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Purpose: This study aimed to investigate the effect of the N6-methyladenosine (m6A) dependent ferroptosis on cisplatin-induced Sertoli cell injury. Materials and Methods: A cisplatin exposure mouse model was established by intraperitoneal injection of cisplatin in our study. TM4 cell lines was used for in vitro study. Ferroptosis was detected according to metabolomic analysis and a series of assays, including malondialdehyde, glutathione, and glutathione disulfide concentration detection, 2',7'-dichlorodihydrofluo-rescein diacetate and BODIPY 581/591 C11 probe detection, and transmission electron microscope imaging. Key ferroptosis-related genes were identified via transcriptomic analysis, western blot and immunohistochemistry. The m6A modification was demonstrated via m6A RNA immunoprecipitation and luciferase reporter assays. Immune cell infiltration was detected by mass cytometry, and verified by flow cytometry and immunofluorescence. Results: Ferroptosis, but not other types of programmed cell death, is a significant phenomenon in cisplatin-induced testis damage and Sertoli cell loss. Ferroptosis induced by cisplatin in Sertoli cell/TM4 cell is GPX4 independent but is regulated by SLC7A11 and ALOX12. Both SLC7A11 and ALOX12 are regulated via m6A dependent manner by METTL3. Furthermore, overexpressed ALOX12-12HETE pathway may result in macrophage polarization and inflammatory response in cisplatin exposure testis. Conclusions: Cisplatin-induced Sertoli cell injury via ferroptosis and promoted ferroptosis in an m6A dependent manner. m6A modification of both SLC7A11 and ALOX12 mRNA could result in ferroptosis in our in vitro model. Further, overexpressed ALOX12 can cause more production of 12-HETE, which may be responsible for testis inflammation caused by cisplatin. [ABSTRACT FROM AUTHOR]

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. • Tas2R143 affected to varying degrees the expression of BTB tight junction proteins (occludin, ZO-1) and NF-κB. • Tas2R143 likely regulates the expression of occludin and ZO-1 through the NF-κB signaling pathway in vitro. • This study proposes for the first time that the bitter receptor Tas2R143 is expressed in mouse Sertoli cells (TM4). • This study provides a theoretical basis for studying the regulatory mechanism of bitter receptors in the reproductive system. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood–testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of glutathione, a powerful antioxidant crucial to protecting against oxidative stress-induced damage. Animal studies have demonstrated the protective effect of L-cysteine in preventing testicular damage caused by chemicals or radiation. This study examines whether L-cysteine enhances the expression of testosterone biosynthesis and the BTB genes in human Leydig cells and THP-1 monocytes. The Leydig cells and THP-1 monocytes were treated with L-cysteine for 24 h. RNA was extracted following treatment, and the gene expression was analyzed using quantitative RT-PCR. Testosterone levels in the cell supernatant were measured using an ELISA kit. L-cysteine treatment in Leydig cells significantly upregulated the expression of CYP11A1 (p = 0.03) and the BTB genes CLDN1 (p = 0.03), CLDN11 (p = 0.02), and TJP1 (p = 0.02). Similarly, L-cysteine significantly upregulated the expression of CYP11A1 (p = 0.03) and CYP19A1 (p < 0.01), and the BTB genes CLDN1 (p = 0.04), CLDN2 (p < 0.01), CLDN4 (p < 0.01), CLDN11 (p < 0.01), and TJP1 (p = 0.03) in THP-1 monocytes. Further, L-cysteine supplementation increased the testosterone secretion levels in human Leydig cells. The findings suggest that L-cysteine supplementation could be used as an adjuvant therapy to promote the integrity of the BTB genes, testosterone biosynthesis and secretion, and the maintenance of testicular functions, which in turn mitigates the risk of male infertility. [ABSTRACT FROM AUTHOR]

Blood–testis barrier (BTB) genes are crucial for the cellular mechanisms of spermatogenesis as they protect against detrimental cytotoxic agents, chemicals, and pathogens, thereby maintaining a sterile environment necessary for sperm development. BTB proteins predominantly consist of extensive tight and gap junctions formed between Sertoli cells. These junctions form a crucial immunological barrier restricting the intercellular movement of substances and molecules within the adluminal compartment. Epithelial tight junctions are complex membrane structures composed of various integral membrane proteins, including claudins, zonula occludens-1, and occludin. Inter-testicular cell junction proteins undergo a constant process of degradation and renewal. In addition, the downregulation of genes crucial to the development and preservation of cell junctions could disrupt the functionality of the BTB, potentially leading to male infertility. Oxidative stress and inflammation may contribute to disrupted spermatogenesis, resulting in male infertility. L-cysteine is a precursor to glutathione, a crucial antioxidant that helps mitigate damage and inflammation resulting from oxidative stress. Preclinical research indicates that L-cysteine may offer protective benefits against testicular injury and promote the expression of BTB genes. This review emphasizes various BTB genes essential for preserving its structural integrity and facilitating spermatogenesis and male fertility. Furthermore, it consolidates various research findings suggesting that L-cysteine may promote the expression of BTB-associated genes, thereby aiding in the maintenance of testicular functions. [ABSTRACT FROM AUTHOR]

Abstract 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.

