[Cadmium induces apoptosis of mouse spermatocytes (GC-2 spd) by promoting mitochondrial fission].

Objective: To study the underlying mechanism of cadmium-induced apoptosis of mouse spermatocytes (GC-2 spd) . Methods: In March 2021, GC-2 spd cells were exposed to different concentrations of CdCl(2) for 24 hours, namely 5 μmol/L CdCl(2) (low-dose) group and 10 μmol/L CdCl(2) (high-dose) group, and unexposed GC-2 spd cells were used as control group. Mitochondrial morphology was observed in the cells stained with Mito-Track Red CMXRos fluorescent probes by confocal microscopy and the mitochrondrial membrane potential was measured by flow cytometry with JC-1 fluorescent probes. Mitochrondrial proteins, cytosolic proteins and total cellular proteins of GC-2 spd cells were extracted using cell mitochondria isolation kit and RIPA buffer, respectively. The expression of mitochondrial homeostasis regulatory proteins (FIS1 and OPA1), and apoptosis-related proteins (Cytochrome c and cleaved Caspase-3) were examined by Western blot. Results: Compared with the cells in the control group, the relative ratio of JC-1 red/green fluorescence signal in the cells of the low-dose and high-dose CdCl(2) groups decreased significantly (0.740±0.071, 0.570±0.028), with a statistically significant difference (P=0.017, 0.004) ; The morphology of mitochondria changed from long tube to point, and the proportion of cells containing point mitochondria increased significantly (45.1%±3.7% and 25.7%±4.9%), the difference was statistically significant (P=0.005, 0.001) ; The relative expression level of mitochondrial FIS1 in cells of low and high dose CdCl(2) groups was significantly higher (1.271±0.120, 1.693±0.155), the difference was statistically significant (P=0.046, 0.000) ; The relative expression level of OPA1 decreased significantly (0.838±0.050, 0.682±0.040), and the difference was statistically significant (P=0.049, 0.001). Compared with the control group, the relative expression level of cytochrome c protein in the cytoplasm of cells in the low dose group of CdCl(2) was not significantly increased (1.249±0.151), and the difference was not statistically significant (P=0.075). However, the relative expression level in the cytoplasm of cells in the high dose group of CdCl(2) was significantly increased (2.355±0.110), and the difference was statistically significant (P=0.000) ; The relative expression level of Cytochrome c in mitochondria of low and high dose CdCl(2) groups decreased significantly (0.681±0.043, 0.619±0.114), with a statistically significant difference (P=0.004, 0.001) ; Moreover, the level of cleaved Caspase-3 protein in cells gradually increased (5.486±0.544, 11.493±1.739), the difference was statistically significant (P=0.004, 0.000) . Conclusion: Cadmium induced cleaved Caspase-3 mediated apoptosis of GC-2 spd cells via promoting mitochrondrial fission and the release of Cytochrome c from the mitochrondria to the cytosol.目的： 探讨镉诱导小鼠精母细胞（GC-2 spd）凋亡的作用机制。 方法： 于2021年3月，分别用5和10 μmol/L氯化镉（CdCl(2)）染毒GC-2 spd细胞24 h，分别为CdCl(2) 5 μmol/L染毒组（CdCl(2)低剂量组）和CdCl(2) 10 μmol/L染毒组（CdCl(2)高剂量组），以未染毒的GC-2 spd细胞作为对照组。采用Mito-Track Red CMXRos线粒体荧光探针染色细胞检测线粒体形态，流式细胞术结合JC-1荧光探针检测线粒体膜电位的变化；使用细胞线粒体分离试剂盒和RIPA裂解液分别提取GC-2 spd细胞的线粒体蛋白、细胞浆蛋白和细胞总蛋白，Western blot检测线粒体稳态调节蛋白[分裂蛋白（FIS1）和融合蛋白（OPA1）]及细胞凋亡相关蛋白（Cytochrome c和cleaved Caspase-3）的表达。 结果： 与对照组细胞比较，CdCl(2)低剂量组和高剂量组细胞中JC-1红色/绿色荧光信号相对比值均明显降低（0.740±0.071、0.570±0.028），差异有统计学意义（P=0.017、0.004）；线粒体形态由长管状变为点状，含点状线粒体的细胞比例明显升高（45.1%±3.7%和25.7%±4.9%），差异有统计学意义（P=0.005、0.001）；CdCl(2)低、高剂量组细胞中线粒体FIS1相对表达水平均明显升高（1.271±0.120、1.693±0.155），差异有统计学意义（P=0.046、0.000）；OPA1相对表达水平明显降低（0.838±0.050、0.682±0.040），差异有统计学意义（P=0.049、0.001）。与对照组细胞比较，CdCl(2)低剂量组细胞的胞浆中Cytochrome c蛋白相对表达水平（1.249±0.151）差异无统计学意义（P=0.075），然而CdCl(2)高剂量组细胞胞浆中其相对表达水平明显升高（2.355±0.110），差异有统计学意义（P<0.001）；CdCl(2)低、高剂量组线粒体中Cytochrome c相对表达水平均明显降低（0.681±0.043、0.619±0.114），差异有统计学意义（P=0.004、0.001）；细胞中cleaved Caspase-3蛋白水平升高（5.486±0.544、11.493±1.739），差异有统计学意义（P=0.004、<0.001）。 结论： CdCl(2)可促进线粒体分裂，影响Cytochrome c蛋白在GC-2 spd细胞内分布并激活Caspase-3蛋白活性，引起细胞凋亡。.