GnRH激动剂主动免疫对GnRHR在腺垂体与子宫表达及分布的作用研究

目的探讨GnRH激动剂主动免疫对绵羊腺垂体与子宫GnRHR表达及分布的影响,为深入研究GnRH-A调节生殖功能的机理及合理应用提供依据。方法 28只5～6月龄健康母绵羊(Ovis aries)随机分为4组(n=7),实验Ⅰ组(EG-Ⅰ)、实验Ⅱ组(EG-Ⅱ)和实验Ⅲ组(EG-Ⅲ)于0 d和14 d分别皮下注射阿拉瑞林抗原200μg、300μg和400μg(0 d和14 d各1次);对照组(CG)皮下注射2.0 ml药物的溶媒(0 d和14 d各1次)。各组于70 d无菌切取腺垂体和子宫。提取腺垂体总RNA,实时荧光定量PCR(FQ-PCR)检测GnRHR mRNA表达的变化,Western blotting分析子宫GnRHR蛋白表达,免疫组织化学SP法染色检测GnRHR表达的变化。结果 EG-Ⅰ、EG-Ⅱ和EG-Ⅲ腺垂体GnRHR mRNA表达量均低于对照组,以EG-Ⅲ最小(P<0.01)。与CG相比,EG-Ⅰ、EG-Ⅱ和EG-Ⅲ子宫GnRHR蛋白表达水平分别减少3.46%、4.90%和24.78%(P<0.05)。子宫组织中有GnRHR分布主要见于子宫内膜细胞和子宫腺上皮细胞的胞质和胞核,EG-Ⅲ灰度值显著低于CG(P<0.05)。结论 GnRH激动剂主动免疫可以剂量依赖性地抑制垂体GnRHR mRNA和子宫组织中GnRHR蛋白的表达。GnRHR主要分布在子宫内膜上皮细胞和腺上皮细胞的胞核和胞质,GnRHR激动剂免疫对子宫中GnRHR的分布具有抑制作用。

GnRH agonist active immunization differentially influences the expression and distribution of GnRHR in pituitary and uterus of ewes

To study the expression of GnRH receptor(GnRHR) mRNA in the pituitary and GnRHR proteins in the uteri,as well as the GnRHR distribution in the uteri of ewes which were actively immunized with alarelin antigen,and also to explore the mechanisms of GnRH agonists regulating reproductive function in ewes.Twenty-eight ewes were randomly divided into four groups(n=7).The eves in the experimental group Ⅰ(EG-Ⅰ),experimental group Ⅱ(EG-Ⅱ) and experimental group Ⅲ(EG-Ⅲ) were injected subcutaneously with 200 μg,300 μg and 400 μg GnRH agonist(Alarelin) antigen twice(on day 0 and 14),respectively.Animals in the control group(CG) were injected 2.0 ml solvent subcutaneously twice(on day 0 and 14).The pituitary and uterine horns in each ewe were collected aseptically on day 70.The fluorescent quantitative PCR(FQ-PCR) was implemented to detect the expression of GnRHR mRNA in the pituitary.The Western blotting was also performed to measure the GnRHR proteins in the uteri.Immuno-histochemistry SP(Streptomyces avidin-peroxidase) method and image analysis were used to locate and analyze the GnRHR distribution in the uteri.The results showed that the expression values of GnRHR mRNA in the EG-Ⅰ,EG-Ⅱ and EG-Ⅲ groups decreased,and were lower than that in CG,with the minimum in EG-Ⅲ(P<0.01).Compared to CG,the values of GnRHR proteins in EG-Ⅰ,EG-Ⅱ and EG-Ⅲ reduced by 3.46%,4.90% and 24.78%(P<0.05),respectively.GnRHR distributed mainly in cytoplasms and nucleus of the uterine endometrial cells and glandular epithelial cells.The gray scales of EG-Ⅲ were lower than that of CG(P<0.05).In conclusion,alarelin antigen immunization could suppress the expression of GnRHR mRNA in the pituitary of the ewes,and inhibit the expression GnRHR proteins in uteri.GnRHR localize mainly in the nucleus and cytoplasm of the endometrial epithelial cells and glandular epithelial cells of the uteri.GnRH agonist immunity could inhibit the distribution of GnRHR in the uteri of the ewes.