RESUMEN
Titanium dioxide nanoparticles (TiO2 NPs) can reduce sperm number, but the mechanisms of defective spermatogenesis induced by TiO2 NPs have not been studied through cell-cell interactions at present. A kind of biomimetic three-dimensional blood-testis barrier microfluidic chip capable of intercellular communication was constructed with soft lithography techniques, including Sertoli cell (TM4), spermatogonia (GC-1) and vascular endothelial cell units, to study the mechanisms of TiO2 NPs-induced defective spermatogenesis. TM4 and GC-1 cells cultured in TiO2 NPs exposure and control chips were collected for transcriptomics and metabonomics analysis, and key proteins and metabolites in changed biological processes were validated. In TM4 cells, TiO2 NPs suppressed glucose metabolism, especially lactate production, which reduced energy substrate supply for spermatogenesis. TiO2 NPs also decreased the levels of key proteins and metabolites of lactate production. In GC-1 cells, TiO2 NPs disturbed chemokine signaling pathways regulating cell proliferation and interfered with glutathione metabolism. The Cxcl13, Stat3 and p-Stat3 levels and cell proliferation rate were decreased, and the GSR, GPX4 and GSH contents were increased in GC-1 cells in chips under TiO2 NPs treatment. The decrease in energy substrate supply for spermatogenesis and inhibition of spermatogonia proliferation could be the main mechanisms of defective spermatogenesis induced by TiO2 NPs.
Asunto(s)
Barrera Hematotesticular , Células de Sertoli , Espermatogénesis , Espermatogonias , Titanio , Masculino , Titanio/toxicidad , Espermatogénesis/efectos de los fármacos , Animales , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Barrera Hematotesticular/efectos de los fármacos , Ratones , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Espermatogonias/patología , Línea Celular , Nanopartículas del Metal/toxicidad , Dispositivos Laboratorio en un Chip , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Comunicación Celular/efectos de los fármacosRESUMEN
Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5⯵M. Exposure to 50⯵M BBP, approximating the IC50 of 53.9⯵M, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50⯵M BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity.
Asunto(s)
Acetilcisteína , Adenina , Apoptosis , Autofagia , Proliferación Celular , Ácidos Ftálicos , Especies Reactivas de Oxígeno , Sesquiterpenos , Masculino , Animales , Ratones , Ácidos Ftálicos/toxicidad , Autofagia/efectos de los fármacos , Apoptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/farmacología , Acetilcisteína/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Adenina/toxicidad , Proliferación Celular/efectos de los fármacos , Línea Celular , Plastificantes/toxicidad , Espermatogonias/efectos de los fármacosRESUMEN
Infertility is intricately linked with alterations in circadian rhythms along with physiological decline and stem cell senescence. Yet, the direct involvement of circadian mechanisms in nicotine-induced injury to the testes, especially the senescence of spermatogonia stem cells (SSCs), is not well comprehended. This study revealed that nicotine exposure induced testis injury by triggering SSCs senescence along with the upregulation of senescence marker genes and senescence-associated secretory phenotype components. Moreover, nicotine treatment caused mitochondrial hyper-fusion, increased oxidative stress, and DNA damage. Exposure to nicotine was found to suppress the expression of sirtuin 6 (SIRT6), which accelerated the senescence of spermatogonia stem cells (SSCs). This acceleration led to increased acetylation of brain and muscle ARNT-like protein (Bmal1), consequently reducing the expression of Bmal1 protein. Conversely, the overexpression of Bmal1 alleviated mitochondrial hyper-fusion and senescence phenotypes induced by nicotine. Overall, this study unveiled a novel molecular mechanism behind nicotine-induced disorders in spermatogenesis and highlighted the SIRT6/Bmal1 regulatory pathway as a potential therapeutic target for combating nicotine-associated infertility.
