银鲫卵母细胞Spindlin基因的表达特征及其功能分析  

EXPRESSION CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF OOCYTE-SPECIFIC SPINDLIN IN GIBEL CARP

作　　者：孙敏[1] 王兰[1] 桂建芳[2] 

机构地区：[1]山西大学生命科学学院,太原030006 [2]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072

出　　处：《水生生物学报》2012年第4期593-599,共7页Acta Hydrobiologica Sinica

基　　金：973项目(2010CB126301);国家自然科学基金(30630050);淡水生态与生物技术国家重点实验室开放基金(2011FB04)资助

摘　　要：在卵母细胞成熟过程中,Spindlin与纺锤体微管蛋白相互作用,在配子到胚胎的过程中具有调节细胞周期的作用。前期研究表明,银鲫Spindlin(CagSpin)与微管蛋白相互作用,并与减数分裂的纺锤体共定位。在成熟过程中,CagSpin被磷酸化,在卵母细胞受精和卵胚转换中发挥重要的作用。研究通过对激素诱导后的卵母细胞进行追踪,采用RT-PCR和Western-blot分析,揭示卵母细胞在完成成熟的过程中,CagSpin持续大量表达。采用体外诱导卵母细胞成熟技术和显微注射的方法,揭示过量表达CagSpin导致胚泡(Germinalvesicle,GV)不能破裂,卵母细胞成熟过程被抑制。这些结果表明,CagSpin在卵母细胞成熟过程中发挥着关键的作用,同时为深入研究CagSpin的功能提供依据。Spindlin, an associated protein with meiotic spindle, had been suggested to regulate the transition from gam- ete to embryo in mouse. In previous studies, we had confirmed that gibel carp （Carassius auratus gibelio） Spindlin （CagSpin） interacted with tubulin and co-localized with meiotic spindle, and found that CagSpin was phosphorylated during oocyte maturation and played important roles in egg fertilization and the oocyte-to-embryo transition. In this study, we further investigated its expression pattern and function role during the hormone-induced oocyte maturation. Firstly, the maturing and matured oocytes were obtained from ovary every two hour interval from the initially induced time until the moment of spawning naturally. RT-PCR analysis revealed that CagSpin transcripts were abundantly, and CagSpin was actively transcribed in these developing oocytes during the whole maturation. Western blot detection demonstrated the corresponding expression pattern of CagSpin in different stages of oocyte maturation. Then, Cag- Spin-GFP-pCS2＋ expression construct was made and used to detect the specific expression and localization of CagSpin in nucleus in CAB cells. Moreover, the CagSpin-GFP-pCS2＋ construct was further used to transcribe CagSpin RNA in vitro for subsequent microinjection analysis. Additionally, the hormone-induced maturation processes could be clearly traced and observed through the dynamic changes of germinal vesicle. Thereby, the combined observation of in vitro oocyte maturation and microinjection assay demonstrated that the injected oocytes with CagSpin RNA could not un- dergo germinal vesicle broken down （GVBD）, and kept in immature status during the whole induced process, which implicates that over-expression of CagSpin inhibits the normal oocyte meiotic maturation. Therefore, the data provide further evidence for CagSpin function, and confirm its significant role during oocyte maturation.

关 键 词：Spindlin 卵母细胞成熟 表达特征 体外诱导 过表达 

分 类 号：Q344.1[生物学—遗传学]