Jia Miao-Miao, Shi Lin, Shi Wen-Hao, Xue Xia, Zhao Zheng-Hao, Liu Gang
Center of Reproduction Medicine, Northwest Women and Children's Hospital, Xi'an, Shannxi 710000, China.
Research Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Sciences, Central South University, Changsha 410078, China.
Zhonghua Nan Ke Xue. 2020 Mar;26(3):228-236.
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.
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.
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.
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 stages Ⅵ－Ⅷ.
研究Dnajb13在隐睾小鼠睾丸中的mRNA和蛋白表达及其定位，以及与生精细胞凋亡的关系。
采用免疫组织化学法检测8周龄小鼠生精细胞中Dnajb13的定位。通过手术建立小鼠单侧隐睾模型，并经TUNEL、流式细胞术及形态学观察进行验证。建模后1、2、3、4、5、6、9和15天，采用定量聚合酶链反应（qPCR）、蛋白质印迹法和免疫组织化学法分析生精细胞凋亡情况，测定隐睾和健康睾丸中Dnajb13的mRNA和蛋白表达。
免疫组织化学显示，Dnajb13定位在健康小鼠睾丸组织精子发生第9 - 16步的长形精子细胞中。TUNEL和流式细胞术表明，第1步的圆形精子细胞和减数分裂期的初级精母细胞对温度升高最为敏感。建模后，首先在第1 - 8步的圆形精子细胞中观察到凋亡，其比例从17.09%降至6.52%（P < 0.05），然后在第9 - 16步变态期的精子细胞中出现凋亡，随后是初级精母细胞。术后3天，隐睾睾丸中Dnajb13 mRNA的表达比健康睾丸高1.6倍（P < 0.05），4天时下降，为正常的1.2倍。Dnajb13蛋白表达在1 - 3天无明显变化，但4天时降低0.68倍（P < 0.05），9天时降低0.4倍。免疫组织化学染色显示术后6天凋亡的多核巨细胞中有Dnajb13蛋白表达。
Dnajb13定位在成年小鼠变态期的精子细胞及成熟精子尾部，参与精子变态和精子鞭毛形成，并在隐睾睾丸凋亡的多核巨细胞中表达，这可能与Ⅵ - Ⅷ期圆形精子细胞凋亡有关。
