鲆鲽鱼类精子成熟及其超活化获能机制研究

The development and maturation of parental gonad and germ cell, and the generation of high quality gametes are the basic material of marine fish aquaculture. Traditional knowledge make people paid more attention to females and ignored the male fish gonads induction and sperm quality monitoring, so that sperm quality and quantity in the mass production often become the crucial factor for the period of the fertilized egg and fry breeding. What is the process of sperm maturation and how does the sperm have biological activity? what are the key factors that determined its capacitation in teleost? All these questions remain yet to be resolved. In this project, we will use the matured males of turbot during breeding season to resolve the following questions: the sperm maturation process after they released to the fine tubular in genital tract; the change of the fine structure and physiological function of germ cell and fine tubular in the sperm maturation process; how the aquaporin family which closely related this process and the expression of progestin receptors on sperm membrane affect the liquid in testicular ducts and promote the formation and liquefied of sperm, sperm maturation and being capacity; in vivo and in vitro induced the sperm activation state and the quality of semen and fertilization ability. The results of this project will illuminate the mechanism of the teleost sperm maturation and hyper motility capacitation and the influence to the sperm production and motility from histology, cellular and internal secretion molecular developmental, enrich the basic theory of marine fish reproduction, development, and optimize the artificial technique for marine fish gonadal development and artificial insemination technique.

亲鱼性腺和生殖细胞发育成熟、优质配子的产生是海水鱼类养殖业发展的基础。传统认识使人们重视雌鱼而忽视雄鱼性腺诱导及精子质量监测，致使在规模化生产中雄鱼精液质量往往成为制约受精卵及苗种生产周期长短的决定性因素。关于硬骨鱼类精子成熟过程、生物学活性以及决定其活性的关键因子等一系列问题都没有明确的认知。因此本项目拟以繁殖期内的大菱鲆为对象研究精子从精小囊释放到生殖道后的成熟过程；此过程中生殖细胞与生殖道精细结构、生理功能的变化；以及与此密切相关的水通道蛋白家族和孕酮膜受体基因的表达调控输精管液体分泌、吸收，促进精液的形成和液化，诱导精子的成熟和超活化获能的机制；并结合在体和离体处理条件下精子的活化状态及精液的质量和受精能力，以期从组织、细胞、内分泌及分子水平阐明鲆鲽鱼类类精子成熟和超活化获能机制，丰富海水鱼类繁殖发育基础理论，指导人工性腺成熟诱导及授精技术。

亲鱼性腺和生殖细胞发育成熟、优质配子的产生是海水鱼类养殖业发展的基础。因此本项目以繁殖期内的大菱鲆为对象，研究精子成熟获能过程；以及与此密切相关的孕酮膜受体基因和水通道蛋白家族的作用；并结合在体和离体处理条件下精子的活化状态及精液的质量和受精能力，从组织、细胞、内分泌及分子水平阐明鲆鲽鱼类精子成熟和超活化获能机制。该项目揭示了大菱鲆精子发生的细胞学动态过程，确定了不同发育期雄性生殖细胞的类型及百分比；发现大菱鲆的精小囊在精原细胞时期，由2-3个Sertoli细胞组成，揭示了不同发育时期Sertoli细胞的结构变化规律。通过繁殖过程中大菱鲆雄性生殖细胞发育动态结合转录组分析，揭示了大菱鲆雄性生殖细胞从增殖到成熟的分子调控网络；将为海洋鱼类精子发生调控机制的阐明提供基础资料。明确了孕酮膜受体的表达与大菱鲆精液质、量的关系：排精期，pgr在精巢中高表达，定位在精子细胞上，表明在繁殖期大菱鲆中，pgr的主要作用是通过介导DHP促进减数分裂和精子细胞变态成熟，在成熟的精子中不发挥作用。mPRα和pgrmc2 在成熟精子中的表达量显著高于在精巢中的表达量，且在质量高的精子组中的表达量显著高于质量低的精子组，这两种膜受体可能通过表达量变化介导孕激素非基因组作用影响精子质量。对大菱鲆水通道蛋白AQP家族进行了系统鉴定，并分析其在精巢繁殖周期中的表达定位模式，同时AQP1a与精子质量具有相关性，体外外源添加水通道蛋白抑制剂或者抗体，均可以显著降低精子超活化的运动情况，从而揭示水通道蛋白家族在大菱鲆精子超活化运动中具有重要的作用。确定了大菱鲆繁殖周期中，激素和pH等微环境变化， P4，DHP和T在血清中的含量随精巢发育显著升高，精巢退化时显著下降。该项目的完成丰富了海水鱼类繁殖发育基础理论，指导人工性腺成熟诱导及授精技术。