神经介素B及其受体在不同发育阶段山羊睾丸组织中的差异表达

The expression of neuromedin B and its receptor in goat testis at different developmental stages

[目的]本试验旨在探究神经介素B(NMB)及其受体NMBR[还包括胃泌素释放肽受体(GRPR)和蛙皮素受体(BRS3)]在山羊不同发育阶段睾丸组织中的差异表达,为解析哺乳动物睾丸发育及功能调节机制提供参考。[方法]选取系谱清晰和体况良好的雄性山羊12只(3月龄和9月龄,各6只)进行阉割并采集睾丸组织,在克隆NMB及其受体基因与分析序列的基础上,采用免疫组化技术检测NMB及其受体在睾丸组织中的表达定位,利用RT-qPCR和Western blot检测NMB及其受体在不同发育阶段山羊睾丸组织中的表达变化。[结果]NMB及其受体基因与绵羊和牛的进化关系最近,NMB及其受体基因编码区的核苷酸序列与牛和绵羊的同源性达96%以上,结构分析发现NMBR、GRPR和BRS3均属于7次跨膜受体。NMB及其受体在3月龄和9月龄山羊睾丸组织中广泛表达,其中在间质细胞、肌样细胞和精子细胞中呈高表达。与3月龄山羊相比,9月龄山羊睾丸组织中NMB与GRPR的mRNA和蛋白表达量显著降低(P<0.05),而NMBR与BRS3的mRNA和蛋白表达量显著增加(P<0.05)。[结论]NMB及其受体可能参与山羊睾丸发育的调节。

[Objectives]The purpose of this experiment was to explore the differential expression of neuromedin B(NMB)and its receptors(neuromedin B receptor,NMBR;gastrin releasing peptide receptor,GRPR;bombesin receptor subtype 3,BRS3)in goat testis at different development stages,which provided reference for analyzing the mechanism of mammalian testicular development and functional regulation.[Methods]Twelve male goats(3-and 9-months-old,six individuals per age group)with clear pedigree and good body conditions were selected for castration and collected the testicular tissue.Based on gene cloning and sequence analysis of NMB and its receptors,immunohistochemistry was used to detect the location of NMB and its receptors in testicular tissue,and RT-qPCR and Western blot were used to detect the expression of NMB and its receptors in goat testis at different development stages.[Results]NMB and its receptors gene had the most recent evolutionary relationship with sheep and cattle.The nucleotide sequence of the coding region of NMB and its receptors gene had more than 96%homology with cattle and sheep,and structural analysis showed that NMBR,GRPR and BRS3 belonged to seven transmembrane receptors.NMB and its receptors were widely distributed in testicular tissues of 3-months-old and 9-months-old goats,and a strong staining intensity in leydig cells,myoid cells and sperm cells.In addition,compared with the 3-months-old goats,the mRNA and protein expression levels of NMB and GRPR in testis of 9-months-old goats significantly decreased(P<0.05),while the mRNA and protein expression levels of NMBR and BRS3 in testis of 9-months-old goats significantly increased(P<0.05).[Conclusions]NMB and its receptors may be involved in the regulation of goat testicular development.