oocytes. When the oocytes with intact germinal vesicles (GV) were cultured in the medium containing various concentrations of demecolcine (0.01 to 0.4 microg.mL-1) for 20 to 22 h, the spindle microtubule organization and first polar body (PB1) extrusion were inhibited by demecolcine in a dose-dependent manner. The highest IE rate (58.1%) was from the treatment with 0.04 microg.mL-1 demecolcine. Demecolcine treatment applied after germinal vesicle breakdown (GVBD) or at metaphase (M) yielded a PB1 extrusion rate and IE efficiency similar to the treatment applied at the onset of maturation. Analysis by immunofluorescence showed that both nonspindle microtubules and spindle microtubules were significantly disorganized by demecolcine. Combination treatment with demecolcine and cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) led to single pronuclear formation rather than PB1 extrusion. When demecolcine-treated oocytes were transferred into demecolcine-free medium, the ability to extrude PB1 was quickly restored and a 72.1% IE rate was obtained following such treatment. These results demonstrate that demecolcine can be used as a potential reagent for induced enucleation of sheep meiotically maturing oocytes and may greatly facilitate research in nuclear transfer.