甘加型藏羊发情周期下丘脑-垂体-卵巢轴中ERβ mRNA及其蛋白的表达

doi:10.3969/j.issn.1004-1524.2022.01.02

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (1): 10-16.DOI: 10.3969/j.issn.1004-1524.2022.01.02

甘加型藏羊发情周期下丘脑-垂体-卵巢轴中ERβ mRNA及其蛋白的表达

包莹莹¹(), 陈卫刚¹, 葛闻博², 何玉琴¹^,^*(), 杨志杰¹, 周凯仁², 杨大鹏², 杨亚文¹, 刘莉莉¹

1.甘肃农业大学生命科学技术学院,甘肃兰州 730070
2.甘肃农业大学动物医学院,甘肃兰州 730070

收稿日期:2020-10-17 出版日期:2022-01-25 发布日期:2022-02-05
通讯作者: 何玉琴
作者简介:* 何玉琴,E-mail: heyq@gsau.edu.cn
包莹莹(1993—),女,甘肃漳县人,硕士研究生,研究方向为动物生物化学分子生物学。E-mail: 1769338582@qq.com
基金资助:
国家自然科学基金(31960689);国家自然科学基金(31360595)

Expression of ERβ in hypothalamic-pituitary-ovarian axis of Ganjia Tibetan sheep in estrus cycle

BAO Yingying¹(), CHEN Weigang¹, GE Wenbo², HE Yuqin¹^,^*(), YANG Zhijie¹, ZHOU Kairen², YANG Dapeng², YANG Yawen¹, LIU Lili¹

1. College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
2. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2020-10-17 Online:2022-01-25 Published:2022-02-05
Contact: HE Yuqin

摘要/Abstract

摘要：

为阐明甘加型藏羊(Ovis arise)发情周期下丘脑-垂体-卵巢(hypothalamic-pituitary-ovarian axis, HPO)轴中雌激素受体β(estrogen receptor β,ERβ)mRNA和蛋白的表达及分布,本试验选取健康且在发情周期内的甘加型藏羊32只,运用qRT-PCR、Western blot 技术和免疫组织化学染色法检测甘加型藏羊发情周期ERβ mRNA及蛋白在HPO轴中的表达和分布差异。结果显示,甘加型藏羊整个发情周期(发情前期、发情期、发情后期及间情期)ERβ mRNA及其蛋白在HPO轴中均有表达和分布。下丘脑中ERβ mRNA及其蛋白在下丘脑中发情前期表达最多,显著高于其他3个时期(P<0.05);垂体中ERβ mRNA及蛋白均在发情前期表达最高,与其他3个时期差异显著(P<0.05);在卵巢中间情期表达最高,显著高于其他3个时期(P<0.05)。免疫组织化学染色结果显示,ERβ阳性表达主要分布在下丘脑大神经元胞体和轴突中;在垂体中多分布在远侧部及中间部嗜酸性细胞胞浆中,胞核中分布较少;在卵巢中,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均有分布。ERβ mRNA及其蛋白在甘加型藏羊发情周期HPO轴的差异性表达表明雌激素受体β参与调节甘加型藏羊的生殖生理活动。

关键词: 甘加型藏羊, ERβ, 下丘脑-垂体-卵巢轴, 发情周期

Abstract:

To evaluate the expression and distribution of estrogen receptor β (ERβ) in the hypothalamus-pituitary-ovarian (HPO) axis of Ganjia Tibetan sheep in estrum cycle, thirty-two sheep with healthy and non-pregnant Ganjia Tibetan sheep during the estrus cycle were collected from the Tibetan sheep farm. Real-time fluorescent quantitative polymerase, Western blot and immunohistochemical staining were used to detect the expression and distribution of ERβ mRNA and its protein in HPO axis of Ganjia Tibetan sheep at different stages of the estrus cycle. The results showed that ERβ mRNA and its protein were expressed and distributed in hypothalamus, pituitary and ovary during the whole estrus cycle (proestrus, estrus, metestrus and diestrus) of Ganjia Tibetan sheep. ERβ mRNA and its protein were significantly higher in the proestrus as compared with the other three stages (P<0.05) in hypothalamus. It was found that ERβ mRNA and protein in proestrus was significantly higher than the other three stages (P<0.05) in pituitary, and the highest expression was found in ovarian tissue in diestrus and significantly higher than the other three stages(P<0.05). The immunohistochemical results reported that the positive expression of ERβ was mainly distributed in large neuronal cytoplasm and axons. The stronger expression was observed in the eosinophilic cytoplasm in distal and intermediate parts of the pituitary, but weaker expression was found in nucleus. In ovary, ERβ positive reaction was distributed in cytoplasm of growth follicular granulosa cells and nuclei, follicular endometrium cells and luteal cells. The different expression of ERβ in the HPO axis in the estrus cycle of Ganjia Tibetan sheep suggests that ERβ has important significance for reproductive physiological activities such as the estrus cycle of Tibetan sheep.

