[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.

PM2.5 mediates mouse testis Sertoli TM4 cell damage by reducing cellular NAD.

OBJECTIVE: This study aims to explore the mechanism of PM2.5 damage to the reproductive system of male mice. METHODS: Mouse testis Sertoli TM4 cells were divided into four groups: a control group (no additional ingredients except for medium), PM2.5 group (medium containing 100 µg/mL PM2.5), PM2.5 + NAM group (medium containing 100 µg/mL PM2.5 and 5 mM NAM), and NAM group (medium containing 5 mM nicotinamide) and cultured in vitro for 24 or 48 h. The apoptosis rate of TM4 cells was measured using flow cytometry, the intracellular levels of NAD+ and NADH were detected using an NAD+/NADH assay kit, and the protein expression levels of SIRT1 and PARP1 were determined by western blotting. RESULTS: Mouse testis Sertoli TM4 cells exposed to PM2.5 demonstrated an increase in the apoptosis rate and PARP1 protein expression, albeit a decrease in NAD+, NADH, and SIRT1 protein levels (p = 0.05). These changes were reversed in the group treated with a combination of PM2.5 and nicotinamide (p = 0.05). CONCLUSION: PM2.5 can cause Sertoli TM4 cell damage in mouse testes by decreasing intracellular NAD+ levels.

Effects of cadmium chloride on testicular autophagy and blood-testis barrier integrity in prepubertal male rats / 中华劳动卫生职业病杂志

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.

Assessment of Zearalenone-Induced Cell Survival and of Global Gene Regulation in Mouse TM4 Sertoli Cells.

Zearalenone (ZEA) is a non-steroidal xenoestrogen mycotoxin produced by many Fusarium fungal species, which are common contaminants of cereal crops destined for worldwide human and animal consumption. ZEA has been reported in various male reproduction dysfonctions, including decreased fertility potential. In this report, the direct effect of ZEA on the immature Sertoli TM4 cell line was evaluated. The results show that high concentrations of ZEA increase reactive oxygen species via the activation of MAPK signaling. Transcriptome analysis was performed on the TM4 cell line treated with ZEA, and genes involved in sex differentiation (Fgfr2, Igf1, Notch1, Sox9) and extracellular matrix (ECM) formation (Ctgf, Fam20a, Fbn1, Mmp9, Postn, Sparcl1, Spp1) were identified at the center of the functional protein association network, suggesting that ZEA could be detrimental to the early steps of Sertoli cell differentiation.

Flurochloridone-induced apoptosis via IRE1α-JNK signaling pathway in mice testicular cells and TM4 cells / 环境与职业医学