Background Material and methods Results and discussion Conclusions The escalating prevalence of fertility problems in the aging population necessitates a comprehensive exploration of contributing factors, extending beyond environmental concerns, work‐related stress, and unhealthy lifestyles. Among these, the rising incidence of testicular disorders emerges as a pivotal determinant of fertility issues. Current treatment challenges are underscored by the limitations of high‐dose and frequent drug administration, coupled with substantial side effects and irreversible trauma inflicted by surgical interventions on testicular tissue.The formidable barrier posed by the blood–testis barrier compounds the complexities of treating testicular diseases, presenting a significant therapeutic obstacle. The advent of nanocarriers, with their distinctive attributes, holds promise in overcoming this impediment. These nanocarriers exhibit exceptional biocompatibility, and membrane penetration capabilities, and can strategically target the blood–testis barrier through surface ligand modification, thereby augmenting drug bioavailability and enhancing therapeutic efficacy.This review concentrates on the transformative potential of nanocarriers in the delivery of therapeutic agents to testicular tissue. By summarizing key applications, we illuminate the strides made in utilizing nanocarriers as a novel avenue to effectively treat testicular diseases.Nanocarriers are critical in delivering therapeutic agents to testicular tissue. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

With the rapid expansion of industrial scale, an increasing number of fine particulate matter (PM2.5) has bringing health concerns. Although exposure to PM2.5 has been clearly associated with male reproductive toxicity, the exact mechanisms are still unclear. Recent studies demonstrated that exposure to PM2.5 can disturb spermatogenesis through destroying the blood-testis barrier (BTB), consisting of different junction types, containing tight junctions (TJs), gap junctions (GJs), ectoplasmic specialization (ES) and desmosomes. The BTB is one of the tightest blood-tissue barriers among mammals, which isolating germ cells from hazardous substances and immune cell infiltration during spermatogenesis. Therefore, once the BTB is destroyed, hazardous substances and immune cells will enter seminiferous tubule and cause adversely reproductive effects. In addition, PM2.5 also has shown to cause cells and tissues injury via inducing autophagy, inflammation, sex hormones disorder, and oxidative stress. However, the exact mechanisms of the disruption of the BTB, induced by PM2.5, are still unclear. It is suggested that more research is required to identify the potential mechanisms. In this review, we aim to understand the adverse effects on the BTB after exposure to PM2.5 and explore its potential mechanisms, which provides novel insight into accounting for PM2.5-induced BTB injury. [ABSTRACT FROM AUTHOR]

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin–related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila , which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood–testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor the Sertoli cell tight junction (TJ) permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with β-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP core protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis. [ABSTRACT FROM AUTHOR]

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 β-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.

Background: It is well known that metabolic disorders, including type 1 diabetes (T1D), are often associated with reduced male fertility, mainly increasing oxidative stress and impairing the hypothalamus--pituitary--testis (HPT) axis, with consequently altered spermatogenesis and reduced sperm parameters. Herein, using a rat model of T1D obtained by treatment with streptozotocin (STZ), we analyzed several parameters of testicular activity. Methods: A total of 10 adult male Wistar rats were divided into two groups of five: control and T1D, obtained with a single intraperitoneal injection of STZ. After 3 months, the rats were anesthetized and sacrificed; one testis was stored at -80°C for biochemical analysis, and the other was fixed for histological and immunofluorescence analysis. Results: The data confirmed that T1D induced oxidative stress and, consequently, alterations in both testicular somatic and germ cells. This aspect was highlighted by enhanced apoptosis, altered steroidogenesis and Leydig cell maturity, and impaired spermatogenesis. In addition, the blood--testis barrier integrity was compromised, as shown by the reduced levels of structural proteins (N-cadherin, ZO-1, occludin, connexin 43, and VANGL2) and the phosphorylation status of regulative kinases (Src and FAK). Mechanistically, the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways was proven, particularly the reduced nuclear translocation of NRF2, affecting its ability to induce the transcription of genes encoding for antioxidant enzymes. Finally, the stimulation of testicular inflammation and pyroptosis was also confirmed, as highlighted by the increased levels of some markers, such as NF-kB and NLRP3. Conclusion: The combined data allowed us to confirm that T1D has detrimental effects on rat testicular activity. Moreover, a better comprehension of the molecular mechanisms underlying the association between metabolic disorders and male fertility could help to identify novel targets to prevent and treat fertility disorders related to T1D. [ABSTRACT FROM AUTHOR]

