小鼠卵母细胞减数分裂过程中活化Pak1的表达及其与染色体分离的相关性

Protein expression of activated Pak1 and its correlation with chromosome segregation in mouse oocyte meiosis

目的研究活化的Pak1在小鼠卵母细胞减数分裂进程中的表达、亚细胞定位及其与染色体分离的相关性。方法用Western blot方法半定量分析活化的Pak1(^(Ser204)位点磷酸化,pPak1^(Ser204))在小鼠卵母细胞减数分裂各个时期的蛋白表达量;用免疫荧光染色法分析减数分裂进程中pPak1^(Ser204)的亚细胞定位模式及其与纺锤体和微管组织中心(MTOC)的时空相关性。结果 pPak1^(Ser204)在小鼠卵母细胞减数分裂重启时(GVBD)开始表达,其表达量随减数分裂进程逐渐升高,在第一次减数分裂中期(MⅠ)时达到峰值并持续保持至第二次减数分裂中期(MⅡ)。在第一次减数分裂前中期(pro-MⅠ)、MⅠ和MⅡ期,pPak1^(Ser204)与MTOC核心蛋白pericentrin和γ-tubulin共同定位于纺锤体两极,pPak1^(Ser204)同时存在于染色体上;在第一次减数分裂后期(AI)到末期(TelI)进程中pPak1^(Ser204)离开MTOC和染色体,聚集在收缩环部位。结论 pPak1^(Ser204)在卵母细胞减数分裂恢复时开始表达,是一种MTOC相关蛋白,可能通过参与纺锤体结构的形成和维持而调控染色体的分离。

Objective To observe the protein expression,subcelluar localization of activated Pak1 and its relationship with chromosome separation during meiosis in mouse oocytes. Methods Western blot was applied to analyze the expression of activated Pak1（ phosphorylated Pak1 at Ser204,pPak1^Ser204） at different stages of meiotic progression in semi-quantitative manner; immunofluorescent staining was employed to detect the sub-cellular localization of pPak1^Ser204 and its spatial-temporal correlation with spindle and microtubule organizing centers（ MTOC） during oocyte meiosis. Results pPak1^Ser204 expression was found upon germinal vesicle breakdown（ GVBD） in mouse oocytes which increased along with the meiotic progression,reaching peak level at metaphase Ⅰ（ MⅠ）,which remained up to meiosis Ⅱ（ MⅡ）. From pro-metaphase Ⅰ（ pro-MⅠ） to MⅠ and at MⅡ stage,pPak1^Ser204 was colocalized with MTOC key components,pericentrin and γ-tubulin on spindle poles,pPak1^Ser204 was also distributed on chromosomes; During anaphase Ⅰ（ AⅠ） to telophase Ⅰ（ Tel Ⅰ） progression,pPak1^Ser204 was detached from chromosomes and MTOC,and mainly concentrated on the cleavage furrow. Conclusions pPak1^Ser204 is expressedupon meiotic resumption in oocytes. It is a MTOC-associated protein which may regulate chromosome separation through participating the formation and maintenance of spindle apparatus.