小鼠睾丸、附睾和前列腺组织细胞外囊泡蛋白质组分的鉴定及功能分析

Proteomic profiling and functional analysis of extracellular vesicles in mouse testis,epididymis and prostate

【目的】对睾丸、附睾和前列腺组织细胞外囊泡(extracellular vesicles,EVs)蛋白质组分进行鉴定,以明确其在精子发生、成熟及功能维持方面所发挥的作用。【方法】用总外泌体分离试剂盒(total exosome isolation,TEI)分别富集小鼠睾丸、附睾和前列腺组织的EVs,用免疫印迹法对EVs进行鉴定。用透射电镜观察EVs的形态,动态光散射粒径分析仪(dynamic light scattering,DLS)分析其直径分布。用液相色谱串联质谱法(LC-MS/MS)分析EVs的蛋白质组分,运用DAVID 6.8在线分析工具从细胞组分、分子功能、生物学过程三方面对筛选出的蛋白质进行基因本体(gene ontology,GO)富集分析。【结果】用TEI法分离纯化的EVs均可检测到分化抗原63(cluster differentiation antigen 63,CD63)和肿瘤易感基因101(tumor susceptibility gene 101,TSG101)标记蛋白,表明EVs分离成功。透射电镜下观察EVs呈凹杯状,睾丸组织来源的EVs以直径30~140 nm的居多,附睾组织EVs直径在105 nm处有峰值,前列腺组织EVs直径在50和90 nm处有峰值。用LC-MS/MS分析3种EVs的蛋白组分,睾丸组织EVs筛选到1148种蛋白,附睾组织EVs筛选到902种蛋白,前列腺组织EVs筛选到651种蛋白。睾丸组织EVs特有蛋白有667种,主要参与RNA加工及剪切、精子发生、DNA损伤应答等生物学过程;附睾组织EVs特有蛋白有311种,主要参与转运、代谢和免疫作用等生物学过程;前列腺组织EVs特有蛋白有142种,主要参与代谢活动等。三者共有蛋白277种,主要发挥蛋白质结合、RNA结合等分子功能,参与蛋白质翻译和运输等生物学过程。【结论】睾丸、附睾和前列腺组织EVs分别在精子发生、精子的睾丸后成熟及功能维持中发挥一定的作用。

【Objective】The objective of this study was to clarify the effects of proteins of testicular,epididymal and prostatic extracellular vesicles(EVs)on sperm development,maturation and functional maintenance.【Method】The EVs of testis,epididymis and prostate were enriched by total exosome isolation(TEI)and identified by Western blotting.Morphology of EVs was observed with transmission electron microscopy(TEM).Distribution of diameter was detected by dynamic light scattering(DLS).The testicular,epididymal and prostatic EVs proteins were analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS).Using Database for Annotation,Visualization and Integrated Discovery(DAVID)6.8 online analysis tools,gene ontology(GO)functional analysis was conducted and identified proteins were categorized based on cellular component,molecular function and biological processes.【Result】Cluster differentiation antigen 63(CD63)and tumor susceptibility gene 101(TSG101)were detected in EVs purified by TEI,indicating that EVs were successfully isolated.Under TEM,EVs showed cup-shaped morphology.By DLS,the majority of testicular EVs was 30-140 nm in diameter and epididymal EVs peaked at 105 nm.Diameters of prostatic EVs peaked at 50 and 90 nm.This experiment identified 1148 testicular EV proteins,902 epididymal EV proteins,and 651 prostatic EV proteins.Among them,667 specific proteins in testicular EVs were mainly mapped to RNA processing and splicing,spermatogenesis and cellular response to DNA damage.A total of 311 specific proteins in epididymal EVs were mainly mapped to transport,multi-metabolic process and immune process,while 142 specific prostatic EV proteins were mainly mapped to metabolic process.The 277 common proteins mainly played molecular function such as protein binding and RNA binding and participated in biological processes such as protein translation and transportation.【Conclusion】The testicular,epididymal and prostatic EVs play roles in spermatogenesis and post-testicular sperm maturation and function maintenance.