Simple Summary: Type 2 diabetes mellitus is a chronic disease afflicting millions of people worldwide. Though metformin is the main pharmacological treatment in children, little is known about the effects of this drug on testicular maturation, which takes place at this stage of life. Sertoli cells (SCs) have a central role in spermatogenesis since they support germ cell development. This is mainly achieved through blood–testis barrier (BTB) formation and the influence of SC metabolites on germ cells. We observed in SC cultures that metformin modifies fatty acid metabolism, ketone body production, and junction formation between SCs. Also, metformin treatment of juvenile rats provokes a mild but significant increase in BTB permeability. However, metformin does not affect testicular histology or meiosis progression. Moreover, BTB function is recovered, and daily sperm production is not affected in adult animals treated with metformin during the juvenile stage, suggesting that the effects on BTB permeability are reversible. Diabetes mellitus incidence is rising at staggering rates in children, and so is the exposure to metformin. Considering that a complete evaluation of the testicular function in children is usually not achievable, the present study seeks to add information about the safety of metformin treatment during this period of life. Sertoli cells (SCs) are essential to maintaining germ cell development. Metformin, the main pharmacologic treatment for pediatric type 2 diabetes, is administered to children during SC maturation. The present study aimed to analyze whether metformin affects SC energy metabolism and blood–testis barrier (BTB) integrity. Primary SC cultures were used for the in vitro studies. In vivo effects were studied in Sprague–Dawley rats treated with 200 mg/kg metformin from Pnd14 to Pnd30. Metformin decreased fatty acid oxidation and increased 3-hydroxybutyrate production in vitro. Moreover, it decreased the transepithelial electrical resistance across the monolayer and induced ZO-1 redistribution, suggesting an alteration of cell junctions. In vivo, a mild but significant increase in BTB permeability and ZO-1 expression was observed in the metformin group, without changes in testicular histology and meiosis progression. Additionally, adult rats that received metformin treatment during the juvenile period showed no alteration in BTB permeability or daily sperm production. In conclusion, metformin exposure may affect BTB permeability in juvenile rats, but this seems not to influence spermatogenesis progression. Considering the results obtained in adult animals, it is possible to speculate that metformin treatment during the juvenile period does not affect testicular function in adulthood. [ABSTRACT FROM AUTHOR]

The protective action of melatonin (MLT) against the harmful effects of cadmium (Cd) on testicular activity in rats has been documented previously; however, the involved molecular mechanisms have yet to be elucidated. Herein, we investigate the involvement of the mammalian target of rapamycin (mTOR) on the ability of MLT to counteract the damage induced by Cd on the rat testicular activity. Our study confirmed that Cd has harmful effects on the testes of rats and the protective action exerted by MLT. We reported, for the first time, that the addition of rapamycin (Rapa), a specific mTOR inhibitor, to animals co‐treated with Cd and MLT completely abolished the beneficial effects exerted by MLT, indicating that the mTOR pathway partially modulates its helpful effects on Cd testicular toxicity. Interestingly, Rapa‐alone treatment, provoking mTOR inhibition, produced altered morphological parameters, increased autophagy of germ and somatic cells, and reduced serum testosterone concentration. In addition, mTOR inhibition also reduced protein levels of markers of steroidogenesis (3β‐Hydroxysteroid dehydrogenase) and blood–testis barrier integrity (occludin and connexin 43). Finally, Rapa altered sperm parameters as well as the ability of mature spermatozoa to perform a proper acrosome reaction. Although further investigation is needed to better clarify the molecular pathway involved in MLT action, we confirm that MLT alleviating Cd effects can be used as a supplement to enhance testicular function and improve male gamete quality. Research Highlights: We confirmed the Cd harmful effects and MLT protective action on rat testes.The mTOR inhibitor Rapa completely abolished the MLT beneficial effects.Results indicate that the mTOR pathway modulates MLT counteractive effects on Cd testicular toxicity. [ABSTRACT FROM AUTHOR]

Cuproptosis, a novel mechanism of programmed cell death, has not been fully explored in the context of spermatogenic cells. This study investigated the potential involvement of cuproptosis in spermatogenic cell death using a mouse model of copper overload. Sixty male Institute of Cancer Research (ICR) mice were randomly divided into four groups that received daily oral gavage with sodium chloride (control) or copper sulfate (CuSO4) at 50 mg kg-1, 100 mg kg-1, or 200 mg kg-1, for 42 consecutive days. Mice subjected to copper overload exhibited a disruption in copper homeostasis. Additionally, significant upregulated expression of key cuproptosis factors was accompanied by a significant rise in the rates of testicular tissue cell apoptosis. Immunohistochemical analysis revealed the presence of ferredoxin 1 (Fdx1) in Sertoli cells, Leydig cells, and spermatogenic cells at various stages of testicular development, and the Fdx1-positive staining area was significantly increased in copper-overloaded mice. Mitochondrial dysfunction and decreased adenosine triphosphate levels were also observed, further implicating mitochondrial damage under cuproptosis. Further analyses revealed pathological lesions and blood-testis barrier destruction in the testicular tissue, accompanied by decreased sperm concentration and motility, in copper-overloaded mice. In summary, our results indicate that copper-overloaded mice exhibit copper homeostasis disorder in the testicular tissue and that cuproptosis participates in spermatogenic cell death. These findings provide novel insights into the pathogenic mechanisms underlying spermatogenic cell death and provide initial experimental evidence for the occurrence of cuproptosis in the testis. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4–8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood–testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, β-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, β-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies. [ABSTRACT FROM AUTHOR]

