GnIH在半滑舌鳎生殖调控中的作用及其分子机制

Gonadotropin-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide which was recently identified in quail. To date, GnIH is the only inhibitor of reproduction in any vertebrate. GnIH acts as an inhibitory factor in the control of reproduction in birds and mammals. However, the role of GnIH in the control of gonadotropin synthesis and release has been debatable in fish. In addition, little information is available regarding the intracellular signal transduction pathways activated by GnIH in vertebrates. Thus, the aims of this study are (1) to obtain the full-length cDNA sequences of GnIH and its receptor GnIH-R in the half-smooth tongue sole (Cynoglossus semilaevis) by RT-PCR coupled to 3’/5’ RACE and perform identity analysis of the deduced amino acid sequences as well as phylogenetic analysis, (2) to examine the expression profiles of GnIH/GnIH-R in various tissues of both sexs and at different states of reproduction by in situ hybridization and real-time quantitative PCR, and (3) to comprehensively evaluate the precise role of GnIH in the regulation of the reproductive axis in tongue sole by in vivo and in vitro methods, with special emphasis on the intracellular signaling mechanisms mediating the regulation of gonadotropin synthesis and release by GnIH in fish. This study will reveal the mode of action of GnIH on the regulation of reproduction in fish for the first time and the differences in reproduction between males and females of tongue sole. In addition, this study will contribute to the technological innovation in artifical breeding and aquaculture.

促性腺激素抑制激素(gonadotropin-inhibitory hormone, GnIH)是新近发现的脊椎动物中唯一的生殖抑制因子，其在鸟类和哺乳类生殖调控中的作用受到了广泛研究，然而功能鉴定尚缺乏来自多样性最为丰富的鱼类群体的证据支持。本项目拟以鲆鲽鱼类代表性物种---半滑舌鳎为研究对象，首先利用同源克隆和RACE的方法获得GnIH及其受体GnIH-R的全长cDNA序列，并对其进行生物信息学分析；其次，采用原位杂交和实时荧光定量PCR技术检测GnIH/GnIH-R在雌雄半滑舌鳎各组织以及卵巢不同发育时期的时空表达特性；第三，通过在体和离体实验系统地研究GnIH对半滑舌鳎生殖轴相关调控因子的影响，重点阐明GnIH调控垂体促性腺激素合成与分泌的信号转导机制。本项目预期成果可为揭示半滑舌鳎生殖双向调控机制和研制新型生殖调控制剂提供理论依据，对半滑舌鳎人工繁育及养殖技术创新具有重要意义。

促性腺激素抑制激素（GnIH，也称作LPXRFa）是目前脊椎动物中唯一一种具有抑制生殖功能的下丘脑神经肽，其在鸟类和哺乳类生殖调控中的作用受到了广泛研究，然而GnIH在鱼类生殖调控中的精确作用及其分子机制尚未完全阐明。本研究首先通过同源克隆及RACE方法获得了半滑舌鳎GnIH及其受体GnIH-R全长cDNA序列，并对其进行生物信息学分析；其次，通过荧光定量PCR方法初步分析了gnih/gnih-r mRNA雌雄组织表达差异、卵巢成熟以及早期发育过程中的时空表达特性；第三，通过下丘脑-垂体离体孵育初步阐明了GnIH对半滑舌鳎生殖调控的作用方式，GnIH能够作用于下丘脑-垂体轴进而影响半滑舌鳎生殖活动；第四，通过细胞转染、双荧光素酶报告系统及药理学方法初步分析了GnIH的下游信号转导机制，并进一步分析了GnIH与其他因子信号互作机制。GnIH通过PKA和PKC通路发挥其生理功能，并阻断了kisspeptin诱导的信号通路；第五，通过下丘脑离体孵育，初步研究了性类固醇激素对gnih等关键生殖因子表达调控的影响。综上所述，本研究首次在鲆鲽鱼类中鉴定出了GnIH系统，初步阐明了其对半滑舌鳎生殖调控的影响及分子机制，这为研制新型生殖调控制剂提供了理论依据，对半滑舌鳎人工繁育及养殖技术创新具有重要意义。

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