【作者】 胡家会；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2005， 博士

【摘要】 精子生物学的快速发展,为保护野生濒危物种和经济动物的人工繁殖研究提供了重要手段,还开辟了体外急性毒性检测的新领域。本文对青岛文昌鱼（Branchiostoma beicheri tsingtauense）和玫瑰无须鲃（Puntius conchonius）精子运动启动的机理和影响因子进行了研究。在此基础上,把玫瑰无须鲃的精子应用于水胺硫磷（isocarbophos）和甲基异柳磷（isofenphos-methyl）的细胞毒性检测中,建立了用鱼类精子体外检测有机磷农药急性毒性的方法体系。 首先,我们探讨了外界环境因素对青岛文昌鱼精子启动及运动的影响,发现:（1）文昌鱼精子的运动启动属于高渗调节类型,低渗或等渗电解质和非电解质溶液均不能激发精子运动;（2）只有Ca²⁺存在的条件下,高渗溶液才能启动精子的运动;（3）当Ca²⁺添加到含有EGTA的无Ca²⁺溶液后,被抑制的精子恢复运动,说明Ca²⁺和高渗是精子运动启动必不可少的外部因素;（4）精子运动的启动需要Ca²⁺,而L-钙离子通道抑制剂异搏啶（verapamil）却不能抑制精子运动的启动;（5）钾离子通道抑制剂奎宁（quinine）和4-氨基吡啶（4-aminopyridine）显著抑制精子的运动,但不影响精子运动的启动,它们的抑制作用是可逆的,说明钾离子通道参与精子运动过程的调节。（6）只有在Ca²⁺存在的情况下,溶液pH的变化才影响精子运动,而不影响运动的启动。基于这些结果,我们认为高渗和Ca²⁺协同作用启动文昌鱼精子运动,钾离子和溶液的pH是维持精子运动必不可少的外部因素。此外,通过计算机辅助精子分析（CASA）系统,首次成功地定量分析了文昌鱼的精子运动。随着精子在海水中激活时间的延长,运动速度快速降低、轨迹的直径变小,可能与精子细胞内ATP的消耗和Ca²⁺的积累有关。 其次,我们研究了外界环境因子对玫瑰无须鲃精子启动及运动的影响,主要观察结果是:（1）无论是水、双蒸水还是其他低渗的电解质（NaCl、KCl）和非电解质（mannitol）溶液,均能激发精子运动的启动,说明精子运动属于低渗调更多还原

【Abstract】 In recently years, sperm biology has developed rapidly, which not only plays important roles in protecting wild species and studying artificial reproduction, but also opens a new field for monitoring acute toxicity in vitro. The study investigated the effects of different environmental factors on the initiation and maintenance of sperm motility in amphioxus （Branchiostoma belcheri tsingtauense） and rosy barb （Puntius conchonius）. It then tested the applicability of the sperm of rosy barb in evaluating in vitro cytotoxicities of isocarbophos and isofenphos-methyl. It is concluded that the rosy barb sperm can be applied to the monitoring acute cytotxicity of the organophosphorus pesticides, isocarbophos and isofenphos-methyl.The effects of different environmental factors on the initiation and maintenance of sperm motility were examined in amphioxus. The main findings are: （1） The initiation of sperm motility in amphioxus belongs to hyperosmolality conformer. Hypoosmolality or equivalent to that of seminal plasma electrolyte and non-electrolyte solutions do not trigger sperm motility. （2） Hyperosmolality triggered sperm motility only in the presence of Ca²⁺. （3） Addition of Ca²⁺ into EGTA-containing Ca²⁺-free ASW, in which none of sperm was motile, was capable of restoring sperm motility. This indicats that Ca²⁺ and hyperosmolality are indispensable for the initiation of sperm motility. （4） Ca²⁺ and hyperosmolality are required for the motility initiation of amphioxus sperm. However, L-type Ca²⁺ channel blocker verapamil did not suppress the initiation of sperm motility. （5） K⁺ channel blockers quinine or 4-aminopyrindine did not suppress the initiation of sperm motility, but they reduced the duration of sperm motility. Their effects were reversible, indicating that the K⁺ channels is essential to the motility mechanism of sperm. （6） The high or low pH reduced obviously the duration of sperm motility in the presence of Ca2+, and changing in pH were not able to cause sperm motility in the solutions isotonic to seawater without Ca²⁺. Based on these findings, it is proposed that theconcerted action between Ca2+ and hyperosmolality is required for the motility initiation of amphioxus sperm, while K+ and pH are indispensable for their motility maintenance.The motility of amphioxus sperm was also monitored by CASA in ASW. It was found that the velocity of sperm motility decreased gradually and the tracks of sperm motility curved with time extended. It is highly likely that the intracellular ATP stored depleted gradually and the intracellular Ca2+ was gradually accumulated after being activated.Secondly, the effects of different environmental factors on the initiation and maintenance of sperm motility were investigated in rosy barb. The main findings are: （1） The motility of sperm can be initiated in