GPER-ERK通路在DES影响小鼠睾丸引带发育中的作用

doi:10.3969/j.issn.1004-616x.2015.02.008

癌变·畸变·突变 ›› 2015, Vol. 27 ›› Issue (2): 116-120,124.doi: 10.3969/j.issn.1004-616x.2015.02.008

GPER-ERK通路在DES影响小鼠睾丸引带发育中的作用

张镟^1,2, 柯松¹, 刘亮¹, 陈凯洪¹, 马廉³, 蒋学武¹

1. 汕头大学医学院第二附属医院小儿外科, 广东汕头 515041;
2. 深圳大学附属妇女儿童医院小儿外科, 广东深圳 518122;
3. 汕头大学医学院第二附属医院儿科, 广东汕头 515041

收稿日期:2014-12-17 修回日期:2015-02-01 出版日期:2015-03-31 发布日期:2015-03-31
通讯作者: 蒋学武,E-mail:xuewujiang@hotmail.com E-mail:xuewujiang@hotmail.com
作者简介:张镟,E-mail:zhxuan2006@126.com。
基金资助:
国家自然科学基金(81341099,81170555)

Effects of DES on mouse gubernaculum testis development via GPER-ERK signaling pathway

ZHANG Xuan^1,2, KE Song¹, LIU Liang¹, CHEN Kaihong¹, MA Lian³, JIANG Xuewu¹

1. Department of Pediatric Surgery, the Second Affiliated Hospital of Shantou University Medical College, Shantou 515041;
2. Department of Pediatric Surgery, the Affiliated Maternal and Child Health Hospital of Shenzhen University Medical College, Shenzhen 518122;
3. Department of Pediatrics, the Second Affiliated Hospital of Shantou University Medical College, Shantou 515041, Guangdong, China

Received:2014-12-17 Revised:2015-02-01 Online:2015-03-31 Published:2015-03-31
About author:10.3969/j.issn.1004-616x.2015.02.008

摘要/Abstract

摘要：

目的:检测G蛋白偶联雌激素受体(GPER)在小鼠睾丸引带上的存在情况,并进一步探讨已烯雌酚(DES)影响小鼠睾丸引带细胞的非基因效应,从睾丸外因素初步了解外源性雌激素影响雄性生殖系统的可能机制。方法:应用免疫组织化学方法了解GPER在不同发育时期(孕17、19 d,生后0、3、7、14和21 d)小鼠睾丸引带上的表达分布情况,并利用免疫电镜进行亚细胞水平的定位。将培养传代的小鼠睾丸引带细胞随机分为3组:实验组(DES组),经典雌激素核受体阻断剂亦是GPER激动剂ICI 182780干预组(ICI 182780-DES组)和GPER阻断剂G15干预组(G15-DES组)。利用Western Blot方法检测不同组别中DES对其ERK通路的影响。结果:GPER存在于不同发育时期的小鼠睾丸引带内层疏松间叶组织区,主要表达于引带细胞的细胞膜及细胞浆上;免疫电镜显示GPER颗粒主要锚定在小鼠睾丸引带细胞的粗面内质网上,表达量少。Western blot方法发现DES对ERK通路上p-ERK1/2的抑制作用可被GPER阻断剂G15阻断,但预先加入ER阻断剂ICI 182780后没有得到类似的反转现象。结论:GPER持续存在小鼠睾丸引带的各个发育阶段,表达于引带细胞的膜性结构上;DES对小鼠睾丸引带的快速非基因效应至少部分是通过GPER介导的。以睾丸引带为研究对象,探讨DES影响雄性生殖系统发育的可能机制,可为其他迫切需要进行效应评价研究的环境雌激素提供一个新的实验参考系统。

关键词: 睾丸引带, G蛋白偶联的雌激素受体, 环境雌激素, 己烯雌酚

Abstract:

OBJECTIVE:To explore the presence of G protein-coupled estrogen receptor (GPER) in the Kunming mouse gubernaculum testis. To explore the rapid effects of diethylstilbestrol (DES) on cultured mouse gubernaculum testis cells via ERK signaling pathway. METHODS:The expression of GPER was assessed by immunohistochemical staining with the development of mouse gubernaculums testis (GD17, GD19, PD0, PD3, PD7, PD14 and PD21). Subcellular localization of GPER was identified by Immunoelectron Microscopy. Cells subcultured from mouse gubernaculum testis were divided into three groups randomly, that is, control group(DES 1.5×10^-8 mol/L), ICI 182780-DES group (pretreated with 1 nmol/L estrogen receptor inhibitor ICI 182780 90 min), and G15-DES group (pretreated with 1 nmol/L GPER inhibitor G15 90 min). p-ERK1/2 was detected in the cells with 1.5×10^-8 mol/L DES in different groups by Western blot. RESULTS:The immunohistochemical staining showed that GPER was expressed in the development of gubernaculums testis, mainly in the unconsolidated mesenchymal tissue of inner gubernaculum testis, and GPER staining was strong both on the membrane and in the cytoplasm. The subcellular localization of GPER protein was mainly in the mesh-like network of cytoplasm, the rough endoplasmic reticulum. G15 could reverse the DES-induced inhibitory effect of p-ERK1/2, but ICI182780 didn't have the same effect. CONCLUSION:GPER was expressed in membrane structure of gubernaculums testis cells in different stages of development. The nongenetic effects of DES on gubernaculums testis cells were likely to be partly mediated by GPER. Therefore, our data provide new insight into the role of EEs in the etiology of male reproductive system and will help develop better approaches for the prevention and therapy of male reproductive malformation.

Key words: gubernaculum testis, G protein-coupled estrogen receptor, environment estrogens, diethylstilbestrol

中图分类号:

R994.6

张镟, 柯松, 刘亮, 陈凯洪, 马廉, 蒋学武. GPER-ERK通路在DES影响小鼠睾丸引带发育中的作用[J]. 癌变·畸变·突变, 2015, 27(2): 116-120,124.

ZHANG Xuan, KE Song, LIU Liang, CHEN Kaihong, MA Lian, JIANG Xuewu. Effects of DES on mouse gubernaculum testis development via GPER-ERK signaling pathway[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(2): 116-120,124.

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GPER-ERK通路在DES影响小鼠睾丸引带发育中的作用

Effects of DES on mouse gubernaculum testis development via GPER-ERK signaling pathway

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