Assessment of the toxicity of different antiretroviral drugs and their combinations on Sertoli and Leydig cells.

The human immunodeficiency virus continues to pose a significant global public health challenge, affecting millions of individuals. The current treatment strategy has incorporated the utilization of combinations of antiretroviral drugs. The administration of these drugs is associated with many deleterious consequences on several physiological systems, notably the reproductive system. This study aimed to assess the toxic effects of abacavir sulfate, ritonavir, nevirapine, and zidovudine, as well as their combinations, on TM3 Leydig and TM4 Sertoli cells. The cell viability was gauged using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays. Reactive oxygen species (ROS) production was assessed via the 2',7'-dichlorofluorescein diacetate (DCFDA) test, and DNA damage was determined using the comet assay. Results indicated cytotoxic effects at low drug concentrations, both individually and combined. The administration of drugs, individually and in combination, resulted in the production of ROS and caused damage to the DNA at the tested concentrations. In conclusion, the results of this study suggest that the administration of antiretroviral drugs can lead to testicular toxicity by promoting the generation of ROS and DNA damage. Furthermore, it should be noted that the toxicity of antiretroviral drug combinations was shown to be higher compared to that of individual drugs.

Adhesion and morphology of mammalian cells on nanoporous and nonporous spherical carbon substrates.

Three spherical activated carbons (SACs) were used as substrates for mammalian cell proliferation. SACs were obtained by carbonizing styrene-co-divinylbenzene ion exchangers 35WET, XAD4, or 1200H. The new materials (XAD_C, WET_C, and H_C) were characterized by adsorption-desorption nitrogen isotherms and mercury intrusion porosimetry. XAD_C and WET_C exhibited well-developed BET surface areas, similar total pore volumes, and highly different pore size distributions. H_C was nonporous spherical material-reference material. The XAD_C was meso-macroporous, but the WET_C was micro-mesoporous. All SACs were not cytotoxic toward Leydig TM3 cells. The differences in porous structure and morphology of the carbon scaffolds led to morphological differences in adhered cells. The monolayer of cells was distributed flat over the entire WET_C and H_C surfaces. Leydig TM3 cells adhered to nonporous SAC but were easily washed out due to weak adhesion. The cells adhered in clusters to XAD_C and proliferated in clusters. As microscopic techniques and viability tests demonstrated, only nanoporous carbons provided a good surface for the attachment and proliferation of eukaryotic cells.

[Analysis the mechanism of cadmium-induced cytotoxicity in mouse testicular mesenchymal cells with miRNA transcriptomic].

OBJECTIVE: To analyze the mechanism of cadmium-induced cytotoxicity in mouse testicular mesenchymal cells(TM3) with transcriptome sequencing and bioinformatics techniques. METHODS: TM3 cells were selected as the cell model and divided into control group(no cadmium treatment) and cadmium-treated group(20 µmol/L CdCl_2). After 24 hours of administration, cells were harvested to extract total RNA, and then miRNA expression profiles were obtained by sequencing program after RNA quality detection. The fold change(FC) >2, P<0.05 was used as the standard to screen for differentially expressed miRNAs. The quantitative real-time polymerase chain reaction(qRT-PCR) was used to verify the differentially expressed miRNAs. Then, their target genes were predicted by miRanda software to construct miRNA-target gene interaction network, and their target genes were enriched by gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) pathway function. RESULTS: A total of 26 differentially expressed miRNAs were identified which may be related to cadmium-induced TM3 cytotoxicity, including 19 up-regulated and 7 down-regulated miRNAs. The result of qRT-PCR were consistent with the miRNA sequencing result. Meanwhile, bioinformatics analysis result showed that the 26 differentially expressed miRNAs predicted 657 target genes. GO enrichment was mainly classified into biological regulation, metabolic process, protein binding and catalytic activity. KEGG pathway analysis showed that target genes were significantly involved in mitogen-activated protein kinase(MAPK) and tumor necrosis factor(TNF) signal pathways closely related to inflammatory response and apoptosis. CONCLUSION: Cadmium can lead to the differential expression of miRNAs in TM3 cells, and its target genes may be involved in Cd-induced TM3 cytotoxicity through signaling pathways such as MAPK and TNF.

