[Ferroptosis is involved in testicular injury induced by TDCIPP in adolescent male mice].

Objective: To investigate the role of ferroptosis in testicular injury in adolescent male mice induced by TDCIPP. Methods: In December 2021, 30 healthy 3-week-old male C57BL/6 mice, with a body weight of (13±2) g, were selected and fed adaptive for one week. They were divided into control group, low-dose group, medium-dose group, high-dose group and iron death inhibitor group according to a random number table, with 6 mice in each group. Mice in low, medium and high dose groups were treated with 5, 25 and 125 mg/ (kg·d) TDCIPP for 28 days, respectively, while the control group was treated with the same amount of corn oil for 28 days. The iron death inhibitor group was given 125 mg/ (kg·d) TDCIPP intragastric administration for 28 days, and 30 mg/kg DFO saline solution was intraperitoneally injected three times a week. After the treatment, the mice were killed, the epididymis was separated, and sperm count was performed. HE staining was used to observe the morphological changes of mouse testis, and iron content in testis was detected by tissue iron detection kit. The level of reactive cxygen species, MDA content, and the mitochondrial membrane potential level of mice were detected. Western blot analysis of testicular glutathione peroxidase (GPX4) and internal cyclooxygenase-2 (COX2) protein expression. Results: Compared with the control group, the spermatogenic cells in the testes of mice treated with medium-and high-dose of TDCIPP were disorderly arranged, showing a vacuolar structure. the number of sperm in the epididymis was significantly reduced (P=0.009, 0.004), while the sperm deformity rate was significantly increased (P=0.010, 0.000). Moreover, the content of ROS, iron ion and MDA in the testes increased significantly (P<0.05), and the mitochondrial membrane potential of mouse testicular cells decreased significantly (P<0.05). The expression of GPX4 proteins decreased (P<0.05). while the expression of COX2 increased significantly (P<0.01). Compared with high-dose group group, spermatogenic cells in ferroptosis inhibitor group were closely arranged and normal, and ROS and Fe contents in testicular tissue were significantly decreased (P<0.01) ; GPX4 protein expression was significantly increased while COX2 protein expression was significantly decreased (P<0.05) . Conclusion: Ferroptosis is involved in TDCIPP-induced testicular damage in male pubertal mice.

[Effects of cadmium chloride on testicular autophagy and blood-testis barrier integrity in prepubertal male rats].

Objective: To study the effects of cadmium chloride (CdCl(2)) exposure on testicular autophagy levels and blood-testis barrier integrity in prepubertal male SD rats and testicular sertoli (TM4) cells. Methods: In July 2021, 9 4-week-old male SD rats were randomly divided into 3 groups: control group (normal saline), low dose group (1 mg/kg·bw CdCl(2)) and high dose group (2 mg/kg·bw CdCl(2)), and were exposed with CdCl(2) by intrabitoneal injection. 24 h later, HE staining was used to observe the morphological changes of testis of rats, biological tracer was used to observe the integrity of blood-testis barrier, and the expression levels of microtubule-associated protein light chain 3 (LC3) -Ⅰ and LC3-Ⅱ in testicular tissue were detected. TM4 cells were treated with 0, 2.5, 5.0 and 10.0 µmol/L CdCl(2) for 24 h to detect the toxic effect of cadmium. The cells were divided into blank group (no exposure), exposure group (10.0 µmol/L CdCl(2)), experimental group[10.0 µmol/L CdCl(2)+60.0 µmol/L 3-methyladenine (3-MA) ] and inhibitor group (60.0 µmol/L 3-MA). After 24 h of treatment, Western blot analysis was used to detect the expression levels of LC3-Ⅱ, ubiquitin binding protein p62, tight junction protein ZO-1 and adhesion junction protein N-cadherin. Results: The morphology and structure of testicular tissue in the high dose group were obvious changed, including uneven distribution of seminiferous tubules, irregular shape, thinning of seminiferous epithelium, loose structure, disordered arrangement of cells, abnormal deep staining of nuclei and vacuoles of Sertoli cells. The results of biological tracer method showed that the integrity of blood-testis barrier was damaged in the low and high dose group. Western blot results showed that compared with control group, the expression levels of LC3-Ⅱ in testicular tissue of rats in low and high dose groups were increased, the differences were statistically significant (P<0.05). Compared with the 0 µmol/L, after exposure to 5.0, 10.0 µmol/L CdCl(2), the expression levels of ZO-1 and N-cadherin in TM4 cells were significantly decreased, and the expression level of p62 and LC3-Ⅱ/LC3-Ⅰ were significantly increased, the differences were statistically significant (P<0.05). Compared with the exposure group, the relative expression level of p62 and LC3-Ⅱ/LC3-Ⅰ in TM4 cells of the experimental group were significantly decreased, while the relative expression levels of ZO-1 and N-cadherin were significantly increased, the differences were statistically significant (P<0.05) . Conclusion: The mechanism of the toxic effect of cadmium on the reproductive system of male SD rats may be related to the effect of the autophagy level of testicular tissue and the destruction of the blood-testis barrier integrity.