The Location And Function Of Centrosomal Protein Family In In Virto Maturation Of Mouse Oocyte This Project Is Supported By The National Natural Science Foundation

Centrosome is an important non-membranse structure in animal cells. It is Responsible for establishing poles to ensure the symmetry cell division, thus the average allocation of genetic material in two daughter cells. It is composed of a pair of centrioles and surrounding dense materials under the electron microscope. These so-called pericentriolar materials are made up of many proteins. The centrosome proteins have many functions including the organization of microtube.There are a special family among these proteins located at centrosome, called Cep X(Xrepresent their molecular weight).They play important roles in mitosis.According to the stage they function, they were classified into 4 groups: 1. the organization of centriol in interphase; 2.the control of G2/M transition; 3.the mediation of the spindle assemble in prophase; 4.the regulation of cytokinesis in telophase. Currently, the location and function of proteins in the mitotic centrosome has achieved a lot. However, there is no centrosome formed in the process of meiosis,it has few data about the location and function of centrosomal proteins.In this study, mice immature oocytes were used to study these 4 types of centrosomal proteins, acting in different stage in mitosis with different functions.Each were picked from one categories: Cep120,which plays a key role in centriol duplication;Cep63, which initiates the G/M transition; Cep70, which regulate the spindle assemble and Cep55, which regulate cytokinesis. Knockdown or overexpression of these proteins was performed to explore their functions in oocyte maturation.Part I To determine the subcellular location and expression modes of Cep proteins at different stages in mouse oocyte maturationObjective:1. Using western blotting technique to learn the expression mode of Cep55,Cep63 and Cep120.2. Using the immnostaining and live cell imaging methods to determine the subcellular location of Cep55, Cep63 and Cep120.Results:1.The expression level are about the same for these 3 proteins in 4 stages in mouse oocyte maturation.2.These 3 proteins are located near the nuclear in GV stage, aggregate to the center of the cell after GVBD. In metaphase, Cep63 mainly located to the spindle pole, whether the Cep55 and Cep120 locate to the whole spindle.3.Cep55 located to the midbody at cytokinesis in live cell image.Conclusion:Different Cep proteins locate to different sites, and not the same with the location in mitosis.Part II To study the effection of gene knockdown or over expression of these 4Cep proteins on GVBD and PB1 extrusion.Objective:1.Depleted the gene of Cep55, Cep63, Cep70 and Cep120,detect the efficiency of depletion.2. using Stereoscopet to calculate the GVBD and PB1 rate after gene knockdown.3.using Stereoscopet to calculate the GVBD and PB1 extrusion rate after gene overexpression.Results:1.the depletion of Cep55,Cep70,Cep63 and Cep120 have no effect on GVBD rate.2. the depletion of Cep55,Cep63 and Cep120 caused the decline of PB1 extrusion rate.3.the overexpression of Cep55 has no effect on GVBD and PB1 extrusion rate.4.the overexpression of Cep120 caused the decline of PB1 extrusion rate.Conclusions:Different Cep proteins may have different functions, thus the different effection of their gene knockdown or over expression.Part III To learn in more depth about the effection of these Gene knockdown or overexpression on mouse oocyte maturation process and the reason for these effection.Objective:1.using the immnostaing technique to observe the changes of chromosome arrangement and spindle morphology after the gene knockdown of Cep55,Cep63,Cep70 and Cep120.2. using the immnostaing technique to observe the changes of chromosome arrangement and spindle morphology after the gene over expression of Cep55,Cep63,Cep70 and Cep120.3. Using western blotting technique to learn the cyclin B1 level in anaphase I after the knockdown of Cep55, Cep63 and Cep120.4..using the chromosome spreading methods to detect the chromosome separation status and the activation of SAC in anaphase I.Results:1.the knockdown of Cep55, Cep63 and Cep120 caused the morphology changes of MI spindle,the knockdown of Cep63 caused the abnormal arrangement of chromosome in MI.3. The knockdown of Cep55,Cep63 and Cep120 caused unseperation of homologous chromosome and the undegradation of cyclin B1 in anaphase.I4.The overexpresiion of Cep120 lead to the abnormal spindle and chromosome arrangement in MI pahse.5.The knockdown of Cep55,Cep63 and Cep120 caused the activation of SAC in M/A transition, thus leading to the decline of PB1 extrusion rate compared with the control group.Conclusions:The knockdown of Cep55, Cep63, Cep120 caused the activation of SAC, thus blocked the G2/M transition, leading to the decline of PB1 extrusion rate.