毛蚶受精和早期卵裂过程核行为的荧光显微镜观察

Cytological observation of nuclear behavior in fertilization and early cleavage in Scapharca subcrenata

利用Hoechst33258染色、荧光显微镜观察方法,对毛蚶受精和早期卵裂过程中核行为的细胞学变化进行了详细观察.结果表明,毛蚶成熟未受精卵呈卵圆形,卵径为52.909±2.092μm,核相处于第一次成熟分裂中期.在水温为26±1℃条件下进行受精,在受精后15~18、20~25 min,受精卵先后排出第一、第二极体,完成第一次和第二次成熟分裂;受精后30 min左右,雌、雄性原核形成;受精后35 min,雌、雄性原核各自形成染色体组,在卵子中央发生联合,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,形成2个大小不等的卵裂球;受精后55~60 min,受精卵完成第二次卵裂,形成1大3小4个卵裂球.另外,在研究中还发现了极少量的多精受精、多极分离等异常细胞学现象,对其成因及机制进行了分析和探讨.

Scapharca subcrenata is distributed from the north to the south coast of China and has become one of the most popular commercially important bivalves in aquaculture as it grows rapidly,has high-quality meat,and is easy to farm.Although there have been numerous studies on the reproductive biology of S.subcrenata,there has been scarce research on fertilization and early development.This paper consecutively investigates cytological changes in nuclear behavior in the process of fertilization and early embryonic development in S.subcrenata to clarify the mechanisms of fertilization and embryonic development and to serve as an important reference for research into its artificial reproduction and chromosome engineering breeding.More than 1 000 fertilized eggs and early embryos in all developmental stages were fixed in 40 mg/g paraformaldehyde solution,stained with a Hoechst 33258 fluorescence solution and observed using an Nikon fluorescence microscope 80i.The results of cytological observation indicate that unfertilized mature eggs were globular（52.909 ± 2.092 μm in diameter） and the nuclear phase remained at the metaphase of the first maturation division.After mixing of sperm and eggs,sperm quickly attached to the surface of the egg.At 6 min after fertilization,sperm had penetrated into cytoplasm of the egg and activated maturation division.The fertilized eggs released the first polar body at 15～18 min and the second polar body 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 and developed into the male and female pronuclei 30 min after fertilization.At about 35 min,the two pronuclei matched into an association nucleus after their chromosomes formed respectively in the center of the egg.The chromosomes of the association nucleus then arranged on the metaphase plate of first mitosis.At 40 min,the chromosomes of the fertilized egg separated equally into two daughter cells that were evidently different in size.The second cleavage formed four daughter cells,one large and three small,at about 55～60 min.Additionally,abnormal phenomena like polyspermy and polyspindles were observed and analyzed.