文章摘要
董迎辉,姚韩韩,张佩云,沈庞幼,刘浩明,林志华.缢蛏受精和早期卵裂过程的细胞学变化观察[J].水产学报,2012,36(9):1400~1409
缢蛏受精和早期卵裂过程的细胞学变化观察
Cytological observation on fertilization and early cleavage in Sinonovaula constricta
投稿时间:2011-11-14  修订日期:2012-04-21
DOI:DOI:10.3724/SP.J.1231.2012.27826
中文关键词: 缢蛏  受精  早期卵裂  多精受精
英文关键词: Sinonovaula constricta  fertilization  early cleavage  polyspermy
基金项目:国家现代贝类产业技术体系专项(nycytx 47);宁波市科技局农业择优委托项目(2010C10011);宁波市自然科学基金(201001A6004021)
作者单位
董迎辉 浙江万里学院生物与环境学院 
姚韩韩 浙江万里学院生物与环境学院 
张佩云 浙江万里学院生物与环境学院 
沈庞幼 宁波市海洋与渔业研究院 
刘浩明 中国海洋大学水产学院 
林志华 浙江万里学院生物与环境学院 
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中文摘要:
      采用普通光镜、荧光显微镜和石蜡切片技术,对缢蛏受精和早期卵裂过程中的精子入卵、减数分裂、雌雄原核形成与结合、早期卵裂以及多精入卵等细胞学事件进行了显微观察。结果表明,缢蛏成熟未受精卵多呈圆球形或卵圆形,少数呈梨形,卵径为82~88 μm,核相处于第一次成熟分裂中期;精子为典型的原生型,全长55~58 μm,头部呈保龄球形,顶体前端的顶体杆呈特别细长的花丝状;在水温21~22 ℃、盐度10的条件下进行人工授精,精、卵混合后,精子迅速附着于卵子表面,启动卵子发育;受精后4~6 min,精子的头部已进入卵内并明显膨胀,卵子外形变圆,卵外附着的精子量明显减少;在受精后12~15 min、20~25 min,受精卵先后排出第一极体、第二极体,完成两次成熟分裂;第二次成熟分裂结束以后,精、卵核体积迅速膨胀,雄原核的膨胀早于雌原核,核膜重建,在受精后约30 min形成雌、雄原核;雌、雄原核均向卵子中央移动,雄原核旁边的精子星光清晰可见,随后二者在卵子中央以染色体联合的方式结合,联合核的染色体共同排列在纺锤体的赤道板上,形成第一次有丝分裂的中期分裂相;受精后40 min左右,受精卵进行第一次卵裂,染色体在纺锤丝的牵引下向两极移动,结果形成2个大小不等的卵裂球;受精后45~50 min,卵子进行第二次卵裂,核相变化与第一次卵裂相同,在与第一次卵裂垂直的纵轴方向上发生不等全裂,最终形成3小1大4个卵裂球;受精后60~70 min,胚胎进行第三次卵裂,仍为不等全裂,但自此次卵裂起已开始进行螺旋分裂。此外,对实验中发现的缢蛏极少量多精入卵、多极分离等异常细胞学现象进行了分析,并探讨了海洋贝类卵子阻止多精入卵发生的机制。
英文摘要:
      The artificially induced spawning and fertilization experiment of the razor clam(Sinonovaula constricta)was carried out at Ningbo Yongsheng Aquatic Seed Corporation Limited in October 2010.More than 1 000 fertilized eggs and early embryos of all developmental stages were collected and more than 100 were carefully observed to investigate a series of cytological events,including sperm penetration,maturation division,formation and chromosome association of the female and male pronuclei,early cleavage and polyspermy.Three techniques,including light microscopy,fluorescence microscopy by Hoechst 33 258 staining,paraffin section and hematoxylin-eosin staining,were used in the experiment to clarify the mechanisms of fertilization and embryonic development,which provided an important reference for research into its artificial reproduction and chromosome engineering breeding.The results of cytological observation indicated that most of unfertilized mature eggs ofS.constrictawere globular or oval(82-88 μm in diameter)and a few were pear shaped and the nuclear phase of them remained at the metaphase of the first maturation division.The sperms were the type of flagellum(55-58 μm in total length),with the bowling shaped head and particularly slender acrosome rod like filament of flower.At water temperature 21-22 ℃ and salinity 10,after mix of sperms and eggs,sperms quickly attached to the surface of the eggs and activated the process of egg development.From 4 min to 6 min after insemination,sperm has penetrated into cytoplasm of egg and significantly expanded,while the shape of eggs become more round and the number of sperms outside the egg was significantly reduced.The fertilized eggs released PB1 at 12-15 min and PB2 at 20-25 min in the process of maturation division.After completion of the second maturation division,the sperm nucleus and the female nucleus expanded immediately,nuclear membrane reconstructed and developed into the male and female pronuclei 30 min after fertilization.Expansion of the sperm nucleus was earlier than that of the female nucleus.At formation of the female and male pronuclei,they approached gradually and all moved to the center of egg.At that time,the spermaster beside the male pronucleus was visible obviously.And then,the two pronuclei matched into an association nucleus after their chromosomes formed respectively.Subsequently,the chromosomes of association nucleus arranged on the metaphase plate of first mitosis.About 40 min,fertilized eggs carried out the first cleavage and the chromosomes were separated equally into two daughter cells which are different evidently in size.45-50 min after fertilization,embryos underwent the second cleavage which was fundamentally similar to the first cleavage,but the cleavage direction was almost vertical to the first cleavage furrow,and ultimately formed four daughter cells,three small and one big.60-70 min,embryos completed the third cleavage.Though the cleavage is also unequal division,embryos began spiral cleavage from the third cleavage.Additionally,the abnormal phenomena,such as polysperm and polyspindles,in the experiment were analyzed and the mechanisms of polyspermy prevention in marine mollusks were discussed and studied.
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