【作者】 胡俊杰；

【导师】 赵兴绪； 张勇；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2012， 博士

【摘要】 温热带哺乳动物繁殖活动有季节性特点。褪黑素是主要由哺乳动物松果腺分泌的一种激素，被认为是调节哺乳动物季节性繁殖的主要影响因子，并通过哺乳动物生殖轴系中分布的褪黑素受体调控动物的生殖。双峰驼是生活在温热带季节性繁殖动物，然而褪黑素受体在双峰驼生殖轴系中的表达和分布特征及调控机理还不清楚。本研究选择繁殖季节（11~12月份）的雌性双峰驼为对象，采用分子生物学、免疫组化、原位杂交、酶联免疫吸附法和细胞培养方法，研究双峰驼生殖轴系褪黑素受体的分布及信号转导，旨在揭示褪黑素与双峰驼的生殖，特别是双峰驼季节性繁殖的关系，以期探讨褪黑素调控动物季节性繁殖的机理，同时也可为研究动物整个生殖活动的调节机理提供新的思路。应用RT-PCR方法从双峰驼松果体及生殖轴系中成功克隆了褪黑素受体MT1(452bp)和MT2(411bp)部分基因，通过在GenBank中查序比对，发现MT1与牛的最为相似（87.5%），MT2与猪的最为相似（91.3%）；对其编码的蛋白进行结构预测和比对，双峰驼MT1和MT2受体是属于G蛋白偶联受体家族的蛋白，双峰驼的MT1和MT2基因为首次报道。原位杂交结果显示MT1和MT2mRNA在下丘脑、垂体和卵巢组织中均有弥散性分布，在白体、初级卵泡卵原细胞和生长卵泡卵母细胞内阳性强于其他区域，分布于胞浆和胞核内。免疫组织化学结果显示MT1和MT2受体在松果体和生殖轴系组织中均表达，但有一定的组织和细胞表达位置的差异性，说明在Mel调节不同的组织器官的生理功能上，MT1和MT2受体既有协同作用也有各自独立性。Western blot实验显示卵巢中分布的MR高于下丘脑、垂体和松果体，这可能是卵巢黄体和白体中褪黑素受体高表达而引起的。成功培养了双峰驼垂体腺细胞和卵巢颗粒细胞，两种细胞都能传代培养，褪黑素可改变卵巢颗粒细胞和垂体细胞内cAMP的累积，说明褪黑素对卵巢颗粒细胞的调控与百日咳毒素敏感的G蛋白依赖的Gi和Gs通路有关，而褪黑素对细胞内cGMP的累积值也有影响，且与cAMP的影响不同，说明褪黑素还通过其他信号转导通路调控细胞。应用实时荧光定量PCR方法研究表褪黑素对双峰驼卵巢颗粒细胞MT1和MT2mRNA的表达有时效关系和浓度依赖性。高浓度的褪黑素（10nmol/mL）可显著的促进MR mRNA的表达。用褪黑素处理1h后mRNA的表达量最高，而且MT1mRNA的表达量高于MT2。通过本研究首次报道了双峰驼的MT1和MT2基因，探索了MT1和MT2受体在繁殖季节的双峰驼生殖轴系和松果体中广泛表达和分布特点及信号转导通路，尤其是研究表明褪黑素对其受体的表达具有促进作用，这为褪黑素调控动物季节性繁殖及褪黑素的作用机理研究提供了新思路。更多还原

【Abstract】 Seasonal breeding is a characteristic for many mammals in temperate and tropical.Melatonin, which secreted by the mammalian pineal gland, is considered to be one ofmain factors affecting and regulating mammalian seasonal reproduction via melatoninreceptors distributing in hypothalamus-pituitary-gonadal axis (HPGA) of the mammalian.Bactrian camel are seasonal breeding animals living in temperate and tropical. However,they are still unclear about the distribution of melatonin receptor in HPGA and regulatingmechanism in Bactrian camel. In order to study these, sample had been taken from femaleBactrian camel in breeding season (November-December), and molecular biology,immunohistochemistry, in situ hybridization, ELISA and cell culture methods had beenused, with the purpose of revealing relationship between melatonin and the seasonalbreeding of the Bactrian camel. This will provide new ideas for exploring the mechanismof animal seasonal breeding with melatonin.The parts of genes of MT1(452bp) and MT2(411bp), which have more than80%similarity with cattle, sheep, human, rat and swine, has been cloned in Bactrian camelreproduction axle tissues with RT-PCR. The similarity of nucleotide is highest87.5%ofMT1with cattle and highest91.3%of MT2with swine in Genbank. Predicting proteinsequences and blasting amino acid of MT1and MT2receptors，the results shou that theyare protein of G protein couple receptors.The in-situ hybridization results show that the mRNA of melatonin receptorsdisperses in tissues of hypothalamus-pituitary-ovary axis of camel and strongly expressesin the cytoplasm and nucleus of granular lutein, oogonia and oocyte.The immunohistochemistry results show that the receptors of MT1and MT2mainlydistributed in membrane, catoplasm and nuclear membrane of neurocytes and cells ofhypothalamus-pituitary-ovary axis and pineal. But they are, not only strongly but alsodifferently, distributing in neurocytes of hypothalamus and pitutary, and the oogonia,oocyte, granular cells, gravid corpus luteum and corpus albicans of ovary. This suggestthat there are synergistic and independent effecting between MT1and MT2receptors onphysiological function of different cells. The western blot results show that melatoninreceptor protein in ovary is highest than other tissues, and suggest that melatonin is vary important for ovary development.Melatonin decreases cAMP and increases cGMP of ovary granular cell in vitro. Theeffect of melatonin decreasing cAMP can be blocked with Pertussis toxin in vitro. Andalso it can weaken Foskolin’s affections. This imply that melatonin regulat the ovariangranulosa cells with pertussis toxin-sensitive G-protein couple receptors signaltransduction pathway. However, melatonin also have an impact on the accumulation ofcGMP, and which is differently with cAMP, indicat that there is another melatoninreceptor signal transduction pathway in cells.It is significant that melatonin regulate MT1and MT2mRNA expression withdifferent times and concentrations to ovary granular cells in vitro. High concentrationmelatonin (10nmol/mL) can significantly promote MR mRNA expression(p<0.01).In conclusions, these results may provide valuable information for mechanism ofmelatonin and Bactrain camel reasonal breeding, and also provide new ideas for exploringthe mechanism of animal seasonal breeding with melatonin.更多还原