阿拉瑞林主动免疫对绵羊垂体GnRHR、FSHR和LHR表达及卵巢GnRHR分布的影响

Effects of alarelin active immunity on expression of GnRHR,FSHR and LHR in pituitary and GnRHR distribution in ovaries of ewes

为探讨促性腺激素释放激素(GnRH)激动剂主动免疫对绵羊腺垂体GnRH受体(GnRHR)、促卵泡激素受体(FSHR)和促黄体激素受体(LHR)表达及分布的影响,将42只5～6月龄母绵羊分为6组(n=7),即EG-Ⅰ组、EG-Ⅱ组、EG-Ⅲ组、EG-Ⅳ组、EG-Ⅴ组和对照组(CG)。EG-Ⅰ、EG-Ⅱ和EG-Ⅲ组绵羊分别皮下注射200μg、300μg和400μg阿拉瑞林抗原,分2次皮下注射(0 d和14 d),EG-Ⅳ和EG-Ⅴ组绵羊分别皮下注射200μg和300μg阿拉瑞林抗原,分4次皮下注射(0 d、7 d、14 d和21 d),对照组(CG)绵羊皮下注射2.0 ml溶剂,分2次(0 d和14 d)。于70 d无菌切取腺垂体、子宫及卵巢,提取垂体总RNA,PCR扩增,反转录,实时荧光定量PCR(FQ-PCR)检测,免疫组织化学SP法染色。结果表明,GnRHR、FSHR和LHR mRNA PCR扩增曲线的起始循环数分别为20、28和24。5个试验组Gn-RHR、FSHR和LHR mRNA值均低于对照,EG-Ⅰ、EG-Ⅱ和EG-Ⅲ组的mRNA值逐渐下降;EG-Ⅳ和EG-Ⅴ组的mRNA值分别低于EG-Ⅰ和EG-Ⅱ组。GnRHR阳性染色主要分布在卵泡细胞以及卵母细胞胞质和胞核及胞膜。各组的染色强度不同,以EG-Ⅲ阳性染色最为明显。阴性对照全部为阴性。显微图像的绿色值和亮度无显著差异,各组灰度值差异显著(P<0.05),以EG-Ⅱ均为最小,EG-Ⅴ最高。EG-Ⅰ的饱和度最小(P<0.05),EG-Ⅲ最高。上述结果说明阿拉瑞林抗原免疫抑制垂体GnRHR、FSHR和LHR mRNA表达,GnRHR阳性染色主要在卵母细胞和卵泡细胞的胞核和胞质。阿拉瑞林免疫可抑制卵巢GnRHR的分布与表达,增加注射剂量和次数作用更明显。

To study the expression of gonadotropin releasing hormone receptor（GnRHR）,follicle stimulating hormone receptor（FSHR） and luteinizing hormone receptor（LHR） mRNA in pituitary and GnRHR distribution in ovaries of ewes actively immunized with different concentrations of alarelin antigen,forty-two ewes（Ovis aries）,five-to-six months old,were randomly divided into six groups（each of 7 eves）.Animals were subcutaneously injected with 200 μg,300 μg and 400 μg GnRH agonist alarelin antigen emulsion in groups EG-Ⅰ,EG-Ⅱ,and EG-Ⅲ twice（0 d and 14 d） respectively.Animals in groups EG-Ⅳ and EG-Ⅴwere subcutaneously injected with 200 μg and 300 μg GnRH agonist alarelin antigen emulsion four times（0 d,7 d,14 d and 21 d） respectively.Control group（CG） was injected subcutaneously with 2.0 ml solvent twice（0 d and 14 d）.The samples of pituitary and ovaries in each ewe were collected aseptically at the end of the experiment（at 70 d）.The primers of mRNAs of GnRHR,FSHR and LHR mRNAs were designed to extract total RNA of the pituitaries.PCR amplification,reverse transcription and fluorescent quantitative RT-PCR（FQ-PCR） were implemented.Immunohistochemistry SP（Streptomyces avidin-peroxidase） method and image analysis were used to locate and analyze GnRH receptor（GnRHR） distribution in ovaries of ewes.Cycle numbers of PCR amplification curve for GnRHR,FSHR and LHR mRNAs were 20,28 and 24 cycles respectively.The expression values of GnRHR,FSHR and LHR mRNAs in five experiment groups（EGs）were lower those that in control group（CG）.The mRNA values in groups EG-Ⅰ,EG-Ⅱ and EG-Ⅲ decreased.Meanwhile,the values of mRNAs in EG-Ⅳ and EG-Ⅴ were lower than those in EG-Ⅰand EG-Ⅱ.The positive staining of GnRHR predominantly distributed in the cytoplasm,nuclei and cytomembrane of follicle cells and oocytes of ewe ovaries.The staining intensities differed among groups.The strongest staining was seen in EG-Ⅲ.The sections in negative control group was negatively stained.There were no significant differences between values of green and brightness in all groups.However,there existed significant differences between gray degrees（P0.05）,with minimum in EG-Ⅱand maximum in EG-Ⅴ.The saturation was the lowest in EG-Ⅰand the highest in EG-Ⅲ（P0.05）.In conclusion,alarelin active immunization improved obviously the production of serum anti-GnRH antibody,and suppressed the expressions of GnRHR,FSHR and LHR mRNAs in pituitaries of ewes.GnRHR positive cells distributed predominantly in the nuclei and cytoplasm of oocytes and follicle cells.Alarelin antigen treatment affected the distribution of GnRHR in ovaries of ewes.The effects were strengthened with increased injection dose and times of alarelin antigen.