Motility Of Spermatozoa In Amphioxus And Rosy Barb And Their Application Of The Acute Toxicity Assay In Vitro

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.