Katanin regulatory subunit B1 (KATNB1) regulates BTB dynamics through changes in cytoskeletal organization.

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology.

Suppressed testicular macrophage M1 polarization by HDAC5 enforces insensitivity to LPS-elicited blood-testis barrier damage.

Infertility caused by lipopolysaccharide (LPS) exposure due to infection is endangering male fertility worldwide, but the mechanism remains unclear. The blood-testis barrier (BTB) is essential for maintaining spermatogenesis and male fertility. In the present study, we showed that LPS (5.0 mg/kg) treatment markedly down-regulated the expression of BTB-related proteins, expanded the biotin penetration distance and caused histopathological injury in seminiferous tubules in mouse testes. Notably, testicular macrophage M1 polarization induced by LPS seems to be related to BTB damage, which was well confirmed by co-culture of RAW264.7 and TM4 cells in vitro. Interestingly, a low-dose LPS (0.1 mg/kg) pretreatment attenuated down-regulation of BTB-related proteins expression and histopathological injury and shorten biotin penetration distance in seminiferous tubules caused by LPS. Correspondingly, a low-dose LPS pretreatment suppresses testicular macrophage M1 polarization induced by LPS in mouse testes. Further experiments revealed that histone deacetylase 5 (HDAC5) was markedly down-regulated at 2 h and slightly down-regulated at 8 h, but up-regulated at 24 h in mouse testes after LPS treatment. Additionally, low-dose LPS pretreatment against the down-regulation of HDAC5 protein caused by LPS treatment. Notably, the suppressed testicular macrophage M1 polarization by low-dose LPS pretreatment was broken by BRD4354, a specific inhibitor of HDAC5 in vitro. These results suggest suppressed testicular macrophage M1 polarization by HDAC5 enforces insensitivity to LPS-elicited BTB damage.

Chronic dietary exposure to glyphosate-induced connexin 43 autophagic degradation contributes to blood-testis barrier disruption in roosters.

Glyphosate (GLY) is the most universally used herbicide worldwide and its application has caused extensive pollution to the ecological environment. Increasing evidence has revealed the multi-organ toxicity of GLY in different species, but its male reproductive toxicity in avian species remains unknown. Thus, in vivo and in vitro studies were conducted to clarify this issue. Data firstly showed that chronic GLY exposure caused testicular pathological damage. Intriguingly, we identified and verified a marked down-regulation gap junction gene Connexin 43 (Cx43) in GLY-exposed rooster testis by transcriptome analysis. Cx43 generated by Sertoli cells acts as a key component of blood-testis barrier (BTB). To further investigate the cause of GLY-induced downregulation of Cx43 to disrupt BTB, we found that autophagy activation is revealed in GLY-exposed rooster testis and primary avian Sertoli cells. Moreover, GLY-induced Cx43 downregulation was significantly alleviated by ATG5 knockdown or CQ administration, respectively, demonstrating that GLY-induced autophagy activation contributed to Cx43 degradation. Mechanistically, GLY-induced autophagy activation and resultant Cx43 degradation was due to its direct interaction with ER-α. In summary, these findings demonstrate that chronic GLY exposure activates autophagy to induce Cx43 degradation, which causes BTB damage and resultant reproductive toxicity in roosters.

The Novel-m0230-3p miRNA Modulates the CSF1/CSF1R/Ras Pathway to Regulate the Cell Tight Junctions and Blood-Testis Barrier in Yak.

The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai-Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood-testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19-25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism.

Protective effect of Cordycepin on blood-testis barrier against pre-puberty polystyrene nanoplastics exposure in male rats.

Plastic pollution is an emerging environmental issue, with microplastics and nanoplastics raising health concerns due to bioaccumulation. This work explored the impact of polystyrene nanoparticle (PS-NPs) exposure during prepuberty on male reproductive function post maturation in rats. Rats were gavaged with PS-NPs (80 nm) at 0, 3, 6, 12 mg/kg/day from postnatal day 21 to 95. PS-NPs accumulated in the testes and reduced sperm quality, serum reproductive hormones, and testicular coefficients. HE staining showed impaired spermatogenesis. PS-NPs disrupted the blood-testis barrier (BTB) by decreasing junction proteins, inducing inflammation and apoptosis. Transcriptomics identified differentially expressed genes related to metabolism, lysosome, apoptosis, and TLR4 signaling. Molecular docking revealed Cordycepin could compete with polystyrene for binding to TLR4. Cordycepin alleviated oxidative stress and improved barrier function in PS-NPs treated Sertoli cells. In conclusion, prepubertal PS-NPs exposure induces long-term reproductive toxicity in male rats, likely by disrupting spermatogenesis through oxidative stress and BTB damage. Cordycepin could potentially antagonize this effect by targeting TLR4 and warrants further study as a protective agent. This study elucidates the mechanisms underlying reproductive toxicity of PS-NPs and explores therapeutic strategies.