Background Flurochloridone (FLC) can induce apoptosis in Sertoli cells, but the specific mechanism remains unknown. Objective To investigate the testicular cell apoptosis in mice as well as apoptosis and activation of endoplasmic reticulum stress in TM4 cell line induced by FLC through in vivo and in vitro study designs respectively, and study the role of inosital-requiring enzyme 1α (IRE1α)-c-Jun N-terminal kinase (JNK) signaling pathway in the process of FLC-induced apoptosis in TM4 cells through intervention study design. Methods Testicular tissues were collected from male C57BL/6 mice which were treated with 3, 15, 75, and 375 mg·（kg·d）−1 FLC by oral perfusion for 28 d. Apoptosis was observed by TUNEL staining, and the levels of apoptosis-related proteins were detected by Western blotting, including B-cell lymphoma-2 (Bcl-2), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2 associated X protein (Bax). In the in vitro study, TM4 cells were treated with different concentrations of FLC (40, 80, and 160 μmol·L−1) for 6 h, then apoptosis rate was detected by flow cytometry, and the levels of apoptosis-related proteins (Bcl-2, Bim, and Bax) and endoplasmic reticulum stress-related proteins [glucose regulated protein 78 (GRP78), phosphorylated-protein kinase R like endoplasmic reticulum kinase (p-PERK), activating transcription factor 6 (ATF6), phosphorylated-inosital-requiring enzyme 1α (p-IRE1α), and phosphorylated-JNK (p-JNK)] were measured by Western blotting. In the intervention study, TM4 cells were pretreated with IRE1α phosphorylation inhibitor 4μ8C and JNK phosphorylation inhibitor SP600125 for 6 h, then treated with 160 μmol·L−1 FLC for 6 h. The levels of apoptosis-related proteins and endoplasmic reticulum stress-related proteins were measured by Western blotting, and cell viability was detected by cell counting kit-8. Results After the male C57BL/6 mice orally exposed to FLC for 28 d, apoptosis occurred in the seminiferous tubule. The protein expression level of Bcl-2, apoptosis inhibitor, was decreased in the 75 and 375 mg·(kg·d)−1 groups (P<0.05), and the protein expression levels of Bim and Bax, apoptosis promoters, were increased in the 75 and 375 mg·(kg·d)−1 groups respectively (P<0.05). The percentages of apoptotic cells in the 0, 40, 80, and 160 μmol·L−1 FLC groups were 2.7%±0.2%, 4.8%±1.3%, 9.4%±0.3%, and 13.2%±0.2%, respectively, increased significantly compared with the control group (P<0.05). The protein expression level of Bcl-2 also was decreased in the 160 μmol·L−1 FLC group (P<0.05), while the levels of Bim and Bax were increased in both of the 80 and 160 μmol·L−1 groups (P<0.05). The expression levels of endoplasmic reticulum stress-related proteins (GRP78, p-PERK, ATF6, p-IRE1α, and p-JNK) were increased (P<0.05) or showed a rising trend in TM4 cells. Pre-treatment with 4μ8C (25 and 50 μmol·L−1) and SP600125 (10 and 20 μmol·L−1) significantly down-regulated the protein expression levels of GRP78, p-IRE1α, p-JNK, and Bax induced by FLC (P<0.05) or in a downward trend. Both of the inhibitors alleviated the decreased cell viability induced by FLC (P<0.05) or in alleviating fashion. Conclusion FLC could induce apoptosis in mice testis and TM4 cell apoptosis through activating endoplasmic reticulum stress and IRE1α-JNK signaling pathway.

Titanium dioxide nanoparticles perturb the blood-testis barrier via disruption of actin-based cell adhesive function.

As one of the most commonly used nanoparticles, titanium dioxide nanoparticles (TiO2-NPs) are widely used as coating reagents in cosmetics, medicine and other industries. The increasing risk of exposure to TiO2-NPs raises concerns about their safety. In this study, we investigated the mechanism by which TiO2-NPs cross the blood-testis barrier (BTB). TM-4 cells were selected as an in vitro Sertoli cell model of BTB. Cell viability, cell morphological changes, apoptosis, oxidative damage, and the expression levels of actin regulatory and tight junction (TJ) proteins were assessed in TM-4 cells treated with 3-nm and 24-nm TiO2-NPs. Cells treated with 3-nm TiO2-NPs exhibited increased cytotoxicity and decreased Annexin II expression, whereas cells treated with 24-nm TiO2-NPs exhibited increased Arp 3 and c-Src expression. Both TiO2-NPs induced significant oxidative stress, decreased the expression of TJ proteins (occludin, ZO-1 and claudin 5), damaged the TJ structure, and exhibited enlarged gaps between TM-4 cells. Our results indicated that both TiO2-NPs crossed the BTB by disrupting actin-based adhesive junctions of TM-4 cells; however, apoptosis was not observed. Our results provide new insights into how TiO2-NPs cross the BTB.

[Deletion of extracellular loops of occludin affects the tight junction of TM4 cells in mice].