Background: The epidemiology of metastases from rare genitourinary cancer and metastases to genitourinary organs from other primary neoplasms remains poorly understood. Objective: To investigate the epidemiology of rare genitourinary metastases from rare genitourinary organ–type cancer and to genitourinary organs using data from a large national autopsy registry in Japan. Design, setting, and participants: A secondary analysis of the data reported in the Annual of the Pathological Autopsy Cases in Japan and the Japanese Mortality Database from 1993 to 2020 was performed. Outcome measurements and statistical analysis: Via a retrospective epidemiologic analysis, we evaluated the frequency (probability of occurrence [number per person]) and proportion (percentage) of metastases from upper urinary tract, adrenal, testicular, urethral, and penile cancers. Moreover, the sites of primary tumors metastasizing to genitourinary organs were examined. Results and limitations: In Japan, the mortality rate of upper urinary tract cancer is increasing rapidly. In the integrated database with 365 099 autopsies and 835 959 metastatic organs, the major metastatic sites (range of frequency ratios) of rare genitourinary organ–type cancers were the lungs (0.38–0.47), liver (0.21–0.56), bone (0.16–0.33), adrenal gland (0.10–0.20), peritoneum (0.0–0.16), and kidneys (0.07–0.22). The major primary sites (range of proportions) of genitourinary organ metastases were the respiratory tract (5.6–34.0%), stomach (4.7–27.0%), hematologic site (0.9–24.9%), lymphoid (2.4–22.2%), bladder (0.8–20.0%), prostate (0.7–14.1%), rectal (2.0–11.7%), and pancreas (2.6–11.0%). The cancers with a high likelihood of genitourinary metastasis were respiratory and stomach cancers. However, the study lacked individual-level information, and there might be a concomitant selection bias in this autopsy study. Conclusions: This large-scale autopsy database analysis identified the epidemiology of metastasis from rare genitourinary organ–type cancer and the origins of metastasis to genitourinary organs. Patient summary: This study provides valuable metastatic epidemiologic data and clinical information that are fundamental to the mechanisms of genitourinary metastasis.

Sertoli cells are unique cells that contribute to the formation of the blood-testis barrier, which is important in sustaining the environment to promote spermatogenesis and to protect immunogenic germ cells from autoimmune destruction. This is achieved through tight junctions and production of regulatory immune factors. These Sertoli cell attributes make them a relevant model for various studies involving male reproduction, autoimmune protection, and even transplantation. RNA sequencing analyses were performed on baseline neonatal porcine Sertoli cells (NPSC) and NPSC after incubation in normal human serum for 90 minutes. We previously analyzed this data for immune-related factors, such as complement components, and for differentially expressed genes related to immune function. Still, these data sets provide insight into understanding how Sertoli cells create an immunoregulatory environment, which has applications in reproduction, transplantation, and autoimmunity. [ABSTRACT FROM AUTHOR]

Sertoli cells are a crucial component of the blood-testis barrier (BTB), which isolates the adluminal compartment of the seminiferous tubules from the rest of the testis thus forming an environment to immunely protect the developing germ cells. The mechanisms of regulating immune responses within this environment are currently under investigation. Here, we focused on Sertoli cell regulation of the complement system. [ABSTRACT FROM AUTHOR]

Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood–testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of glutathione, a powerful antioxidant crucial to protecting against oxidative stress-induced damage. Animal studies have demonstrated the protective effect of L-cysteine in preventing testicular damage caused by chemicals or radiation. This study examines whether L-cysteine enhances the expression of testosterone biosynthesis and the BTB genes in human Leydig cells and THP-1 monocytes. The Leydig cells and THP-1 monocytes were treated with L-cysteine for 24 h. RNA was extracted following treatment, and the gene expression was analyzed using quantitative RT-PCR. Testosterone levels in the cell supernatant were measured using an ELISA kit. L-cysteine treatment in Leydig cells significantly upregulated the expression of CYP11A1 (p = 0.03) and the BTB genes CLDN1 (p = 0.03), CLDN11 (p = 0.02), and TJP1 (p = 0.02). Similarly, L-cysteine significantly upregulated the expression of CYP11A1 (p = 0.03) and CYP19A1 (p < 0.01), and the BTB genes CLDN1 (p = 0.04), CLDN2 (p < 0.01), CLDN4 (p < 0.01), CLDN11 (p < 0.01), and TJP1 (p = 0.03) in THP-1 monocytes. Further, L-cysteine supplementation increased the testosterone secretion levels in human Leydig cells. The findings suggest that L-cysteine supplementation could be used as an adjuvant therapy to promote the integrity of the BTB genes, testosterone biosynthesis and secretion, and the maintenance of testicular functions, which in turn mitigates the risk of male infertility.

