Spindlin1 Proteins Function In The Mouse Egg

In mammalian,the spindle assembly checkpoint(SAC)is an important safety device which prevents chromosome mis-segregation and cell death in mitosis.The presence of a single chromosome erroneously attached the biopolar-spindle can activated the SAC and lead to a metaphase arrest to permit the reparation of the 'damage'.In meiosis,the SAC control has been verified to be functional and some SAC components,such as MAD and BUB,are necessary for correct timing of premetaphase in mouse oocytes.However,the SAC mechanism in oocyte meiosis is still ambiguous.In this research,we have discovered a potential oocyte-specific protein involving in SAC control—spindlinl.In previous research,we utilized the proteomics to determine the expression of proteins in mouse oocyte and generated a proteome reference database for MetⅡ(metaphase of the second meiotic division) mouse oocyte,which can be accessed over the Internet (http://reprod.njmu.edu.cn/2d).Among these,ten protein spots were identified as spindlinl by mass spectrometry,which distribute at different molecular mass(Mr)and isoelectric point(pI),suggesting its functional complexity in mouse oocyte.When the oocytes were activated by strontium chloride for three hours,five protein spots vanished from the image.The result implied that the protein may be involved in some biological events at the specific stage.Then we stained the 2D gel of MetⅡoocytes with a fluorescently labeling Pro-Q Diamond dye and visualized the phosphorylated proteins.The image shows that all vanished protein spots were modified by phosphorylation,which means that phosphorylation is the functional pattern of spindlinl at metaphase stage.To study the function of spindlinl protein,we prepared anti-spin antibody and verified its specificity by Western blot, immunohistochemistry and immunofluorescence.The result of Western blot displayed that there were a single band at the position of expected molecular mass.At the same time,the consequences of immunohistochemistry and immunofluorescence showed that spindlinl protein was present in the cytoplasm of oocytes and localized to the metaphase spindle,which were concordant with the researches reported before.The data above proven that anti-spin antibody is specific and effective.The current research was focus on the localization variation of spindlinl protein and its associated function in mouse oocyte.To confirm the spatial relationship between spindlin1 andα-tubulin,double immunostaining was performed.The result showed that spindlin1 protein localized to the metaphase spindle,colocalizing withα-tubulin protein, but not anaphase spindle.To explore the role of spindlin1 directly,MetⅡ oocytes were microinjected with anti-spin antibody,which leading to obvious spindle abnormalities and chromosomes congression failure.The result demonstrates strongly that spindlinl protein is very important for maintaining normal spindle appearance in MetⅡ.Then the microinjected oocytes were activated with strontium chloride and the division process was investigated by time lapse video.The anaphase onset and second polar body extrusion were sped up conspicuously due to reduction of spindlinl levels.Base on the consequences above,we considered that spindlinl protein is very important for maintaining normal metaphase spindle.Besides, spindlin1 may be involved in the SAC control;the vanished protein spots from the gel when the oocytes were activated may be associated with the proper function binding to the spindle.