Generation of mouse testicular organoids with highly compartmentalized tubular lumen structure and their cryopreservation.

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.

Thiram exposure: Disruption of the blood-testis barrier and altered apoptosis-autophagy dynamics in testicular cells via the Bcl-2/Bax and mTOR/Atg5/p62 pathways in mice.

Thiram, a prevalent dithiocarbamate insecticide in agriculture, is widely employed as a crop insecticide and preservative. Chronic exposure to thiram has been linked to various irreversible damages, including tibial cartilage dysplasia, erythrocytotoxicity, renal issues, and immune system compromise. Limited research exists on its effects on reproductive organs. This study investigated the reproductive toxicology in mouse testes exposure to varying concentrations (0, 30, 60, and 120 mg/kg) of thiram. Our study uncovered a series of adverse effects in mice subjected to thiram exposure, including emaciation, stunted growth, decreased water intake, and postponed testicular maturation. Biochemical analysis in thiram-exposed mice showed elevated levels of LDH and AST, while ALP, TG, ALT, and urea were decreased. Histologically, thiram disrupted the testis' microarchitecture and compromised its barrier function by widening the gap between spermatogenic cells and promoting fibrosis. The expression of pro-apoptotic genes (Bax, APAF1, Cytc, and Caspase-3) was downregulated, whereas Bcl-2 expression increased in thiram-treated mice compared to controls. Conversely, the expression of Atg5 was upregulated, and mTOR and p62 expression decreased, with a trend towards lower LC3b levels. Thiram also disrupted the blood-testis barrier, significantly reducing the mRNA expression of zona occludens-1 (ZO-1) and occludin. In conclusion, chronic exposure to high thiram concentrations (120 mg/kg) caused testicular tissue damage, affecting the blood-testis barrier and modulating apoptosis and autophagy through the Bcl-2/Bax and mTOR/Atg5/p62 pathways. This study contributes to understanding the molecular basis of thiram-induced reproductive toxicity and underscores the need for further research and precautions for those chronically exposed to thiram and its environmental residuals.

FTO mediates bisphenol F-induced blood-testis barrier impairment through regulating ferroptosis via YTHDF1/TfRc and YTHDF2/SLC7A11 signal axis.

Bisphenol F (BPF) has been extensively utilized in daily life, which brings new hazards to male reproductive health. However, the specific functional mechanism is still unclear. Both cell and animal models were utilized for exploring the role of RNA methylation and ferroptosis and its underlying mechanisms in male reproductive injury induced by BPF. In animal model, BPF severely destroyed the integrity of the blood-testis barrier (BTB) and induced ferroptosis. Furthermore, BPF significantly affected the barrier function of TM4 cells and promoted ferroptosis. Importantly, ChIP assays revealed that BPF inhibited AR transcriptional regulation of FTO and FTO expression was downregulated in TM4 cells. Overexpression of FTO prevented the impairment of BTB by inhibiting ferroptosis in TM4 cells. Mechanistically, FTO could significantly down-regulate the m6A modification level of TfRc and SLC7A11 mRNA through MeRIP experiment. RIP experiments showed that YTHDF1 can bind to TfRc mRNA and promote its translation while YTHDF2 could bind to SLC7A11 mRNA and reduce its mRNA stability. Therefore, our results suggest that FTO plays a key role in BPF induced male reproductive toxicity through YTHDF1-TfRc axis and YTHDF2-SLC7A11 axis and may provide new ideas and methods for the prevention and treatment of male reproductive diseases associated with environmental pollutants.

Tas2R143 regulates the expression of the Blood-Testis Barrier tight junction protein in TM4 cells through the NF-κB signaling pathway.