Due to the increasing trend of delayed childbirth, the age-related decline in male reproductive function has become a widely recognized issue. Sertoli cells (SCs) play a vital role in creating the necessary microenvironment for spermatogenesis in the testis. However, the mechanism underlying Sertoli cell aging is still unclear. In this study, senescent Sertoli cells showed a substantial upregulation of miR-143-3p expression. miR-143-3p was found to limit Sertoli cell proliferation, promote cellular senescence, and cause blood–testis barrier (BTB) dysfunction by targeting ubiquitin-conjugating enzyme E2 E3 (UBE2E3). Additionally, the TGF-β receptor inhibitor SB431542 showed potential in alleviating age-related BTB dysfunction, rescuing testicular atrophy, and reversing the reduction in germ cell numbers by negatively regulating miR-143-3p. These findings clarified the regulatory pathways underlying Sertoli cell senescence and suggested a promising therapeutic approach to restore BTB function, alleviate Sertoli cell senescence, and improve reproductive outcomes for individuals facing fertility challenges. [ABSTRACT FROM AUTHOR]

The blood-testis barrier (BTB) is constituted by tight junctions between adjacent Sertoli cells (SCs) that create a specialized adluminal microenvironment to foster the development of spermatocytes and spermatids. The BTB is a well-studied target of numerous environmental toxicants, including di-(2-ethylhexyl) phthalate (DEHP), a compound widely used in various consumer products. Mono-(2-ethylhexyl) phthalate (MEHP) is the active toxic metabolite of DEHP that has long been recognized in postnatal rodents to disrupt SC function. This study evaluates the impact of MEHP on the integrity of the BTB in both pubertal and adult rats and the signal transduction pathways known to be involved in the disruption of the BTB. Treatment of prepubertal rats with 700 mg/kg MEHP for 24 h functionally disrupted the BTB integrity. A similar treatment of adult rats with MEHP did not disrupt the integrity of the BTB. The observed disruption of the BTB integrity in the MEHP-treated prepubertal rats occurred concomitantly with a decreased expression and mislocalization of both the ZO1 and occludin tight junction-associated proteins, as well as sloughing of spermatocytes and spermatids. At this same time, MEHP treatment induced a transient surge of p44/42 mitogen-activated protein kinase (MAPK) pathway. Interestingly, after a recovery period of 5 weeks, the BTB recovered and was functionally intact. This is the first report to indicate that acute MEHP exposure of prepubertal rats, but not adult rats, disrupts the functional integrity of the BTB and that this effect on the BTB is reversible. [ABSTRACT FROM AUTHOR]

As a pivotal player in spermatogenesis, the blood-testis barrier (BTB) made from junction apparatus coexisting in Sertoli cells (SCs) is impaired with an increase in age and ultimately induces spermatogenic dysfunction or even infertility. It has been corroborated that bone marrow mesenchymal stem cell (BMSC) transplantation can efficiently repair and regenerate the testicular function. As vital mediators of cell-to-cell communication, MSC-derived exosomes (Exos) can directly serve as therapeutic agents for tissue repair and regeneration. However, the therapeutic value of BMSC-Exos in aging-induced BTB damage remains to be confirmed. In this study, we explored that the old porcine testes had defective autophagy, which aggravated BTB disruption in SCs. BMSC-Exos could decrease ROS production and NLRP3 inflammasome activation but enhanced autophagy and tight junction (TJ) function in D-gal-triggered aging porcine SCs and mouse model testes, according to in vitro and in vivo experiments. Furthermore, rapamycin, NAC, MCC950, and IL-1Ra restored the TJ function in D-gal-stimulated aging porcine SCs, while BMSC-Exos' stimulatory effect on TJ function was inhibited by chloroquine. Moreover, the treatment with BMSC-Exos enhanced autophagy in D-gal-induced aging porcine SCs by means of the AMPK/mTOR signal transduction pathway. These findings uncovered through the present study that BMSC-Exos can enhance the BTB function in aging testes by improving autophagy via the AMPK/mTOR signaling pathway, thereby suppressing ROS production and NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Simple Summary: Cryptorchidism is believed to be one of the leading causes of infertility in male yaks. In this study, we compared the morphology of the normal testis of the yak with that of the cryptorchidism, and found dysplasia of the seminiferous tubules, impaired tightness of the Sertoli cells, and a disruption of the integrity of the blood–testis barrier (BTB) in the cryptorchidism. Based on RNA-seq, bioinformatics analysis and biological experiments, CAV1 up-regulates or down-regulates the expression levels of ZO-1, occludin and claudin-11 via the FAK/ERK pathway in vitro cell model assays. Our results reveal a novel mechanism by which CAV1 modulates tight junctions and BTB, suggesting that CAV1 may be involved in the regulation of the occurrence of yak cryptorchidism. Yaks, a valuable livestock species endemic to China's Tibetan plateau, have a low reproductive rate. Cryptorchidism is believed to be one of the leading causes of infertility in male yaks. In this study, we compared the morphology of the normal testis of the yak with that of the cryptorchidism, and found dysplasia of the seminiferous tubules, impaired tightness of the Sertoli cells, and a disruption of the integrity of the blood–testis barrier (BTB) in the cryptorchidism. Previous studies have shown that CAV1 significantly contributes to the regulation of cell tight junctions and spermatogenesis. Therefore, we hypothesize that CAV1 may play a regulatory role in tight junctions and BTB in Yaks Sertoli cells, thereby influencing the development of cryptorchidism. Additional analysis using immunofluorescence, qRT-PCR, and Western blotting confirmed that CAV1 expression is up-regulated in yak cryptorchidism. CAV1 over-expression plasmids and small RNA interference sequences were then transfected in vitro into yak Sertoli cells. It was furthermore found that CAV1 has a positive regulatory effect on tight junctions and BTB integrity, and that this regulatory effect is achieved through the FAK/ERK signaling pathway. Taken together, our findings, the first application of CAV1 to yak cryptorchidism, provide new insights into the molecular mechanisms of cell tight junctions and BTB. This paper suggests that CAV1 could be used as a potential therapeutic target for yak cryptorchidism and may provide insight for future investigations into the occurrence of cryptorchidism, the maintenance of a normal physiological environment for spermatogenesis and male reproductive physiology in the yak. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEA) is present worldwide as a serious contaminant of food and feed and causes male reproductive toxicity. The implication of paraptosis, which is a nonclassical paradigm of cell death, is unclear in ZEA-induced male reproductive disorders. In this study, the toxic effects of ZEA on the blood-testis barrier (BTB) and the related mechanisms of paraptosis were detected in goats. ZEA exposure, in vivo, caused a significant decrease in spermatozoon quality, the destruction of seminiferous tubules, and damage to the BTB integrity. Furthermore, ZEA exposure to Sertoli cells (SCs) in vitro showed similar dysfunction in structure and barrier function. Importantly, the formation of massive cytoplasmic vacuoles in ZEA-treated SCs corresponded to the highly swollen and dilative endoplasmic reticulum (ER), and paraptosis inhibition significantly alleviated ZEA-induced SC death and vacuolization, which indicated the important contribution of paraptosis in ZEA-induced BTB damage. Meanwhile, the expression of ER stress marker proteins was increased after ZEA treatment but decreased under the inhibition of paraptosis. The vacuole formation and SC death, induced by ZEA, were remarkably blocked by ER stress inhibition. In conclusion, these results facilitate the exploration of the mechanisms of the SC paraptosis involved in ZEA-induced BTB damage in goats. [ABSTRACT FROM AUTHOR]

