文蛤受精及早期胚胎发育过程的细胞学观察

Cytological observation on fertilization and early embryonic development in the clam Meretrix meretrix

用普通光镜、荧光显微镜技术和石蜡切片技术三种方法,对文蛤卵在受精及早期胚胎发育过程中的外形和核相变化进行了详细观察。结果表明:文蛤成熟未受精卵呈圆球形,直径90.06μm±5.59μm,核相处于第一次成熟分裂中期;精子为鞭毛型,全长48.05μm±1.60μm,头部呈狭茧形,长度为3.06μm±0.17μm;精卵混合后,精子迅速附着于卵子表面,受精后5min-10min,精子进入卵内并明显膨胀,激活卵子启动两次成熟分裂;分别在受精后20min、30min,受精卵完成第一次和第二次成熟分裂,先后排出第一、第二极体;成熟分裂完成之后,精、卵核体积迅速膨胀到最大,核膜重新出现,形成弥散状的雌、雄原核;受精后35min左右,雌、雄原核在卵子中央发生染色体联合,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40min-45min,在纺锤丝的牵引下染色体被拉向两极,结果形成2个大小不等的卵裂球;受精后55min-60min,第二次卵裂结束,形成1大3小4个卵裂球,卵裂过程中的核相变化与第一次卵裂基本相同,只是卵裂方向是与第一次卵裂的卵裂沟呈基本垂直的纵裂;受精后80min-90min,第三次卵裂完成,仍为不等全裂,但自此次起开始进行螺旋分裂。此外,实验中也发现了少量的多精入卵、多极分离和天然三倍体等异常现象。

The fertilization experiment of Meretrix meretrix was carried out three times at Qingjiang Field Research Station of Zhejiang Mari-culture Research Institute, in July 2006. More than 100 fertilized eggs and early embryos of Meretrix meretrix in every developmental stage were observed to study the cytological changes of nuclear behavior and external shape. Three methods, including optical microscopy, fluorescence microscopy after Hoechst 33258 staining, parafim section （5 μm） and hematoxylineosin staining, were used for this experiment. The results of cytological observation indicated that unfertilized mature eggs of Meretrix meretrix were globular （90.06 μm ± 5.59 μm in diameter） and remained at the metaphase of the first maturation division, while the sperms were the type of flagellum （48.05 μm ±1.60 μm in total length）, with the head like cocoon （3.06 μm ± 0.17 μm in length）. After mix of sperms and eggs, sperms quickly attached to the surface of the eggs and induced acrosome reaction. At 5 min - 10 min after fertilization, sperm has penetrated into cytoplasm of egg and activated the maturation division. The fertilized eggs released the first and the second polar bodies for about the 20th min and the 30th min respectively. At completion of the second maturation division, sperm nucleus and the haploid female nucleus expanded to their maximum and developed into the male and female pronuclei which are loose matched into an association nucleus after their chromosomes and incompact. About 35 rain, the male and female pronuclei formed respectively in the center of egg. Subsequently, thechromosomes of association nucleus arranged on the metaphase plate of first cleavage. 40 min - 45 min, the chromosomes were separated equally into two daughter cells which are different evidently in size. 55 min - 60 min, the second cleavage finished and formed four daughter cells, one big and three small. The process of the second cleavage was fundamentally similar to the first cleavage, but the cleavage direction was almost vertical to the first cleavage furrow. 80 min - 90 min after fertilization, the third cleavage has completed. Though the cleavage is also unequal division, embryos began spiral cleavage from the third cleavage. In addition, the abnormal phenomena, like polysperm, polyspindles and natural triploid, in the experiment also observed and analyzed .