甘加型藏羊发情周期血浆雌激素测定及HPO轴ERα的表达

Determination of Plasma Estrogen and ERαin HPO Axis Expression in Estrus Cycle of Ganjia Tibetan Sheep

为研究甘加型藏羊(Ovis arise)发情周期内血浆雌激素的分泌规律及其受体α(Estrogen receptorα,ERα)在下丘脑-垂体-卵巢(Hypothalamic-pituitary-ovarian axis,HPO)组织中的表达及分布,选取发情周期内健康且未孕甘加型藏羊32只,运用酶联免疫分析法(ELISA)研究其血浆雌激素分泌规律,用实时荧光定量PCR(RT-qPCR)、蛋白免疫印迹技术(Western Blot)和免疫组织化学染色法(IHC)检测HPO组织中ERαmRNA及蛋白的表达与分布。结果显示,发情周期内血浆雌激素含量呈脉冲式和波动式交替变化,发情期最高,发情后第10小时达到第1个峰值(97.21 ng/mL);ERαmRNA及蛋白在HPO中均有表达。下丘脑组织中发情后期最高,显著高于间情期;垂体组织中发情期最高,与其他3个时期差异显著;卵巢组织中在发情前期最高,各时期表达差异显著。免疫组织化学试验结果显示,ERα阳性表达主要在下丘脑促垂体区神经胶质细胞胞核和大神经元胞体及轴突分布;在垂体远侧部及中间部嗜酸性细胞胞核中高表达,胞浆中表达较弱,在卵巢的生长卵泡颗粒细胞胞浆和胞核、卵泡内膜细胞及黄体细胞胞浆中分布。甘加型藏羊发情周期内血浆雌激素及其受体在HPO组织中呈动态表达变化,表明雌激素及其受体α参与调节甘加型藏羊的生殖生理活动。

We evaluated the plasma Estrogen secretion,the expression and distribution of its receptorα(Estrogen receptorα,ERα)in the hypothalamus-pituitary-ovarian(HPO)axis organization of Ganjia Tibetan sheep(Ovis arise)in the estrus cycle.32 healthy and non-pregnant Ganjia Tibetan sheep during the estrous cycle were collected from the Tibetan sheep farms.Enzyme-linked immunosorbent assay,real-time fluorescent quantitative polymerase chain reaction,western blot and immunohistochemistry were applied for examining the estrogen secretion in plasma,the expression and distribution of ERαin HPO axis tissue.The results revealed that the plasma estrogen content in the estrus cycle changed alternately in pulse and fluctuation,and it was the highest in estrus period and reached its first peak of 97.21 ng/mL after the estrus 10 h.Meanwhile,the ERαmRNA and its protein were expressed in the HPO axis of the entire estrus cycle of Ganjia Tibetan sheep.The relative expression of ERαmRNA and protein in the hypothalamus was significantly higher in the metestrus compared to the diestrus.We found that ERαmRNA and protein in estrus were significantly higher than the other three periods in pituitary tissues.The highest expression was found in the ovarian tissue in the proestrus and showed significant differences in different circles.The immunohistochemical results reported that the positive expression of ERαwas mainly distributed in the nuclei of glial cells and the axons of macro-neurons in the pituitary-promoting region of the hypothalamus.The stronger expression was observed in the eosinophilic nuclei of distal and intermediate parts of the pituitary,while the weaker expression was found in the cytoplasm.In the ovary,the ERαpositive expression was distributed in the cytoplasm of growing follicular granulosa cells,the cytoplasm of nuclei,endometrial cells of follicles,and cytoplasm of luteal cells.The constant expression of plasma estrogen and its receptor in the HPO axis indicated that estrogen and its receptor were involved in the regulation of reproductive physiological activities in Ganjia Tibetan sheep.