Ca2+通道在体内受精硬骨鱼类许氏平鮋精子激活中的作用机制研究

Sperm activation is a prerequisite for the completion of fertilization in animals. Spermatozoa are terminally differentiated cells. Sperm activation is dependent on ion channels in the plasma membrane of spermatozoa and regulates changes in intracellular pH (pHi), membrane potential, and intracellular Ca2+ concentration, which in turn causes the spermatozoa to enter a fast-moving state from a resting state. In particular, intracellular Ca2+ is the most crucial element that drives flagellar vibration and regulates sperm motility to complete fertilization. Previous studies have focused on internal fertilization mammals and external fertilization teleost, but little information related to internal fertilization teleost about how sperm senses environmental Ca2+, and transmit signals to the interior of cells, thereby regulating the process of sperm activation. This project intends to use Sebastes schlegelii as the research object. Therefore, this project intends to identify sperm-specific Ca2+ channels，analyze the electrophysiological characteristics and the role during spermatozoa activation in vivo by combining bioinformatics analysis, CASA sperm analysis system and patch clamp electrophysiology technology. Clarify the mechanism of Ca2+ channel in the spermatozoa activation in vivo of Sebastes schlegelii. The present study provides basic data on sperm capacitation and fertilization for internal fertilization teleost and improves the basic theory of adaptive evolution of sperm.

精子激活是动物受精完成的先决条件。精子是终端分化细胞，精子激活依赖于质膜上离子通道，调控细胞内pH(pHi)、膜电势及胞内Ca2+浓度变化，进而使精子从静止状态进入快速运动状态。尤其胞内Ca2+是驱动鞭毛振动，调节精子运动行为以完成受精作用的最关键要素。以往Ca2+通道在精子激活中作用的研究，多集中于体内受精哺乳动物及体外受精硬骨鱼类，而有关体内受精硬骨鱼类精子如何感知环境Ca2+变化，并将信号传导至细胞内部，进而调控精子激活运动的过程等相关研究匮乏。因此，本项目拟以许氏平鮋为研究对象，结合生物信息学分析，CASA精子分析系统和膜片钳电生理技术，通过鉴定精子特异Ca2+通道，分析Ca2+通道电生理特性及在体内精子激活中的作用，解析Ca2+通道在许氏平鮋体内精子激活中的作用机制，为体内受精硬骨鱼类精子获能及受精提供基础资料，完善精子适应性进化的基础理论。

精子激活是动物受精完成的先决条件。精子是终端分化细胞，精子激活依赖于质膜上离子通道，调控细胞内pH(pHi)、膜电势及胞内Ca2+浓度变化，进而使精子从静止状态进入快速运动状态。尤其胞内Ca2+是驱动鞭毛振动，调节精子运动行为以完成受精作用的最关键要素。因此，本项目以许氏平鮋为研究对象，结合生物信息学分析，CASA精子分析系统和生理技术，鉴定许氏平鮋基因组中Ca2+通道；分析Ca2+通道结构特点、时空表达特性及在精子激活中的作用；解析了卵巢液的离子和小分子构成；体外实验验证了Ca2+和Ca2+通道对精子激活和运动特征的影响。该项目揭示了许氏平鮋精子成熟变态过程及超微结构特征，具有延伸棒状精子头部，符合典型体内受精物种的精子特征；通过许氏平鮋基因组生物信息分析，鉴定了Ca2+通道成员如下：Catsper缺失；存在电压依赖性钙通道；非电压依赖性瞬时感受器电位通道；环核苷酸门控通道；同时存在钙离子结合蛋白等家族成员；结合组织特异性和精子变态成熟过程转录组，筛选获得精子特异高表达基因Efcab2；通过体外不同浓度Ca2+溶液及Ca2+通道抑制剂处理，结合激活前后精子细胞内部Ca2+，pH的荧光染色结果，进一步明确Ca2+及Ca2+通道在精子激活中起重要的作用。通过卵巢液对许氏平鮋精子激活运动特征分析，表明卵巢液可以显著提高许氏平鮋精子运动率和运动速率。通过代谢小分子和微量元素分析，鉴定了卵巢液中与精子激活紧密相关的重要离子Ca2+，Zn2+；和运动寿命相关的糖酵解和糖异生代谢小分子。为体内受精硬骨鱼类精子获能及受精提供基础资料，完善精子适应性进化的基础理论。