体外诱导日本蟳精子顶体反应的形态变化与诱导条件

Morphological changes and induced conditions of acrosome reaction in vitro induction of sperm from marine crab (Charybdis japonica)

用光镜和透射电镜观察了日本蟳精子顶体反应过程的形态变化,采用正交实验法分析了体外诱导精子顶体反应的最佳诱导条件,并对比了离子载体A23187与卵水的诱导效果以及取自雄蟹贮精囊的精子顶体反应诱导情况。日本蟳精子的顶体反应过程可分为4个阶段:第Ⅰ阶段为头帽隆起;第Ⅱ阶段为头帽破裂,顶体囊外翻,辐射臂收缩;第Ⅲ阶段为穿孔器前伸,顶体颗粒聚集于顶体管后段;第Ⅳ阶段为穿孔器断裂,顶体囊外层脱落,顶体丝形成,顶体颗粒向外释放,核体积缩小。取自雌蟹纳精囊的精子,在pH为8.0、CaCl2浓度为0.30%的人工海水中,用离子载体A23187(60μg/mL)诱导50min,可以得到最大的精子顶体反应率(80.41%),而经卵水诱导得到的顶体反应率为73.87%。用所得出的最佳诱导条件对取自雄蟹贮精囊的精子进行处理,可得到64.37%的顶体反应率。

Charybdis japonica is an important marine crab widely distributed in China sea area. The main purpose of the study was to provide a theory basic for the research of reproduction biology and promote the progress of artificial breeding technolagy of C. japonica. The morphological changes of sperm during the acrosome reaction in C. japonica were studied in detail with light and transmission electron microscope,and the optimal induced conditions of acrosome reaction in vitro was obtained by orthogonal experiments. Then the induced effect of ionophore A23187 and egg-water was investigated by contrast experiment,and the acrosome reaction rate of the sperm collected from male seminal receptacle has been analysed induced by treatment with ionophore A23187. The process of acrosome reaction consists of four consecutive stages：（1） swelling of the apical cap,（2） the apical cap ruptures to form an aperture which acrosomal vesical can evert through it and radial arms contract,（3） elongation forward of perforator and aggregation of acrosomal granule in the posterior segment of acrosomal tubule,（4） rupture of perforator and shedding of the acrosomal vesicle outer layer result in formation of the acrosomal filament,then acrosomal granules release out and nuclear size reduce. Most of the acrosome reaction of C. japonica sperm were complete in 50 minutes. Three factors with five levels including a series of pH（6. 0,7. 0,8. 0,9. 0 and 10. 0） ,a series of different concentrations in Ca2 ＋（0. 10%,0. 15%,0. 20%,0. 25% and 0. 30%） and A23187（30,40,50,60 and 70 μg/mL） were chosen to assay the optimal induced conditions for the acrosome reaction in sperm of with an orthogonal experiment. The highest rate（80. 41%） of the acrosome reaction in sperm collected from female spermatheca can be achieved when sperm exposed to 60 μg/mL A23187,0. 30% CaCl2 at pH = 8. 0 in Ca2 ＋ free artificial seawater for 50 minutes,but the acrosome reaction rate of the sperm is 73. 87% induced by egg-water. 64. 37% of acrosome reaction rate can be achieved by treating the sperm taken from male seminal receptacle with the optimal induced conditions.