外源激素诱导大鲵雌雄配子同步发育成熟的优化模式及其分子机制研究

Taking the Chinese giant salamader (Andrias davidianus Blanchard),an endangered species, as the research object, in view of the lower spawning,fertilization,hatching and survival rates in Chinese giant salamader artificial breeding as well as the gamete asynchronous developmental maturation in males and females,which caused the unsolved bottleneck problems of Chinese giant salamander artificial breeding, we strive to make technological breakthrough and conduct the corresponding basic research. By the use of the combination methods of histology, reproductive physiology and molecular biology, we concentrate on the induction of gonadal developmental maturation of Chinese giant salamander by exogenous hormones such as gonadotropin releasing hormone analogue (GnRHa), frog pituitary, human chorionic gonadotropin(HCG) and 17α-progesterone(17α-P), so as to establish the optimal model of gamete sychronous developmental maturation of Chinese giant salamander induced by exogenous hormones mentioned above. Meanwhile, we explore the changes of hypothalamus-pituitary-gonad axis under the induction of exogenous hormones. Through the determinations of endogenous steroid hormone levels as well as the cloning, expression and manipulation of gonadotropin hormone(GtH) gene,progesterone receptor gene and maturation induction hormone production key enzyme gene, 20β-hydroxysteroid dehydrogenase(20β-HSD) gene, we explore the manipulating molecular mechanisms of gamete developmental maturation of Chinese giant salamander. This research not only rich on manipulation mechanisms of amphibian repoductive endocrinology in theory, but also can explore the key technology of promotiong gamete developmetal maturation, so as to provide the scientific ways and theoretical basis of germplasm resource propogation and protection of Chinese giant salamander.

以我国频危物种中国大鲵为研究对象，针对大鲵人工繁殖中催产率、授精率、孵化率、成活率低以及大鲵雌雄配子发育成熟不同步等引起的大鲵人工繁殖瓶颈问题，展开技术攻关和相应基础研究，采用组织学、繁殖生理学和分子生物学技术相结合的方法，围绕外源激素GnRHa、蛙脑垂体、HCG以及17α-孕激素诱导雌雄大鲵性腺发育成熟这一重点，建立外源激素诱导大鲵雌雄配子同步发育成熟的优化模式，探讨大鲵"下丘脑-垂体-性腺"这一生殖神经内分泌调控轴在外源激素作用下的变化情况，通过检测内源性类固醇激素水平变化，以及通过GtH基因、孕激素受体基因和成熟诱导激素关键酶基因20β-HSD基因的克隆、表达及其调控的研究，探讨调控大鲵雌雄配子发育成熟的分子机制。本研究在理论上丰富了两栖类生殖内分泌的调控机制，在实践上又可探索大鲵人工繁殖中促进配子发育成熟的关键技术，从而为大鲵种质资源增殖和保护提供科学的途径和理论依据。

大鲵人工繁殖中催产率、受精率、孵化率、成活率低以及大鲵雌雄配子发育成熟不同步等问题一直是中国大鲵人工繁殖、大鲵产业化发展和大鲵生物多样保护的一个瓶颈问题。本研究以我国频危物种中国大鲵为研究对象，采用组织学、繁殖生理学和分子生物学技术相结合的方法，展开技术攻关和相应基础研究，以解决人工饲养条件下，人工催产中雌雄大鲵同步产卵的问题，建立外源激素诱导大鲵雌雄配子发育成熟的最佳诱导程序。结果表明GnRHa剂量为2µg/kg、HCG剂量为200IU/ kg单独使用都具有较好的诱导产卵效果，催产率分别为92.45±2.52%和90.62±3.68%，受精率达93.46±2.43%和96.82±1.36%。在仿生态条件下，水温16±2℃进行人工孵化，孵化期为45天，孵化率和成活率分别为96.38±2.32%和95.25±2.62%%。人工催产后，雌性大鲵的效应期为11±6天，雄性大鲵排精要比雌性大鲵产卵延迟10±4天，依此，我们建立了提前10天给雄性大鲵注射启动针，再给雌雄大鲵同时注射催产针的优化同步产卵模式。并探讨大鲵“下丘脑-垂体-性腺”这一生殖神经内分泌调控轴在外源激素作用下的变化情况。本研究在理论上丰富了两栖类生殖内分泌的调控机制，在实践上建立了大鲵人工繁殖中促进配子发育成熟的关键技术，从而为大鲵种质资源增殖和保护提供了科学的途径。