Melatonin inhibits autophagy in TM3 cells via AKT/FOXO1 pathway.

BACKGROUND: Melatonin can regulate apoptosis and autophagy of mouse Leydig cells, but its specific mechanism is still unclear. METHODS: In this study, we used the TM3 cell line as the research object, and used H2O2 to induce autophagy. After adding 10 ng/ml melatonin, we used qRT-PCR and western-blot to detect autophagy-related gene and protein expression, and flow cytometry to detect cellular ROS level. RESULTS: The results showed that melatonin can significantly inhibit the occurrence of autophagy, accompanied by a significant decrease in the expression of Becn1, LC3, and FOXO1 (P < 0.05), a significant increase in the expression of p62 and pAKT (P < 0.05), and a significant decrease in ROS level (P < 0.05). After added the inhibitor of AKT perifosine, the effect of melatonin on inhibiting autophagy was reversed. On this basis, we used small RNA interference technology to knock down the expression of FOXO1, and found that there was no significant change of the expression of genes and proteins related to autophagy and ROS level. CONCLUSIONS: In summary, melatonin can inhibit H2O2-induced autophagy in TM3 cells through the AKT/FOXO1 pathway.

Biogenic silver nanoparticles (AgNp-Bio) restore testosterone levels and increase TNF-α and IL-6 in Leydig cells infected with Toxoplasma gondii.

Toxoplasma gondii, a protozoan parasite, is responsible for toxoplasmosis. The available therapy for patients with toxoplasmosis involves a combination of pyrimethamine and sulfadiazine, which have several adverse effects, including bone marrow suppression, megaloblastic anemia, leukopenia, and granulocytopenia. The development of therapeutic alternatives is essential for the management of toxoplasmosis, emphasizing the recent advances in nanomedicine. This study aimed to evaluate the in vitro effects of biogenic silver nanoparticles (AgNp-Bio) on tachyzoite forms and Leydig cells infected with T. gondii. We observed that the AgNp-Bio reduced the viability of the tachyzoites and did not exhibit cytotoxicity against Leydig cells at low concentrations. Additionally, treatment with AgNp-Bio reduced the rate of infection and proliferation of the parasite, and lowered the testosterone levels in the infected cells. It increased the levels of IL-6 and TNF-α and reduced the levels of IL- 10. Among the morphological and ultrastructural changes, AgNp-Bio induced a reduction in the number of intracellular tachyzoites and caused changes in the tachyzoites with accumulation of autophagic vacuoles and a decrease in the number of tachyzoites inside the parasitophorous vacuoles. Collectively, our data demonstrate that the AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and inflammatory mechanisms and could be a potential alternative treatment for toxoplasmosis.

Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells.

The purpose of this study was to verify whether the nanosecond pulsed electric field, not eliciting thermal effects, permanently changes the molecular processes and gene expression of Leydig TM3 cells. The cells were exposed to a moderate electric field (80 quasi-rectangular shape pulses, 60 ns pulse width, and an electric field of 14 kV/cm). The putative disturbances were recorded over 24 h. After exposure to the nanosecond pulsed electric field, a 19% increase in cell diameter, a loss of microvilli, and a 70% reduction in cell adhesion were observed. Some cells showed the nonapoptotic externalization of phosphatidylserine through the pores in the plasma membrane. The cell proportion in the subG1 phase increased by 8% at the expense of the S and G2/M phases, and the DNA was fragmented in a small proportion of the cells. The membrane mitochondrial potential and superoxide content decreased by 37% and 23%, respectively. Microarray's transcriptome analysis demonstrated a negative transient effect on the expression of genes involved in oxidative phosphorylation, DNA repair, cell proliferation, and the overexpression of plasma membrane proteins. We conclude that nanosecond pulsed electric field affected the physiology and gene expression of TM3 cells transiently, with a noticeable heterogeneity of cellular responses.

