甘加藏羊发情周期和乏情期HPO轴中THRβ mRNA和蛋白的表达分布

doi:10.3969/j.issn.1004-1524.2022.03.08

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (3): 480-488.DOI: 10.3969/j.issn.1004-1524.2022.03.08

甘加藏羊发情周期和乏情期HPO轴中THRβ mRNA和蛋白的表达分布

刘莉莉¹(), 高何璇², 杨亚文¹, 包莹莹¹, 何玉琴¹^,^*(), 杨志杰¹, 陈卫刚¹, 葛文博³

1.甘肃农业大学生命科学技术学院,甘肃兰州 730070
2.兰州银行网络金融部,甘肃兰州 730070
3.甘肃农业大学动物医学院,甘肃兰州 730070

收稿日期:2021-05-06 出版日期:2022-03-25 发布日期:2022-03-30
通讯作者: 何玉琴
作者简介:*何玉琴,E-mail: heyq@gsau.edu.cn
刘莉莉(1991—),女,甘肃宁县人,硕士研究生,研究方向为动物生物化学与分子生物学。E-mail: 1748362947@qq.com
基金资助:
国家自然科学基金(31960689)

Expression and distribution of THRβ mRNA and protein in HPO axis of Ganja Tibetan sheep during estrus cycle and anestrus

LIU Lili¹(), GAO Hexuan², YANG Yawen¹, BAO Yingying¹, HE Yuqin¹^,^*(), YANG Zhijie¹, CHEN Weigang¹, GE Wenbo³

1. College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
2. Network Finance Department of Lanzhou Bank, Lanzhou 730070, China
3. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2021-05-06 Online:2022-03-25 Published:2022-03-30
Contact: HE Yuqin

摘要/Abstract

摘要：

甲状腺激素(TH)通过与其靶细胞受体特异性结合发挥其生物学效能。为阐明松果体、下丘脑-垂体-卵巢轴(Hypothalamic-pituitary-ovary axis, HPOA)甲状腺激素受体β(Thyroid hormone receptor β, THRβ)基因和蛋白的表达及分布对季节性繁殖动物甘加藏羊(Ovis aries)生殖活动的调节。本研究以处于发情周期和乏情期的甘加藏羊为研究对象,应用RT-qPCR、Western blot 和免疫组织化学方法检测和分析了松果体、HPO轴中THRβ mRNA及其蛋白的表达和分布。结果显示,THRβ mRNA及其蛋白在松果体和HPO轴组织中均有表达和分布。在松果体中,THRβ mRNA及其蛋白的表达量均在乏情期最高,显著(P<0.05)高于发情周期;在下丘脑中,THRβ mRNA表达量在发情后期显著(P<0.05)高于其他时期,蛋白表达量在间情期显著(P<0.05)高于其他时期;在垂体中,THRβ mRNA及其蛋白的表达量在间情期显著(P<0.05)高于其他时期;在卵巢中,THRβ mRNA及其蛋白的表达量在发情后期显著(P<0.05)高于其他时期。免疫组织化学结果显示:THRβ免疫阳性产物主要分布在松果体细胞的细胞浆,下丘脑的神经胶质细胞、大神经元胞体和椎体细胞胞浆,腺垂体的嗜酸性和嗜碱性细胞胞浆,卵巢的颗粒细胞胞浆、卵泡内膜细胞及黄体细胞。甘加藏羊发情周期和乏情期松果体、HPOA组织中THRβ mRNA和蛋白的差异性表达,表明THRβ参与调控甘加藏羊的生殖活动。

关键词: 甘加藏羊, 下丘脑-垂体-卵巢轴, 发情周期, 乏情期

Abstract:

Thyroid hormone (TH) exerted its biological effects by specifically binding to its target cell receptor. In order to clarify the regulation of thyroid hormone receptor β (THRβ) gene and its protein in the pineal gland and Hypothalamic-pituitary-ovary (HPO) axis of the reproductive activity of seasonal breeding animals, Ganjia Tibetan sheep (Ovis aries). In this study, the healthy Ganjia Tibetan sheep during the estrus cycle and anestrus were used as the research object. RT-qPCR, Western blot and immunohistochemical staining were used to detect and analyze the expression and distribution of THRβ mRNA and its protein in the pineal gland and HPOA. The results showed that THRβ mRNA and its protein were expressed and distributed in the pineal gland and HPOA tissues. The expression of THRβ mRNA and its protein in the pineal gland in the anestrus were significantly (P<0.05) higher than that in the estrus cycle. In hypothalamus, the expression of THRβ mRNA in the metestrus were significantly (P <0.05) higher than that in other stages while the expression of its protein in the disestrus were significantly (P<0.05) higher than that in other stages; In pituitary gland, the expression of THRβ mRNA and its protein in the diestrus were significantly (P<0.05) higher than that in other stages. In ovary, the expression of THRβ mRNA and its protein in the metestrus were significantly (P<0.05) higher than that in other stages. The immunohistochemical results showed that THRβ was mainly distributed in the eosinophilic cytoplasm of the pineal, glial cells, large neuron cell bodies and vertebral cell cytoplasm of the hypothalamic, eosinophilic and basophilic cytoplasm of adenohypophysis, granulosa cell cytoplasm, follicular inner membrane cells and luteal cells of the ovary. THRβ mRNA and its protein in the pineal gland and HPOA showed different changes during the estrus and anestrus, which indicated that THRβ gene and its protein were involved in the regulation of the reproductive activities of the Ganjia Tibetan sheep.

