尼罗罗非鱼Smoothened的鉴定、表达模式及在精巢中的作用  

Identification and expression pattern of Smoothened in Nile tilapia(Oreochromis niloticus)and its role in testicular cells

作　　者：黄琴 张泽明 白孝明 赵长乐 代向燕 陶文静 周林燕 王德寿 魏静 HUANG Qin;ZHANG Zeming;BAI Xiaoming;ZHANG Changle;DAI Xiangyan;TAO Wenjing;ZHOU Linyan;WANG Deshou;WEI Jing(Key Laboratory of Freshwater Fish Reproduction and Development,Ministry of Education,School of Life Sciences,Southwest University,Chongqing 400715,China)

机构地区：[1]西南大学生命科学学院,淡水鱼类资源与生殖发育教育部重点实验室,重庆400715

出　　处：《水产学报》2023年第3期37-46,共10页Journal of Fisheries of China

基　　金：国家自然科学基金(32172969,31972776,31972778,32072957);重庆市自然科学基金(cstc2020jcyjmsxmX1045)。

摘　　要：为探究Smoothened(Smo)信号在精巢不同细胞增殖与存活中的作用,实验分离鉴定了尼罗罗非鱼smo(命名为Onsmo),检测了其在不同组织中的表达分布及在精巢中的细胞表达模式,在尼罗罗非鱼精巢组织的体外培养体系中,用Smo特异性激动剂SAG或抑制剂环巴胺分别进行处理,EdU掺入法及TUNEL法检测了处理后生殖细胞(Vasa^(+))、Sertoli细胞(Amh^(+))与Leydig细胞(Cyp17a1^(+))增殖或凋亡情况。结果显示,Onsmo开放阅读框全长2478 bp,编码825个氨基酸,含有7次跨膜结构域,与人SMO氨基酸一致性达77%;Onsmo表达于包括精巢在内的多个组织;在精巢中,Onsmo在多种不同类型细胞表达,包括精原细胞、精母细胞、Sertoli细胞以及Leydig细胞;在精巢组织的体外培养体系中,SAG处理对精原细胞增殖具有显著促进作用,而环巴胺处理对Sertoli细胞、Leydig细胞凋亡具有显著促进作用。研究表明,On Smo信号在尼罗罗非鱼精巢精原细胞增殖与体细胞存活中具有重要作用。该研究首次证实了Smo信号在鱼类精巢不同细胞增殖与存活中的作用,为进一步研究其作用机制奠定了重要基础。Smoothened(SMO)is a seven-transmembrane protein related to the G protein-coupled receptor and plays a central role as transducer in the Hedgehog(Hh)signaling pathway,which has been demonstrated to play an important role in the differentiation and development of gonadal cells in mammals.However,the role of Smo signaling in fish testis development remains unknown by now.To elucidate the role of Smo signaling in Oreochromis niloticus testis,the O.niloticus smo(Onsmo)cDNA was cloned and characterized.Its expression profile in different tissues was investigated by RT-PCR and in testis by fluorescence in situ hybridization(FISH).After treatment with Smo specific agonist SAG and inhibitor cyclopamine,the proliferation and apoptosis of germ cells and somatic cells in an ex vivo testicular organ culture system were evaluated by EdU incorporation and TUNEL staining,respectively.The full open reading frame of Onsmo is 2478 bp in length,it encodes a putative 825 amino acids protein,including seven transmembrane domains.The identity of amino acid sequence with human SMO is up to 77%.Onsmo was expressed in multiple examined tissues including testis.According to FISH results,Onsmo in testis was expressed in spermatogonia,spermatocytes,Sertoli cells(SC)and Leydig cells(LC).In the testicular organ culture system,our experimental data show that SAG significantly promoted the proliferation of spermatogonia,whilst cyclopamine significantly promoted the apoptosis of testicular SC and LC.Taken together,our study suggests that Smo signaling plays an important role in the proliferation of spermatogonia and the survival of testicular somatic cells in O.niloticus.To our best knowledge,this study firstly reports the role of Smo signaling in fish testicular cells.This study not only enriches our understanding of the role of Smo signaling in fish testicular cells,but also lays an important foundation for further elucidation of its mechanism.

关 键 词：尼罗罗非鱼 SMOOTHENED 精巢 EdU掺入法 TUNEL法 增殖 存活 

分 类 号：Q786[生物学—分子生物学] S917.4[农业科学—水产科学]