[Role of autophagy in cadmium-induced damage to the blood-testis barrier in mice].

OBJECTIVE: To investigate the role of autophagy in cadmium chloride (CdCl2)-induced damage to the blood-testis barrier (BTB) in mice. METHODS: Twenty four-week-old male C57BL/6 mice were randomly divided into four groups and intraperitoneally injected with CdCl2 at 0 mg/kg/d (the control), 0.5 mg/kg/d (low-dose), 1.0 mg/kg/d (medium-dose) and 2.0 mg/kg/d (high-dose) respectively for 28 consecutive days. Then the morphological changes of the testis tissue was observed by HE staining, the integrity of BTB measured with the biotracer, and the expressions of the BTB components ZO-1 and N-Cadherin proteins detected by Western blot. The TM4 Sertoli cells were treated with CdCl2at 0, 2.5, 5 and 10 µmol/L respectively for 24 hours, followed by determination of the expression levels of ZO-1 and N-Cadherin as well as the autophagy-related proteins LC3II and p62. Then the cells were again treated with CdCl2 in the presence of the autophagy inhibitor chloroquine (CQ) at 5 µmol/L or the autophagy inducer rapamycin (Rap) at 50 nmol/L for 24 hours, followed by measurement of the expressions of LC3II, p62, ZO-1 and N-Cadherin by Western blot. RESULTS: Compared with the control group, the cadmium-exposed mice showed increased interstitial space in the seminiferous tubules, formation of intracellular cavitation in the germ cells with decreased layers and disordered arrangement, and damaged integrity of the BTB. The expressions of the ZO-1 and N-Cadherin proteins were significantly down-regulated in the testis tissue of the mice in the medium- and high-dose CdCl2 groups (P < 0.05), and even more significantly in the CdCl2-exposed cells in comparison with those in the control mice (P < 0.01), while the expressions of the LC3II and p62 proteins were remarkably up-regulated (P < 0.05). The expressions of ZO-1, N-Cadherin, LC3II and p62 were also up-regulated in the cells co-treated with CQ and CdCl2 (P < 0.01), those of ZO-1, N-Cadherin and p62 down-regulated (P< 0.05) and that of LC3II up-regulated (P < 0.05) in the cells co-treated with Rap and CdCl2. CONCLUSION: CdCl2 can damage the integrity of the mouse BTB, which may be attributed to its ability to enhance the autophagy in Sertoli cells and regulate the expressions of BTB proteins.

Non-Lethal Concentrations of CdCl2 Cause Marked Alternations in Cellular Stress Responses within Exposed Sertoli Cell Line.

Cadmium is a component of ambient metal pollution, which is linked to diverse health issues globally, including male reproductive impairment. Assessments of the acute effects of cadmium on male reproduction systems, such as testes, tend to be based on frank adverse effects, with particular molecular pathways also involved. The relationship between cytotoxicity potential and cellular stress response has been suggested to be one of the many possible drivers of the acute effects of cadmium, but the link remains uncertain. In consequence, there is still much to be learned about the cellular stress response induced by a non-lethal concentration of cadmium in male reproductive cells. The present study used temporal assays to evaluate cellular stress response upon exposure to non-lethal concentrations of Cadmium chloride (CdCl2) in the Sertoli cell line (TM4). The data showed alternations in the expression of genes intimated involved in various cellular stress responses, including endoplasmic reticulum (ER) stress, endoplasmic unfolded protein stress (UPRmt), endoplasmic dynamics, Nrf2-related antioxidative response, autophagy, and metallothionein (MT) expression. Furthermore, these cellular responses interacted and were tightly related to oxidative stress. Thus, the non-lethal concentration of cadmium perturbed the homeostasis of the Sertoli cell line by inducing pleiotropic cellular stresses.

[Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells].

OBJECTIVE: To study the effect of reactive oxygen species(ROS) in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells) and explore the underlying molecular mechanisms. METHODS: TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 µmol/L) for 24 h. CCK-8 assay was used to detect the effect of CdCl_2 on TM3 cell activity. Hoechst33342 staining was performed to explore the formation of apoptotic bodies. DCFH-DA probe was used to detect the level of ROS in the cells. TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10 µmol/L CdCl_2 for 24 h. The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot; RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3. RESULTS: After exposure to CdCl_2 for 24 h, viability of TM3 cells decreased and the number of apoptotic bodies increased. Western blot result showed that the protein level of Caspase-9 in the 10 µmol/L CdCl_2 treatment group was increased to 0.86±0.10(P<0.05) compared with the control group(0.56±0.07). Compared with the control group(0.37±0.11), the protein level of cleaved Caspase-3 in the 5 and 10 µmol/L CdCl_2 treatment groups were increased to 0.65±0.03 and 1.05±0.13(P<0.05). Compared with the control group(46.80±1.24), the intracellular ROS content in the 5 and 10 µmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P<0.05). Compared with the cadmium-treated group, NAC inhibited Caspase-9(CdCl_2 group: 0.89±0.07; CdCl_2+NAC group: 0.28±0.02)and cleaved Caspase-3(CdCl_2 group: 1.53±0.21; CdCl_2+NAC group: 0.66 ±0.07), the difference was statistically significant(P<0.05). At the same time, NAC decreased the ROS level(62.64±0.93) in the CdCl_2 exposure group(80.13±0.94)(P<0.05). In addition, RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10 µmol/L CdCl_2 treatment groups were 1.40±0.14 and 1.90±0.12(P<0.05), compared with the control group(0.97±0.10). Compared with the control group(0.88±0.08), the cleaved Caspase-3 mRNA levels in the 5 and 10 µmol/L CdCl_2 treatment groups were increased to 1.42±0.11 and 1.59±0.12(P<0.05). While in the 5 and 10 µmol/L CdCl_2-treated group, compared with the control group(0.94±0.02), the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P<0.05). Compared with the cadmium treatment group(0.57±0.06), NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03), and Caspase-9(CdCl_2 group: 1.96±0.07; CdCl_2+NAC group: 1.04±0.02) and Caspase-3(CdCl_2 group: 1.65±0.02; CdCl_2+NAC group: 0.66±0.04) were decreased(P<0.05). CONCLUSION: The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_2, and inhibition of ROS production can significantly reduce the CdCl_2-induced apoptosis of TM3 cells.