Asunto(s)
Factores de Transcripción ARNTL , Senescencia Celular , Ritmo Circadiano , Dinámicas Mitocondriales , Nicotina , Sirtuinas , Sirtuinas/metabolismo , Sirtuinas/genética , Masculino , Animales , Nicotina/farmacología , Nicotina/efectos adversos , Senescencia Celular/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Ratones , Factores de Transcripción ARNTL/metabolismo , Factores de Transcripción ARNTL/genética , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Homeostasis/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/efectos de los fármacosRESUMEN
Diquat dibromide (DQ) is a globally used herbicide in agriculture, and its overuse poses an important public health issue, including male reproductive toxicity in mammals. However, the effects and molecular mechanisms of DQ on testes are limited. In vivo experiments, mice were intraperitoneally injected with 8 or 10â¯mg/kg/ day of DQ for 28 days. It has been found that heme oxygenase-1 (HO-1) mediates DQ-induced ferroptosis in mouse spermatogonia, thereby damaging testicular development and spermatogenesis. Histopathologically, we found that DQ exposure caused seminiferous tubule disorders, reduced germ cells, and increased sperm malformation, in mice. Reactive oxygen species (ROS) staining of frozen section and transmission electron microscopy (TEM) displayed DQ promoted ROS generation and mitochondrial morphology alterations in mouse testes, suggesting that DQ treatment induced testicular oxidative stress. Subsequent RNA-sequencing further showed that DQ treatment might trigger ferroptosis pathway, attributed to disturbed glutathione metabolism and iron homeostasis in spermatogonia cells in vitro. Consistently, results of western blotting, measurements of MDA and ferrous iron, and ROS staining confirmed that DQ increased oxidative stress and lipid peroxidation, and accelerated ferrous iron accumulation both in vitro and in vivo. Moreover, inhibition of ferroptosis by deferoxamine (DFO) markedly ameliorated DQ-induced cell death and dysfunction. By RNA-sequencing, we found that the expression of HO-1 was significantly upregulated in DQ-treated spermatogonia, while ZnPP (a specific inhibitor of HO-1) blocked spermatogonia ferroptosis by balancing intracellular iron homeostasis. In mice, administration of the ferroptosis inhibitor ferrostatin-1 effectively restored the increase of HO-1 levels in the spermatogonia, prevented spermatogonia death, and alleviated the spermatogenesis disorders induced by DQ. Overall, these findings suggest that HO-1 mediates DQ-induced spermatogonia ferroptosis in mouse testes, and targeting HO-1 may be an effective protective strategy against male reproductive disorders induced by pesticides in agriculture.
Asunto(s)
Diquat , Ferroptosis , Hemo-Oxigenasa 1 , Herbicidas , Especies Reactivas de Oxígeno , Espermatogonias , Testículo , Animales , Masculino , Ferroptosis/efectos de los fármacos , Ratones , Espermatogonias/efectos de los fármacos , Espermatogonias/patología , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Testículo/efectos de los fármacos , Testículo/patología , Diquat/toxicidad , Herbicidas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Proteínas de la MembranaRESUMEN
The aim of this study was to investigate the potential mechanism through which Yishen Tongluo decoction (YSTL) repairs DNA damage caused by benzo(a)pyrene diol epoxide (BPDE) in mouse spermatocytes (GC-2). The GC-2 cells were divided randomly into the control group, BPDE group, and low-, medium-, and high-dose YSTL groups of YSTL decoction. A comet assay was used to detect the DNA fragment index (DFI) of cells in each group. Based on the DFI results, whole transcriptome sequencing was conducted, followed by trend analysis, gene ontology (GO) enrichment analysis, kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, and ceRNA network analysis. Compared with the control group, the BPDE group reported a significant increase in the DNA fragmentation index (DFI) (p < .05). Compared with the BPDE group, the low-, high- and medium-dose YSTL groups had a significantly reduced DFI (p < .05). Whole-transcriptome sequencing revealed seven differentially expressed circRNAs, 203 differentially expressed miRNAs, and 3,662 differentially expressed mRNAs between the control group and the BPDE group. There was a total of 12 differentially expressed circRNAs, 204 miRNAs, and 2150 mRNAs between the BPDE group and the traditional Chinese medicine group. The pathways involved include DNA repair pathway, nucleotide excision repair pathway, base excision repair pathway, etc. The ceRNA network reported that Hmga2 was the core protein involved, novel_cir_000117 and mmu-miR-466c-3p were located upstream of Hmga2, and they were regulatory factors associated with Hmga2. Finally, we conclude that YSTL decoction may repair sperm DNA damage caused by BPDE through the novel_cir_000117-mmu-miR-466c-3p-Hmga2 pathway.