Although bitter receptors, known as Tas2Rs, have been identified in the testes and mature sperm, their expression in testicular Sertoli cells (SCs) and their role in recognizing harmful substances to maintain the immune microenvironment remain unknown. To explore their potential function in spermatogenesis, this study utilized TM4 cells and discovered the high expression of the bitter receptor Tas2R143 in the cells. Interestingly, when the Tas2R143 gene was knocked down for 24 and 48 h, there was a significant downregulation (P < 0.05) in the expression of tight junction proteins (occludin and ZO-1) and NF-κB. Additionally, Western blot results demonstrated that the siRNA-133+NF-κB co-treatment group displayed a significant downregulation (P < 0.05) in the expression of occludin and ZO-1 compared to both the siRNA-133 transfection group and the NF-κB inhibitors treatment group. These findings suggest that Tas2R143 likely regulates the expression of occludin and ZO-1 through the NF-κB signaling pathway and provides a theoretical basis for studying the regulatory mechanism of bitter receptors in the reproductive system, aiming to attract attention to the chemical perception mechanism of spermatogenesis.

LncRNA SNHG5/IGF2BP1/Occludin axis regulates Nd2O3 induced blood-testis barrier disruption.

Neodymium oxide (Nd2O3) is a rare earth element that can lead to various type of tissue and organ damage with prolonged exposure. The long noncoding RNA small nucleolar ribonucleic acid host gene 5 (lncRNA SNHG5) plays a role in disease progressiong. However, its connection with Nd2O3 induced reproductive harm in males has not been thoroughly investigated. Our research discovered that exposure to Nd2O3 increases the expression of SNHG5 in the testes of mice, which in turn binds directly to and reduces in the protein levels of insulin like growth factor 2 mRNA-binding protein 1 (IGF2BP1) both in vivo and in vitro. This process disrupts the cytoskeleton of blood-testis barrier(BTB) by impacting the stability of the tight junction protein Occludin (Ocln) mRNA structure and the permeability of the BTB. In summary, our study elucidates the regulatory mechanism of SNHG5/IGF2BP1/Occludin axis in Nd2O3-induced BTB injury, providing valuable insights for the treatment of male infertility.

4-methylimidazole exposure impairs sperm mobility by reducing the expression of blood-testis barrier junction protein in mouse testes.

4-methylimidazole (4-MI), a derivative of imidazole, is a widely used component in caramel-colored food products such as soy sauce, beer and other soft drinks. The present study is aimed to investigate the effects of 4-MI on the male reproduction. The results revealed that 8 weeks of 4-MI exposure did not significantly alter the body weight and testicular weight of male mice. However, testicular morphology and computer-assisted sperm analysis showed that exposed to 4-MI caused irregular arrangement of spermatogenic cells in the testes and weakened sperm motility. Consistently, we observed the decreased fertilization ability in vivo of 4-MI-treated male mice. We further demonstrated that 4-MI disrupted the blood-testis barrier (BTB) integrity by decreasing the protein expression of BTB-related junction with permeability assay and western blot. In addition, the apoptosis of Sertoli cells (TM4) occurred in 4-MI treated mice, which might be caused by the generation of oxidative stress. Collectively, our findings document that 4-MI exposure damages the sperm mobility via disruption of BTB integrity.

Study on the mechanisms of defective spermatogenesis induced by TiO2 NPs based on 3D blood-testis barrier microfluidic chip.

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.

ERK/CREB and p38 MAPK/MMP14 Signaling Pathway Influences Spermatogenesis through Regulating the Expression of Junctional Proteins in Eriocheir sinensis Testis.

The hemolymph-testis barrier (HTB) is a reproduction barrier in Crustacea, guaranteeing the safe and smooth process of spermatogenesis, which is similar to the blood-testis barrier (BTB) in mammals. The MAPK signaling pathway plays an essential role in spermatogenesis and maintenance of the BTB. However, only a few studies have focused on the influence of MAPK on crustacean reproduction. In the present study, we knocked down and inhibited MAPK in Eriocheir sinensis. Increased defects in spermatogenesis were observed, concurrently with a damaged HTB. Further research revealed that es-MMP14 functions downstream of ERK and p38 MAPK and degrades junctional proteins (Pinin and ZO-1); es-CREB functions in the ERK cascade as a transcription factor of ZO-1. In addition, when es-MMP14 and es-CREB were deleted, the defects in HTB and spermatogenesis aligned with abnormalities in the MAPK. However, JNK impacts the integrity of the HTB by changing the distribution of intercellular junctions. In summary, the MAPK signaling pathway maintains HTB integrity and spermatogenesis through es-MMP14 and es-CREB, which provides insights into the evolution of gene function during barrier evolution.