hypoosmolality solutions including freshwater, distilled water and other electrolyte （NaCl） or non-electrolyte （mannitol） solutions, suggesting that the initiation of sperm motility belongs to hypoosmolality conformer in rosy barb. （2） As the motility of sperm was initiated / inhibited in hypoosmolality / isoosmolality solutions alternatively, the initiation / inhibition of sperm motility are only related to osmolality of solutions. （3） The percent and duration of the sperm motility gradually decreased with increasing of osmolality in hypoosmolality solutions. （4） K+ channel blockers, quinine not only suppressed the initiation of sperm motility, but also reduced the duration of sperm motility at the higher concentration （^50 uM）. Initiation of sperm motility was not suppressed at the lower quinine, but it reduced the duration of sperm motility. （5） K+ channel blockers, 4-AP can not suppress the initiation of sperm motility, but can reduce the duration of sperm motility at all concentrations tested; （6） Ca2+ channel blockers, verapamil did not affect the initiation of sperm motility, but had negative effects on sperm motility. The result presumed that Ca2+ channel has not taken a role in the initiation of sperm motility, but it may be a regulator of sperm motility. （7） Changes in pH can not trigger the initiation of sperm motility, but can reduce the duration of sperm motility. The optimum pH for the movement of sperm ranged from 7 to 8. Based on these findings, it is proposed that the extracellular osmolality is required for the motility initiation of rosy barb sperm, while K+ and Ca2+ channels were involved in sperm motility, and pH are indispensable for their motility maintenance.Thirdly, the sperm of rosy barb was used to evaluate in vitro the cytotoxicity of isocarbophos of which effects were measured by different endpoints. Findings are: （1） the incubation of isocarbophos （5-500 uM） caused conspicuous decreasing on the motility rate of rosy barb sperm in a dose and time-dependent manner. （2） The ultrastructures and morphology of the sperm was also impaired by treatment with isocarbophos, such as distortion of mid-piece, swelling of mitochondria and dissociation of plasma membrane, reduced smoothness of plasma membrane, appearance of numerous vacuoles in the cytoplasm and dilated of mitochondria cristae, etc. Showing that isocarbophos damaged the structures of sperm and reduced the motility of sperm.Finally, in present study, the sperm of rosy barb was used to evaluate the acute cytotoxicity of isofenphos-methyl by both morphological and biochemical methods. Our results demonstrate that: （1） The incubation of isofenphos-methyl （30-240 uM） caused conspicuous decrease in the motility rate of sperm in a dose and time-dependent manner. （2） The ultrastructures of the sperm treated with isofenphos-methyl were also impaired, such as dilation of mitochondria, broken membrane, distortion of mid-piece, swelling of mitochondria and disappearance of plasma membrane as well as the damage of flagella end etc. （3） The MDA accumulation in sperm treated by isofenphos-methyl was accelerated significantly in a dose and time-dependent manner. （4） Compared to a control group, the activity of SOD and LDH increased slightly in the groups of spermatozoa incubated in 30 u M of isofenphos-methyl for 10 min, but their activity was obviously inhibited the other concentration （60,120 and 240 uM） groups in all the recorded periods.Based on these findings, it is proposed that rosy barb sperm is the sensitive to the cytotoxicity of isofenphos-methyl and isocarbophos, and that mitochondria and membrane are probably the most prominent sites of organophosphorous pesticides （OPI） cytotoxicity in rosy barb sperm that is an excellent candidate for rapidly measuring in vitro cytotoxicity of OPI.更多还原