Key words: Ganga Tibetan sheep, ERβ, hypothalamus-pituitary-ovarian axis, estrus cycle

中图分类号:

S826

包莹莹, 陈卫刚, 葛闻博, 何玉琴, 杨志杰, 周凯仁, 杨大鹏, 杨亚文, 刘莉莉. 甘加型藏羊发情周期下丘脑-垂体-卵巢轴中ERβ mRNA及其蛋白的表达[J]. 浙江农业学报, 2022, 34(1): 10-16.

BAO Yingying, CHEN Weigang, GE Wenbo, HE Yuqin, YANG Zhijie, ZHOU Kairen, YANG Dapeng, YANG Yawen, LIU Lili. Expression of ERβ in hypothalamic-pituitary-ovarian axis of Ganjia Tibetan sheep in estrus cycle[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 10-16.

图/表 4

表1 引物信息

Table 1 Information of primer

基因 Gene	引物序列(5'~3') Primer sequences	产物大小 Product size/bp	Tm/℃	用途 Application	登录号 Login ID
ERβ	F: GCTCTGGTCTGGGTGATT R: TGTTCCATGCCCTTGTTA	119	58	目的基因 Target gene	NM 0010009737.1
β-actin	F:CTTCCAGCCTTCTCCTTCCTGG R:GCCAGGGCAGTGATCTCTTT	180	58	内参基因 Reference gene	HM067830.1

图1 甘加型藏羊发情周期HPO轴中ERβ mRNA相对表达量 A,下丘脑;B,垂体;C,卵巢。没有相同小写字母表示差异显著(P<0.05)。下同。

Fig.1 Relative expression levels of ERβ mRNA in HPO axis of Ganjia Tibetan sheep during the estrus cycle A, Hypothalamic;B, Pituitary;C, Ovarian. PE, Proestrus; E, Estrus; ME, Metestrus; DE, Diestrus. The different lowercase indicated significant difference (P<0.05). The same as below.

图2 甘加型藏羊发情周期HPO轴中ERβ蛋白相对表达量

Fig.2 Relative expression levels of ERβ protein in in HPO axis of Ganjia Tibetan sheep during the estrus cycle

图3 甘加型藏羊发情周期HPO轴ERβ阳性反应分布 A、F、K,发情前期;B、G、L,发情期;C、H、M,发情后期;D、I、N,间情期;E、J、O,阴性对照。GC,神经胶质细胞;NC,神经元;Nu,神经细胞核;AC,嗜酸性细胞;BC,嗜碱性细胞;CC,嫌色细胞;PD,远侧部;PI,中间部;FA,卵泡腔;GV,卵母细胞核;LC,黄体细胞;TF,卵泡膜;SG,颗粒层。

Fig.3 The location of positive reaction of ERβ in HPO axis of Ganjia Tibetan sheep during the estrus cycle A, F, K, Proestrus; B, G,L, Estrus; C, H,M, Metestrus; D,I,N, Diestrus; E,J,O, Negative control. GC, Glial cell; NC, Nerve cell; Nu, Neuronuclei; AC, Acidophilic cell; BC, Basophilic cell; CC, Chromophobe cell; PD, Pars distalis; PI, Pars intermedie; FA, Follicular antrum; GV, Germinal vesicle; LC, Luteal cell; TF, Theca folliculi; SG, Stratum granulosum.

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甘加型藏羊发情周期下丘脑-垂体-卵巢轴中ERβ mRNA及其蛋白的表达

Expression of ERβ in hypothalamic-pituitary-ovarian axis of Ganjia Tibetan sheep in estrus cycle

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