Aucubin supplementation alleviate diabetes induced-disruption of blood-testis barrier and testicular damage via stabilizing cell junction integrity.

Disruption of blood-testis barrier (BTB) was a crucial pathological feature of diabetes induced-testicular injury at early phase. Aucubin (AU), a main active component in Eucommiae Cortex, has drawn attention for its benefits against male reproductive system disease. The current study was aimed at investigating the protective role of AU and exploring the underlying mechanism in diabetic model. A murine model of type 2 diabetes mellitus (T2DM) was induced by high-fat diet (HFD) combined with streptozocin (STZ). Testicular weight index and morphology, sperm quality, integrity of BTB and protein levels were analyzed. The underlying mechanism of the protective effect of AU was further explored in Sertoli cells (SCs) cultured with high glucose (HG). Our results showed AU inhibited testicular structural destruction, restored disruption of BTB and improved abnormal spermatogenic function in diabetic mice. Consistent with in vivo results, HG induced decreased transcellular resistance and increased permeability in SCs monolayers, while AU exposure reverses this trend. Meanwhile, reduced expression of Zonula occludin-1(ZO-1) and Connexin43(Cx43) in testicular tissue diabetic mice and HG-induced SCs was prominently reversed via AU treatment. Mechanistic studies suggested a high affinity interaction between AU and c-Src protein was identified based on molecular docking, and the activation of c-Src was significantly inhibited in AU treatment. Furthermore, AU significantly increased the expression of Cx43 and ZO-1 proteins HG-induced SCs, which can be further enhanced in gene-silenced c-Src cells to some extent. Our results suggested that AU ameliorated disruption of BTB and spermatogenesis dysfunction in diabetic mice via inactivating c-Src to stabilize cell junction integrity.

Qiangjing tablets repair of blood-testis barrier dysfunction in rats via regulating oxidative stress and p38 MAPK pathway.

BACKGROUND: The blood-testis barrier (BTB) is a physical barrier of the testis to prevent various exogenous substrates from entering apical compartments and provides immune privilege for spermatogenesis, which is essential for normal spermatogenic function of testis. It has been shown that oxidative stress can damage BTB by activating the p38 MAPK pathway. In Traditional Chinese Medicine, Qiangjing tablets (QJT) improve spermatogenesis and increase pregnancy rates. Previous studies have confirmed that QJT can improve sperm quality and have obvious antioxidant effects. In this study, we explore whether QJT contributes to recovery from BTB dysfunction in rats. METHODS: BTB dysfunction was induced in rats by 1% Cyclophosphamide (CP). The CP-induced rats in the treatment group were given a dose of QJT (0.45 g/kg·d) by gavage. Testis tissues were collected for histopathological and biochemical analysis, and the testis weight was estimated. Levels of BTB-related proteins and antioxidant enzyme were analyzed in the testis tissues. RESULTS: QJT resolved the pathological injury of rats testis induced by CP. Furthermore, MDA levels were significantly reduced, and the levels of SOD markedly increased in the testicular tissue after QJT treatment. In addition, QJT down-regulated the expression of p38 protein in rat testis and up-regulated the expressions of key proteins ZO-1, occludin and F-actin in BTB. CONCLUSION: These results demonstrate that QJT exerts protective effects on CP-induced rats with BTB dysfunction, likely by regulating the oxidative stress-mediated p38 MAPK pathway.

Omega-3 polyunsaturated fatty acids alleviate hydrogen sulfide-induced blood-testis barrier disruption in the testes of adult mice.

Hydrogen sulfide (H2S), a gaseous intracellular signal transducer, participates in multiple physiological and pathological conditions, including reproductive conditions, and disrupts spermatogenesis. The blood-testis barrier (BTB) plays a vital role in spermatogenesis. However, the effect of H2S on the BTB and the underlying mechanism remain unclear. Herein, we examined the effect of H2S and omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on the BTB and testicular functions. ICR male mice were randomly divided into the following groups: control, H2S exposure, and H2S exposure with ω-3 PUFAs intervention. The sperm parameters (sperm concentration and sperm motility) declined in the H2S group and improved in the ω-3 intervention group. BTB integrity was severely disrupted by H2S, and the BTB-related gene levels (ZO-1, Occludin, Claudin 11) decreased; ω-3 supplementation could alleviate BTB disruption by upregulating BTB-related genes, and TM4 Sertoli cells had a similar trend in vitro. p38 MAPK phosphorylation was upregulated in the Na2S treatment group and downregulated after ω-3 cotreatment. These findings suggest that H2S can impair the BTB and that ω-3 PUFAs supplementation can attenuate H2S toxicity in the male reproductive system. Our study elucidated the relationship between a gasotransmitter (H2S) and the BTB and identified the potential therapeutic effect of ω-3 PUFAs.

