类胰岛素生长因子（IGFs）促鱼类卵巢成熟的功能研究

Reproduction and growth, the most basic characteristics of organism, are closely related to and distinguished from each other. IGFs are the key factors conjuncting growth and reproduction axis. Recent research showed that gonad development and maturation were associated with cell differentiation and tissue growth, traditional growth factors IGF-1, IGF-2 and a recently discovered IGF subtype (IGF-3) played an important role on ovary maturation of fish. By taking orange-spotted grouper (Epinephelus coioides) as the research object, using the technologies of bioinformatics, histology, physiology and molecular biology, we cloned 3 types of IGFs gene. Then, the genetic spatiotemporal expression patterns in ovary and ocycle and physiological roles of IGFs will be investigated in the grouper ovary. Based on the above research results, one of the IGFs which Plays the most important role to ovary maturation will be selected for further research. Constructing recombinant expression vector encoding orange-spotted grouper IGF to express IGF mature peptides and preparing specific antibody will be carried out. Finally, the effects of recombinant grouper IGF on the ovary and oocyte maturation in the orange-spotted grouper and the regulatory mechanism of IGF synthesis and release will be investigated by in vivo and in vitro experiments respectively. The results will aim to preliminarily clarify the molecular mechanism of IGF which are traditional growth factors on ovary and oocyte maturation, and offer a theoretical basis for the endocrine regulation of fish reproduction.

生殖和生长是生物体最基本的特征，两者既密切相关又相互区别，IGFs是生长轴和生殖轴相交联的关键因子。最近研究表明：鱼类性腺的发育及成熟伴随着细胞分化和组织生长，传统的生长因子IGF-1、IGF-2和最近发现的IGF-3，对鱼类卵巢成熟发挥着重要作用。本研究以斜带石斑鱼为研究对象，采用生物信息学、组织学、生理学和分子生物学技术相结合的方法，克隆3种IGFs基因，通过研究斜带石斑鱼3种IGFs在卵巢的精确时空表达模式，初步比较IGF-1、IGF-2、IGF-3在卵巢成熟中的作用差异，选取其中一个关键因子作为目的基因，通过构建关键IGF因子的蛋白表达体系并制备抗体，然后通过离体和在体实验研究人工重组斜带石斑鱼IGF对成鱼卵巢和卵母细胞成熟的机制以及卵巢IGF合成与释放的调控机理，初步阐明促生长因子IGF对卵巢成熟的分子机理，为深入研究鱼类生殖内分泌调控提供理论依据。

本研究基于斜带石斑鱼3种IGFs在卵巢的精确时空表达模式，初步比较IGF-1、IGF-2、IGF-3在卵巢成熟中的作用差异，发现IGF-3是影响斜带石斑鱼卵巢成熟的关键作用因子。后续研究围绕关键作用因子IGF-3开展。.硬骨鱼中近几年新发现的类胰岛素样生长因子亚型为IGF-3，在性腺的特异性表达表明了其在鱼类生殖功能中的重要作用。研究表明： IGF-3是调控生物体生长和生殖活动的关键因子，IGF-3的调控机制一直是生长生殖研究的热点。本研究中以斜带石斑鱼为对象，初步探索IGF-3在斜带石斑鱼卵巢中的定位、基因表达模式和生理调控作用。实时荧光定量PCR研究IGF-3基因对斜带石斑鱼卵巢的精确表达模式，研究斜带石斑鱼卵巢滤泡不同发育阶段IGF-3 mRNA的表达变化。利用重组表达的斜带石斑鱼IGF-3蛋白对卵巢不同时期滤泡进行时间和浓度梯度离体孵育，检测胚泡破裂的变化，探讨重组蛋白对滤泡成熟的作用。IGF-3相对应的发挥作用的类胰岛素生长因子受体IGFRs、其完整的信号通路、以及其与哪些转录因子相互作用, 如何影响转录因子和生殖、性别分化相关因子来调控鱼类的生长和生殖发育。初步探索了IGF-3对斜带石斑鱼卵巢发育成熟的功能影响，进一步研究鱼类IGF-3的表达调控与生产实践的关系。