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.

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.

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.

Triptolide exposure triggers testicular vacuolization injury by disrupting the Sertoli cell junction and cytoskeletal organization via the AKT/mTOR signaling pathway.

BACKGROUND: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure. METHODS: Male mice were subjected to TP at doses of 15, 30, and 60 µg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining. RESULTS: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells. CONCLUSION: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes.

The blood-testis barrier disruption is a prerequisite for toxicant-induced peritubular macrophage increases in the testis of peripubertal rats.

Peritubular macrophages (PTMφ) are predominantly localized near spermatogonial stem cells in the testis. We previously revealed that exposure of peripubertal male Fischer rats to mono-(2-ethylhexyl) phthalate (MEHP) leads to increased PTMφs in the testis. The mechanisms that trigger increases in PTMφs in the testis are poorly understood. However, MEHP exposure is known to both induce spermatocyte apoptosis and to perturb the blood-testis barrier (BTB). This study aims to elucidate the association between the disruption of BTB and the increases of PTMφs in the testis by comparing the effects observed with MEHP to 2 other testicular toxicants with variable effects on the BTB and subtype of germ cell undergoing apoptosis. Methoxyacetic acid (MAA) acts directly on spermatocytes and does not affect BTB function, whereas cadmium chloride (CdCl2) induces profound injury to BTB. The results indicated that MAA exposure significantly increased spermatocyte apoptosis, whereas no significant changes in the numbers of PTMφs in the testis occurred. In contrast, CdCl2 exposure disrupted BTB function and increased the abundance of PTMφs in the testis. To further investigate whether MEHP-induced changes in BTB integrity accounted for the increase in PTMφs, a plasmid for LG3/4/5, the functional component of laminin-alpha 2, was overexpressed in the testis to stabilize BTB integrity before MEHP exposure. The results showed that LG3/4/5 overexpression substantially reduced the ability of MEHP to compromise BTB integrity and prevented the increase in PTMφ numbers after MEHP exposure. These results indicate that BTB disruption is necessary to increase PTMφs in the testis induced by toxicants.

Prenatal dexamethasone exposure impairs rat blood-testis barrier function and sperm quality in adult offspring via GR/KDM1B/FSTL3/TGFß signaling.

Both epidemiological and animal studies suggest that adverse environment during pregnancy can change the offspring development programming, but it is difficult to achieve prenatal early warning. In this study we investigated the impact of prenatal dexamethasone exposure (PDE) on sperm quality and function of blood-testis barrier (BTB) in adult offspring and the underlying mechanisms. Pregnant rats were injected with dexamethasone (0.1, 0.2 and 0.4 mg·kg-1·d-1, s.c.) from GD9 to GD20. After weaning (PW4), the pups were fed with lab chow. At PW12 and PW28, the male offspring were euthanized to collect blood and testes samples. We showed that PDE significantly decreased sperm quality (including quantity and motility) in male offspring, which was associated with impaired BTB and decreased CX43/E-cadherin expression in the testis. We demonstrated that PDE induced morphological abnormalities of fetal testicle and Sertoli cell development originated from intrauterine. By tracing to fetal testicular Sertoli cells, we found that PDE dose-dependently increased expression of histone lysine demethylases (KDM1B), decreasing histone 3 lysine 9 dimethylation (H3K9me2) levels of follistatin-like-3 (FSTL3) promoter region and increased FSTL3 expression, and inhibited TGFß signaling and CX43/E-cadherin expression in offspring before and after birth. These results were validated in TM4 Sertoli cells following dexamethasone treatment. Meanwhile, the H3K9me2 levels of FSTL3 promoter in maternal peripheral blood mononuclear cell (PBMC) and placenta were decreased and its expression increased, which was positively correlated with the changes in offspring testis. Based on analysis of human samples, we found that the H3K9me2 levels of FSTL3 promoter in maternal blood PBMC and placenta were positively correlated with fetal blood testosterone levels after prenatal dexamethasone exposure. We conclude that PDE can reduce sperm quality in adult offspring rats, which is related to the damage of testis BTB via epigenetic modification and change of FSTL3 expression in Sertoli cells. The H3K9me2 levels of the FSTL3 promoter and its expression in the maternal blood PBMC can be used as a prenatal warning marker for fetal testicular dysplasia.

