小鼠卵母细胞减数分裂过程中LIMK1参与调控Aurora-A在纺锤体的定位

LIMK1 takes part in the regulation of Aurora-A-localization on spindle during mouse oocyte meiosis

目的研究小鼠卵母细胞减数分裂进程中活化的LIMK1(pLIMK1^(Thr508))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pLIMK1^(Thr508)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),pLIMK1^(Thr508)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,pLIMK1^(Thr508)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,pLIMK1^(Thr508)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,pLIMK1^(Thr508)与Aurora-A共同定位于纺锤体两极;在从AI期到Tel I期进程中pLIMK1^(Thr508)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pLIMK1^(Thr508)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（ pLIMK1^（Thr508）） 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 pLIMK1^Thr508 and 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,pLIMK1^Thr508 was weaklydetected 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,pLIMK1^Thr508 left 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）,pLIMK1^Thr508 and Aurora-A remained overlapped and concentrated as multi foci around the condensed chromosomes; at metaphase Ⅰ（ MⅠ） and metaphase Ⅱ（ MⅡ）,pLIMK1^Thr508 was co-localized with Aurora-A on spindle poles; During anaphase Ⅰ（ AⅠ） to telophase Ⅰ（ Tel Ⅰ） progression,pLIMK1^Thr508 was detached from spindle poles and mainly concentrated on the cleavage furrow,while Aurora-A loosely congressed on spindle. In addition,LIMK1 inhibition with BMS-3 destroyed Aurora-A polar location and spindle formation. Conclusions pLIMK1^Thr508 is a microtubule organizing center（ MTOC）-associated protein,may participate in spindle assembly and maintenance through regulating Aurora-A in mouse oocytes during meiotic progression.