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小鼠体细胞核移植重构胚中γ-微管蛋白的动态变化

张庆华, 雷蕾

牡丹江市第二人民医院病理科，牡丹江 157005；哈尔滨医科大学组织学与胚胎学教研室，胚胎与干细胞工程重点实验室，哈尔滨 150081

摘要

本文旨在研究小鼠体细胞核移植(somatic cell nuclear transfer, SCNT)重构胚中γ-微管蛋白的动态变化。采用免疫荧光化学染色与激光共聚焦显微镜观察的方法，对去核前后第二次减数分裂中期(metaphase II of meiosis, MII)的卵母细胞和核移植后的重构胚进行γ-微管蛋白的定位观察，分析γ-微管蛋白的作用。结果显示，MII期卵母细胞去核后，γ-微管蛋白主要分布于皮层；卵母细胞激活后随着胞质星体的逐渐增多，皮层γ-微管蛋白明显减少，但没有新的纺锤体形成。卵丘细胞注入去核MII期卵母细胞后，经SrCl2激活能形成一个纺锤体，在分裂前期，斑点状的γ-微管蛋白散布在染色体之间；在分裂中期，γ-微管蛋白沿着纺锤体分布或聚集于纺锤体两极；在分裂后期或末期，γ-微管蛋白迁移到分开的染色单体之间。部分重构胚发生提前激活，形成异常纺锤体，主要表现在γ-微管蛋白和染色体分布异常。卵丘细胞注入未去核的MII期卵母细胞后，经SrCl2激活后能形成两个纺锤体，γ-微管蛋白的分布与正常核移植相类似。同样，部分卵母细胞发生提前激活，形成两个小梭形纺锤体或一个宽而大的桶状纺锤体。以上结果提示，供体细胞核内的γ-微管蛋白在SCNT重构胚纺锤体组装过程中发挥着关键作用；SCNT重构胚易被提前激活形成异常纺锤体，γ-微管蛋白的异常与染色体分布失常和纺锤体结构异常密切相关。

关键词： γ-微管蛋白; 核移植; 有丝分裂; 小鼠

分类号：R329.2+4

[Dynamic changes of γ-tubulin in mouse somatic cell nuclear transferred embryos.] [Article in Chinese]

ZHANG Qing-Hua, Lei Lei

Department of Pathology, Mudanjiang Second People's Hospital, Mudanjiang 157005, China； Department of Histology and Embryology, Key Laboratory of Embryo and Stem Cell Engineering, Harbin Medical University, Harbin 150081, China

Abstract

The aim of the present study is to observe the dynamic changes of γ-tubulin in mouse somatic nuclear transferred (SCNT) embryos. The γ-tubulin was detected and analyzed in the enucleated oocyte and SCNT embryos by immunofluorescence and laser confocal microscopy. The results showed that γ-tubulin distributed in the cortex of the enucleated MII oocytes, and decreased in this area during the activation of oocytes. Meanwhile cytoplasmic asters appeared, but there was no spindle formed. Spindle formation could be observed in the enucleated oocytes which were injected with cumulus cells and activated by SrCl2. The spots-like γ-tubulin signals spread between chromosomes at the prophase, and the signals arrayed with spindle or aggregated at two poles of the spindle at the early metaphase. Furthermore, γ-tubulin signals were localized between the segregated sister chromatids at anaphase or telophase. Some reconstructed embryos exhibited advanced activation, showing abnormal spindles and aberrant distribution of γ-tubulin and chromosomes. Two spindles would be formed when the cumulus cell was injected into an intact oocyte, and the distribution of γ-tubulin was similar to that of the normal SCNT. Moreover, advanced activation also occurred in this case and formed either two small spindles or one big barrel-shaped spindle. These results suggest that γ-tubulin plays a pivotal role in spindle assembling in mouse SCNT embryos. The reconstructed oocytes were easily to be activated, and aberrant distribution of γ-tubulin is associated with formation of abnormal spindles and chromosome misalignment.

Key words: γ-tubulin; nuclear transfer; meiosis; mouse

收稿日期：2012-07-04　　录用日期：2012-08-30

通讯作者：雷蕾　　E-mail: LeiL086@yahoo.com.cn

引用本文：

张庆华, 雷蕾. 小鼠体细胞核移植重构胚中γ-微管蛋白的动态变化[J]. 生理学报 2013; 65 (2): 158-164.

ZHANG Qing-Hua, Lei Lei. [Dynamic changes of γ-tubulin in mouse somatic cell nuclear transferred embryos.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (2): 158-164 (in Chinese with English abstract).