The FAK/occludin/ZO-1 complex is critical for cadmium-induced testicular damage by disruption of the integrity of the blood-testis barrier in chickens.

Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and reproductive function. However, the mechanism of Cd-induced disruption of BTB and testicular damage has not been fully elucidated. Here, our study investigates the effects of Cd on BTB integrity and testicular dysfunction. 80 (aged 1 day) Hy-Line white variety chickens were randomly designed into 4 groups and treated for 90 days, as follows: control group (essential diet), 35 Cd, 70 Cd and 140 Cd groups (35, 70 and 140 mg/kg Cd). The results found that Cd exposure diminished volume of the testes and induced histopathological lesions in the testes. Exposure to Cd induced an inflammatory response, disrupted the structure and function of the FAK/occludin/ZO-1 protein complex and disrupted the tight junction and adherens junction in the BTB. In addition, Cd exposure reduced the expression of steroid-related proteins and inhibited testosterone synthesis. Taken together, these data elucidate that Cd disrupts the integrity of the BTB and further inhibits spermatogenesis by dissociating the FAK/occludin/ZO-1 complex, which provides a basis for further investigation into the mechanisms of Cd-induced impairment of male reproductive function and pharmacological protection.

Busulfan impairs blood-testis barrier and spermatogenesis by increasing noncollagenous 1 domain peptide via matrix metalloproteinase 9.

BACKGROUNDS: Sterility induced by anti-cancer treatments has caused significant concern, yet the mechanism and treatment exploration are little for male infertility after cancer therapy. Busulfan, the antineoplastic that was widely applied before bone marrow transplantation, was known to induce male reproductive disorder. OBJECTIVES: To investigate the effect of busulfan on blood-testis barrier function in adult rats and determine whether noncollagenous 1 domain peptide, the biologically active fragment proteolyzed from the collagen α3 chain (IV) by matrix metalloproteinase 9, was involved during this process. MATERIALS AND METHODS: Adult male rats were treated with one-dose or double-dose of busulfan (10 mg/kg) before euthanized at day 35. Blood-testis barrier integrity assay, HE staining, immunofluorescence, and Western blot were used to validate the effect of busulfan on blood-testis barrier permeability and spermatogenesis. JNJ0966 was applied to specifically inhibit the matrix metalloproteinase 9 activity. The polymerization activity of F-actin/G-actin and microtubule/tubulin in the testis were assessed by using commercial kits. RESULTS: A noteworthy blood-testis barrier injury and significant up-regulation of matrix metalloproteinase 9 activity and noncollagenous 1 level after a single-dose busulfan (10 mg/kg) treatment in adult rat testis were revealed. The application of JNJ0966 was found to decrease noncollagenous 1 level and rescue the busulfan-induced blood-testis barrier injury including the mis-localization of junction proteins across the seminiferous epithelium, by recovering the organization and polymerization of both F-actin and microtubule. The busulfan-induced spermatogenesis impairment was also improved by JNJ0966. CONCLUSION: These findings thus demonstrate that the elevation in matrix metalloproteinase 9 and noncollagenous 1 might participate in busulfan-induced blood-testis barrier disruption in adult male rats. As such, busulfan-induced male infertility could possibly be managed through interventions on noncollagenous 1 production.

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

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

Zearalenone Exposure Disrupts Blood-Testis Barrier Integrity through Excessive Ca2+-Mediated Autophagy.

Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood-testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5-7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood-testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca2+ and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca2+ by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca2+-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.