Background: Testis play role as endocrine organ by regulate and synthesis of hormones, and genital role as germ sells production (spermatogenesis) for reproduction process. The Sertoli cells in the seminiferous tubule with basal lamina and endothelium of vesicles, form a special structure called blood-testis-barrier (BTB) withe protection function and specific properties that similar to blood tissue barriers of other body organs. Methods: Forty male healthy Swiss albino mice weighing 24-30 g will be kept in 2 groups (20 for each) in the labs of Al-Nahrain University College of medicine for 6 weeks from 1 December 2022_15 Jan 2023. Regular rodent food and water will be provided and Grouping as: Group A food minced with concentration of 400mg/kg of Bifenthrin insecticides and Group B have normal food. Results: The histological study showed partial depletion testis cells in all mice testes with non-significant decrease in interstitial space that has Leydig cells between seminiferous tubule (ST) in testis of male mice that were fed with Bifenthrin diet. There were less cadherin expression in the BTB indicating that the Bifenthrin has stressful stimulus to potentiate apoptosis in the genital and somatic (Sertoli) cells of the testis that eventually impact the cadherin protein. The Bifenthrin has as stimulant chemical factor for more apoptosis in the ST cells death with subsequently testicular atrophy and subsequently infertility. The Bifenthrin treated male mice shows significant diminish positive values of N-cadherin expression that appear as less brown coloration of immunohistochemical biomarker expression of N-cadherin in the ST cells of the testis that was best evaluated by applying Aperio software program. Conclusions: The N-cadherin protein has pivotal role in regulation the adhesiveness of the germinativeepithelial cells in ST of the testis. The Bifenthrin insecticides as an endocrine disrupter material has great effects on spermatogenesis and tissue damage. [ABSTRACT FROM AUTHOR]