Lead acetate induces apoptosis in Leydig cells by activating PPARγ/caspase-3/PARP pathway.

This study was designed to investigate the cytotoxicity of lead acetate (Pb(AC)2, a representative air pollutant) by focusing on PPARγ/caspase-3/PARP apoptotic signaling pathway and to explore the inhibitory effect of PPARγ antagonist on apoptosis of TM3 Leydig cells. MTT assay was utilized to examine cell viability. Cell apoptosis was analyzed using a flow cytometry by staining with Annexin V-PE/7AAD staining and a fluorescence microscope by staining with Hoechst 33,258. The levels of apoptosis-related proteins were examined using western blot. From the results, Pb reduced significantly TM3 cell proliferation in concentration- and time-dependent manner. It increased significantly apoptosis; increased the PPARγ, Bax, procaspase-3, cleaved caspase-3, proPARP, cleaved PARP levels; and decreased Bcl-2 level in Pb-treated TM3 cells as compared to control cells. Furthermore, pretreatment with PPARγ antagonist significantly attenuated the apoptosis and cleavage of caspase-3 and PARP induced by Pb. Our results suggested that Pb induced cytotoxicity on TM3 Leydig cells, at least in part, by increasing PPARγ expression, stimulating cleavage of caspase-3 and PARP, and then induced cell apoptosis.

Cadmium-induced apoptosis through reactive oxygen species-mediated mitochondrial oxidative stress and the JNK signaling pathway in TM3 cells, a model of mouse Leydig cells.

Cadmium (Cd) is a heavy metal that widely exists in the environment and industry, and which causes serious damages to reproductive system. Recent studies have reported that cadmium induces apoptosis of various germ cells in testes, resulting in male infertility. However, the exact mechanism of cadmium-induced apoptosis remains unclear. In this study, we hypothesized that reactive oxygen species (ROS)-mediated c-jun N-terminal kinase (JNK) signaling pathway was involved in cadmium-induced apoptosis in TM3 cells, a model of mouse Leydig cells. TM3 cells were exposed for various times to a range of cadmium concentrations. We found that cadmium reduced TM3 cell viability and increased apoptosis in a time- and dose- dependent manner. Moreover, the levels of ROS generation and the phosphorylation of JNK were elevated by cadmium treatment. In addition, the nuclear transcription factor c-jun was significantly activated, which led to increased expression of downstream c-jun targets and Bcl-2 was decreased, accompanied with downstream activation of apoptosis-related proteins such as Cleaved-Caspase3 and Cleaved-PARP. However, pretreatment with the ROS inhibitor N-acetyl-L-cysteine (NAC) and JNK inhibitor JNK-IN-8, ROS, JNK and cadmium-induced TM3 cell apoptosis were remarkably suppressed. Based on above-mentioned results, this study provides a mechanistic understanding of cadmium induced TM3 cell apoptosis through the ROS/JNK signaling pathways.

MicroRNA profiling reveals the role of miR-133b-3p in promoting apoptosis and inhibiting cell proliferation and testosterone synthesis in mouse TM3 cells.

