阿拉瑞林免疫对FSHR表达与子宫发育的影响

Effects of alarelin active immunity on FSHR expression and uterine development in ewes

为研究GnRHa主动免疫对绵羊子宫FSHR表达与分布及子宫发育的作用,探讨GnRHa调节生殖功能的机制,将42只5～6月龄母绵羊(Ovis aries)随机分为6组(n=7)。EG-Ⅰ、EG-Ⅱ和EG-Ⅲ动物分别皮下注射阿拉瑞林(alarelin)抗原200、300和400μg,0 d和14 d各1次;EG-Ⅳ和EG-Ⅴ动物皮下注射alarelin抗原200μg和300μg,0、7、14和21 d各1次;对照组皮下注射药物溶媒,0 d和14 d各1次。于70 d在颈动脉放血处死绵羊,无菌切取两侧子宫角。免疫组织化学SP法染色并进行图像分析,光学显微镜和电子显微镜分别观察子宫显微和超微结构变化。Western blot分析FSHR蛋白表达。结果发现各试验组FSHR蛋白表达量随着alarelin免疫剂量和次数的增多而逐渐增加。EG-Ⅲ和EG-Ⅴ极显著高于对照组(P<0.01)。FSHR主要分布于子宫内膜细胞和子宫腺上皮细胞的胞质和胞核;EG-Ⅰ、EG-Ⅱ和EG-Ⅲ灰度值逐渐升高,EG-Ⅲ和EG-V(P<0.01)显著高于CG,证明alarelin主动免疫能增强子宫FSHR的分布与表达,加大注射剂量和次数作用更明显。EG-I、EG-Ⅱ和EG-Ⅲ的子宫质量、UWT和EET均变小,EG-Ⅲ和EG-Ⅴ显著小于对照组(P<0.05)。试验组出现不同程度的子宫壁和上皮变薄,细胞胞质减少,腺体数量减少,腺腔缩小,平滑肌细胞及核均变小。线粒体和线粒体嵴减少,微绒毛变短,以EG-Ⅲ最明显。结果表明,Alarelin主动免疫母羊能增强子宫FSHR的分布与蛋白表达,使子宫超微和超微结构明显改变,抑制子宫发育,尤其是子宫内膜的生长显著受阻。

The experiment was undertaken to evaluate the effects of active immunization against the gonadotropin releasing hormone agonist（GnRHa） on the expression and immuno-localization of FSHR in uteri,as well as uterine development in the ewes.Forty two ewes（Ovis aries）,five-to-six months old,were randomly assigned into six groups（n=7）.The animals in the experimental groups EG-Ⅰ,EG-Ⅱ and EG-Ⅲ were subcutaneously injected with 200 μg,300 μg and 400 μg alarelin antigens twice（on day 0 and 14）,respectively,in groups EG-Ⅳ and EG-Ⅴ were subcutaneously injected with 200 μg and 300 μg alarelin antigens four times（on day 0,7,14 and 21）,respectively and in control group（CG） were injected subcutaneously with 2.0 mL solvent twice（on day 0 and 7）.The samples of the uterine horns of each ewe were collected aseptically on day 70 after killed by bloodletting from carotid.The FSHR distribution in the uteri was studied by the methods of Immunohistochemistry SP（streptomyces avidin-peroxidase） and image analysis,the mirostructure and ultrastructure changes of the uteri were observed under the optical and electron microscope,respectively and the expression of FSHR was detected by Western blot.The results showed that the expression of FSHR increased with the increasing of alarelin dose and injection times and the expression levels of FSHR in EG-Ⅲ and EG-Ⅴ were significantly higher than（P＆lt;0.01） in CG.The FSHR were predominantly concentrated in the cytoplasm and nuclei of the uterine endometrial cells and glandular epithelium cells.The gray values of microscopy images in EG-Ⅰ,EG-Ⅱ and EG-Ⅲ increased gradually and were significantly higher in EG-Ⅲ and EG-Ⅴ than in CG（P＆lt;0.01）.The uterine weights,endometrial epithelium thickness（EET） and uterine wall thickness（UWT） in all experimental groups reduced and were significantly lower in EG-Ⅲ and EG-Ⅴ than in CG（P＆lt;0.05）.The uterine cavities and glandular lumen volumes narrowed,and the uterine wall became clearly thinner in all experimental groups.The quantities of mitochondria and mitochondria cristaes decreased and the microvilli became shorter,especially in EG-Ⅲ.In conclusion,alarelin active immunity enhanced distribution of FSHR,promoted the expression of FSHR protein in the uteri of the ewes,changed the mirostructure and ultrastructure of the uteri,inhibited the uterine development of ewes,and especially suppressed the growth of uterine endometrium.