Study On Multicolor Imaging And Super-resolution Imaging Of Oocyte Spindle

Oocyte aneuploidy is the most common type of human chromosome abnormality,which means that the number of chromosomes is more or less than that of diploid.The main reason for the abnormal type of human autosomal aneuploidy is the wrong meiosis of oocytes during maturation.This abnormality is the main internal factors that lead to the related diseases such as female infertility and fetal congenital malformation.Due to the complexity of oocyte growth and the limitation of research methods,although a lot of studies have been carried out on the important process of chromosome meiosis,the exact mechanism of oocyte abnormality has not been clarified.Oocyte spindle is a kind of organelle which is like a spindle.Its main components include microtubule,the dynamic molecular motor attached to microtubule and some complex supramolecular structures.It plays an important role in the process of meiosis of oocyte.Therefore,it is of great academic value and clinical significance to study the fine structure of the spindle,the fine location of key molecules and the causes of its abnormalities.Due to the limitation of optical diffraction limit,traditional fluorescence imaging methods can not image and co locate many important proteins at the same time.In this study,three important molecules or structures of tubulin,dynein and chromosome were imaged by confocal microscopy to explore their characteristics in the event of abnormality;the feasibility of high-resolution imaging of oocyte spindle was explored preliminarily by stochastic optical reconstruction microscopy（STORM）.The results will provide an effective technical means for the formation mechanism of aneuploidy of oocyte.The main contents are as follows:1.Establishment of culture system in vitro of oocytesIn this paper,we established the normal culture system of oocytes in vitro,and then established the abnormal culture system by using three concentartions of the sodium orthovanadate（SOV）,concluding 5μmol/L,50μmol/L and 500μmol/L.The oocytes development was observed after 18 hours incubation at 37°C and 5%CO₂,the results showed that the proportion of the first polar body decreased with the increase of SOV concentration,In the normal culture system,maturation rate was 70%,when SOV concentration was 5μmol/L,50μmol/L and 500μmol/L,maturation rate was 61%,43%and 19%respectively.2.Three dimensional morphology of oocytes by scanning electron microscopeAfter 18 hours of culture,oocytes were fixed and dehydrated.Then obtaining their three-dimensional morphology with scanning electron microscope（SEM）.It showed that SEM can obtain the fine appearance of oocytes and accurately judge whether they are discharged from the first polar body,but it can not observe the morphology of the spindle and chromosome inside the oocytes.3.Imaging of oocytes with confocal microscopeAfter 18 hours of culture in vitro,the oocytes were labeled with tubulin,dynein and chromosome by immunofluorescence staining.The normal and abnormal morphology of spindle and chromosome,and the co-localization of tubulin and dynein were imaged by confocal microscope,and the colocalization of tubulin and dynein is analysed quantificationally by colocalization.The results showed that in the normal culture system,the spindle was a standard bipolar type,the chromosomes were arranged regularly on the equatorial plate,and the dynein was mainly distributed on the two poles;in the abnormal culture system,the spindle was in various abnormal forms,the chromosomal arrangement was disordered,the distribution of dynein was relatively uniform,not concentrated on the two poles.These showed that SOV affected the normal formation of spindle and the co-localization of tubulin and dynein.4.Imaging of oocytes with stochastic optical reconstruction microscopeAfter 18 hours of culture in vitro,the oocytes were labeled with tubulin by immunofluo-rescence staining,and the spindle was imaged by STORM.The imaging results showed that the diameters of spindle microtubule bundles were between 100 nm and 200 nm,compared with the results of conventional wide field fluorescence imaging,the imaging resolution was higher,and the distribution and trend of abnormal spindle microtubule can be clearly seen.The results showed that STORM super-resolution imaging technology can provide more detailed localization and resolution in the study of intracellular microstructure,and can provide strong technical support for the study of oocyte aneuploidy.In this paper,the abnormal development model of oocytes in vitro can be successfully established with SOV,which can inhibit the maturation of oocytes.The higher the concentration is,the more obvious the inhibition effect is,the greater the impact on the localization of tubulin and dynein on the spindle.The spatial localization of tubulin,dynein and chromosome in spindle can be realized by laser confocal microscopy using immunofluorescence labeling and provide powerful research means for revealing the mechanism of aneuploidy formation of oocytes;STORM super-resolution imaging can observe the more fine structure of tubulin,and it is expected to provide better help for the research of meiosis abnormality of oocytes by optimizing the labeling method and expanding the imaging function in the future.