OBJECTIVE: To analyze the functions of the two extracellular loops of occludin in the tight junction of TM4 cells in mice. METHODS: Using genetic engineering, we separately or simultaneously deleted two extracellular loops of occludin, cloned the three occludin genes without extracellular loops into the pcDNA3.1 expression vector, and transfected them into TM4 cells. Then we determined the expression of occludin by RT-PCR and Western blot, and analyze the effects of the extracellular loops of occludin on the tight junction of the TM4 cells with the in vitro cell line model. RESULTS: The results of sequencing showed that the expression vector of pcDNA3.1 - occludin Δ OCC1, pcDNA3.1 - occludin Δ OCC2 and pcDNA3.1 - occludin Δ OCC1 + OCC2 was constructed successfully. The mRNA and protein expressions of occludin in the non-extracellular loop groups were significantly higher than in the control group. Both the extracellular loops of occludin increased the tight junction of the TM4 cells. The macromolecular permeability in the TM4 cells was significantly lower in the pcDNA3.1 - occludin Δ OCC1 than in the pcDNA3.1 - occludin Δ OCC2 group (P < 0.05), indicating a higher impact of the second than the first extracellular loop on the tight junction of the TM4 cells. CONCLUSIONS: Both of the two extracellular loops of occludin can affect the tight junction of TM4 cells, the second even more significantly than the first one.

Protective Mechanism of Sulforaphane on Cadmium-Induced Sertoli Cell Injury in Mice Testis via Nrf2/ARE Signaling Pathway.

The present study evaluated the mechanism underlying the protective effect of sulforaphane (SFN) on cadmium (Cd)-induced Sertoli cell (TM4 cells) injury in mice. The apoptosis rate of cells in each group was detected by flow cytometry. It was determined the effect of SFN on the expression of downstream molecular targets of Nrf2/ARE axis and on the lipid peroxide content. The related genes involved in the nuclear factor E2-related factor 2(Nrf2)/antioxidant response element (ARE) signaling pathway were evaluated by RT-PCR; for example, the mRNA expression levels of Nrf2, heme oxygenase-1 (HO-1), glutathione peroxidase (GSH-Px), quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS), while the protein expression levels were assessed by Western blot. Our results showed that the mRNA and protein expression levels of Nrf2, HO-1, NQO1, GSH-Px, and γ-GCS were increased in various degree when the Sertoli cells were to added different concentrations of SFN. Our results also showed that SFN reduced the apoptosis rate, increased the activity of T-SOD, inhibited the increase of the MDA content caused by Cd. Meanwhile, SFN could increase the mRNA and protein expression levels of Nrf2, HO-1 and NQO1 and reduced the mRNA and protein expression levels of GSH-Px and γ-GCS caused by Cd in Sertoli cells (p < 0.01). Taken together, SFN could improve the antioxidant capacity of Sertoli cells, and exert a protective effect on the oxidative damage and apoptosis of Cd-induced Sertoli cells through the activation of Nrf2/ARE signal transduction pathway.

Protective effect of proanthocyanidin on mice Sertoli cell apoptosis induced by zearalenone via the Nrf2/ARE signalling pathway.

This study evaluated the protective effect of proanthocyanidin (PC) on the cytotoxicity of the Sertoli cell TM4 of mice, as induced by zearalenone (ZEA). Flow cytometry was used to detect the apoptosis rate of cells in each group. The activities of antioxidant enzymes and the content of antioxidant substances were detected by using a proprietary kit; the RT-PCR method was used to detect the expression level of mRNA, the related genes of Nrf2/ARE signal pathway, the nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase (GSH-Px), quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase (γ-GCS) and the expression level of mRNA, the apoptosis-related genes, Bcl-2 and Bax; the Western-blot method was used to detect the protein expression levels of Nrf2, GSH-Px, HO-1, γ-GCS and NQO1 in each group. Our results showed that PC could reduce the apoptosis rate of the TM4 cells exposed to ZEA (p < 0.01); PC could enhance the decrease in the activities of T-SOD and GSH-Px induced by ZEA (p < 0.05), reduce the increase in the content of MDA, as caused by ZEA; PC could significantly up-regulate the down-regulation levels of the mRNA and protein of Nrf2, GSH-Px, HO-1, γ-GCS and NQO1 induced by ZEA. PC could enhance the decrease in the mRNA expression level of Bcl-2 and down-regulate the mRNA expression of Bax induced by ZEA (p < 0.05). These results demonstrated that PC conferred protective effects against oxidative damage and apoptosis of TM4 cells induced by ZEA. The protection mechanism of PC on TM4 cells might act through the activation of the Nrf2/ARE signalling pathway.