2,2',4,4'-Tetrabromodiphenyl ether exposure disrupts blood-testis barrier integrity through CMA-mediated ferroptosis.

2,2',4,4'-Tetrabromodiphenyl ether (PBDE-47), being the most prevalent congener of polybrominated diphenyl ethers (PBDEs), has been found to accumulate greatly in the environment and induce spermatogenesis dysfunction. However, the specific underlying factors and mechanisms have not been elucidated. Herein, male Sprague-Dawley (SD) rats were exposed to corn oil, 10 mg/kg body weight (bw) PBDE-47 or 20 mg/kg bw PBDE-47 by gavage for 30 days. PBDE-47 exposure led to blood-testis barrier (BTB) integrity disruption and aberrant spermatogenesis. Given that Sertoli cells are the main toxicant target, to explore the potential mechanism involved, we performed RNA sequencing (RNA-seq) in Sertoli cells, and the differentially expressed genes were shown to be enriched in ferroptosis and lysosomal pathways. We subsequently demonstrated that ferroptosis was obviously increased in testes and Sertoli cells upon exposure to PBDE-47, and the junctional function of Sertoli cells was restored after treatment with the ferroptosis inhibitor ferrostatin-1. Since glutathione peroxidase 4 (GPX4) was dramatically reduced in PBDE-47-exposed testes and Sertoli cells and considering the RNA-sequencing results, we examined the activity of chaperone-mediated autophagy (CMA) and verified that the expression of LAMP2a and HSC70 was upregulated significantly after PBDE-47 exposure. Notably, Lamp2a knockdown not only inhibited ferroptosis by suppressing GPX4 degradation but also restored the impaired junctional function induced by PBDE-47. These collective findings strongly indicate that PBDE-47 induces Sertoli cell ferroptosis through CMA-mediated GPX4 degradation, resulting in decreased BTB-associated protein expression and eventually leading to BTB integrity disruption and spermatogenesis dysfunction.

Porcine reproductive and respiratory syndrome virus infects the reproductive system of male piglets and impairs development of the blood-testis barrier.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease that threatens the global swine industry. Recent studies have focused on the damage that PRRSV causes to the reproductive system of male pigs, although pathological research is lacking. Therefore, we examined the pathogenic mechanisms in male piglets infected with PRRSV. Gross and histopathological changes indicated that PRRSV affected the entire reproductive system, as confirmed via immunohistochemical analysis. PRRSV infected Sertoli cells and spermatogonia. To test the new hypothesis that PRRSV infection in piglets impairs blood - testis barrier (BTB) development, we investigated the pathology of PRRSV damage in the BTB. PRRSV infection significantly decreased the quantity and proliferative capacity of Sertoli cells constituting the BTB. Zonula occludens-1 and ß-catenin were downregulated in cell - cell junctions. Transcriptome analysis revealed that several crucial genes and signalling pathways involved in the growth and development of Leydig cells, Sertoli cells, and tight junctions in the testes were downregulated. Apoptosis, necroptosis, inflammatory, and oxidative stress-related pathways were activated, whereas hormone secretion-related pathways were inhibited. Many Sertoli cells and spermatogonia underwent apoptosis during early differentiation. Infected piglets exhibited disrupted androgen secretion, leading to significantly reduced testosterone and anti-Müllerian hormone levels. A cytokine storm occurred, notably upregulating cytokines such as tumour necrosis factor-α and interleukin-6. Markers of oxidative-stress damage (i.e. H2O2, malondialdehyde, and glutathione) were upregulated, whereas antioxidant-enzyme activities (i.e. superoxide dismutase, total antioxidant capacity, and catalase) were downregulated. Our results demonstrated that PRRSV infected multiple organs in the male reproductive system, which impaired growth in the BTB.

3-chloro-1,2-propanediol induces oxidative stress and promotes testicular damage and infertility in rats through CYP2C9.

