银鲫卵母细胞体外诱导成熟技术的建立  

DEVELOPMENT OF IN VITRO MATURATION TECHNOLOGY FOR GIBEL CARP OOCYTES

作　　者：李志[1] 汪洋[1] 周莉[1] 李熙银 张晓娟[1] 周剑[1] 桂建芳[1] LI Zhi;WANG Yang;ZHOU Li;LI Xi-Yin;ZHANG Xiao-Juan;ZHOU Jian;GUI Jian-Fang(State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China)

机构地区：[1]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》2017年第5期984-991,共8页Acta Hydrobiologica Sinica

基　　金：中国科学院战略性先导科技专项(XDA08030201)资助~~

摘　　要：研究以银鲫为材料,根据银鲫(Carassius auratus gibelio)卵母细胞生发泡(Germinal vesicle,GV)边移程度及剥离GV中减数分裂前期染色体的凝集状态,将银鲫Ⅳ时相的卵母细胞分为GV0、GV1、GV2和GV3四个时期;并进一步比较了分别处于这4个时期银鲫卵母细胞体外诱导培养的成熟率、卵裂率和孵化率。结果表明,GV1期之后的卵母细胞均可有效进行体外诱导成熟,可正常受精发育,由于GV1期卵母细胞有较长时间用于显微操作,因此GV1期卵母细胞被选为进行体外诱导的最早时期的卵母细胞。以GV1期卵母细胞为研究材料,摸索了银鲫卵母细胞体外诱导成熟的适宜条件:取GV1期的Ⅳ时相卵母细胞,放置于pH 8.5、加有1μg/m L孕酮激素(17α,20β-dihydroxy-4-pregnen-3-one,DHP)的格氏平衡盐溶液(Gey’s balanced salt solution,GBSS)中,在23℃培养箱中体外诱导12h后,将滤泡膜剥离后再进行人工体外授精,其所获胚胎的孵化率可达55.5%。此外,将体外转录合成的带GFP标签的h2af1o m RNA注射到GV1期卵母细胞,发现经显微操作和体外诱导后不仅可以通过GFP绿色荧光信号活体观察GVBD、受精、卵裂和早期胚胎发育的全过程,而且诱导成熟的卵子仍可正常受精和胚胎发育。研究建立的银鲫卵母细胞体外诱导成熟技术为银鲫和其他鱼类卵母细胞发育过程研究及其相关基因和细胞显微操作提供了技术平台。In vitro oocytes maturation is a key and efficient technology for gene and cellular micromanipulation in oocytesof fish.In this study,we used gibel carp oocytes as studying materials to develop an efficient in vitro inductionmaturation technology.Based on previously established germinal vesicle(GV)isolation and premeiotic chromosomepreparation methods,four different stage oocytes including GV0,GV1,GV2and GV3were firstly distinguished byGV migration and premeiotic chromosome condensation status,and the maturation rate(%),cleavage rate(%)andhatching rate(%)were analyzed.GV1,GV2and GV3oocytes had the capacity for in vitro induction maturation,andGV1oocytes were the optimal stage oocytes because they have longer time for micromanipulation.We found that theoptimal induction conditions for GV1oocytes in vitro were12hours at23℃in pH8.5Gey’s balanced saltsolution(GBSS)with1μg/ml DHP(17α,20β-dihydroxy-4-pregne-3-one),by which up to55.5%hatching rate wasreached from these stripped follicle membrane oocytes.Moreover,we injected the GFP-zfh2af1o mRNA into the GV1oocytes,and found that the whole process of in vitro maturation,fertilization and embryo development of the micromanipulatedGV1oocytes could be tracked by the expression of GFP,and normal fertilization and embryo developmentcould be produced from the induced mature eggs.Therefore,we established an efficient platform for studying oocytedevelopment and gene and cellular micromanipulation in this fish.

关 键 词：银鲫 卵母细胞成熟 体外诱导 生发泡 显微注射 

分 类 号：Q-366[生物学]