In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.     

斑马鱼卵母细胞的体外成熟及成熟卵的受精发育

采用体外培养的方法,研究斑马鱼卵母细胞的成熟过程。Ⅳ时相初级卵母细胞在o．5μg／m1 17a－羟基孕酮的EM－199培养液中,80％氧气,25℃的体外培养条件下,在40min内,胚泡(GV)逐渐由卵母细胞中央至动物极边缘l／2处移到动物极边缘,进八V时相卵母细胞。30min后胚泡破裂(GVBD),胚泡破裂率为59％。此种卵母细胞继续培养2h才完全成熟。成熟卵不能从滤泡膜中自然排出。冷开水中剥离其外边的滤泡膜后加入具有受精能力的精子,即能使成熟卵受精,受精膜举起,胚盘在动物极形成。其后受精卵的分裂、发育等与自然成熟受精卵相同。以发育至囊胚为受精标准,这种体外成熟卵受精率为78％。这是斑马鱼卵母细胞体外培养成熟的首例报道。鱼类卵母细胞体外成熟技术的建立,为外源基因卵母细胞胚泡内转移奠定了基础。