过刊浏览

小鼠孤雌胚早期发育过程中#gamma#--微管蛋白的动态变化

张庆华, 单智焱, 关娜, 徐燕宁, 申景岭, 钟淑琦, 雷蕾

哈尔滨医科大学组织学与胚胎学教研室.黑龙江,哈尔滨 150081

摘要

微管蛋白是构成微管的主要蛋白,其中#alpha#、#beta#亚单位形成异二聚体,而#gamma#--微管蛋白在微管组装中起作用。为了研究小鼠早期孤雌胚中#gamma#--微管蛋白的动态变化,该实验采用了免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法,在SrCl_(2)激活的卵母细胞减数分裂以及早期孤雌胚有丝分裂过程中对#gamma#--微管蛋白进行了定位观察。结果显示,SrCl_(2)和细胞松弛素B(cytochalasin B,CB)诱导的第二次减数分裂中期(metaphaseⅡ of meiosis,MⅡ)小鼠卵母细胞恢复减数分裂,并且纺锤体始终与质膜平行,表明纺锤体旋转被抑制,但核分裂不受影响。减数分裂过程中#gamma#--微管蛋白主要定位于中期纺锤体两极和后期分开的染色单体之间;孤雌活化两雌原核形成以后,#gamma#--微管蛋白聚集在两雌原核周围。在早期孤雌胚有丝分裂间期无定形的#gamma#--微管蛋白均匀分布于核;前中期#gamma#--微管蛋白向两极移动,遍布于整个纺锤体区。有丝分裂中期、后期和末期#gamma#--微管蛋白的分布变化与减数分裂相似。结果表明,SrCl_(2)和CB激活的MⅡ卵母细胞产生杂合二倍体;#gamma#--微管蛋白具有促微管负极帽形成和稳定微管的功能,从而促进纺锤体的形成;分裂后期和末期#gamma#--微管蛋白的重新分布可能是由纺锤体牵引同源染色体分离所诱导的;#gamma#--微管蛋白负责两雌原核的迁移靠近。

关键词： #gamma#-微管蛋白; 孤雌激活; 减数分裂; 有丝分裂; 小鼠

Dynamic changes of #gamma#--tubulin in preimplantation development of parthenogenetic mouse embryos

Zhang Qinghua, Shan Zhiyan, Guan Na, Xu Yanning, Shen Jingling, Zhong Shuqi, Lei Lei

Department of Histology and Embryology,Harbin Medical University.Harbin 150081,Heilongjiang

Abstract

Tubulin is the major protein of microtubule.#alpha#-and #beta#-tubulins form heterodimers,while #gamma#-tubulin regulates microtubule organization.The present study aimed to observe the dynamic changes of #gamma#-tubulin in preimplantation development of parthenogenetic mouse embryos.Immunofluorescence and laser confocal microscopy were used to detect the location of #gamma#-tubulin in preimplantation parthenogenetic embryos activated by SrCl_(2).The oocytes were collected at 13-14 h after hCG injection,and then activated with 10 mmol/L SrCl_(2) in Ca~(2+)-free CZB medium with 5 mmol/L cytochalasin B(CB),fixed at 1 h intervals until 6 h after activation.The results showed that spindle was paralleled with the cell membrane all the time,when the meiosis of MⅡ mouse oocytes resumed.The rotation of spindle was inhibited,but karyokinesis was not influenced.At 0 h after activation,i.e.at metaphase,#gamma#-tubulin was distributed mainly on the two poles of spindle.At 1 h after activation,i.e.at anaphase,following the separation of chromosomes,#gamma#-tubulin was transformed from dense to disperse.At 2 h after activation,#gamma#-tubulin was localized between the segregated sister chromatids at telophase.However,at 3-6 h after activation,#gamma#-tubulin concentrated around the two female pronuclei during their formation and juxtaposition.Moreover,another group of MⅡ oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO_(2) in air at 37 oC to permit parthenogenetic development.The embryos were collected and fixed at 3 h,14 h,16 h,and 18 h of culture.At 3 h after culture,i.e.at mitotic interphase,it was shown that amorphous #gamma#-tubulin distributed around the nuclei of early parthenogenetic embryos.At 24 h after culture,i.e.at prometaphase,#gamma#-tubulin migrated along the spindle microtubule to the two poles.Our results showed that #gamma#-tubulin had similar location patterns at metaphase,anaphase and telophase in meiosis and mitosis.It was concluded that #gamma#-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule,thus promoting the spindle formation at meiosis and mitosis.The relocation of #gamma#-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle.#gamma#-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.

Key words: #gamma#-tubulin;parthenogenetic activation;meiosis;mitosis;Mouse

收稿日期：　　录用日期：

通讯作者：　　E-mail:

引用本文：

张庆华, 单智焱, 关娜, 徐燕宁, 申景岭, 钟淑琦, 雷蕾. 小鼠孤雌胚早期发育过程中#gamma#--微管蛋白的动态变化[J]. 生理学报 2008; 60 (6): 777-782.

Zhang Qinghua, Shan Zhiyan, Guan Na, Xu Yanning, Shen Jingling, Zhong Shuqi, Lei Lei. Dynamic changes of #gamma#--tubulin in preimplantation development of parthenogenetic mouse embryos. Acta Physiol Sin 2008; 60 (6): 777-782 (in Chinese with English abstract).