降钙素基因相关肽在牦牛附睾中的定位与表达

Location and expression of calcitonin gene-related peptide in yak epididymis

【目的】分析降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在牦牛附睾中的转录水平及蛋白分布特点,探究其对精子运输及成熟的功能。【方法】采集6头3~4岁健康成年牦牛的附睾,将其解剖为头、体、尾三部分,应用实时荧光定量PCR(RT-qPCR)、Western blotting分别检测CGRP mRNA和蛋白在附睾的头、体、尾中的表达水平;采用H.E染色法分析附睾组织结构特点;采用免疫组化和免疫荧光技术分析附睾各部分CGRP的分布及表达情况。【结果】RT-qPCR结果表明,CGRP在牦牛附睾头的相对表达量显著高于附睾体和附睾尾(P<0.05),附睾尾表达量最低(P<0.05);Western blotting检测结果表明,CGRP蛋白水平在附睾尾中的表达水平显著高于附睾头和附睾体(P<0.05)。显微镜下观察表明,牦牛附睾组织纤毛长度从头部至尾部逐渐降低,差异显著;附睾头间质组织的间质平滑肌层厚度显著低于附睾体和附睾尾(P<0.05);基细胞和晕细胞在附睾尾分布显著,上皮厚度亦在附睾尾显著降低。免疫组化及免疫荧光分析发现,CGRP主要分布于附睾上皮、基底膜及附睾间质小血管,附睾上皮晕细胞的表达强度最高。【结论】CGRP主要分布于牦牛附睾上皮和间质小血管,提示CGRP对牦牛附睾间质小血管的收缩发挥调节作用。牦牛附睾尾晕细胞数量最多,晕细胞中CGRP的高强度表达提示其可能参与附睾尾的免疫调节。

【Objective】This study analyzed the transcription level and protein distribution characteristics of calcitonin gene-related peptide(CGRP)in epididymis of plateau yak and explored its function on sperm transport and maturation.【Method】The epididymis of 6 adult yaks at the age of 3 to 4 years were collected and dissected into three parts of caput,corpus and cauda.The expression levels of CGRP mRNA and protein in caput,corpus and cauda of epididymis were detected by RT-qPCR and Western blotting.The structure characteristics of epididymis were analyzed by H.E staining.Immunohistochemistry and immunohistofluorescence were used to analyze the distribution and expression of CGRP in epididymis.【Result】RT-qPCR showed that the expression of CGRP in yak caput was significantly higher than that in corpus and cauda(P<0.05),and the expression in cauda was the lowest(P<0.05).Western blotting showed that the expression level of CGRP protein in cauda was higher than that in caput and corpus(P<0.05).Microscopic observations showed that the distribution of basal cells and halo cells in cauda was significantly higher than that in caput and corpus.The length of cilia from caput to cauda of yak decreased significantly.The thickness of cauda epithelium was significantly lower than that of caput and corpus.The interstitial tissue thickness of caput was significantly lower than that of corpus and cauda(P<0.05).The interstitial smooth muscle layer thickened gradually.Immunohistochemistry and immunofluorescence analysis showed that CGRP was mainly distributed in epididymal epithelium,basal lamina and small vessels of epididymal interstitium.The positive expression intensity of CGRP was different in epididymal epithelium,and the highest in halo cells.【Conclusion】CGRP mainly distributed in epididymal epithelium and small interstitial vessels in yaks,indicating that CGRP played a regulatory role in the contraction of small interstitial vessels in yaks.The number of cauda halo cells was the largest in yak epididymis.The high expression of CGRP in the cauda of yak suggested that CGRP may be involved in the immune regulation of cauda.