Dnajb13在隐睾小鼠睾丸中的表达及其与生精细胞凋亡的关系

Expression of Dnajb13 and its involvement in the apoptosis of spermatogenic cells in the testis of the mouse with cryptorchidism

目的:通过分析热休克蛋白40家族成员Dnajb13在成年小鼠生精细胞中的定位,研究其mRNA和蛋白在隐睾小鼠睾丸中表达量和定位的变化情况,初步探讨其在生精细胞凋亡中的作用。方法:手术建立单侧隐睾小鼠模型,采用免疫组化方法研究Dnajb13在小鼠生精细胞中的定位;采用TUNEL、流式细胞检测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qPCR、Western印迹以及免疫组化分析隐睾手术后第1、2、3、4、6、9和15天与正常侧相比隐睾侧Dnajb13 mRNA和蛋白的表达及定位变化情况。结果:免疫组化结果显示在成熟小鼠正常睾丸组织中,Dnajb13表达于生精上皮周期Ⅸ~ⅩⅥ期长形精子中;成功构建隐睾小鼠模型。TUNEL结合流式细胞检测表明I期圆形精子以及分裂期初级精母细胞对温度升高最敏感,隐睾模型手术后最早发生凋亡的生精细胞是圆形精子细胞(Ⅰ~Ⅷ期),从17.09%降为6.52%(P<0.05),随后是变形期精子细胞(Ⅸ~ⅩⅥ期),最后为初级精母细胞;qPCR结果显示术后第3天Dnajb13 mRNA表达量轻微上升,约为正常侧的1.6倍(P<0.05),第4天开始下降,约为正常侧的1.2倍;Western印迹结果显示术后第1~3天Dnajb13蛋白表达量无显著变化,第4天开始下降为正常侧的0.68倍(P<0.05),至第9天均急剧降下降为正常侧的0.4倍,Dnajb13在术后第6天已经凋亡的多核巨细胞中开始表达。结论:Dnajb13定位于成熟小鼠变形期精子细胞以及成熟精子尾部,参与了精子变形过程以及精子鞭毛的形成。隐睾状态下Dnajb13在凋亡的多核巨细胞中表达,可能参与了Ⅵ~Ⅷ阶段的圆形精子细胞的凋亡。

Objective:To study the mRNA and protein expressions of Dnajb13 and its localization in the testis of the mouse with cryptorchidism and its association with the apoptosis of spermatogenic cells.Methods:The localization of Dnajb13 in the spermatogenic cells of 8-week-old mice was detected by immunohistochemistry.The model of unilateral cryptorchidism was surgically established in the mice and verified by TUNEL,flow cytometry and morphological observation.The apoptosis of the spermatogenic cells was analyzed and the mRNA and protein expressions of Dnajb13 in both cryptorchid and healthy testes were determined by quantitative polymerase chain reaction(qPCR),Western blot and immunohistochemistry at 1,2,3,4,5,6,9 and 15 days after modeling.Results:Immunohistochemistry showed that Dnajb13 was localized in the elongated spermatids at steps 9-16 of spermiogenesis in the testis tissue of the healthy mice.TUNEL and flow cytometry manifested that the round spermatids at step 1 and primary spermatocytes in miosis were most sensitive to elevated temperature.After modeling,apoptosis was first observed in the round spermatids at steps 1-8,which were decreased from 17.09%to 6.52%(P<0.05),then in the spermatids during metamorphosis at steps 9-16,and then in the primary spermatocytes.At 3 days after surgery,the expression of Dnajb13 mRNA in the cryptorchid testis was 1.6 times higher than that in the healthy one(P<0.05)and decreased at 4 days,1.2 times that of the normal.The expression of the Dnajb13 protein exhibited no significant change at 1-3 days,but a 0.68-fold reduction at 4 days(P<0.05)and a 0.4-fold reduction at 9 days.Immunohistochemical staining revealed the expression of the Dnajb13 protein in the apoptotic multinucleated giant cells at 6 days.Conclusion:Dnajb13 is localized in the spermatids during metamorphosis and in the tails of mature sperm in adult mice,involved in sperm metamorphosis and sperm flagellum formation,and expressed in apoptotic multinucleated giant cells in the cryptorchid testis,which may be associated with the apoptosis of round spermatids at stagesⅥ-Ⅷ.