Asunto(s)
Daño del ADN , Reparación del ADN , Medicamentos Herbarios Chinos , Animales , Masculino , Ratones , Medicamentos Herbarios Chinos/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726.
Asunto(s)
Cobre , Espermatogénesis , Testículo , Tretinoina , Masculino , Animales , Espermatogénesis/efectos de los fármacos , Tretinoina/farmacología , Cobre/toxicidad , Ratones , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/patología , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Meiosis/efectos de los fármacos , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Epidídimo/patologíaRESUMEN
Microcystins (MCs) and nitrites are coexisted in the environment and have reproductive toxicity. The combined toxic effect and mechanism of MCs and nitrite on spermatogenesis remain largely unclear. In the present study, co-exposure to microcystin-leucine arginine (MC-LR) and sodium nitrite (NaNO2) aggravated testicular damage of Balb/c mice and mitochondrial impairment of spermatogonia, Sertoli cells, and sperm. Furthermore, MC-LR and NaNO2 reduced sperm density with a synergistic effect. In addition, MC-LR and NaNO2 synergistically induced oxidative stress in the reproductive system by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels and increasing levels of mitochondrial reactive oxygen species (mtROS) and reactive oxygen species (ROS). More importantly, mitoquidone mesylate (MitoQ), an inhibitor of mtROS, blocked MC-LR and NaNO2-induced spermatogonia and Sertoli cell apoptosis by inhibiting high expression of Bax, Fadd, Caspase-8, and cleaved-Caspase-3. On the other hand, MitoQ suppressed pyroptosis of Sertoli cells by inhibiting the expression of NLRP3, N-GSDMD, and cleaved-Caspase-1. Additionally, MitoQ alleviated co-exposure-induced sperm density reduction and organ index disorders in F1 generation mice. Together, co-exposure of MC-LR and NaNO2 can enhance spermatogenic disorders by mitochondrial oxidative impairment-mediated germ cell death. This study emphasizes the potential risks of MC-LR and NaNO2 on reproduction in realistic environments and highlights new insights into the cause and treatment of spermatogenic disorders.
Asunto(s)
Apoptosis , Ratones Endogámicos BALB C , Microcistinas , Piroptosis , Especies Reactivas de Oxígeno , Espermatogénesis , Microcistinas/toxicidad , Animales , Masculino , Ratones , Apoptosis/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Piroptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Testículo/efectos de los fármacos , Testículo/metabolismo , Espermatozoides/efectos de los fármacos , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Nitrito de Sodio , Toxinas Marinas , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismoRESUMEN
Cadmium (Cd) is a common pollutant with reproductive toxicity. Our previous study revealed that Cd triggered spermatogonia ferroptosis. However, the underlying mechanisms remain unclear. Nuclear receptor coactivator 4 (NCOA4) mediates ferritinophagy and specific degradation of ferritin through lysosomes, resulting in the release of ferrous ions. Excessive autophagy can lead to ferroptosis. This study investigated the role of autophagy in Cd-triggered ferroptosis using GC-1 spermatogonial (spg) cells which exposed to CdCl2 (5⯵M, 10⯵M, or 20⯵M) for 24 without/with CQ. The cells which transfected with Ncoa4-siRNA were used to explore the role of NCOA4-mediated ferritinophagy in Cd-triggered ferroptosis. The results revealed that Cd caused mitochondrial swelling, rupture of cristae, and vacuolar-like changes. The Cd-treated cells exhibited more autophagosomes. Simultaneously, Cd increased intracellular iron, reactive oxygen species, and malondialdehyde concentrations while decreasing glutathione content and Superoxide Dismutase-2 activity. Moreover, Cd upregulated mRNA levels of ferritinophagy-associated genes (Ncoa4, Lc3b and Fth1), as well as enhanced protein expression of NCOA4, LC3B, and FTH1. While Cd decreased the mRNA and protein expression of p62/SQSTM1. These results showed that Cd caused ferritinophagy and ferroptosis. The use of chloroquine to inhibit autophagy ameliorated Cd-induced iron overload and ferroptosis. Moreover, Ncoa4 knockdown in spermatogonia significantly reduced intracellular iron concentration and alleviated Cd-triggered ferroptosis. In conclusion, our findings demonstrate that Cd activates the ferritinophagy pathway mediated by NCOA4, resulting in iron accumulation through ferritin degradation. This causes oxidative stress, ultimately initiating ferroptosis in spermatogonia. Our results may provide new perspectives and potential strategies for preventing and treating Cd-induced reproductive toxicity.