BackgroundIt is well known that metabolic disorders, including type 1 diabetes (T1D), are often associated with reduced male fertility, mainly increasing oxidative stress and impairing the hypothalamus–pituitary–testis (HPT) axis, with consequently altered spermatogenesis and reduced sperm parameters. Herein, using a rat model of T1D obtained by treatment with streptozotocin (STZ), we analyzed several parameters of testicular activity.MethodsA total of 10 adult male Wistar rats were divided into two groups of five: control and T1D, obtained with a single intraperitoneal injection of STZ. After 3 months, the rats were anesthetized and sacrificed; one testis was stored at -80°C for biochemical analysis, and the other was fixed for histological and immunofluorescence analysis.ResultsThe data confirmed that T1D induced oxidative stress and, consequently, alterations in both testicular somatic and germ cells. This aspect was highlighted by enhanced apoptosis, altered steroidogenesis and Leydig cell maturity, and impaired spermatogenesis. In addition, the blood–testis barrier integrity was compromised, as shown by the reduced levels of structural proteins (N-cadherin, ZO-1, occludin, connexin 43, and VANGL2) and the phosphorylation status of regulative kinases (Src and FAK). Mechanistically, the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways was proven, particularly the reduced nuclear translocation of NRF2, affecting its ability to induce the transcription of genes encoding for antioxidant enzymes. Finally, the stimulation of testicular inflammation and pyroptosis was also confirmed, as highlighted by the increased levels of some markers, such as NF-κB and NLRP3.ConclusionThe combined data allowed us to confirm that T1D has detrimental effects on rat testicular activity. Moreover, a better comprehension of the molecular mechanisms underlying the association between metabolic disorders and male fertility could help to identify novel targets to prevent and treat fertility disorders related to T1D.

Sertoli cells (SCs) support and nourish germ cells (GCs) through their crosstalk during spermatogenesis. However, the underlying epigenetic mechanism that ensures SCs' functions in this process remains unclear. Here, we report that UHRF1, a critical epigenetic regulator, is mainly expressed in human and mouse pre-mature SCs, and is essential for establishing Sertoli-Germ cell crosstalk. SC-specific UHRF1 knockout mice exhibit complete sterility with Sertoli cell (SC) proliferation and differentiation aberrance, blood-testis barrier (BTB) disruption, and immature germ cell (GC) sloughing. RNA sequencing and Whole Genome Bisulfite Sequencing (WGBS) revealed that many extracellular matrix (ECM)-related genes (e.g., Timp1, Trf, and Spp1) appeared upregulated with the DNA hypomethylation status in UHRF1-deficient SCs. Strikingly, overexpression of Timp1, Trf, and Spp1 in SCs in vitro and in vivo could phenocopy the SC-specific UHRF1-deficient mice. Our data demonstrated that UHRF1 regulates the transcriptional program of ECM-related genes in SCs and establishes SC-GC crosstalk.

A major change in the transcriptome of testicular Sertoli cells (Scs) at the onset of puberty enables them to induce robust spermatogenesis. Through comprehensive literature mining, we generated a list of genes crucial for Sc functioning and computationally predicted the microRNAs regulating them. Differential expression analysis of microRNAs in infant and pubertal rat Scs showed that miR-382-3p levels decline significantly in pubertal Scs. Interestingly, miR-382-3p was found to regulate genes like Ar and Wt1, which are crucial for functional competence of Scs. We generated a transgenic (Tg) mouse model in which pubertal decline of miR-382-3p was prevented by its overexpression in pubertal Scs. Elevated miR-382-3p restricted the functional maturation of Scs at puberty, leading to infertility. Prevention of decline in miR-382-3p expression in pubertal Scs was responsible for defective blood-testis barrier (BTB) formation, severe testicular defects, low epididymal sperm counts and loss of fertility in these mice. This provided substantial evidence that decline in levels of miR-382-3p at puberty is the essential trigger for onset of robust spermatogenesis at puberty. Hence, sustained high levels of miR-382-3p in pubertal Scs could be one of the underlying causes of idiopathic male infertility and should be considered for diagnosis and treatment of infertility.

Introduction: Male infertility is a multifactorial pathological condition and may be a harbinger of future health. Phthalates are ubiquitous environmental contaminants that have been implicated in the global decline in male fertility. Among them, di-(2-ethylhexyl) phthalate (DEHP) is the most prevalently used. Lycopene (LYC) is a possible preventive and therapeutic agent for male infertility owing to its antioxidant properties. The blood-testis barrier (BTB) is formed between Sertoli cells where it creates a unique microenvironment for spermatogenesis. Objectives: We hypothesize that phthalate caused male infertility and LYC plays an important role in phthalate-induced male fertility disorders. Methods: Hematoxylin-eosin (H&E) staining, ultrastructure observation, and fluorescence microscopy were used to examine the morphological changes. RNA-Seq, and western blotting were conducted to detect gene and protein levels. Routine testing for sperm morphology and sperm-egg binding assay were conducted to examine the morphological structure and function of sperm. Cell scratch assay and transepithelial electrical resistance (TER) were used to detect cell migration capacity and barrier integrity. Results: In vivo experiments, we showed that LYC prevented DEHP-induced impairment of BTB integrity, which provided a guarantee for the smooth progress of spermatogenesis. LYC improved DEHP-induced change in sperm parameters and fertilization ability. Subsequent in vitro experiments, LYC alleviated MEHP-induced disruption of intercellular junctions in mouse Spermatogonia cells (GC-1 cells) and mouse Sertoli cells (TM4 cells). In MEHP-induced BTB impairment models of Sertoli cells, treatment with LYC or overexpressing connexin-43 (Cx43) promoted cell migration capacity and normalized BTB integrity. Cx43 knockdown inhibited cell migration capacity and perturbed BTB reassembly in LYC preventing DEHP-induced BTB impairment. Conclusion: Our study provides evidence for the role of LYC in phthalates-induced spermatogenic disorders and points to Cx43 as a potential target for male fertility.

