荧光显微成像用于研究卵母细胞减数分裂过程

Application of Fluorescence Microscopy in Research of Oocyte Meiosis

动力蛋白、微管蛋白以及染色体在卵母细胞减数分裂过程中起着重要作用,但目前传统荧光成像方法受到衍射极限的限制,分辨率较低,无法达到卵母细胞减数分裂对成像的要求。首先构建了卵母细胞体外正常成熟体系和原钒酸钠(SOV)作用下的异常成熟体系,然后基于共聚焦显微技术和随机光学重建显微技术(STORM)两种荧光成像方法,研究卵母细胞减数分裂过程中动力蛋白和微管蛋白的定位以及染色体的形态与结构。荧光显微成像结果表明:在正常成熟体系中,动力蛋白分布、微管蛋白形成的纺锤体以及染色体的形态与结构能反映卵母细胞发育的不同阶段;在SOV作用下,纺锤体结构异常率增加,出现桶状、细长、团缩、杂乱、梨状、多极等典型异常纺锤体结构,染色体结构异常率也相应增加。STORM结果更清晰地展现了纺锤体结构信息,三维STORM结果揭示了异常纺锤体的紊乱情况,该方法为卵母细胞减数分裂过程研究提供了精度更高的成像手段。

Dynein,tubulin,and chromosome play important roles in oocyte meiosis.However,the traditional fluorescence microscopy is limited by the diffraction limit and has low resolution,which cannot meet the imaging requirements of oocyte meiosis.Firstly,the normal maturation system of oocyte in vitro and abnormal maturation system under the action of sodium orthovanadate（SOV）are established.Then the confocal fluorescence microscopy and stochastic optical reconstruction microscopy（STORM）are applied to study the localization of the two important proteins of dynein and tubulin and the morphology and structure of chromosome during the oocyte meiosis.The fluorescence imaging results show that the co-localization of dynein,spindle assembled by tubulin,and the morphology and structure of chromosome can correctly reflect the different stages of oocyte meiosis in the normal system.In the SOV treated groups,the abnormality rate of spindle structure increases,and there are typical spindle structures such as barrel,slender,regiment,disorderly,piriform,and multipolar.The abnormality rate of chromosome structure also increases.The STORM results clearly present the structural information of the spindle,and the 3 DSTORM results reveal the disorder level of abnormal spindle.This method provides an imaging approach with higher resolution for the research of the oocyte meiosis.