Asunto(s)
Autofagia , Cadmio , Ferritinas , Ferroptosis , Coactivadores de Receptor Nuclear , Espermatogonias , Ferroptosis/efectos de los fármacos , Coactivadores de Receptor Nuclear/metabolismo , Coactivadores de Receptor Nuclear/genética , Masculino , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Ferritinas/metabolismo , Autofagia/efectos de los fármacos , Cadmio/toxicidad , Animales , Línea Celular , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Tripterygium glycosides (TG) is extracted from the roots of Chinese herbal medicine named Tripterygium wilfordii Hook F (TwHF). TG tablets are the representative TwHF-based agents with anti-inflammatory and immunomodulatory activities for treating rheumatoid arthritis. Although the curative effect of TG is remarkable, the clinical application is limited by a variety of organ toxicity. One of the most serious side-effects induced by TG is damage of the male reproductive system and the toxic mechanism is still not fully elucidated. TG-induced testicular injury was observed in male mice by treated with different concentrations of TG. The results showed that TG induced a significant decrease in testicular index. Pathological observation showed that spermatogenic cells were obviously shed, arranged loosely, and the spermatogenic epithelium was thin compared with control mice. In addition, the toxic effect of TG on mouse spermatogonia GC-1 cells was investigated. The results displayed that TG induced significant cytotoxicity in mouse GC-1 cells. To explore the potential toxic components that triggered testicular injury, the effects of 8 main components of TG on the viability of GC-1 cells were detected. The results showed that celastrol was the most toxic component of TG to GC-1 cells. Western blot analysis showed that LC3-II and the ratio of LC3-II/LC3-I were significantly increased and the expression level of p62 were decreased in both TG and celastrol treated cells, which indicated the significant activation of autophagy in spermatogonia cells. Therefore, autophagy plays an important role in the testicular injury induced by TG, and inhibition of autophagy is expected to reduce the testicular toxicity of TG.
Asunto(s)
Autofagia , Glicósidos , Triterpenos Pentacíclicos , Espermatogonias , Testículo , Tripterygium , Triterpenos , Animales , Masculino , Tripterygium/química , Tripterygium/toxicidad , Autofagia/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/patología , Glicósidos/toxicidad , Glicósidos/farmacología , Espermatogonias/efectos de los fármacos , Ratones , Triterpenos/farmacología , Triterpenos/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacosRESUMEN
Microplastics have emerged as environmental hazards in recent years. This study was intended to prove the toxic effects of microplastics on the male reproductive system and further elucidate its mechanism. C57bl/6 mice were exposed to ultrapure water or different doses (0.25, 0.5 and 1 mg/d) of 5 µm polystyrene microplastics (PS-MPs) for 4 weeks, and the GC-1 mouse spermatogonium was treated with different concentrations of PS-MPs. The results showed that sperm count and motility were decreased, and sperm deformity rate was increased after exposure to PS-MPs. The morphology of testes in PS-MPs groups exhibited pathological changes, such as abnormal development of spermatogenic tubules, and inhibited spermatogonium function. Furthermore, the fluorescence intensity of TUNEL staining and the BAX/BCL2 ratio were increased. Exposure to PS-MPs resulted in impaired mitochondrial morphology of spermatogonium, decreased activity of GSH-px and SOD, and increased the MDA level. In vitro, after treatment with PS-MPs, the cell apoptosis rate of spermatogonium was significantly increased, mitochondrial membrane potential was decreased, mitochondrial morphology was damaged, and exposure to PS-MPs increased mitochondrial reactive oxygen species, inducing an oxidative stress state in spermatogonia. In summary, PS-MPs induced a decrease in sperm quality by activating spermatogonium mitochondrial oxidative stress and apoptosis, offering novel insights into mitigating the reproductive toxicity of microplastics.
