双氢睾酮通过调控孕酮、雌二醇和细胞凋亡参与绵羊子宫功能

doi:10.11843/j.issn.0366-6964.2022.09.016

摘要/Abstract

摘要： 旨在探究双氢睾酮（dihydrotestosterone，DHT）是否通过调节孕酮（progesterone，P₄）、雌二醇（oestrogen，E₂）和细胞凋亡参与影响绵羊子宫功能，以揭示其在绵羊生殖生理中的潜在作用。本试验以1.5岁左右的雌性小尾寒羊为试验动物，检测卵泡期、黄体期和妊娠期子宫中DHT合成酶和雄激素受体（androgen receptor，AR）的表达变化。随后，体外培养绵羊子宫内膜上皮细胞，并用DHT （10^-10~10^-7 mol·L^-1）和AR拮抗剂氟他胺（Flu，10^-8 mol·L^-1）处理（n=3）。通过酶联免疫吸附试验、细胞免疫荧光、蛋白质印迹法和实时荧光定量PCR检测P₄和E₂水平、合成酶和受体表达。此外，还检测经DHT和Flu处理后，绵羊子宫内膜上皮细胞中凋亡因子Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）、B细胞淋巴瘤蛋白2（B cell lymphoma protein 2，Bcl-2）、半胱天冬酶3（caspase 3，CASP3）和活化半胱天冬酶3（active-caspase 3，Act-CASP3）的表达变化。结果表明，绵羊子宫不同时期DHT的合成和AR的表达存在差异，妊娠期子宫DHT合成及AR表达显著低于卵泡期和黄体期（P<0.05）。10^-10~10^-7 mol·L^-1DHT处理后，P₄合成酶表达显著上调（P<0.05），在10^-8~10^-7 mol·L^-1 DHT时P₄合成显著增加（P<0.05），在10^-10~10^-8 mol·L^-1 DHT时孕酮受体蛋白表达显著增加（P<0.05），但10^-7mol·L^-1 DHT时孕酮受体蛋白表达显著下降（P<0.05）；在10^-8~10^-7 mol·L^-1 DHT时E₂相关合成酶显著减少，并且E₂水平显著下降（P<0.05），在10^-9和10^-7 mol L^-1 DHT时E₂受体ERα蛋白显著下调（P<0.05），在10^-10~10^-7 mol·L^-1 DHT时ERβ和GPER显著增加（P<0.05）。经Flu处理后部分解除DHT对P₄和E₂的调控。此外，10^-10~10^-7 mol·L^-1 DHT显著促进子宫内膜上皮细胞凋亡（P<0.05）。本研究证实DHT至少部分通过AR调节P₄和E₂合成及受体表达，影响细胞凋亡，参与调节子宫功能，这为进一步阐明雄激素参与调节子宫功能提供了新的基础和相关数据。

关键词: 双氢睾酮, 孕酮, 雌二醇, 凋亡, 子宫功能

Abstract: The study aimed to investigate whether dihydrotestosterone (DHT) regulates sheep uterine function through progesterone (P₄), oestrogen (E₂) and apoptosis, and to reveal its potential role in sheep reproductive physiology. In this experiment, female Small Tailed Han sheep about 1.5 years old were used as experimental animals to detect the expression changes of DHT synthase and androgen receptor (AR) in the uterus during follicular phase, luteal phase and pregnant phase. Subsequently, sheep uterus endometrial epithelial cells were cultured in vitro and treated with DHT (10^-10-10^-7mol·L^-1) and AR antagonist flutamide (Flu, 10^-8mol·L^-1, n=3). P₄ and E₂ levels, synthase and receptors expression were detected by enzyme-linked immunosorbent assay, cellular immunofluorescence, Western blot and real-time fluorescent quantitative PCR. In addition, after treatment with DHT and Flu, the expression of apoptosis factor B cell lymphoma protein 2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase 3 (CASP3) and active-caspase 3 (Act CASP3) in sheep endometrial epithelial cells were detected. The results showed that there were differences in DHT synthesis and AR expression in sheep uterus at different stages. The DHT synthesis and AR expression in uterus at pregnant phase were significantly lower than those at follicular and luteal stages (P<0.05). After 10^-10-10^-7 mol·L^-1 DHT treatment, the level of P₄ significantly up-regulated (P<0.05), P₄ synthesis significantly increased at 10^-8-10^-7 mol·L^-1DHT (P<0.05), the expression of progesterone receptor protein significantly increased at 10^-10-10^-8 mol·L^-1DHT (P<0.05), but the expression of progesterone receptor protein significantly decreased at 10^-7mol·L^-1 DHT (P<0.05). The expression of E₂ synthase and the level of E₂ significantly decreased at 10^-8-10^-7 mol·L^-1 DHT (P<0.05), E₂ receptor ERα protein was significantly down-regulated at 10^-9 and 10^-7 mol·L^-1 DHT (P<0.05). ERβ and GPER significantly increased at 10^-10-10^-7 mol·L^-1 DHT (P<0.05). The regulation of P₄ and E₂ by DHT was partially relieved after Flu treatment. In addition, 10^-10-10^-7 mol·L^-1 DHT significantly promoted apoptosis of endometrial epithelial cells (P<0.05). This study confirmed that DHT at least partially regulates P₄ and E₂ synthesis and receptors expression through AR, affects cell apoptosis to participates in the regulation of uterine function, which provides a new basis and relevant data for further elucidating the role of androgens in the regulation of uterine function.

Key words: dihydrotestosterone, progesterone, oestrogen, apoptosis, uterine function

中图分类号:

S826.2

丁自强, 葛闻博, 段宏伟, 吕建树, 曾建林, 王文娟, 牛甜, 张勇, 赵兴绪, 胡俊杰. 双氢睾酮通过调控孕酮、雌二醇和细胞凋亡参与绵羊子宫功能[J]. 畜牧兽医学报, 2022, 53(9): 2993-3005.

DING Ziqiang, GE Wenbo, DUAN Hongwei, LÜ Jianshu, ZENG Jianlin, WANG Wenjuan, NIU Tian, ZHANG Yong, ZHAO Xingxu, HU Junjie. Dihydrotestosterone Affects Sheep Uterine Function through Regulating Progesterone, Oestrogen and Apoptosis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2993-3005.

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