摘要
本试验旨在研究RNA干扰抑制素α亚基(Inhibinα-subunit,INHα)基因和添加抑制素A(InhibinA)对绵羊颗粒细胞雌激素(Estrogen,E2)和孕酮(Progesterone,P)分泌及相关基因表达的影响,以探索抑制素在绵羊颗粒细胞E2和P分泌中的作用。从1.0~1.5岁小尾寒羊的卵泡(3~7mm)中分离培养颗粒细胞,分为2个处理组:干扰组(脂质体介导法转染颗粒细胞)和InhibinA添加组(200ng·mL^(-1))。利用ELISA试剂盒检测E2和P的分泌,荧光定量RT-PCR检测E2和P的分泌相关基因(CYP11、3β-HSD和CYP19)的表达量。结果表明,与对照组相比,干扰组siRNA转染颗粒细胞48h后,INHα基因的抑制率达87%,E2和P的分泌量显著降低(P<0.05);InhibinA添加组E2和P的分泌水平显著升高(P<0.05);干扰组3β-HSD和CYP19的mRNA表达量显著降低(P<0.05),CYP11的表达量显著升高(P<0.05);InhibinA添加组CYP19、CYP11和3β-HSD的mRNA表达水平均显著升高(P<0.05)。综上表明,抑制素在绵羊颗粒细胞中起关键调控作用,通过调节绵羊颗粒细胞类固醇激素的分泌参与调控卵泡发育和排卵过程。
The aim of this study was to investigate the effects of silencing the inhibin α-subunit （INHα） gene or treatment with inhibinA on the secretion of estrogen （E2）, progesterone （P） and expressions of related genes in sheep granulosa cells （GCs）, explore the role of inhibin in the secretion of E2 and P. GCs were isolated from antral follicles （3-7mm） obtained from the ovaries of small-tailed Han sheep （1.0-1.5 years old）. GCs were transfected with siRNA or treated with inhibinA （200 ng·mL-1）. The concentrations of E2 and P were tested by ELISA. The mRNA levels of related-genes （CYP11, 3β-HSD and CYP19） were analyzed by quantitative real-time PCR （qRT-PCR）. The results showed that siRNA was transfected into GCs at 48 h and the silencing efficiency of INHa was 87%. Knockdown of INHct by siRNA decreased the concentrations of E2 and P at 48 h compared with the control （P〈0.05）. Secretion of E2 and P were increased in the group treated with inhihinA （P〈0.05）. Knockdown of INHα by siRNA decreased the mRNA expressions of 3β-HSD and CYP19, and increased the mRNA expression of CYP11 （P〈0.05）. The mRNA expressions of 3β-HSD, CYP11 and CYP19 were increased in the group treated with inhibinA （P〈0. 05）. These results indicated that inhibin was a key regulator and involved in the process of follicular growth and ovulation by regulating the secretion of steroid hormone in sheep GCs.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第9期1648-1653,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家现代肉羊产业技术体系项目(CARS-39)
关键词
抑制素
雌激素
孕酮
绵羊
颗粒细胞
基因表达
inhibin
estrogen
progesterone
sheep
granulosa cells
gene expression