Long-Term Maintenance of Viable Adult Rat Sertoli Cells Able to Establish Testis Barrier Components and Function in Response to Androgens.

A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-ß3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions.

Influence of hormonal imbalance on the integrity of seminiferous epithelium in the testes of adult rats chronically exposed to letrozole and rats exposed to soya isoflavones during the prenatal period, lactation, and up to sexual maturity.

The structural integrity of the germ cells in the seminiferous epithelium and the correct process of spermatogenesis are made possible by proteins that participate in the formation of different types of junctions. This study was performed on samples of the testes of 4 groups (2 experimental and 2 corresponding control) of male Wistar rats. In the first experimental group, the adult rats received letrozole - a nonsteroidal inhibitor of cytochrome P450 aromatase (P450arom). The second experimental group was exposed to soya isoflavones during the prenatal period, lactation, and up to sexual maturity. The aim of this study was to examine the immunoexpression of ß-catenin, N-cadherin, occludin, connexin43, annexin V, and advanced glycation end products (AGE) in the seminiferous epithelium of rat testes with chronic estrogen deficiency and of rats exposed to soya isoflavones. Series of sections of the testes were stained using PAS and silver impregnation. Moreover, immunohistochemistry tests were performed. A semi-quantitative determination of protein immunoexpression was performed using Image J. The number of annexin V positive Sertoli cells per tubule were counted manually. Comparisons between the experimental and corresponding control groups were performed using a non-parametric Mann-Whitney U test. The most common alterations were prematurely sloughed germ cells in the lumen of the seminiferous tubules and invaginations of the seminiferous tubules. We observed a lower number of annexin V positive Sertoli cells and a lower expression of N-cadherin and occludin in the seminiferous epithelium of both groups of rats with hormonal imbalances. Moreover, a higher expression of AGE, a lower expression of connexin 43 and a lower amount of reticular fibers in the basal lamina of seminiferous tubules was present in rats treated with letrozole and a higher expression of ß-catenin was found in rats exposed to soya isoflavones. The hormonal imbalance between androgens and estrogens resulted in a decreased number of annexin V positive Sertoli cells. This may be associated with a failed clearance of apoptotic germ cells that leads to disturbances in the blood-testis-barrier (BTB) by affecting the expression of junctional proteins in the seminiferous epithelium. Moreover, a decreased level of estrogens was also associated with an increased expression of AGEs and with a changed composition of basal lamina in the seminiferous tubules of rats. These changes could lead to germ cell sloughing and invaginations of the seminiferous tubules.

Differential Effects of Viral Nucleic Acid Sensor Signaling Pathways on Testicular Sertoli and Leydig Cells.

The human testis can be infected by a large number of RNA and DNA viruses. While various RNA virus infections may induce orchitis and impair testicular functions, DNA virus infection rarely affects the testis. Mechanisms underlying the differential effects of RNA and DNA viral infections on the testis remain unclear. In the current study, we therefore examined the effects of viral RNA and DNA sensor signaling pathways on mouse Sertoli cells (SC) and Leydig cells (LC). The local injection of viral RNA analogue polyinosinic-polycytidylic acid [poly(I:C)] into the testis markedly disrupted spermatogenesis, whereas the injection of the herpes simplex virus (HSV) DNA analogue HSV60 did not affect spermatogenesis. Poly(I:C) dramatically induced the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 in SC and LC through Toll-like receptor 3 and interferon ß promoter stimulator 1 signaling pathways, impairing the integrity of the blood-testis barrier and testosterone synthesis. Poly(I:C)-induced TNF-α production thus plays a critical role in the impairment of cell functions. In contrast, HSV60 predominantly induced the expression of type 1 interferons and antiviral proteins via the DNA sensor signaling pathway, which did not affect testicular cell functions. Accordingly, the Zika virus induced high levels of TNF-α in SC and LC and impaired their respective cellular functions, whereas Herpes simplex virus type 2 principally induced antiviral responses and did not impair such functions. These results provide insights into the mechanisms by which RNA viral infections impair testicular functions.

