Molecular Biological Studies on the Regulation of Sperm Motility

我们发现，在鲑鱼精子从睾丸到精子管道的过程中，外部pH和HCO_3的增加导致pH_i和cAMP的增加，从而影响鞭毛轴索的运动器官，导致精子运动潜能的获得。关于鲑科鱼类在产卵时精子运动的启动，我们首次发现鞭毛运动的控制发生在鞭毛的有限部分，即基部，使用制备的分子量为15kDa的运动启动磷酸化蛋白（MIPP）单克隆抗体。在淡水鲤科鱼类和海洋鱼类中，我们发现了不同的精子运动起始机制；外部渗透压的减少或增加引起K^+_i的增加或减少，引起轴索的起始运动，赋予精子运动能力。在激活精子活力方面，利用凝胶过滤和等电聚焦色谱对鲱鱼卵释放的激活精子活力的物质进行了纯化，鉴定出5种分子量为8.1 KDa、Pl为4.9 ~ 5.4的蛋白质。克隆了pl5.1型HSAP的cDNA，确定了包含73个氨基酸残基的氨基酸序列。HSAP类似于typsin抑制剂，提示胰蛋白酶抑制剂样HSAP与精子表面胰蛋白酶样受体相互作用，引起鲱鱼精子运动激活的信号转导机制。用HPLC法对Ciona卵中释放的精子激活和吸引因子（SAAF）进行了纯化，发现该分子具有激活精子和吸引精子两种功能。进一步的研究结果表明，精子激活需要Ca^<2+>和cAMP，但精子趋化只需要Ca^<2+>，这表明一个分子可以触发两个不同的信号转导系统。

We found that during transit of sperm from the testis to the sperm duct of salmonid fishes, increases in external pH and HCO_3 cause increases in pH_i and cAMP which affect the motile apparatus of flagellum, axoneme, causing the acquisition of the potential for sperm motility. Concerning the initiation of sperm motility in salmonid fishes at spawning, we obtained the first finding that control of flagellar movement occurs at the limited portion i.e.basal part of flagellum using prepared monoclonal antibodies of the 15kDa motility initiating phosphoprotein (MIPP) with a molecular weight of 15KDa. In the freshwater cyprinid fishes and marine fishes, we showed the different sperm motility initiation mechanisms ; Decrease or increase in external osmolalities causes increase or decrease in K^+_i causing the initiation axonemal movement to confer motility to spermatozoa. In the activation of sperm motility, the substance released from herring egg which activate sperm motility were purified by using gel-filtration and isoelectric focusing chromatograpies and identified 5 proteins with MW of 8.1 KDa and with Pl of 4.9-5.4. cDNA of the HSAP with Pl 5.1 was cloned and amino acid sequence with 73 amino acid residues was defined. The HSAP is resemble to typsin inhibitors, suggesting that interaction between trypsin inhibitor-like HSAPs and trypsin-like receptor on the surface of spermatozoa causes the signal transduction mechanism which triggers the activation of herring sperm motility. The sperm activating and attracting factor (SAAF) released from the egg of Ciona was purified using several HPLC chromatographies and it was concluced that one molecule has two functions, sperm activation and sperm attraction. Further findings suggest that sperm activation requires both Ca^<2+> and cAMP,but sperm chemotaxis needs only Ca^<2+>, suggesting that one molecule triggers two different signal transduction systems.