CYP17A1基因敲低对山羊睾丸间质细胞睾酮合成和睾丸发育相关基因表达的影响  

Effect of CYP17A1 Gene Knockdown on Expression of Genes Related to Testosterone Synthesis and Testicular Development in Goat(Capra hircus)Leydig Cells

作　　者：张远 陈祥 张艳[1,2] 付开斌 唐文 陈家靖 嵇桃桃 杨沛方 ZHANG Yuan;CHEN Xiang;ZHANG Yan;FU Kai-Bin;TANGWen;CHEN Jia-Jing;JI Tao-Tao;YANG Pei-Fang(College of Animal Science,Guizhou University/Key Laboratory of Animal Geneties,Breeding and Reproduction in the Plateau Mountain Region,Ministry of Education,Guiyang 550025,China;Guizhou Key Laboratory of Animal Genetics and Breeding,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [2]贵州省动物遗传育种与繁殖重点实验室,贵阳550025

出　　处：《农业生物技术学报》2023年第12期2535-2544,共10页Journal of Agricultural Biotechnology

基　　金：贵州省科技支撑计划(黔科合支撑[2021]一般151);贵州省高层次创新型“百”层次人才项目(黔科合平台人才-GCC[2022]021-1);贵州省牛羊发展科研项目(QNY20221101)。

摘　　要：细胞色素P450c17(cytochrome P450c17,CYP17A1)是胆固醇合成雄激素途径中的限速酶,在雄激素生物合成中具有关键作用。明确CYP17A1对山羊(Capra hircus)睾酮合成和睾丸发育的影响,对于提高公羊繁殖力具有重要意义。本研究以黔北麻羊为研究对象,构建CYP17A1的RNA干扰载体并转染至睾丸间质细胞,以qRT-PCR和Western blot技术检测CYP17A1基因敲低效果,以CCK-8(Cell Counting Kit-8)检测CYP17A1敲低对睾丸间质细胞增殖的影响;利用qRT-PCR检测增殖相关基因、睾酮合成相关基因以及睾丸发育相关基因的表达水平。结果显示,CYP17A1在各月龄的睾丸和附睾组织中均有表达,其基因敲低效果明显,CYP17A1基因和蛋白表达均极显著下调(P<0.01),睾丸间质细胞的增殖受到极显著抑制(P<0.01);CYP17A1基因敲低使间质细胞增殖相关基因增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和细胞周期蛋白E(cyclin E),睾酮合成相关基因CYP11A1、3β-羟基类固醇脱氢酶1型(3β-hydroxysteroid dehydrogenase type 1,HSD3B1)、HSD17B3和CYP19A1以及睾丸发育相关基因—类无精症缺失基因(deleted in azoospermia like,DAZL)和胰岛素样生长因子2(insulin like growth factor 2,IGF2)均发生显著的下调表达(P<0.01)。本研究为深入探讨CYP17A1基因对黔北麻羊繁殖性状的影响提供基础资料。Cytochrome P450c17(CYP17A1)is a rate-limiting enzyme in the pathway of cholesterol synthesis and plays a key role in androgen biosynthesis.It is of great significance to clarify the effect of CYP17A1 on testosterone synthesis and testicular development in goats(Capra hircus)for improving the fertility of rams.In this study,RNA interference vector of CYP17A1 was constructed and transfected into Leydig cells of Qianbei Ma goat.The knockdown effect of CYP17A1 gene was detected by qRT-PCR and Western blot.The effect of CYP17A1 knockdown on the proliferation of Leydig cells was detected by CCK-8(Cell Counting Kit-8).The expression levels of proliferation-related genes,testosterone synthesis-related genes and testicular development-related genes were detected by qRT-PCR.The results showed that CYP17A1 was expressed in the testis and epididymis of each month old,and its gene knockdown effect was obvious,in which the expression of CYP17A1 gene and protein was significantly down-regulated(P<0.01),and the proliferation of Leydig cells was significantly inhibited(P<0.01).CYP17A1 gene knockdown significantly down-regulated the expression of proliferating cell nuclear antigen(PCNA)and cyclin E,CYP11A1,3β-hydroxysteroid dehydrogenase type 1(HSD3B1),HSD17B3 and CYP19A1,deleted in azoospermia like(DAZL)and insulin like growth factor 2(IGF2)(P<0.01).This study provides basic data for further exploring the effect of CYP17A1 gene on the reproductive traits of Qianbei Ma goat.

关 键 词：细胞色素P450c17(CYP17A1) 黔北麻羊 睾丸间质细胞 细胞增殖 睾丸发育 

分 类 号：S827[农业科学—畜牧学]