Environmentally relevant perinatal exposure to DBP disturbs testicular development and puberty onset in male mice.

Di-n-butyl phthalate (DBP) is considered as a potential modifier of puberty. However, different results indicate that DBP plays an accelerated, delayed, or neutral role in the initiation of puberty. Furthermore, whether the effect of DBP on puberty will disrupt the function of reproductive system in the adults is still ambiguous. Therefore, we aimed to investigate the effect of maternal exposure to DBP on the onset of puberty in male offspring mice and the subsequent changes in the development of reproductive system. Here, pregnant mice were treated with 0 (control), 50, 250, or 500 mg/kg/day DBP in 1 mL/kg corn oil administered daily by oral gavage from gestation day (GD) 12.5 to parturition. Compared with the control group, the 50 mg/kg/day DBP group accelerated puberty onset and testicular development were quite remarkable in male offspring mice during early puberty. Furthermore, in 22-day male offspring mice, 50 mg/kg/day DBP induced increased levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone in serum, and promoted the expression of steroidogenesis-related genes in the testes. Testicular Leydig cells (LCs) were isolated from the testes of 3-week-old mice and treated with 0 (control), 0.1, 1 mM monobutyl phthalate (MBP, the active metabolite of DBP) for 24 h. Consistent with the in vivo results, the expression of steroidogenesis-related genes and testosterone production were increased in LCs following exposure to 0.1 mM MBP. In adulthood, testes of the male offspring mice exposed to all doses of DBP exhibited adverse morphology compared with the control group. These results demonstrated that maternal exposure to 50 mg/kg/day DBP induced earlier puberty and precocious development of the testis, and eventually damaged the reproductive system in the later life.

Wu-Zi-Yan-Zong-Wan protects mouse blood-testis barrier from Tripterygium wilfordii Hook. f. multiglycoside-induced disruption by regulating proinflammatory cytokines.

ETHNOPHARMACOLOGICAL RELEVANCE: Wu-Zi-Yan-Zong-Wan (WZYZW) is a classical traditonal Chinese herbal formula and a Chinese patent medicine used to treat male infertility. However, the chemical components of WZYZW and its mechanism are not yet fully clarified. AIM OF THE STUDY: The purpose of this study is to observe the effect and underlying mechanism of WZYZW on ameliorating blood-testis barrier (BTB) dysfunction in mice with spermatogenic dysfunction induced by administration of Tripterygium wilfordii Hook. f. multiglycosides (GTW). MATERIALS AND METHODS: WZYZW was administered by gavage to mice with GTW-induced spermatogenic dysfunction (kidney essence deficiency pattern) for 40 days. Testis tissues were obtained for subsequent histopathological analysis. Biotin tracing was used to evaluate the permeability of Sertoli cell tight junctions. The levels of proinflammatory cytokines including interleukin (IL)-6, IL-17A, IL-1α and tumor necrosis factor (TNF)-α were analyzed by ELISA. The expression levels of proteins related to tight junction including ZO-1, JAM-A and occludin were analyzed by western blotting. The ultrastructures of tight junctions were observed by transmission electron microscopy. RESULTS: WZYZW ameliorated GTW-induced testicular spermatogenic dysfunction. Levels of IL-6, IL-17A, IL-1α, and TNF-α in the groups receiving low, medium, and high doses of WZYZW decreased in a dose-dependent manner. WZYZW impeded a biotin tracer from permeating the BTB, protecting its integrity in GTW-treated mice. In addition, our results showed no significant changes in the protein expressions of ZO-1, JAM-A, and occludin after WZYZW administration compared with the GTW group. Meanwhile, WZYZW exhibited a linear arrangement and restored the typical "sandwich" structure of BTB. No acute poisoning incidences were observed in all groups during the experiment. CONCLUSIONS: Our findings demonstrate that WZYZW may ameliorate some GTW-induced BTB dysfunction, possibly by regulating proinflammatory cytokine levels. In vitro studies on the regulation of BTB permeability by WZYZW and its active components are further required.