Late-onset hypogonadism (LOH) is an age-related clinical and biological syndrome in which serum testosterone deficiency is an important characteristic and diagnostic indicator. In this study, we firstly analyzed the difference in the expression level of three miR-133 s (including miR-133a-3p, miR-133a-5p, and miR-133b-3p) in rat testis samples, blood samples from mice before and 1 wk after testis removal, and mouse TM3 cells. Secondly, the mimics and inhibitors corresponding to the three miR-133 s of mouse were transfected into TM3 cells separately to determine the correlation between the three miRNAs. Finally, using mouse TM3 cells to analyze the effect of miR-133b overexpression or inhibition on the proliferation and apoptosis of mouse testicular Leydig cells, the effect on genes related to testosterone synthesis, and the effect on the level of testosterone in the culture medium. We found that, compared with the testis tissue of newborn rats, miR-133a-5p was increased in adult rats, and miR-133a-3p and miR-133b-3p were decreased. In addition, 1 wk after the testis was removed, the expression levels of these three miRNAs in the blood of adult mice decreased. The correlation of the three miRNAs was summarized, and it was found that miR-133b-3p played an important role in it. In TM3 cells, overexpression of miR-133b-3p suppressed the proliferation and promotes apoptosis of cells, suppressed the expression level of most genes related to cell proliferation and testosterone synthesis, and the concentration of testosterone in the culture medium decreased while these phenomena can be reversed by the inhibition of miR-133b-3p expression. It was found that miR-133b-3p can regulate testosterone production in TM3 cells at least by targeting FSCN1. The above results suggest that miR-133b-3p plays an important role in regulating testosterone synthesis. These findings also provide new candidate diagnostic indicators for late-onset hypogonadism in men and provide new clues for the further study of pathogenesis.

Development of a chronic focal equine arteritis virus infection of a male reproductive tract cell line.

Equine arteritis virus (EAV) is an Alphaarterivirus (family Arteriviridae, order Nidovirales) that frequently causes an influenza-like illness in adult horses, but can also cause the abortions in mares and death of newborn foals. Once primary infection has been established, EAV can persist in the reproductive tract of some stallions. However, the mechanisms enabling this persistence, which depends on testosterone, remain largely unknown. We aimed to establish an in vitro model of non-cytopathic EAV infection to study viral persistence. In this work, we infected several cell lines originating from the male reproductive tract of different species. EAV infection was fully cytopathic for 92BR (donkey cells) and DDT1 MF-2 (hamster cells) cells, and less cytopathic for PC-3 cells (human cells); ST cells (porcine cells) seemed to eliminate the virus; LNCaP (human cells) and GC-1 spg (murine cells) cells were not permissive to EAV infection; finally, TM3 cells (murine cells) were permissive to EAV infection without any overt cytopathic effects. Infected TM3 cells can be maintained at least 7 days in culture without any subculture. They can also be subcultured over 39 days (subculturing them at 1:2 the first time at 5 dpi and then every 2-3 days), but in this case, the percentage of infected cells remains low. Infected TM3 cells may therefore provide a new model to study the host-pathogen interactions and to help determine the mechanisms involved in EAV persistence in stallion reproductive tract.

Nesfatin-1 regulates steroidogenesis in mouse Leydig cells.

Nesfatin-1 is a polypeptide hormone known to regulate appetite and energy metabolism and is derived from the precursor protein nucleobindin 2 (NUCB2). Recent studies have shown that nesfatin-1 is expressed in many peripheral tissues in mice, including the reproductive organs. However, its function and regulation in the testis remain unknown. In this study, we investigated the expression of Nucb2 mRNA and nesfatin-1 protein in mouse Leydig cells and the Leydig cell line, TM3 cells. We also examined whether Nucb2 mRNA expression is regulated by gonadotropins and whether exogenous nesfatin-1 affects steroidogenesis in primary Leydig cells isolated from the testis and TM3 cells. We found that Nucb2 mRNA and nesfatin-1 protein were present in primary Leydig cells and TM3 cells, and nesfatin-1 binding sites were also found in both cell types. Nucb2 mRNA expression in testis, primary Leydig cells, and TM3 cells was increased after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin. After nesfatin-1 treatment, the expression of steroidogenesis-related enzyme genes Cyp17a1 and Hsd3b was upregulated in primary Leydig cells and TM3 cells. Our results suggest that NUCB2/nesfatin-1 expression in mouse Leydig cells may be regulated through the hypothalamic-pituitary-gonadal axis and that nesfatin-1 produced by Leydig cells may locally regulate steroidogenesis in an autocrine manner. This study provides insight into the regulation of NUCB2/nesfatin-1 expression in Leydig cells and the effect of nesfatin-1 on steroidogenesis, which may have implications for male reproductive health.