Nucleoside Reverse Transcriptase Inhibitor Interaction with Human Equilibrative Nucleoside Transporters 1 and 2.

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 µM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 µM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Selenium (Na2SeO3) Upregulates Expression of Immune Genes and Blood-Testis Barrier Constituent Proteins of Bovine Sertoli Cell In Vitro.

Sertoli cells were isolated from newborn calves and cultured in a medium supplemented with 0, 0.25, 0.50, 0.75, and 1.00 mg/L of sodium selenite to study their immune stimulatory effect, influence on cell's viability, and expression of blood-testis barrier proteins (occludin, connexin-43, zonula occluden, E-cadherin) using quantitative PCR and western blot analyses. Results showed that medium supplemented with 0.50 mg/L of selenium significantly (P < 0.05) promoted cell viability, upregulated toll-like receptor gene (TLR4), anti-inflammatory cytokines (IL-4, IL-10, TGFß1), and expressions of blood-testis barrier proteins, and modulated expressions of pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-γ). Sertoli cells grown in culture medium supplemented with 0.25 mg/L of selenium significantly upregulated TLR4, IL-4, IL-10, TGFß1, and blood-testis barrier proteins compared to the control group. Sodium selenite supplementation at 0.75 and 1.00 mg/L levels was cytotoxic and temporarily downregulated the expression of blood-testis barrier protein within 24 h after culture; however, commencing from 72 h post culture, increased cell viability and upregulation of expression of blood-testis barrier proteins were observed. In conclusion, the results of this study showed that selenium supplementation in the culture medium up to 0.50 mg/L concentration upregulates immune genes and blood-testis barrier constituent proteins of bovine Sertoli cells.

Endocrine effects of lifelong exposure to low-dose depleted uranium on testicular functions in adult rat.

Environmental toxicant exposure can induce disorders in sex steroidogenesis during fetal gonad development. Our previous study demonstrated that chronic adult exposure to a supra environmental concentration of depleted uranium (DU) does not impair testicular steroidogenesis in rats. In this study, we investigated the effects of lifelong exposure (embryo - adult) to low-dose DU (40 or 120mgL-1) on adult rat testicular steroidogenesis and spermatogenesis. A significant content of uranium was detected in testis and epididymis in the DU 120mgL-1 group and the assay in epididymal spermatozoa showed a significant content in both groups. No major defect was observed in testicular histology except a decrease in the number of basal vacuoles in the DU groups. Moreover, plasma Follicle-Stimuling Hormone [FSH] and Luteinizing Hormone [LH] levels were increased only in the DU 120mgL-1 group and intratesticular estradiol was decreased in both groups. Testosterone level was reduced in plasma and testis in the DU 40mgL-1 group. These modulations could be explained by an observed decrease in gene expression of luteinizing hormone receptor (LHR), and enzymes involved in steroid production and associated signal transduction (StAR, cyp11a1, cyp17a1, 3ßhsd, 17ßhsd, TGFß1, AR). Several genes specific to germ cells and cell junctions of the blood-testis barrier were also modulated. In conclusion, these data show that fetal life is a critical window for chronic uranium exposure and that the endocrine activities of low-dose uranium could disrupt steroidogenesis through the hypothalamic-pituitary-testicular axis. Further investigation should be so useful in subsequent generations to improve risk assessment of uranium exposure.

Garlic (Allium sativum) feeding impairs Sertoli cell junctional proteins in male Wistar rat testis: microscopy study.

Sertoli cell junctions, such as adhesion junction (AJ), gap junction (GJ) and tight junction (TJ), are important for maintaining spermatogenesis. In previous studies, we showed the inhibitory effect of crude garlic (Allium sativum, As) on spermatogenesis and steroidogenesis. The aim of this work was to complete our investigation on the impact of this plant, especially on Sertoli cell junctional proteins (SCJPs). During 1 month, 24 male rats were divided into groups: group control (0% of As) and treated groups fed 5%, 10% and 15% of As. Light and electron microscopy observations were performed to localise junctional proteins: connexin-43, Zona Occluding-1 and N-cadherin (immunohistochemistry) and to describe junctions. We showed that the specific cells involved in the localisation of the SCJP were similar in both control and treated groups, but with different immunoreactivity intensity between them. The electron microscopy observation focused on TJs between Sertoli cells, constituting the blood-testis barrier, showed ultrastructural changes such as fragmentation of TJs between adjacent Sertoli cell membranes and dilatation of rough endoplasmic reticulum saccules giving an aspect of scale to these junctions. We concluded that crude garlic consumption during 1 month induces perturbations on Sertoli cell junctions. These alterations can explain apoptosis in testicular germ cells previously showed.