3-chloro-1,2-propanediol (3-MCPD) is a newly discovered food process pollutant with nephrotoxicity. And the mechanism by which 3-MCPD affects male spermatogenesis has not been fully studied. Cell viability, blood-testis barrier (BTB) related protein, progesterone content, reactive oxygen species (ROS) generation, and cell apoptosis were determined by a CCK8 assay, western blot, ELISA, flow cytometry, and TUNEL staining, respectively. Wistar rats were divided into three groups: low-dose 3-MCPD, high-dose 3-MCPD, and control. Sperm parameters, hormonal levels, and biomarkers of oxidative stress in the testis and epididymis were detected by ELISA. Multiple molecular experiments including molecular docking and western blot were used to elucidate the underlying mechanisms. 3-MCPD affects testicular cell activity, and promotes ROS production and apoptosis. Disrupting the integrity of BTB in the body, downregulating sex hormones and sperm quality, and promoting apoptosis. 3-MCPD may function through CYP2C9. This study preliminarily explores the mechanism by which 3-MCPD affects spermatogenesis. It was found that 3-MCPD destroys the structure and function of BTB and damages the testicular function of male mice, thus affecting the process of spermatogenesis via CYP2C9.

Nerve growth factor alleviates arsenic-induced testicular injury by enhancing the function of Sertoli cells.

Sertoli cells (SCs) maintain testicular homeostasis and promote spermatogenesis by forming the blood-testis barrier (BTB) and secreting growth factors. The pro-proliferative and anti-apoptotic effects of nerve growth factor (NGF) on SCs have been proved previously. It is still unclear whether the damage effect of arsenic on testis is related to the inhibition of NGF expression, and whether NGF can mitigate arsenic-induced testicular damage by decreasing the damage of SCs induced by arsenic. Here, the lower expression of NGF in testes of arsenic exposed mice (freely drinking water containing 15 mg/l of NaAsO2) was observed through detection of Western blot and Real-time PCR. Subsequently, hematoxylin and eosin (HE) staining, Evans blue staining and transmission electron microscopy were used to evaluate the pathology, BTB permeability and tight junction integrity in testes of control mice, arsenic exposed mice (freely drinking water containing 15 mg/l of NaAsO2) and arsenic + NGF treated mice (freely drinking water containing 15 mg/l of NaAsO2 + intraperitoneal injection with 30 µg/kg of NGF), respectively. Evidently, spermatogenic tubule epithelial cells in testis of arsenic exposed mice were disordered and the number of cell layers was reduced, accompanied by increased permeability and damaged integrity of the tight junction in BTB, but these changes were less obvious in testes of mice treated with arsenic + NGF. In addition, the sperm count, motility and malformation rate of mice treated with arsenic + NGF were also improved. On the basis of the above experiments, the viability and apoptosis of primary cultured SCs treated with arsenic (10 µM NaAsO2) or arsenic + NGF (10 µM NaAsO2 + 100 ng/mL NGF) were detected by Cell counting kit-8 (CCK8) and transferase-mediated DUTP-biotin nick end labeling (TUNEL) staining, respectively. It is found that NGF ameliorated the decline of growth activity and the increase of apoptosis in arsenic-induced SCs. This remarkable biological effect that NGF inhibited the increase of Bax expression and the decrease of Bcl-2 expression in arsenic-induced SCs was also determined by western blot and Real-time PCR. Moreover, the decrease in transmembrane resistance (TEER) and the expression of tight junction proteins ZO-1 and occludin was mitigated in SCs induced by arsenic due to NGF treatment. In conclusion, the above results confirmed that NGF could ameliorate the injury effects of arsenic on testis, which might be related to the function of NGF to inhibit arsenic-induced SCs injury.

Octanoic acid mitigates busulfan-induced blood-testis barrier damage by alleviating oxidative stress and autophagy.

BACKGROUND: The management of male infertility continues to encounter an array of challenges and constraints, necessitating an in-depth exploration of novel therapeutic targets to enhance its efficacy. As an eight-carbon medium-chain fatty acid, octanoic acid (OCA) shows promise for improving health, yet its impact on spermatogenesis remains inadequately researched. METHODS: Mass spectrometry was performed to determine the fatty acid content and screen for a pivotal lipid component in the serum of patients with severe spermatogenesis disorders. The sperm quality was examined, and histopathological analysis and biotin tracer tests were performed to assess spermatogenesis function and the integrity of the blood-testis barrier (BTB) in vivo. Cell-based in vitro experiments were carried out to investigate the effects of OCA administration on Sertoli cell dysfunction. This research aimed to elucidate the mechanism by which OCA may influence the function of Sertoli cells. RESULTS: A pronounced reduction in OCA content was observed in the serum of patients with severe spermatogenesis disorders, indicating that OCA deficiency is related to spermatogenic disorders. The protective effect of OCA on reproduction was tested in a mouse model of spermatogenic disorder induced by busulfan at a dose 30 mg/kg body weight (BW). The mice in the study were separated into distinct groups and administered varying amounts of OCA, specifically at doses of 32, 64, 128, and 256 mg/kg BW. After evaluating sperm parameters, the most effective dose was determined to be 32 mg/kg BW. In vivo experiments showed that treatment with OCA significantly improved sperm quality, testicular histopathology and BTB integrity, which were damaged by busulfan. Moreover, OCA intervention reduced busulfan-induced oxidative stress and autophagy in mouse testes. In vitro, OCA pretreatment (100 µM) significantly ameliorated Sertoli cell dysfunction by alleviating busulfan (800 µM)-induced oxidative stress and autophagy. Moreover, rapamycin (5 µM)-induced autophagy led to Sertoli cell barrier dysfunction, while OCA administration exerted a protective effect by alleviating autophagy. CONCLUSIONS: This study demonstrated that OCA administration suppressed oxidative stress and autophagy to alleviate busulfan-induced BTB damage. These findings provide a deeper understanding of the toxicology of busulfan and a promising avenue for the development of novel OCA-based therapies for male infertility.