Downregulation of microRNA-96-5p protects TM3 cells against zearalenone toxicity via targeting ATG9A.

Male infertility factor accounts for ~50% of all infertility cases, and traditional treatments for male infertility are limited. The association between the dysfunction of Leydig cells and hypospermatogenesis is essential for developing novel treatment methods for male infertility. It was previously stated that elevated expression of microRNA (miR)-96-5p was associated with the toxicological response of Leydig cells to treatment with zearalenone (ZEN). However, the exact role of miR-96-5p in Leydig cells remains to be illustrated. The mouse Leydig cell line TM3 was used in the present study to investigate the role of miR-96-5p. ZEN was used to induce cell injury in TM3 cells. Cell Counting Kit-8 assay and the Ki67 staining method were used to evaluate cell viability. Reverse transcription-quantitative PCR was used to determine the expression levels of miR-96-5p. In addition, a dual luciferase assay was used to investigate the target of miR-96-5p. Annexin V/propidium iodide staining was performed to detect cell apoptosis. Western blot analysis was used to detect the expression levels of certain proteins. Finally, monodansylcadaverine (MDC) and LC3 staining were applied for monitoring the level of autophagy. ZEN inhibited the proliferation of TM3 cells in a dose-dependent manner. In addition, the level of miR-96-5p were significantly increased in ZEN-treated TM3 cells. Meanwhile, inhibition of miR-96-5p could reverse ZEN-induced decrease in viability in TM3 cells. Moreover, ZEN notably inhibited autophagy in TM3 cells and this phenomenon was reversed by the application of the miR-96-5p inhibitor. Autophagy related 9A (ATG9A) was identified as the biological target of miR-96-5p. The results derived from MDC and LC3 staining demonstrated that downregulation of miR-96-5p expression levels protected TM3 cells against ZEN toxicity by regulating autophagy. Inhibition of miR-96-5p expression protected TM3 cells against ZEN via targeting ATG9A. Therefore, miR-96-5p may serve as a potential biomarker for male infertility.

Zinc attenuates ecstasy-induced apoptosis through downregulation of caspase-3 in cultured TM3 cells: An experimental study.

BACKGROUND: 3, 4-Methylenedioxymethamphetamine (MDMA) is commonly known as the most famous amphetamine derivative. OBJECTIVE: To evaluate the influence of zinc on MDMA-induced apoptosis and caspase- 3 gene expression in Leydig cell line (TM3). MATERIALS AND METHODS: Leydig cells were studied in differenet treatment groups regarding MDMA (0, 0.5, 1, 3, 5 mM) and zinc (0, 4, 8, 16, 32 µM). By the way, the effective concentration was determined to be 5 mM for MDMA and 8 µM for zinc. Then, TM3 cells were cultured in free medium as control (group I), medium containing MDMA (5 mM) (group II), zinc (8 µM) (group III), and zinc (8 µM) prior to MDMA (5 mM) (group IV) as well as in an untreated group (control). Cell viability was assessed at different times after cell culture by MTT assay. The mRNA expression level of caspase-3 was analyzed using real-time quantitative polymerase chain reaction. RESULTS: The cellular viability was significantly reduced in TM3 cells after 24 hr and 48 hr exposure time regarding different concentrations of MDMA as well as high concentration of zinc (16 and 32 µM). Cell viability was increased in the group that received zinc (8 µM) before addition of MDMA (5 mM) compared to the control and MDMA groups. The mean ± SE of fold was 22.40 ± 7.5, 0.06 ± 0.02, and 0.009 ± 0.003 in MDMA, zinc, and zinc + MDMA groups, respectively. The mean of caspase-3 mRNA level was significantly increased in the MDMA-treated group (5 mM), while the relative expression of caspase-3 gene was significantly decreased in the zinc (8 µM) + MDMA (5 mM) group compared with the MDMA (5 mM) group (p = 0.001). CONCLUSION: Dietary intake of zinc has a protective effect against MDMA consumption in mouse.