Regulation of blood-testis barrier (BTB) dynamics during spermatogenesis via the "Yin" and "Yang" effects of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2.

In mammalian testes, haploid spermatozoa are formed from diploid spermatogonia during spermatogenesis, which is a complicated cellular process. While these cellular events were reported in the 1960s and 1970s, the underlying molecular mechanism(s) that regulates these events remained unexplored until the past ∼10 years. For instance, adhesion proteins were shown to be integrated components at the Sertoli cell-cell interface and/or the Sertoli-spermatid interface in the late 1980s. But only until recently, studies have demonstrated that some of the adhesion proteins serve as the platform for signal transduction that regulates cell adhesion. In this chapter, a brief summary and critical discussion are provided on the latest findings regarding these cell-adhesion proteins in the testis and their relationship to spermatogenesis. Moreover, antagonistic effects of two mammalian target of rapamycin (mTOR) complexes, known as mTORC1 and mTORC2, on cell-adhesion function in the testis are discussed. Finally, a hypothetic model is presented to depict how these two mTOR-signaling complexes having the "yin" and "yang" antagonistic effects on the Sertoli cell tight junction (TJ)-permeability barrier can maintain the blood-testis barrier (BTB) integrity during the epithelial cycle while preleptotene spermatocytes are crossing the BTB.

Toxicant-induced leakage of germ cell-specific proteins from seminiferous tubules in the rat: relationship to blood-testis barrier integrity and prospects for biomonitoring.

Evaluation of testicular toxicity during drug development is currently based on histopathological evaluation. A sensitive biomarker for testicular toxicology could provide an in-life and "early warning" measurement. Previous studies suggested that disruption of spermatogenesis induced leakage of germ cell proteins from seminiferous tubules (STs) into interstitial fluid (IF); such proteins have potential for use as biomarkers. To investigate this possibility further, adult male rats were treated with three testicular toxicants thought to have differing sites of action; cadmium chloride affects the blood-testis barrier (BTB), methoxyacetic acid (MAA) disrupts pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) targets Sertoli cells. IF proteins were assessed by Coomassie-based dye-stained gels. Immunostaining was used to identify toxicant-induced damage (DAZL) and BTB integrity (ZO-1, occludin, N-cadherin, and ß-catenin) and function (biotin). Cadmium chloride induced dose-dependent leakage of proteins from STs into IF coincident with loss of integrity and function of the BTB. Two of the "leaked" proteins were identified on Westerns as being germ cell specific, namely VASA and fatty acid-binding protein 9 (FABP9). In contrast, similar protein leakage was not evident after either MAA-induced or DNB-induced disruption of spermatogenesis and neither of these treatments affected BTB integrity or function. These results suggest that loss of BTB integrity is required for germ cell-specific proteins to leak from STs into IF, implying that use of such biomarkers has very limited potential for noninvasive monitoring of compound-induced disruption to spermatogenesis.

Size-selective junctional barrier and Ca(2+)-independent cell adhesion in the testis of Cynops pyrrhogaster: expression and function of occludin.

In urodeles which has testicular structure different from that in mammals, blood-testis barrier was reported to exist like in mammals. However, molecular and functional analyses of the components of the blood-testis barrier in urodeles have not been reported yet. Toward elucidation of the barrier functions and their molecular components in newt testis, we aimed to isolate occludin cDNAs and obtained two kinds of occludin partial cDNAs (occludin 1 and 2) encoding the putative second extracellular loop. Immunoblot and immunofluorescence studies using antibodies against peptides each corresponding to a part of the second extracellular loop of occludin 1 and 2, and those against beta-catenin and zonula occludens-1 (ZO-1) showed that occludin, as well as beta-catenin and ZO-1, was expressed not only in Sertoli cells but also in germ cells throughout all the stages from spermatogonia to elongate spermatids. Tracer experiments revealed a size-selective barrier which allows small molecules ( approximately 500 Da) to get into cysts through Sertoli cells' barrier, but not larger ones (>1.9 kDa) in the stages from spermatogonia to almost mature sperm. No occludin peptides corresponding to a part of the second extracellular loop destroyed the junctional barrier, while both the peptides and antibodies significantly inhibited reaggregation of the dissociated testicular cells which was to a large extent Ca(2+)-independent. These results indicate that the second extracellular loop of occludin is involved in cell adhesion rather than in size-selective barrier in newt testis, though the possibility cannot be excluded that the peptides were not long enough to inhibit the barrier function.