敲低TPX2对猪卵母细胞纺锤体组装及凋亡的影响  

Effects of TPX2 knockdown on spindle assembly and apoptosis of porcine oocytes

作　　者：贺依静 李桥 彭磊 李佳 初亚婕 林其昕 芮荣[1] 剧世强[1] HE Yijing;LI Qiao;PENG Lei;LI Jia;CHU Yajie;LIN Qixin;RUI Rong;JU Shiqiang(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2023年第1期92-98,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31972759,31572589);江苏高校优势学科建设工程资助项目。

摘　　要：[目的]本研究旨在探究Xklp2靶蛋白(TPX2)在猪卵母细胞减数分裂过程中的表达定位及其潜在功能。[方法]采集猪卵丘-卵母细胞复合体(COC)并随机分组后进行体外成熟培养,通过间接免疫荧光染色与蛋白免疫印迹等方法检测TPX2在卵母细胞减数分裂过程中的亚细胞定位与动态表达情况;通过生发泡期(germinal vesicle, GV)显微注射特异性siRNA以敲低TPX2,研究其对卵母细胞减数分裂进程、纺锤体结构以及早期凋亡的影响。[结果]TPX2在第一次减数分裂中期(metaphaseⅠ,MⅠ)表达量显著上升,其亚细胞定位与α-微管蛋白(α-tubulin)的分布特点相似,并主要富集在纺锤体两极。TPX2被敲低后卵母细胞第一极体(PB1)排出率显著下降(P<0.05),且大多数细胞被阻滞在生发泡破裂期(GVBD)和第一次减数分裂后末期(ATⅠ),同时纺锤体结构异常比例显著升高(P<0.05),并伴随染色体排列紊乱。与对照组相比,敲低TPX2后卵母细胞早期凋亡率急剧增加(P<0.05),凋亡相关基因Caspase 3表达量以及Bax/Bcl2值显著升高(P<0.05)。[结论]TPX2与猪卵母细胞减数分裂过程中的纺锤体组装及细胞周期调控密切相关,敲低TPX2引起卵母细胞纺锤体结构异常,并诱导早期细胞凋亡,最终导致卵母细胞成熟失败。[Objectives]This experiment aimed to investigate the localization, expression and potential roles of targeting protein for Xklp2(TPX2)in porcine oocytes during meiosis. [Methods]The cumulus oocyte complexe(COC)were collected and cultured in vitro,then the localization and expression of TPX2 were detected by immunofluorescence staining and Western blot. After TPX2-siRNA microinjection, the cell cycle, spindle morphology, and early apoptosis were analyzed to investigate the effects of TPX2-depleted. [Results]The expression level of TPX2 significantly increased after metaphaseⅠ(MⅠ),the localization characteristic was as same as α-tubulin and enriched at spindle poles. After TPX2-depletion, the first polar bady(PB1)extrusion significantly decreased(P<0.05),and most oocytes were arrested in germinal vesicle breakdown(GVBD)and anaphaseⅠand telophaseⅠ(ATⅠ)stages, with an increase in spindle abnormal rate and misaligned chromosomes(P<0.05). In addition, the rate of early apoptosis obviously increased(P<0.05),and the mRNA expression level of apoptosis related genes(Caspase 3 and Bax/Bcl2)also increased(P<0.05)after TPX2 depletion. [Conclusions]The results indicated that TPX2 plays an indispensable role in the regulation of spindle assembly and cell cycle of porcine oocytes. TPX2-depleted can induce abnormal spindle structure and early apoptosis.

关 键 词：Xklp2靶蛋白(TPX2) 猪卵母细胞 减数分裂 纺锤体组装 凋亡 

分 类 号：S857.2[农业科学—临床兽医学]