Fluorescence Super-resolution Microscopy Imaging Was Used To Study Oocyte Meiosis

Aneuploidy is the most common type of human chromosome abnormalities.The aneuploidy of reproductive cells is mainly caused human mental retardation,congenital malformations and pregnancy failure,and somatic aneuploidy is closely related to tumorigenesis.Although people have done a lot of research on the occurrence of aneuploidy over the years,the exact mechanism has not yet been elucidated.Anopheplastic children and the incidence of cancer often bring a heavy burden to families and society.Therefore,it is important to study the molecular regulation mechanism of aneuploidy in order to reduce the aneuploidy risk of birth and tumor of incidence.About 30% of human autosomal aneuploidy is caused by meiosis errors during oocyte maturation.Oocyte maturation is closely related to cytoplasm,and the cytoplasmic kinetic protein(Dynein)plays an important role in chromosome segregation and spindle formation due to its unique physiological function.However,at present,the traditional fluorescence imaging method for Dynein and tubulin and chromosome imaging studies is limited by the diffraction limit,which the resolution is low,our group tried to use the confocal microscopy and random optical reconstruction microscopy(STORM)imaging method to observe the morphological structure of Dynein,tubulin and chromosome during oocyte meiosis,which could provide technical support for the mechanism of molecular regulation of the aneuploidy of the oocyte.In view of this study,our paper is based on three-dimensional random optical reconstruction super-resolution fluorescence microscope to carry out research work,the specific contents are as follows:1?The background of this research and the present situation and existing problems of oocytes were introduced in detail.The far-field super-resolution fluorescence microscopy was highlighted,including the working principle and advantages and disadvantages of stimulated emission depletion microscopy(STED),saturated structural illumination microscopy(SSIM),light activation localization microscopy(PALM)and random optical reconstruction microscopy(STORM).2?The basic theory and technical problems of STORM are discussed,and thethree-dimensional super-resolution fluorescence microscopy system is introduced in detail.3?Study on somatic cell fluorescence labeling method of super resolution imaging,selecting mouse myoblasts(C2C12)and human embryonic fibroblast cells(MRC-5)and mouse fibroblast cells(NIH-3T3)to label because of their microtubule structures rich and spreading.In the imaging process of discontinuous problems,we optimized from fixative,antibody concentration and antibody,and got the stable marking method and imaging condition.4?Study on oocyte of super resolution imaging.First,the normal and abnormal maturation system of oocytes were successfully established.Then the confocal fluorescence images showed different meiosis period accoring to the Dynein distribution,the spindle formed by microtubules and the morphology and structure of the chromosome and the fluorescence localization.And the STORM images presented the clearer structure of the spindles,which provided the technical support for the mechanism of oocytes' aneuploidy at a higher spatiotemporal resolution.5?Finally,the summary and prospect are summarized.Based on the existing experimental results,the suggestions and prospects of optimizing the experimental design are put forward.The fluorescence images showed that in the normal system,the co-localization of Dynein,spindle and chromosome could reflect the different stages of oocyte meiosis.In the treated oocyte groups,the dynein and spindle didn't depict the spindle-shaped during meiosis.In addition,the rate of spindle's structural abnormalities increased,typical abnormal structure of spindle appeared,such as slender,barrel,regiment,disorderly,piriform and multipolar.The rate of chromosome's structural abnormalities increased too.The STORM images presented the clearer structure of the spindles,which provided the technical support for the mechanism of oocytes' aneuploidy at a higher spatiotemporal resolution.