Asunto(s)
Apoptosis , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Microplásticos , Mitocondrias , Estrés Oxidativo , Poliestirenos , Motilidad Espermática , Espermatogonias , Testículo , Animales , Masculino , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Microplásticos/toxicidad , Poliestirenos/toxicidad , Poliestirenos/química , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Testículo/efectos de los fármacos , Testículo/patología , Testículo/metabolismo , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Espermatogonias/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Recuento de Espermatozoides , Superóxido Dismutasa/metabolismoRESUMEN
Titanium dioxide nanoparticles (TiO2 NPs) are widely used in consumer products, raising concerns about their impact on human health. This study investigates the effects of TiO2 NPs on male germ cells while focusing on cell proliferation inhibition and underlying mechanisms. This was done by utilizing mouse GC-1 spermatogonia cells, an immortalized spermatogonia cell line. TiO2 NPs induced a concentration-dependent proliferation inhibition with increased reactive oxygen species (ROS) generation. Notably, TiO2 NPs induced autophagy and decreased ERK phosphorylation. Treatment with the ROS inhibitor N-Acetyl-l-cysteine (NAC) alleviated TiO2 NPs-induced autophagy, restored ERK phosphorylation, and promoted cell proliferation. These findings call attention to the reproductive risks posed by TiO2 NPs while also highlighting NAC as a possible protective agent against reproductive toxins.
Asunto(s)
Acetilcisteína , Autofagia , Proliferación Celular , Nanopartículas del Metal , Especies Reactivas de Oxígeno , Titanio , Titanio/toxicidad , Masculino , Autofagia/efectos de los fármacos , Animales , Acetilcisteína/farmacología , Ratones , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Espermatogonias/efectos de los fármacos , Nanopartículas/toxicidadRESUMEN
The estrogen-like effect of bisphenol A (BPA) disrupting the maintenance of functional male germ cells is associated with male sub-fertility. This study investigated toxicity of male germ cells induced by four bisphenol analogs: BPA, BPAF, BPF, and BPS. The investigation of bisphenol analogs' impact on male germ cells included assessing proliferation, apoptosis induction, and the capacity to generate reactive oxygen species (ROS) in GC-1 spermatogonia (spg) cells, specifically type B spermatogonia. Additionally, the therapeutic potential and protective effects of N-Acetyl Cysteine (NAC) and NF-κB inhibitor parthenolide was evaluated. In comparison to BPA, BPF and BPS, BPAF exhibited the most pronounced adverse effect in GC-1 spg cell proliferation. This effect was characterized by pronounced inhibition of phosphorylation of PI3K, AKT, and mTOR, along with increased release of cytochrome c and subsequent cleavages of caspase 3, caspase 7, and poly (ADP-ribose) polymerase. Both NAC and parthenolide were effective reducing cellular ROS induced by BPAF. However, only NAC demonstrated a substantial recovery in proliferation, accompanied by a significant reduction in cytochrome c release and cleaved PARP. These results suggest that NAC supplementation may play an effective therapeutic role in countering germ cell toxicity induced by environmental pollutants with robust oxidative stress-generating capacity.
Asunto(s)
Acetilcisteína , Apoptosis , Compuestos de Bencidrilo , Proliferación Celular , Fenoles , Especies Reactivas de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Masculino , Fenoles/toxicidad , Animales , Compuestos de Bencidrilo/toxicidad , Acetilcisteína/farmacología , Ratones , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Sesquiterpenos/farmacología , Línea Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , FN-kappa B/metabolismoRESUMEN
Cadmium, an environmental toxicant, severely impairs male reproductive functions and currently lacks effective clinical treatments. Mesenchymal stem cell-derived exosomes (MSC-Exos) are increasingly recognized as a potential alternative to whole-cell therapy for tissue injury and regeneration. This study aims to investigate the protective effects of MSC-Exos against cadmium toxicity on male reproduction. Our findings reveal that MSC-Exos treatment significantly promotes spermatogenesis, improves sperm quality, and reduces germ cell apoptosis in cadmium-exposed mice. Mechanistically, MSC-Exos dramatically mitigate cadmium-induced cell apoptosis in a spermatogonia cell line (GC-1 spg) in vitro by reducing DNA damage and promoting autophagic flux. These results suggest that MSC-Exos have a protective effect on cadmium-induced germ cell apoptosis by ameliorating DNA damage and autophagy flux, demonstrating the therapeutic potential of MSC-Exos for cadmium toxicity on male reproduction.
