温度调控黑线仓鼠生殖活动的分子网络机制研究

Temperature is a main environmental factor regulating animal reproductive activities. The study of the molecular pathway for temperature regulation of reproductive activities in the striped hamster is critical to understand the molecular mechanisms that lead to rodent outbreaks. In this proposal, adult striped hamsters will be fed at high temperature (28 ± 1℃), suitable temperature (14 ± 1℃) and low temperature (5 ± 1℃) respectively for four weeks. Then the hypothalamus, pituitary, and ovaries or testes will be collected from hamsters in estrus for transcriptome sequencing, GO function and KEGG pathway analysis so as to examine the pathway on temperature regulation of reproductive activity. Screening of phage library and yeast hybridization will be used to detect the signaling pathway of temperature-regulating synthesis of GnRH and molecular pathway of temperature-controlling development of follicular in the striped hamsters. The methods of follicle culture and protein injection will be carried out to verify the pathway. The quantity of gene expressions will be measured by using real-time quantitative PCR. Additionally, the concentration of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in blood will be examined by using the enzyme immunoassay method, and the number of follicles in estrus will be counted by using HE staining method. The correlation analysis will be carried out between the expression quantity of genes and the number of follicle or the concentration of the hormones. The results are expected to reveal the molecular pathways for temperature regulation of reproductive activity in the striped hamsters. We will also exhibit the effect of each gene or protein on the reproductive activity, which will provide the ideal target(s) for regulating hamsters’ reproductive activity. The goal of this research is to expand our understanding about the molecular mechanisms and put forward a new platform with integrated methods for rodent control in agriculture.

温度是调控动物生殖活动的主要环境因子，研究温度调控黑线仓鼠生殖活动的分子作用路径，有利于掌握鼠害暴发的分子机制。成体黑线仓鼠分别在高温（28±1℃）、适宜温度(14±1℃)和低温(5±1℃)处理4周，取发情期黑线仓鼠的下丘脑、垂体、卵巢或睾丸，转录组测序，GO功能和KEGG Pathway分析；用噬菌体文库筛选、酵母杂交检测温度调控黑线仓鼠GnRH合成的信号传递途径，及温度调控黑线仓鼠卵泡发育的分子作用路径；用卵泡培养、蛋白注射，验证作用路径；用实时定量PCR，检测基因的表达量；用酶联免疫反应检测血液卵泡刺激素(FSH)和黄体生成素(LH)的浓度；用HE染色，统计卵泡数量；用SPSS软件相关性分析的双变量过程分析卵泡数量与基因表达量和激素浓度的关联性。揭示温度调控黑线仓鼠生殖活动的分子网络机制，及各基因的效应，为调控黑线仓鼠的生殖活动提供理想靶点，为农业害鼠治理措施的制定提供新思路或方法。

鼠类在维持生态系统平衡中发挥重要作用，但当种群数量暴发时，鼠类不仅盗食粮食、啃咬树木、破坏草场，还会传播疾病，对人们的生产和生活造成一定的危害，所以研究鼠类种群数量波动机制为合理控制鼠害发生具有重要意义。鼠类生殖活动强度是影响鼠害发生的主要因素之一，而温度是调控动物生殖活动强度的主要环境因子，所以本项目探究温度调控黑线仓鼠生殖活动的分子机制，为科学有效防治鼠害的发生具有一定的指导意义。本课题分别以低温（LT: 8℃)、适宜温度（MT: 21℃)和高温（HT: 29℃)三个温度水平处理成体黑线仓鼠8周，研究不同温度条件下黑线仓鼠的卵巢和睾丸发育情况，血液卵泡刺激素（FSH）和黄体生成素（LH）浓度的变化，下丘脑、卵巢和睾丸在不同温度条件下显著性差异表达的基因，基因间的相互作用网络，及其对黑线仓鼠繁殖活动的调控效应。研究结果表明，在适宜温度（MT）条件下黑线仓鼠的性腺发育、雌性个体卵泡数量、雄性个体睾丸重量、性腺重量与体重的比值、以及血液FSH和LH的浓度均显著高于低温组（LT）和高温组（HT）(P<0.05)的个体；下丘脑Kiss-1/GPR54、RFRP-3/GPR147、Dio2/Dio3、PACAP/PAC1R、PRLR和ESRα等基因的表达水平，卵巢和睾丸Tac3/Tacr3、PACAP/PAC1R和ESRα/ESRβ等基因的表达水平均存在显著(P<0.05)或极显著(P<0.01)的差异，基因间也存在一定的互作，且对黑线仓鼠的繁殖活动存在显著(P<0.05)或极显著(P<0.01)的调控效应。因此，下丘脑Kiss-1/GPR54、RFRP-3/GPR147、Dio2/Dio3、PACAP/PAC1R、PRLR和ESRα和卵巢和睾丸Tac3/Tacr3、PACAP/PAC1R和ESRα/ESRβ等在温度调控黑线仓鼠繁殖活动中发挥着重要作用，为调控黑线仓鼠生殖活动提供理想靶点，为农业害鼠综合治理措施的制定提供新思路和方法。