Cytological observation on fertilization and early cleavage in Sinonovaula constricta

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 of S.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.