氧化应激相关基因Nme5在小鼠隐睾中参与生精细胞凋亡

摘要

目的 观察氧化应激相关基因Nme5在隐睾中的表达变化,探讨其在生精细胞凋亡中的作用.方法 建立单侧隐睾小鼠模型,采用TUNEL结合形态学观察确认小鼠隐睾模型是否建立成功并分析生精细胞凋亡情况.采用Real-time PCR、Western blot以及免疫组化分析隐睾手术后第1、2、3、4、5、6、9和15天与正常侧相比隐睾侧氧化应激相关基因Nme5 mRNA和蛋白表达情况.结果 免疫组化结果显示正常睾丸组织中,Nme5在生精上皮周期的Ⅱ～Ⅻ阶段均表达于粗线期初级精母细胞、圆形精子以及长形精子中;TUNEL结合形态学观察表明小鼠隐睾模型构建成功;Real-time PCR结果显示术后第4天Nme5 mRNA表达量轻微上升,第6天显著下降;Western blot结果显示术后第4天Nme5蛋白开始下降,至第9天急剧降低;免疫组化结果显示术后第4～6天隐睾侧Nme5在多核巨细胞中表达上调.结论 正常睾丸组织中Nme5在粗线期初级精母细胞、圆形精子以及长形精子中都有表达,参与了从减数分裂到精子变形的全部过程,在精子发生中起重要作用.隐睾状态下Nme5在新生圆形精子和由退行性圆形精子细胞组成的多核巨细胞中表达上调,可能通过调控氧化应激相关基因Gpx5来调控圆形精子细胞凋亡.%Objective We aimed to assess the functional significance of Nme5 in testis of mice with cryptorchidism.Methods Experimentalunilateral cryptorchidism mouse model was established.Whether the model was established successfully was confirmed by DNA end labeling(TUNEL)and morphological observation,and apoptotic characteristics of spermatogenic cells were analyzed.The expression levels of Nme5 mRNA and protein were spatiotemporally analyzed in testes 1,2,3,4,5,6,9 and 15 day (s)after modeling in mice by quantitative real-time PCR and Western blotting.Immunohistochemical analysis was used to detect changes of Nme5 in response to heat-stress from cryptorchidism.Results Morphological studies have shown that at the normal side of testes Nme5 are localized in primary spermatocytes and spermatids at stage Ⅱ ～ Ⅻ of eminiferous tubules.Under cryptorchidism,Nme5 mRNA was slightly increased at the 4th day and significantly decreased at the 6th day while its protein was slightly reduced in testes at the 4th day and greatly reduced at the 9th day.However,immunohistochemical staining showed that Nme5 protein was up-regulated in multinucleated giant cells and degenerative spermatids in testes at the 4-6th day.Conclusion Nme5 is expressed in first spermatocyte,round spermatid and long sperm at pachytene stage,is involved in all the process from meiosis to sperm deformation,and plays an important role in spermatogenesis.In mice with cryptorchidism,Nme5 is up-regulated in multinuclear giant cells(neonatal round sperm and degenerative round sperm cells);apoptosis of round sperm cells is regulated probably through regulating oxidative stress related gene Gpx5.