Key words: Ganga Tibetan sheep, hypothalamus-pituitary-ovary axis, estrus cycle, anestrus

中图分类号:

S826

刘莉莉, 高何璇, 杨亚文, 包莹莹, 何玉琴, 杨志杰, 陈卫刚, 葛文博. 甘加藏羊发情周期和乏情期HPO轴中THRβ mRNA和蛋白的表达分布[J]. 浙江农业学报, 2022, 34(3): 480-488.

LIU Lili, GAO Hexuan, YANG Yawen, BAO Yingying, HE Yuqin, YANG Zhijie, CHEN Weigang, GE Wenbo. Expression and distribution of THRβ mRNA and protein in HPO axis of Ganja Tibetan sheep during estrus cycle and anestrus[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 480-488.

图/表 4

表1 THRβ和β-actin基因引物序列

Table 1 Primers sequences of THRβ and β-actin gene

基因 Gene	引物序列 Primer sequences(5'-3')	产物大小 Product size/bp	退火温度 Annealing temperature/℃	登录号 Login ID
THRβ	F:ACCGCCATCTCCCCGTTCAG R:TTGTGAGCTGCCATGTGAAGACC	129	60	NM001190391.1
β-actin	F:CCATTGAGCACGGCATTGT R: GCAGGGGTGTTGAAGGTCTC	184	58	HM067830.1

图1 甘加藏羊发情周期和乏情期松果体和HPO轴组织中THRβ mRNA和蛋白相对表达量 A,松果体;B,下丘脑;C,垂体;D,卵巢。同一系列柱上无相同字母的表示各时期mRNA或蛋白的相对表达量差异显著(P<0.05)。

Fig.1 Relative expression of THRβ mRNA and protein in Pineal gland and HPO axis during estrus cycle and anestrus of Ganjia Tibetan sheep A,Pineal gland;B,Hypothalamus;C,Pituitary;D, Ovary. Bars marked without the same letters on the same series indicated significant differences in the relative expression of mRNA or protein at different periods at P<0.05.

图2 甘加藏羊发情周期和乏情期松果体、HPO轴中THRβ的蛋白含量 PE,发情前期;E,发情期;ME,发情后期;DE,间情期;AE,乏情期。

Fig.2 Content of THRβprotein in pineal gland and HPO axis during estrus cycle and anestrus of Ganjia Tibetan sheep PE, Proestrus; E, Estrus; ME, Metestrus; DE, Diestrus; AE, Anestrus.

图3 甘加藏羊发情周期和乏情期松果体和 HPO轴组织中THRβ免疫阳性反应产物分布 A、F、K、P,发情前期;B、G、L、Q,发情期;C、H、M、R,发情后期;D、I、N、S,间情期;E、J、O、T,乏情期。P,松果体细胞;BV,血管;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;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 Location of THRβ immunoreactive products of in pineal and HPO axis of Ganjia Tibetan sheep during the estrus cycle and anestrus A, F, K, P, Proestrus; B, G, L, Q, Estrus; C, H, M, R, Metestrus; D, I, N, S, Diestrus; E, J, O, T, Anestrusl. P, Pineal cell; BV, Blood vessel; GC, Glial cell; NC, Nerve cell; Nu, Neuronuclei; AC, Acidophilic cell; BC, Basophilic cell; CC, Chromophobe cell; FA, Follicular antrum; GV, Germinal vesicle; LC, Luteal cell; TF, Theca folliculi; SG, Stratum granulosum.

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甘加藏羊发情周期和乏情期HPO轴中THRβ mRNA和蛋白的表达分布

Expression and distribution of THRβ mRNA and protein in HPO axis of Ganja Tibetan sheep during estrus cycle and anestrus

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[1]	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.
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