Effects of bisphenols on Blood-Testis Barrier protein expression in vitro: A systematic review and meta-analysis.

Bisphenols are a group of environmental endocrine-disrupting chemicals that produce alterations in the expression of intercellular junction proteins of the Blood-Testis Barrier (BTB) involved in spermatogenesis. The association between bisphenol exposure and BTB protein expression is controversial. Therefore, we performed this systematic review and meta-analysis to clarify bisphenol effects on Sertoli cell BTB protein expression in vitro. The Standardized Mean Difference (SMD) with a 95 % confidence interval (95 % CI) was used to evaluate the association between alterations in the BTB protein expression and bisphenol exposure in vitro. Six articles were included in the meta-analysis. Bisphenol-A (BPA) exposure at 200 µM was associated with significant decrease in BTB protein expression (SMD = -2.70, 95 %CI: -3.59, -1.80, p het = 0.46, p = <0.00001). In the moderate (40-50 µM) and low dose (<25 µM), no significant associations were obtained. We also found a non-monotonic dose-response curve of bisphenol effect in ZO-1 protein expression; low and high doses presented a significant decrease compared to control, while moderate dose presented no change. The current temporary Tolerable Daily Intake (tTDI) of BPA is 4 µg/kg bw/day. The 5-25 µM doses of BPA are equivalent to ∼1-5 mg/kg bw, respectively. Although the low dose group (<25 µM) assessed doses below the previous NOAEL value, these doses are above the current tTDI. Thus, it is necessary to conduct more studies with lower bisphenol concentrations to avoid underestimating the potential adverse effects of bisphenols at doses below tTDI.

Low Doses of Glyphosate/Roundup Alter Blood-Testis Barrier Integrity in Juvenile Rats.

It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.

Semen Cuscutae-Fructus Lycii improves spermatogenic dysfunction by repairing the blood-testis barrier in rats according to in silico and in vitro methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae and Fructus Lycii (SC-FL) is a commonly used herbal pair for male infertility treatment. Studies have found that the mechanism of SC-FL treatment may be related to repairing the blood-testis barrier (BTB). The application of network pharmacology can be used to explore the correlation between medicines and diseases and predict the potential pharmacological mechanisms of SC-FL. AIM OF THE STUDY: This study aimed to explore the specific effects and mechanisms of SC-FL in repairing the BTB and initially revealed the mechanism of Chinese medicine treating male infertility through network pharmacology and animal experiments. MATERIALS AND METHODS: We searched databases using the network pharmacology method and performed mass spectrometry analysis. We analyzed and predicted the active ingredients, targets and key pathways of SC-FL in male infertility treatment. Then, we designed animal experiments to verify the results. Thirty-six Sprague-Dawley rats were randomly divided into the normal control group (NC group), spermatogenic dysfunction group (SD group) and SC-FL treatment group (SCFL group). Glucosides of Tripterygium wilfordii Hook. F (GTW) (40 mg/kg/d) was administered for 4 weeks to generate a spermatogenic dysfunction model. The rats in the SCFL group were given the SC-FL suspension (6 g/kg/d) daily. After 4 weeks of treatment, we detected the sperm quality of each group of rats and observed the cell morphology. Western blotting and qRT-PCR were used to detect the expression of BTB-related proteins in testicular tissues. RESULTS: 213 chemical ingredients of SC and FL were retrieved from the TCMSP database, and 54 effective chemical ingredients were obtained. Mass spectrometry analysis showed the above results were credible. Then, we identified 44 potential targets for the treatment of male infertility, and we plotted a network diagram of the interaction network between the core targets and a diagram of herbal medicine-active ingredient-target-disease interactions. The target genes were enriched according to biological functions, and 22 biological processes, 49 cellular components, 1487 molecular functions, and 122 signaling pathways were obtained. The results of the animal experiments showed that the sperm concentration and motility of the SCFL group were significantly improved compared with those of the SD group. Compared with those in the SD group, the structure and morphology of the Sertoli cells and seminiferous tubules of rats in the SCFL group improved, and the number of spermatogenic cells increased significantly. Western blotting and qRT-PCR results showed that compared with that in the SD group, the expression of p38 MAPK decreased significantly, and the expression of c-Jun, Occludin, ZO-1 and connexin 43 increased significantly in the SCFL group. CONCLUSION: We predicted that the active ingredients of SC-FL can treat male infertility by interacting with the core targets JUN, IL6, MAPK1, TP53, MYC, CCND1, AR, EGF, FOS, and MAPK8, and the possible mechanism is related to the MAPK signaling pathway. SC-FL can regulate the MAPK pathway and affect the expression of Occludin, ZO-1 and connexin 43 to repair damaged BTB and improve spermatogenic dysfunction induced by GTW, which may be one of the possible mechanisms.

