GPX5在成年绵羊附睾中的表达与蛋白定位

Study on Expression of Glutathione Peroxidase type-5(GPX5) and Protein Localization in Epididymis of Adult Sheep

【目的】研究谷胱甘肽过氧化物酶-5(glutathione peroxidase type-5,GPX5)在成年绵羊附睾的表达特征以及GPX5蛋白在附睾中的定位,为绵羊精子在附睾中抗氧化机制的研究提供理论依据。【方法】采取年龄相近的成年蒙古绵羊的附睾、睾丸和输精管。每一只公羊的附睾分别按照附睾头、附睾体和附睾尾进行分割。样品保存于-80℃冰箱,采用实时荧光定量PCR和Western blotting的方法,对成年绵羊的附睾、睾丸和输精管的GPX5表达量进行分析。将新鲜的组织样品各部分切取适合大小浸泡于4%多聚甲醛溶液中24h,按照石蜡切片的方法制作组织切片。用GPX5特异性抗体孵育组织切片,利用DAB显色试剂盒对阳性信号进行标记。【结果】实时荧光定量PCR结果显示,成年绵羊附睾头GPX5基因的表达量极显著高于附睾体和附睾尾(P<0.01),而在输精管和睾丸几乎不表达。Western blotting的结果显示,GPX5蛋白在成年绵羊附睾头高表达,在附睾体和附睾尾有微量蛋白存在,睾丸和输精管中未发现GPX5蛋白。这表明GPX5主要在成年绵羊附睾头表达;经过免疫组化染色分析,GPX5蛋白主要位于附睾头上皮细胞质以及静纤毛,附睾体和附睾尾中的GPX5蛋白主要集中在静纤毛。在附睾头、附睾体和附睾尾中的精子上以及附睾腔都可以观察到GPX5蛋白,表明GPX5蛋白从附睾上皮分泌到附睾腔中,随着精子在附睾中的运输与精子结合。【结论】在成年绵羊附睾中,GPX5主要由附睾头上皮细胞合成和分泌,在附睾管腔中与精子结合,为精子功能的正常发挥提供保护。

【Objective】 The objective of this study was to investigate the expression pattern of GPX5（Glutathione peroxidase type-5）and GPX5 protein localization in adult sheep epididymis to provide a theoretical basis for studying antioxidative mechanisms of sperm in sheep epididymis. 【Method】 Epididymis, testis and deferent duct of adult Mongolia sheep of similar age were collected. The epididymis of each ram were selected as epididymal caput, epididymal corpus and epididymal cauda, respectively. Samples were stored at-80 ℃. The expression of GPX5 in epididymis, testis and deferent duct were detected by real-time fluorescent quantitative PCR and Western blot. The fresh tissue samples of different segments were cut to suitable size and immersed in 4% paraformaldehyde solution for 24 hours; then paraffin embedded tissue sections were made and incubated with GPX5 specific antibody. The positive signals of GPX5 protein were detected by DAB kit. 【Result】The result of real-time fluorescent quantitative PCR showed that the expression level of GPX5 of caput was significantly higher than that of corpus and cauda（P〈0.01）, but almost no expression were found in testis or deferent duct. We showed that GPX5 protein was highly expressed in caput, and a small amount of GPX5 protein was present in corpus and cauda; however no GPX5 protein was found in testis nor deferent duct. Collectively, these studies suggested that GPX5 expressed mainly in the epididymal caput of adult sheep. As assessed by immunohistochemical staining, the GPX5 protein was localized mainly in the epithelium and stereocilia of caput, while in corpus and cauda the GPX5 protein localized mainly in stereocilia. The GPX5 protein was detected in sperm located in caput, corpus, cauda as well as epididymal lumen. The GPX5 protein secreted from epididymis epithelium into epididymal lumen and combined gradually with spermatozoa during epididymal transit.【Conclusion】GPX5 was synthesized and secreted mostly in the epididymal caput epithelium of adult sheep and combined with spermatozoa in epididymal lumen, thereby providing protection for their normal physiological functions.