Asunto(s)
Autofagia , Cadmio , Daño del ADN , Exosomas , Células Madre Mesenquimatosas , Masculino , Animales , Exosomas/metabolismo , Cadmio/toxicidad , Autofagia/efectos de los fármacos , Ratones , Células Madre Mesenquimatosas/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular , Espermatozoides/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2⯵g/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.
Asunto(s)
Toxinas Marinas , Microcistinas , Sirtuinas , Espermatogonias , Animales , Masculino , Ratones , Apoptosis , Proliferación Celular , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN , Toxinas Marinas/metabolismo , Toxinas Marinas/toxicidad , Ratones Endogámicos ICR , Microcistinas/metabolismo , Microcistinas/toxicidad , Semen , Sirtuinas/efectos de los fármacos , Sirtuinas/metabolismo , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismoRESUMEN
Bisphenol A (BPA), one of the main components of industrial products, is clinically associated with the increased male infertility rate. However, the underlying molecular mechanism of the BPA-resulted reproductive toxicity is not fully elucidated. Voltage-dependent anion channel 1 (VDAC1) is a pore protein and located at the outer mitochondrial membrane. As a mitochondrial gatekeeper, VDAC1 controls the release of reactive oxygen species (ROS) and the metabolic and energetic functions of mitochondria, and serves as a critical player in mitochondrial-mediated apoptosis. Herein, we explored the role of VDAC1 in BPA-induced apoptosis of spermatogonia. The results showed that BPA increased spermatogonia cell line GC-1 spg cell apoptosis and intracellular ROS level, and suppressed AMPK/mTOR signaling pathway at a dose of 80 µM for 48 hr. Lentivirus-mediated short hairpin RNA targeting VDAC1 (Lv-shVDAC1) silenced VDAC1 expression and enhanced BPA-restricted cell viability. Knockdown of VDAC1 inhibited the apoptosis of BPA-treated GC-1 spg cells determined by with changes of the expressions of pro-apoptotic and anti-apoptotic proteins. Knockdown of VDAC1 also alleviated the BPA-triggered intracellular ROS generation and oxidative stress. Moreover, silence of VDAC1 increased AMPKα1/2 phosphorylation and suppressed mTOR phosphorylation under BPA exposure. Dorsomorphin, an AMPK inhibitor, partially abolished the effects of VDAC1 gene silencing on BPA-stimulated GC-1 spg cells. In conclusion, inhibition of VDAC1 attenuated the BPA-induced oxidative stress and apoptosis and promoted the cell viability in spermatogonia through modulating AMPK/mTOR signaling pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Apoptosis , Estrés Oxidativo , Espermatogonias , Canal Aniónico 1 Dependiente del Voltaje , Masculino , Especies Reactivas de Oxígeno , Transducción de Señal , Espermatogonias/efectos de los fármacos , Serina-Treonina Quinasas TOR , Animales , RatonesRESUMEN
Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of melatonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3-6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 µM) and stored for 1 month. Finally, the cell viability, colonization rate, expression of Bcl-2 and BAX gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and Bcl-2 gene expression. It also diminished BAX gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic.
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Antioxidantes , Melatonina , Espermatogonias , Animales , Animales Recién Nacidos , Antioxidantes/farmacología , Apoptosis , Proliferación Celular , Criopreservación/métodos , Congelación , Masculino , Melatonina/farmacología , Ratones , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Especies Reactivas de Oxígeno , Espermatogonias/efectos de los fármacos , Células Madre/efectos de los fármacos , Proteína X Asociada a bcl-2RESUMEN
Zearalenone (ZEN), a widely known mycotoxin, is mainly produced by various Fusarium species, and it is a potent estrogenic metabolite that affects reproductive health in livestock and humans. In this study, the molecular mechanisms of toxicity and cell damage induced by ZEN in GC-1 spermatogonia (spg) cells were evaluated. Our results showed that cell viability decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to ZEN. In addition, the key proteins involved in apoptosis, cleaved caspase-3 and -8, BAD, BAX, and phosphorylation of p53 and ERK1/2, were significantly increased in ZEN-exposed GC-1 spg cells for 24 h, and cytochrome c was released from mitochondria by ZEN. Interestingly, ZEN also triggered autophagy in GC-1 spg cells. The expression levels of the autophagy-related genes Atg5, Atg3, Beclin 1, LC3, Ulk1, Bnip 3, and p62 were significantly higher in ZEN-treated GC-1 spg cells, and the protein levels of both LC3A/B and Atg12 were remarkably increased in a dose-dependent manner in ZEN-exposed GC-1 spg cells compared to the control. In addition, immunostaining results showed that ZEN-treated groups showed a remarkable increase in LC 3A/B positive puncta as compared to the control in a dose-dependent manner based on confocal microscopy analysis in GC-1 spg cells. Our findings suggest that ZEN has toxic effects on tGC-1 spg cells and induces both apoptosis and autophagy.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Zearalenona/toxicidad , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , RatonesRESUMEN
Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinß1, the specific marker protein of SSCs cells, and the integrinß1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinß1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.
