缝隙连接蛋白43（Cx43）调控大菱鲆精细胞变态的机制研究

Spermiogenesis is a highly organized developmental process of spermatids structural rebuilt and functional maturation. Its key stage is the autophagy of residual body, which is critical to the quality and quantity of sperm production. Connexin 43 (Cx43) functions as intercellular communication, and it is the key medium in Sertoli cell regulating the spermiogenesis and sperm maturity. Meanwhile, Cx43 is also discovered to control spermiogenesis directly by participating in the autophagosome formation. However, the process of spermiogenesis and its regulatory mechanism in fish remains unclear. Our previous research has found the turbot (Scophthalmus maximus) existed a high percentage of sperm abnormality, quite a lot of gap junctions and autophagosomes in the seminiferous epithelium, and the expression of Cx43 up-regulated during spermiogenesis. In this project, we intend to investigate the ultrastructural changes of the seminiferous epithelium, and the effect of Cx43 RNAi, inhibitor and enhance agent on the formation, distribution and function of gap junctions and autophagosomes during spermiogenesis, by which we aim to uncover the regulatory mechanism of Cx43 in fish spermiogenesis. All these results will enrich the knowledge of marine fish sexual maturity, and provide the theoretical foundation of sperm abnormality in cultured fish.

精细胞变态（spermiogenesis）是精子结构重塑和功能完善的精密过程，其中Sertoli细胞对精细胞残余体的自噬回收是核心环节，决定了精子的质和量。缝隙连接蛋白43（Cx43）在生精上皮发挥细胞间通讯功能，是Sertoli细胞调控精细胞发育成熟的关键媒介。近期哺乳动物中研究还发现Cx43可通过诱导自噬直接参与精细胞变态。然而，有关鱼类精细胞变态过程及调控机制尚不明确。申请人前期研究发现大菱鲆精子存在大量畸形现象，精巢具备缝隙连接和自噬小体，且Cx43的表达在精细胞变态时显著升高。本项目拟在此基础上研究生精上皮超微结构变化过程，敲降、抑制和增效Cx43对缝隙连接和自噬小体形成、分布及功能的影响，从而揭示鱼类精细胞变态生精上皮的结构变化规律，阐明Cx43通过细胞间通讯和诱导自噬调控精细胞变态的作用机制。以期丰富海水鱼类性腺成熟相关理论，为探明部分养殖鱼类精子畸形的内在原因提供理论依据。

养殖业中雄性亲鱼诱导不成熟、精子畸形、活力低下等现象往往由精细胞变态过程紊乱所引起。然而，有关海水鱼类的精细胞变态和成熟过程机制仍不明确。本项目以大菱鲆为研究对象，系统解析了大菱鲆精子发生的细胞学过程，鉴定到大菱鲆雄性生殖细胞的23种发育形态及精细胞变态的7个主要时期，发现精子尾部在变态早期出现，精子活力与精子畸形呈负相关，推测残余体吸收障碍是导致大菱鲆精子畸形的主要原因。首次系统揭示了鱼类精子发生过程中Sertoli细胞的发育和成熟过程，鉴定大菱鲆精子成熟类型为半囊型，精原时期的精小囊由1-2个Sertoli细胞组成。克隆并比较了Sertoli细胞潜在分子标记Sox9/Amh/Gsdf的表达和分布特征，确立了三者在标记Sertoli细胞功能上的差异和适用范围。揭示了生精上皮的细胞间链接方式，发现缝隙连接是生殖细胞-体细胞之间的重要连接形式，同时发现自噬在精细胞变态时期发挥重要作用。阐明了Cx43在精子发生过程中的表达和分布规律，初步揭示了Cx43在精子发生过程中可能兼具细胞间通讯和自噬诱导功能，雄激素可能参与调控。研究结果丰富了硬骨鱼类精细胞变态成熟的认知，有助于从生殖细胞-体细胞互作的全新角度理解鱼类精子发生和成熟机制，为鱼类生殖调控提供科学依据和有益探索。