PKA、PKC信号通路介导维生素E调控半滑舌鳎亲鱼性腺发育的机制研究

Activation or inhibition of PKA and PKC signaling pathways plays an important role in regulating the secretion of sex hormones in vertebrates. The previous study of our group found that vitamin E (VE) promoted the gene expression of pituitary gonadotropin in Cynoglossus semilaevis broodstock. However, it is not clear whether VE regulates gonadal development through the PKA or PKC signaling pathway acting on the sex hormones secretion in pituitary and gonad. In this project, PKA and PKC were taken as the starting point, and the parent fish of Cynoglossus semilaevis as the research object, the effects of VE on the main markers of signaling pathways were analyzed using the cultured pituitary and gonadal cells in vitro, to clarify the correlation between VE and PKA or PKC;the action target of VE were identified with RNA interference and gene knockout technique; the effects of VE on the gene expression and protein content of gonadotropin and sex steroid hormone biosynthesis were analyzed to clarify the mechanism of PKA or PKC mediated VE regulating the secretion of sex hormone; correlation analysis of the above markers and genes with gonadal development and gamete quality at different stages (initial, middle and late) by raising the fish with graded levels of dietary VE, and comparing the difference between male and female, to systematically clarify the signal communication mechanism of VE regulating gonadal development in broodstock. This study can help to regulate the development of sex gland and improve the reproductive performance of fish through nutritional pathway.

PKA、PKC信号通路的激活或抑制在调控脊椎动物性激素分泌过程中发挥重要作用。本项目组前期研究发现维生素E（VE）促进半滑舌鳎亲鱼垂体促性腺激素的基因表达。但VE是否通过上述信号通路影响垂体-性腺的性激素分泌而调控性腺发育，尚不清晰。本研究以PKA、PKC为切入点，以半滑舌鳎亲鱼为研究对象，利用体外培养的垂体和性腺细胞，分析VE对信号通路标志物的影响，明确VE与PKA、PKC的相关性；利用RNA干扰、基因敲除等技术，分析VE对促性腺激素、性类固醇激素合成相关的基因表达和蛋白含量影响，确定VE作用靶点，解析PKA、PKC介导VE调控性激素分泌的作用机理；利用亲鱼的VE梯度饲养实验，关联分析上述标志物和基因与不同时期（初、中、末）性腺发育、配子质量的相关性，并比较雌雄异同，系统阐明VE调控亲鱼性腺发育过程中PKA、PKC发挥的媒介作用，为利用营养途径调控亲鱼性腺发育、提升繁殖性能提供科学依据。

PKA、PKC信号通路的激活或抑制在调控脊椎动物性激素分泌过程中发挥重要作用。本项目组前期研究发现维生素E（VE）促进半滑舌鳎亲鱼垂体促性腺激素的基因表达。在此基础上，本研究探讨VE是否通过上述信号通路影响垂体-性腺的性激素分泌而调控性腺发育。本项目严格按照项目计划书中的研究内容开展研究，所得到的主要成果包括：首先利用体外培养的垂体和性腺细胞，摸清了VE和信号通路抑制剂的互相作用浓度，并明确了VE与PKA、PKC信号通路是相关联的；其次利用RNA干扰和基因敲除等技术，证实了VE是绕过细胞膜受体直接通过G蛋白作用于PKA信号通路，明确了VE作用靶点；第三，利用亲鱼的VE梯度饲养实验，验证了在性腺发育过程中性激素（促性腺激素、性类固醇激素）相关基因表达（或分泌含量）及配子质量与饲料中VE含量响应。本项目研究结果明确了PKA、PKC信号通路介导维生素E对性激素基因表达和分泌的调控机制，解析了维生素E发挥生殖调控作用的信号通讯机制，为利用营养途径调控亲鱼性腺发育、提升繁殖性能提供科学依据。