Adverse effects and potential mechanisms of polystyrene microplastics (PS-MPs) on the blood-testis barrier.

Microplastics (MPs) are defined as plastic particles or fragments with a diameter of less than 5 mm. These particles have been identified as causing male reproductive toxicity, although the precise mechanism behind this association is yet to be fully understood. Recent research has found that exposure to polystyrene microplastics (PS-MPs) can disrupt spermatogenesis by impacting the integrity of the blood-testis barrier (BTB), a formidable barrier within mammalian blood tissues. The BTB safeguards germ cells from harmful substances and infiltration by immune cells. However, the disruption of the BTB leads to the entry of environmental pollutants and immune cells into the seminiferous tubules, resulting in adverse reproductive effects. Additionally, PS-MPs induce reproductive damage by generating oxidative stress, inflammation, autophagy, and alterations in the composition of intestinal flora. Despite these findings, the precise mechanism by which PS-MPs disrupt the BTB remains inconclusive, necessitating further investigation into the underlying processes. This review aims to enhance our understanding of the pernicious effects of PS-MP exposure on the BTB and explore potential mechanisms to offer novel perspectives on BTB damage caused by PS-MPs.

Perfluorooctane sulfonate-induced Sertoli cell injury through c-Jun N-terminal kinase: a study by RNA-Seq.

Per- and polyfluoroalkyl substances (PFASs) are a family of "forever chemicals" including perfluorooctane sulfonate (PFOS). These toxic chemicals do not break down in the environment or in our bodies. In the human body, PFOS and perfluoroctanoic acid (PFOA) have a half-life (T1/2) of about 4-5 yr so low daily consumption of these chemicals can accumulate in the human body to a harmful level over a long period. Although the use of PFOS in consumer products was banned in the United States in 2022/2023, this forever chemical remains detectable in our tap water and food products. Every American tested has a high level of PFAS in their blood (https://cleanwater.org/pfas-forever-chemicals). In this report, we used a Sertoli cell blood-testis barrier (BTB) model with primary Sertoli cells cultured in vitro with an established functional tight junction (TJ)-permeability barrier that mimicked the BTB in vivo. Treatment of Sertoli cells with PFOS was found to perturb the TJ-barrier, which was the result of cytoskeletal disruption across the cell cytoplasm, disrupting actin and microtubule polymerization. These changes thus affected the proper localization of BTB-associated proteins at the BTB. Using RNA-Seq transcriptome profiling, bioinformatics analysis, and pertinent biochemical and cell biology techniques, it was discovered that PFOS -induced Sertoli cell toxicity through the c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase, SAPK) and its phosphorylated/active form p-JNK signaling pathway. More importantly, KB-R7943 mesylate (KB), a JNK/p-JNK activator, was capable of blocking PFOS-induced Sertoli cell injury, supporting the notion that PFOS-induced cell injury can possibly be therapeutically managed.NEW & NOTEWORTHY PFOS induces Sertoli cell injury, including disruption of the 1) blood-testis barrier function and 2) cytoskeletal organization, which, in turn, impedes male reproductive function. These changes are mediated by JNK/p-JNK signaling pathway. However, the use of KB-R7943, a JNK/p-JNK activator was capable of blocking PFOS-induced Sertoli cell injury, supporting the possibility of therapeutically managing PFOS-induced reproductive dysfunction.