目的 研究小鼠卵母细胞减数分裂进程中活化的LIMK1(pLIMK1Thr508)与Aurora-A亚细胞分布的相关性, LIMK1活性变化对Aurora-A和纺锤体组装的影响.方法 收集21日龄CB6F1小鼠卵母细胞,分别体外培养至生发泡期(GV)、生发泡破裂期(GVBD)、第1次减数分裂中期(MI)、第1次减数分裂后期(AI)和末期(Tel I)以及第2次减数分裂中期(MII),固定并利用免疫荧光染色法分析减数分裂进程中pLIMK1Thr508的亚细胞定位模式及其与纺锤体组装调控因子Aurora-A的时空相关性;利用抑制剂BMS-3处理MI期细胞,结合免疫荧光染色分析LIMK1活性缺失对于Aurora-A空间分布和纺锤体形成的影响.结果 在小鼠卵母细胞减数分裂前期(prophase)(即GV期), pLIMK1Thr508信号微弱并主要聚集于生发泡,此时 Aurora-A 在胞内没有特殊聚集;在临近减数分裂重启时, pLIMK1Thr508离开生发泡,Aurora-A 信号出现,两者呈高度致密的点状聚集并紧密重合;在生发泡完全破裂后, pLIMK1Thr508和Aurora-A又呈多个点片状聚集在凝集的染色体周围;在MI期和MII期,pLIMK1Thr508与Aurora-A共同定位于纺锤体两极;在从AI期到Tel I期进程中pLIMK1Thr508离开纺锤体两极,聚集在收缩环部位,Aurora-A在纺锤体两极有微弱聚集.LIMK1抑制剂BMS-3能够破坏 Aurora-A在纺锤体两极的聚集,并影响纺锤体结构.结论 pLIMK1Thr508在卵母细胞减数分裂中是一种微管组织中心(MTOC)相关蛋白,可能通过调控Aurora-A而参与纺锤体结构的形成和维持.%Objective To investigate the sub-cellular distribution correlation between activated LIMK1 (pLIMK1Thr508) and Aurora-A in mouse oocyte meiosis,and changes in Aurora-A location and spindle structure in condition of LIMK1 inhibition.Methods Immunofluorescence staining was employed to detect the sub-cellular localization of pLIMK1Thr508and its spatial-temporal correlation with spindle organizing regulator Aurora-A in mouse oocyte meiosis; BMS-3, the specific inhibitor to LIMK1 activity, was applied to analyze the effects of LIMK1 inhibition on Aurora-A distribution and spindle formation. Results At meiotic prophase,pLIMK1Thr508was weakly detected and concentrated in the germinal vesicle(GV) in oocytes,with no signal of Aurora-A across the cytoplasm and nuclear area;as meiotic assumption approaching,pLIMK1Thr508left nuclear,aggregating as a single dense dote in the vicinity of nuclear, and being co-localized with the emerging Aurora-A; After germinal vesicle broke down (GVBD), pLIMK1Thr508and Aurora-A remained overlapped and concentrated as multi foci around the condensed chromosomes;at metaphase Ⅰ(MⅠ) and metaphase Ⅱ(MⅡ), pLIMK1Thr508was co-localized with Aurora-A on spindle poles;During anaphase Ⅰ(AⅠ) to telophase Ⅰ(Tel Ⅰ) progression, pLIMK1Thr508was detached from spindle poles and mainly concentrated on the cleavage furrow,while Aurora-A loosely congressed on spindle. In ad-dition, LIMK1 inhibition with BMS- 3 destroyed Aurora-A polar location and spindle formation. Conclusions pLIMK1Thr508is a microtubule organizing center (MTOC)-associated protein, may participate in spindle assembly and maintenance through regulating Aurora-A in mouse oocytes during meiotic progression.
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