甘加型藏羊血浆E_2动态变化及其受体GPR30在HPO轴的表达及定位研究  

Study on the Dynamic Changes of Plasma E2 and Expression and Location of Its Receptor GPR30 in HPO Axis of Ganjia Tibetan Sheep

作　　者：蔡永强 高何璇 包莹莹 陶乐凯 何玉琴[1] 杨亚文 刘莉莉 张光敏 孙晓煜 CAI Yongqiang;GAO Hexuan;BAO Yingying;TAO Lekai;HE Yuqin;YANG Yawen;LIU Lili;ZHANG Guangmin;SUN Xiaoyu(College of life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;College of Information Science and Technology,Lanzhou 730070,China;College of Biology and Geographical Sciences,Yili Normal University,YiLi,Xinjiang 835000,China)

机构地区：[1]甘肃农业大学生命科学技术学院,甘肃兰州730070 [2]甘肃农业大学信息科学技术学院,甘肃兰州730070 [3]新疆伊犁师范大学生物与地理科学学院,新疆伊犁835000

出　　处：《江西农业大学学报》2023年第3期695-704,共10页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31960689)。

摘　　要：[目的]旨在探讨雌二醇(E_(2))与其膜受体(GPR30)对甘加型藏羊(Ovis arise)生殖活动的调控作用,以期为甘加型藏羊生殖生理活动规律提供研究依据。[方法]以甘加型藏羊为试验材料,采用酶联免疫吸附法、实时荧光定量PCR法、免疫印迹法和免疫组织化学染色法分别检测甘加型藏羊繁殖季节不同发情阶段(发情前期、发情期、发情后期和间情期)与非繁殖季节的乏情期血浆中E_2含量的动态变化、GPR30 mRNA及其蛋白在HPO轴的表达水平和分布情况。[结果]繁殖季节不同发情阶段与非繁殖季节乏情期血浆中E_(2)浓度呈波动式变化,波峰出现在发情后10,24,36 h和发情前期的第16天早晨,波谷出现在发情后22,39,60 h、第5天早晨和发情前期的第14天早晨,E_(2)平均浓度在发情期最高[(73.269±0.241) pg/mL],间情期最低[(57.919±0.547) pg/mL],除发情后期外,且与其他各时期差异显著(P<0.05);下丘脑和卵巢中GPR30 mRNA和蛋白的最高表达量均出现在其发情后期(P<0.05),但垂体中GPR30 mRNA表达量在间情期最高,蛋白表达量在发情前期最高(P<0.05);GPR30免疫阳性细胞主要在下丘脑大神经元胞体、胞核及轴突、神经胶质细胞胞浆,垂体嗜酸性和嗜碱性细胞胞浆以及卵巢卵泡颗粒层细胞、卵泡膜细胞和黄体细胞胞浆上表达。[结论]甘加型藏羊发情周期血浆E_(2)浓度在发情期最高,间情期最低;发情周期各时期GPR30 mRNA、GPR30蛋白及其免疫阳性细胞在下丘脑、垂体和卵巢中均有差异性表达,其中下丘脑和卵巢中GPR30 mRNA和蛋白的表达呈正相关,垂体中呈负相关,表明E_(2)通过与分布在HPO轴上的GPR30受体结合,调节甘加型藏羊周期性发情和生殖器官功能活动。[Objective]This study aims to explore the regulation of estradiol(E_(2))and membrane receptor(GPR30)on the reproductive activity of Ganjia Tibetan sheep(Ovis arise),thus providing research basis for the law of reproductive physiological activities of Ganjia Tibetan sheep.[Method]Taking Ganjia Tibetan sheep as the test material,enzyme-linked immunosorbent assay,real-time fluorescence quantitative PCR,immunoblotting and immunohistochemical staining were used to detect the dynamic changes of plasma E_(2) content,GPR30 mRNA and its protein expression and distribution in the HPO axis of Ganjia Tibetan sheep at different stages of estrus(Pre-estrus,estrus,metestrus and diestrus)and anestrus in the non-breeding season.[Result]There are fluctuating changes in plasma E_(2) concentration at different estrus stages and non-breeding seasons during anestrus.The peak appeared at 10 h,24 h,36 h after estrus and the morning of 16 days after proestrus.The trough appeared at 22 h,39 h,60 h,the morning of 5 days after estrus and the morning of 14 days after proestrus.The average concentration of E_(2) was the highest at estrus(73.269±0.241)pg/mL and the lowest at diestrus(57.919±0.547)pg/mL,except for the metestrus period,which was significantly different from other periods(P<0.05).The highest expression levels of GPR30 mRNA and protein appeared in hypothalamus and ovary in metestrus(P<0.05),the highest expression level of GPR30 mRNA in pituitary was in estrus,and the highest expression level of protein was in proestrus(P<0.05).GPR30 immunoreactive cells were mainly expressed in the cytoplasm of hypothalamic large neurons,nuclei and axons,glial cells,eosinophilic and basophilic cells of pituitary,granulosa cells,theca cells and luteal cells of ovary.[Conclusion]The expression of GPR30 mRNA,GPR30 protein and their immunoreactive cells were different in hypothalamus,pituitary and ovary at different stages of estrous cycle.There was a positive correlation between GPR30 mRNA and protein expression in hypothalamus and ovary,and a nega

关 键 词：甘加型藏羊 雌激素 雌激素膜受体30 下丘脑-垂体-卵巢轴 发情周期 

分 类 号：S852.2[农业科学—基础兽医学] S826.91[农业科学—兽医学]