Mitogen-activated protein kinase and Akt pathways are involved in 4-n-nonyphenol induced apoptosis in mouse Sertoli TM4 cells.

Nonylphenol (NP) is considered an important environmental toxicant, which may disrupt male reproductive system. The aim of this study was to investigate 4-n-nonylphenol (4-n-NP) induced apoptosis and its related mechanism in mouse Sertoli cell line, TM4 cells. Our results showed that NP treatment (0.1, 1, 10, 20 and 30 µM) decreased cell viability and induced apoptosis in the cells, accompanied by alteration of Bcl-2 family mRNA expression, activation of caspases-3, release of Ca(2+), and increase of reactive oxygen species (ROS) generation. Subsequently, it was found that the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in the cells were markedly decreased, and maleic dialdehyde (MDA) content was increased by NP treatment. Then activation of the mitogen-activated protein kinases (MAPKs) pathways and inhibition of Akt pathway were simultaneously detected in NP challenged TM4 cells. Taken together, it was concluded that NP induced cytotoxicity and apoptosis in TM4 cells, and the apoptosis may be mediated via MAPKs and Akt pathways in addition to Ca(2+) release and ROS generation.

Effects of 4-nonylphenol isomers on cell receptors and mitogen-activated protein kinase pathway in mouse Sertoli TM4 cells.

In the present study, experiments were performed to investigate the effects of nonylphenol (NP) isomers (4-[1,2, 4-trimethylhexyl]-phenol (NP41), 4-[1,2, 5-trimethylhexyl]-phenol (NP42)) on Sertoli TM4 cells. NP41 decreased mRNA expression levels of androgen receptor and toll-like receptor (TLR)-4 in 20-40µM (P<0.05), and increased mRNA levels of estrogen receptor (ER)-α and progesterone receptor in 1-40µM (P<0.05). NP42 treatment only evoked significant decrease in mRNA expression levels of ER-α in 20-40µM (P<0.05). Similarly, NP41 (1-40µM) drastically increased the protein expression of ER-α, which was significantly decreased in 20-40µM NP42 groups (P<0.01). Both NP41 and NP42 showed no effect on the expression of ER-ß. Protein levels of follicle stimulating hormone receptor were increased significantly in high concentrations of NP41 (40µM) and NP42 (10-40µM) challenged cells. Furthermore, NP41 and NP42 showed various effects on the expression of junction-associated molecules and inhibin B secretion in TM4 cells. Additionally, activation of JNK1/2 pathway was induced by NP41 and NP42. However, ERK1/2 and p38 pathways were inhibited in TM4 cells exposed to low concentrations of NP41 (0.1-20µM) and NP42 (0.1-1µM), and high concentrations of NP41 (40µM) and NP42 (10-40µM) resulted in a return of p-ERK1/2 and p-p38 to control levels. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signaling pathways, and the effects may be related to the structure of NP isomer.

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: a comparative proteomic analysis.

BACKGROUND: Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study. METHODS: We utilized proteomic study to identify the protein expression changes of Sertoli TM4 cells treated with 10(-8)M and 10(-5)M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis. RESULTS: Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10(-8)M and 10(-5)M BPA, respectively, for 24h. Exposure to 10(-5)M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10(-8)M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages. CONCLUSIONS: Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism. GENERAL SIGNIFICANCE: Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.