| 摘要: |
| 为探究雄激素受体(Androgen receptor，AR)及共调节因子对绵羊附睾上皮细胞(Epididymal epithelial cells，EECs)谷胱甘肽过氧化物酶5(Glutathione peroxidase 5，GPX5)的调节机制，本研究采用CCK-8检测睾酮对EECs增殖的影响；利用qRT-PCR、免疫荧光法和 Western Blot法分别检测AR、GPX5、甾体激素受体共激活子 1(Steroid receptor coactivator 1，SRC-1)、CREB结合蛋白(cAMP-response element-binding protein，CBP)、p300和核受体共抑制因子2(Nuclear receptor corepressor 2，NCOR2)的mRNA水平和蛋白表达情况，采用双荧光素酶报告基因测定干扰SRC-1或p300后EECs的荧光素酶活性。结果表明:1)与对照组相比，100 nmol/L睾酮组细胞中的GPX5 mRNA和蛋白表达量极显著升高(P<0.01)，1 000 nmol/L睾酮组细胞中的GPX5 mRNA和蛋白表达量差异不显著(P>0.05)，但分别极显著(P<0.01)和显著(P<0.05)低于100 nmol/L睾酮组；100 nmol/L睾酮组细胞中的AR mRNA和蛋白表达量分别呈极显著(P<0.01)和显著(P<0.05)高于对照组，然而1 000 nmol/L睾酮组细胞中的AR mRNA和蛋白的表达量显著低于对照组(P<0.05)；1 000 nmol/L睾酮组细胞中的CBP、p300、SRC-1蛋白表达极显著高于对照组(P<0.01)，特别是核内的表达量明显增高。2)与对照组相比，siRNA-AR组细胞中的GPX5 mRNA和蛋白的表达量差异不显著(P>0.05)，而SRC-1和p300蛋白表达量极显著升高(P<0.01)。pcDNA3.1-AR组GPX5 mRNA和蛋白表达量较对照组呈极显著(P<0.01)和显著(P<0.05)升高。3)siRNA-SRC-1 和siRNA-p300组细胞中的GPX5 mRNA和蛋白表达量较对照组均显著降低(P<0.05)，而AR的蛋白表达量与对照组差异不显著(P>0.05)，AR的转录活性显著降低(P<0.05)。综上，睾酮对绵羊EECs GPX5、AR及其共调节因子的表达具有浓度依赖性的调节效应，睾酮通过AR及其共调节因子SRC-1和p300/CBP的协同调节GPX5表达。本研究为探究GPX5在绵羊附睾中的调节机制提供理论依据。 |
| 关键词: 附睾上皮细胞 谷胱甘肽过氧化物酶5 睾酮 雄激素受体 共调节因子 |
| DOI：10.11841/j.issn.1007-4333.2023.03.09 |
| 投稿时间：2022-05-03 |
| 基金项目:内蒙古自治区科技重大专项(2020ZD0003)；内蒙古自治区自然科学基金项目(2022QN08016) |
|
| Regulation of GPX5 expression in sheep epididymis by androgen receptor and coregulators |
|
LUAN Zhaojin1,2,ZHAO Yongchao1,XU Jiaoxia1,LIU Yongbin3,ZHANG Jiaxin1*
|
| (1.Inner Mongolia Key Laboratory of Animal Genetics, Breeding and Reproduction, College of Animal Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China;2.College of basic medicine and forensic medicine, Baotou Medical College, Baotou, Inner Mongolia 010040, China;3.Academy of agriculture and animal husbandry of Inner Mongolia Autonomous Region, Hohhot, Inner Mongolia 010018, China) |
| Abstract: |
| The purpose of this study was to investigate the regulatory mechanism of androgen receptor(AR)and co-regulatory factors in the regulation of testosterone on glutathione peroxidase 5(GPX5)in sheep epididymal epithelial cells(EECs). CCK-8 was used to detect the effect of testosterone on the proliferation of EECs. The mRNA levels and protein expressions of AR, GPX5, steroid receptor coactivator 1(SRC-1), cAMP-response element-binding protein(CBP), P300 and Nuclear receptor corepressor 2(NCOR2)were detected by qRT- PCR, immunofluorescence and Western blot; The luciferase activity of EECs after siRNA interference with SRC-1 or P300 was measured by double luciferase reporter gene. The results showed that: 1)Compared with the control group, the expression of GPX5 mRNA and protein in 100 nmol/L testosterone group was significantly increased(P<0. 01), while the expression of GPX5 mRNA and protein in 1 000 nmol/L testosterone group was not significantly different from that in the control group(P>0. 05), but it was significantly(P<0. 01)and significantly(P<0. 05)lower than that in 100 nmol/L testosterone group. The expression of AR in the nucleus was significantly increased(P<0. 05), and the expression of AR mRNA and protein were significantly increased(P<0. 01)and significantly(P<0. 05), respectively. The expression of AR mRNA and protein in 1 000 nmol/L testosterone group was significantly lower than that in the control group(P<0. 05), and the expression of CBP, P300 and SRC-1 protein in 1 000 nmol/L testosterone group increased significantly(P<0. 01), especially in the nucleus. 2)There was no significant difference in the expression of GPX5 mRNA and protein between siRNA-AR group and control group(P>0. 05), while the expression of SRC-1 and P300 protein was significantly higher(P<0. 01). The expression of GPX5 mRNA and protein in pcDNA 3. 1-AR group was significantly higher than that in the control group(P<0. 01)and significantly(P<0. 05). 3)The expression of GPX5 mRNA and protein in siRNA-SRC-1 group or siRNA-p300 group was significantly lower than that in the control group(P<0. 05), and there was no significant difference in the expression of AR protein between siRNA-SRC-1 and siRNA-p300 groups and the control group(P>0. 05), the transcriptional activity of AR in siRNA-SRC-1 and siRNA-p300 groups decreased significantly(P<0. 05). In conclusion, testosterone has a concentration dependent regulatory effect on the expression of GPX5, AR and coregulators in sheep EECs. Testosterone regulates the expression of GPX5 through AR and its coregulators SRC-1 and p300/CBP. This study provides a theoretical basis for exploring the regulation mechanism of GPX5 in epididymis. |
| Key words: epididymal epithelial cells glutathione peroxidase 5 testosterone androgen receptor coregulators |