T-2 toxin-induced DRP-1-dependent mitophagy leads to the apoptosis of mice Leydig cells (TM3).

T-2 toxin, one member of the type A trichothecene family, induces the apoptosis of human hepatocytes (L02) and murine Leydig cells (TM3), as well as mitochondrial dysfunctions. In the present study, we attempted to investigate whether T-2 toxin toxicity is related to mitochondrial dysfunction and mitophagy. We found that T-2 toxin might induce autophagy and mitophagy in TM3 cells (TM3) in a concentration-dependent manner. In addition, T-2 toxin could induce mitochondrial dysfunction, depolarization, and fission concentration-dependently. The inducible effects of T-2 toxin on mitophagy, mitochondrial dysfunction, and cell apoptosis could all be significantly reversed by autophagy inhibitor, 3 MA. Finally, DRP-1 participated in the inducible effects of T-2 toxin on TM3 cell mitophagy, mitochondrial dysfunction, and cell apoptosis. In summary, mitophagy and mitochondrial dysfunction are essential mechanisms of the toxicity induced by T-2 toxin. Thus, our findings provide a rationale for further studies on selectively targeting mitophagy to improve mitochondrial dysfunction and to protect cells from T-2 toxin-induced toxicity.

Regulation of oncogenes and gap junction intercellular communication during the proliferative response of zearalenone in TM3 cells.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced by Fusarium species. The exposure risk to humans and animals is the consumption of contaminated food and animal feeds. The aim of this study was to investigate ZEA-induced effects and its tumorigenic mechanism in TM3 cells (mouse Leydig cells). Cell proliferation, apoptosis, and gap junction intercellular communication (GJIC) were assessed in this study. Results showed that low concentrations of ZEA could significantly promote the growth of TM3 cells. The percentage of cell distribution was decreased significantly in G1/G0 phase and was increased significantly in S phase with 10 and 20 µg/L of ZEA for 72 h ( p < 0.05, p < 0.01). The expressions of cyclin D1 and Cdk4 were significantly increased in the exposure groups compared with the control group ( p < 0.05, p < 0.01). Compared with the control group, the apoptosis was significantly decreased in 10 and 20 µg/L groups ( p < 0.01), and the ratio of Bax/Bcl-2 protein level was significantly decreased in a dose-dependent manner. The protein levels of proto-oncogene c-Myc, c-Jun, and c-Fos were significantly elevated and the protein levels of anti-oncogene p53 and phosphatase and tensin homolog (PTEN) were decreased obviously compared with the control group ( p < 0.05, p < 0.01). ZEA affected the expressions of connexins and inhibited the activity of GJIC. These results demonstrated that ZEA can disturb the dynamic balance between proliferation and apoptosis and causes abnormal regulation of oncogenes, GJIC, and connexins in TM3 cells, which may easily induce the translation of normal cells into tumor cells.

Endogenous inhibins regulate steroidogenesis in mouse TM3 Leydig cells by altering SMAD2 signalling.

This study tested the hypothesis that inhibins act in an autocrine manner on Leydig cells using a pre-pubertal Leydig cell line, TM3, as a model of immature Leydig cells. The expression of Inha, Inhba, and Inhbb in TM3 cells was determined by RT-PCR and the production of the inhibin-alpha subunit was confirmed by western blot. Knockdown of Inha expression resulted in significant decreases in the expression of Leydig cell markers Cyp17a1, Cyp11a1, Nr5a1, and Insl3. Western blot showed that activin A, TGFß1 and TGFß2 activated SMAD2, and that knockdown of Inha expression in TM3 cells enhanced both activin A- and TGFß-induced SMAD2 activation. SB431542, a chemical inhibitor of the TGFß/activin type I receptors, blocked ligand-induced SMAD2 activation and the downregulation of Cyp17a1 expression. Our findings demonstrate that TGFßs and activin A negatively regulate steroidogenic gene expression in TM3 cells via ALK4/5 and SMAD2 and endogenous inhibins can counter this regulation.