Aflatoxin B1 disrupts blood-testis barrier integrity by reducing junction protein and promoting apoptosis in mice testes.

Aflatoxin B1 (AFB1) is an unavoidable food and environmental contaminant, which can lead to disorders in spermatogenesis and its mechanism remains unclear. The blood-testis barrier (BTB) is responsible for ensuring normal spermatogenesis in testes. Therefore, we hypothesized that disruption of the BTB was involved in AFB1-induced spermatogenesis disorders. To confirm our hypothesis, male Kunming mice were orally gavaged AFB1 (0, 0.375, 0.75, or 1.5 mg/kg) for 30 days. Primarily, we first proved that AFB1 disrupted the BTB integrity. Then, AFB1 decreased BTB-related junction protein expression and elevated Sertoli cell apoptosis, which were associated with oxidative stress. Additionally, AFB1 upregulated the p-p38 MAPK/p38 MAPK ratio. These results collectively indicated that AFB1 disrupted the BTB via reducing the expression of BTB-related junction protein and promoting apoptosis in mice testes, which were associated with the oxidative stress-mediated p38 MAPK signaling pathway.

Blood testis barrier revisited-Analysis of post-chemotherapy germ cell tumor orchidectomy and retroperitoneal lymph node dissection specimens.

OBJECTIVE: To assess the response of chemotherapy on the primary tumor, compare it with the response in retroperitoneal disease, and study factors associated with pathological complete response. METHODS: We conducted a retrospective audit of all high inguinal orchidectomies (HIOs) performed after chemotherapy between 2012 and 2019 at a tertiary cancer center in India. Patient characteristics and histopathological response were extracted from electronic medical records, and predictors of testicular disease response were assessed. RESULTS: Of the 260 retroperitoneal lymph node dissections (RPLNDs) performed in the study period, 37 HIOs (14.23%) were carried out after chemotherapy. The median age of presentation was 28 years (16-41). Histopathology was divided into a viable tumor, mature teratoma, and necrosis/scarring. Residual disease was seen in 17 RPLND (46.0%) and 18 HIO (48.6%) specimens respectively. Of these 18, three patients had a residual viable tumor in the testis, and the remaining had a mature teratoma. Clinico-radiological assessment showed an average reduction of 61% in testicular disease size following chemotherapy. On orchidectomy histopathological assessment, the median tumor size was 9, 4, and 1.5 cm in specimens with a viable tumor, mature teratoma, and necrosis/scarring, respectively. CONCLUSIONS: A low threshold for upfront chemotherapy in patients with a high disease burden may be considered as tumors within the testis respond to chemotherapy in more than half of the patients. Discordance rates of residual cancer in RPLND and HIO specimens exist but post-chemotherapy tumor size in testis correlates with the presence of a residual viable tumor.