能量代谢因子MCH在鲤鱼生殖中的功能研究

Reproduction is tightly linked to energy metabolism. It is very important to unveil the function of energy balance neuroendocrine factors in reproduction for mechanisim of pathways mediating reproduction and metabolic regulation. MCH, which is an important endocrine factor, is well established for its effects on metabolic regulation. No studies have ever reported the function of ?sh MCH with respect to reproduction. The results of our previous research showed that MCH is involved in the regulation of reproduction in common carp. In this project, the following researchs will be conducted as follows:(1) MCH and its variant receptor will be cloned, their distributions in HPG axis and connections between MCH and GnRH, and Leptin receptor will be studied. These results may provide morphological evidence for the role of MCH in the control of reproduction. (2) Transgenic carp that over-express MCH will be produced to investigate the effects of MCH on gonad development and expression of key factors involved in reproduction . These results may elucidate the role of MCH in reproduction. (3) The effects of exogenous MCH administration on the level of LH and FSH in pituitary cells, and LH-R, FSH-R and E2(female)/T(male) in germ cells will be studied in vitro. These results may reveal the targets of MCH in reproduction. These studies will provide basis for mechanism of MCH in mediating reproduction and energy homeostasis and protection of fish reproduction healthy.

鱼类生殖活动与能量代谢紧密相关，阐明能量代谢因子在生殖活动中的功能是解析生殖与能量代谢间调控机制的关键。本课题组前期研究发现，作为调节能量代谢的重要内分泌因子黑色素聚集激素MCH，与鲤鱼生殖活动相关。在此基础上，本课题拟开展MCH在鲤鱼生殖中的功能研究。具体内容包括：（1）克隆MCH及其受体，研究其组织分布、亚细胞定位以及与GnRH、Leptin受体细胞之间的联系，为MCH在鲤鱼生殖中的功能提供形态学依据；（2）研制转MCH基因鲤鱼，研究MCH对鲤鱼性腺发育、HPG轴生殖关键因子的作用，揭示MCH在鲤鱼生殖中的功能；（3）研究MCH对体外培养的垂体细胞LH、FSH，性腺细胞LH-R、FSH-R、雌二醇(雌)/睾酮(雄)合成和释放的影响，揭示MCH在生殖调控中的作用靶点及机理。MCH在鲤鱼生殖中的功能研究可为阐明鱼类生殖和能量代谢间的调控机制提供科学依据，为保护鱼类生殖健康奠定理论基础。

鱼类生殖活动与能量代谢紧密相关，探索能量代谢因子在生殖中的功能是解析生殖与能量代谢间调控机制的关键。本项目以能量代谢关键因子黑色素聚集激素MCH为研究对象，探索了其在鲤鱼生殖中的功能，取得了如下结果：.（1）分别克隆了鲤鱼MCH前体、两种MCH受体MCHR1和MCHR2的全编码序列，三种cDNA衍生出的氨基酸残基数分别是124、359和346aa，氨基酸序列同源性分析显示三种基因均与金鱼有最高的同源性，其次是斑马鱼，与哺乳动物的同源性均较低。.（2）分析了MCH、MCHR1和MCHR2的组织表达模式。MCH、MCHR1和MCHR2基因在中枢和外周组织中均有表达，MCH在脑区和垂体的表达量高于其他组织；MCHR1在肌肉、皮肤、垂体、肾、延髓的表达量显著高于其他组织及脑区；MCHR2在皮肤、肾、性腺、脑、小脑、延髓的表达量显著高于其他组织及脑区。三种基因的组织表达模式为MCH的功能提供了形态学依据。.（3）研制了MCH转基因鲤鱼，转MCH基因鲤鱼GnRH mRNA的表达量是对照鱼的1.25±0.25倍，垂体水平GtHα、LHβ、FSHβ mRNA的表达量分别是对照鱼的2.56±0.55、2.67±0.34和4.25±0.50倍，性腺水平FSH-R和LH-R mRNA 的表达量分别是对照鱼的3.56±0.46和2.15±0.26倍，该结果表明，MCH对鲤鱼性腺发育具有促进作用。.（4）分离培养了鲤鱼垂体细胞和卵母细胞，利用人工合成的鲤鱼MCH成熟肽刺激分离的细胞。经MCH刺激后，垂体中GtHα、LHβ和FSHβ mRNA的表达量显著升高，分别提高1.90±0.13、2.36±0.25和2.89±0.21倍，而卵母细胞中的LH-R、FSH-R mRNA的表达量没有显著变化。该结果提示，MCH通过垂体发挥对性腺发育的调控作用。.（5）构建了pEGFP-C1-MCH真核表达载体，经菌落PCR筛选及双酶切和测序鉴定后，转染至HEK293细胞，转染成功的细胞在荧光显微镜下表达较强的绿色荧光，RT-PCR结果显示细胞转染后MCH基因稳定表达。该重组细胞的构建为后续研究MCH基因在性腺发育中的分子机制提供了细胞模型。