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Células Madre Germinales Adultas/efectos de los fármacos , Células Madre Germinales Adultas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Tretinoina/farmacología , Animales , Diferenciación Celular , Pollos , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , MasculinoRESUMEN
Perinatal exposures to endocrine disrupting chemicals (EDCs) alter the male reproductive system. Infants are exposed to genistein (GEN) through soy-based formula, and to Mono(2-ethylhexyl) Phthalate (MEHP), metabolite of the plasticizer DEHP. Spermatogonial stem cells (SSCs) are formed in infancy and their integrity is essential for spermatogenesis. Thus, understanding the impact of EDCs on SSCs is critical. Prostaglandins (PGs) are inflammatory mediators synthesized via the eicosanoid pathway starting with cyclooxygenases (Coxs), that regulate physiological and pathological processes. Our goal was to study the eicosanoid pathway in SSCs and examine whether it was disrupted by GEN and MEHP, potentially contributing to their adverse effects. The mouse C18-4 cell line used as SSC model expressed high levels of Cox1 and Cox2 genes and proteins, and eicosanoid pathway genes similarly to levels measured in primary rat spermatogonia. Treatments with GEN and MEHP at 10 and 100 µM decreased Cox1 gene and protein expression, whereas Cox2, phospholipase A2, prostaglandin synthases transcripts, PGE2, PGF2a and PGD2 were upregulated. Simultaneously, the transcript levels of spermatogonia progenitor markers Foxo1 and Mcam and differentiated spermatogonial markers cKit and Stra8 were increased. Foxo1 was also increased by EDCs in primary rat spermatogonia. This study shows that the eicosanoid pathway is altered during SSC differentiation and that exposure to GEN and MEHP disrupts this process, mainly driven by GEN effects on Cox2 pathway, while MEHP acts through an alternative mechanism. Thus, understanding the role of Cox enzymes in SSCs and how GEN and MEHP exposures alter their differentiation warrants further studies.
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Células Madre Germinales Adultas/efectos de los fármacos , Dietilhexil Ftalato/análogos & derivados , Eicosanoides/metabolismo , Disruptores Endocrinos/toxicidad , Genisteína/toxicidad , Espermatogonias/efectos de los fármacos , Células Madre Germinales Adultas/metabolismo , Animales , Línea Celular , Dietilhexil Ftalato/toxicidad , Masculino , Ratones , Prostaglandina-Endoperóxido Sintasas/genética , Ratas , Transducción de Señal/efectos de los fármacos , Espermatogonias/metabolismoRESUMEN
Glyphosate is the most used herbicide in the world. Controversial studies exist on its effect on the male reproductive system. We used the validated BioAlter® model to test the effects of low concentrations of Glyphosate. Pubertal rat seminiferous tubules were treated with Glyphosate 50 nM, 500 nM, 5 µM or 50 µM over a 3-week culture period. The Trans-Epithelial Electrical Resistance was not modified by any of the concentrations. The decrease of Clusterin mRNAs suggested that glyphosate would target the integrity of Sertoli cells. The decrease of the numbers of germ cells from day 14 onward highlighted the chronic effect of glyphosate at 50 nM, 500 nM or 5 µM. No consistent effect of glyphosate was observed on the numbers of spermatogonia or on their specific mRNA levels. However, those low concentrations of glyphosate targeted young spermatocytes and middle to late pachytene spermatocytes resulting in a decrease of the numbers of round spermatids, the direct precursors of spermatozoa. This study underlines that the effect of a toxicant should be also studied at low doses and during the establishment of the blood-testis barrier.