This study investigated the protective effect of rutin on reproductive and blood–testis barrier (BTB) damage induced by perfluorooctanoic acid (PFOA) exposure. In this study, male ICR mice were randomly divided into three groups, Ctrl group (ddH2O, 5 mL/kg), PFOA group (PFOA, 20 mg/kg/d, 5 mL/kg), PFOA + rutin group (PFOA, 20 mg/kg/d, 5 mL/kg; rutin, 20 mg/kg/d, 5 mL/kg). Mice were exposed to PFOA for 28 days by gavage once daily in the presence or absence of rutin. Histopathological observations demonstrated that rutin treatment during PFOA exposure can reduce structural damage to testis and epididymis such as atrophy of spermatogenic epithelium and stenosis of epididymal lumen, while increase in the number and layers of spermatogenic cells. Biochemical detection demonstrated that rutin can reduce 8-hydroxy-2′-desoxyguanosine (8-OHdG) concentration in the serum and testis tissues. Rutin can also ameliorate glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) content, and reduce malondialdehyde (MDA) and total cholesterol (TC) content in testis tissues. Biotin tracking immunofluorescence and transmission electron microscopy demonstrated that rutin can ameliorate BTB structural damage during PFOA exposure. Rutin ameliorated the stress expression of tight junction proteins occludin and claudin-11. In conclusion, our findings suggested that rutin has a degree of protection in reproductive and BTB damage, which could put forward a new perspective on the application of rutin to prevent reproductive damage. [ABSTRACT FROM AUTHOR]

Asthenozoospermia, characterized by poor sperm motility, is a common cause of male infertility. Improving energy metabolism and alleviating oxidative stress through drug regimens are potential therapeutic strategies. In this study, we observed upregulated miR-24-3p levels in asthenozoospermia spermatozoa, contributing to energy metabolism disorder and oxidative stress by reducing GSK3β expression. Thus, reducing miR-24-3p levels using drugs is expected to improve sperm motility. The blood-testis barrier (BTB) protects the testis from xenobiotics and drugs. In this study, we found that Sertoli cell-derived small extracellular vesicles (SC-sEV) can traverse the BTB and enter germ cells. We successfully loaded miR-24-3p inhibitor into SC-sEV, creating the nano-drug SC-sEV@miR-24-3p inhibitor, which effectively delivers miR-24-3p inhibitor into germ cells. In a gossypol-induced mouse asthenozoospermia model, administration of SC-sEV@miR-24-3p inhibitor significantly improved sperm motility, in vitro fertilization success, and blastocyst formation rates. As anticipated, it also improved the litter size of asthenozoospermia mice. These results suggest that SC-sEV@miR-24-3p inhibitor holds promise as a potential clinical treatment for asthenospermia. Sertoli cell-derived sEV-encapsulated miR-24-3p inhibitor traverse the blood-testis barrier and enter the germ cells to rescue asthenozoospermia. [Display omitted] • SC-sEV can traverse the BTB and enter germ cells. • SC-sEV did not exert any toxic effect on male mice. • Upregulated miR-24-3p contributes to reduced sperm motility. • SC-sEV@miR-24-3p inhibitor significantly improved sperm motility. [ABSTRACT FROM AUTHOR]

Cadherins (CDH) are crucial intercellular adhesion molecules, contributing to morphogenesis and creating tissue barriers by regulating cells' movement, clustering and differentiation. In the testis, classical cadherins such as CDH1, CDH2 and CDH3 are critical to gonadogenesis by promoting the migration and the subsequent clustering of primordial germ cells with somatic cells. While CDH2 is present in both Sertoli and germ cells in rodents, CDH1 is primarily detected in undifferentiated spermatogonia. As for CDH3, its expression is mainly found in germ and pre-Sertoli cells in developing gonads until the establishment of the blood--testis barrier (BTB). This barrier is made of Sertoli cells forming intercellular junctional complexes. The restructuring of the BTB allows the movement of early spermatocytes toward the apical compartment as they differentiate during a process called spermatogenesis. CDH2 is among many junctional proteins participating in this process and is regulated by several pathways. While cytokines promote the disassembly of the BTB by enhancing junctional protein endocytosis for degradation, testosterone facilitates the assembly of the BTB by increasing the recycling of endocytosed junctional proteins. Mitogen-activated protein kinases (MAPKs) are also mediators of the BTB kinetics in many chemically induced damages in the testis. In addition to regulating Sertoli cell functions, follicle stimulating hormone can also regulate the expression of CDH2. In this review, we discuss the current knowledge on regulatory mechanisms of cadherin localisation and expression in the testis. [ABSTRACT FROM AUTHOR]

Daily, people are exposed to chemicals and environmental compounds such as bisphenols (BPs). These substances are present in more than 80% of human fluids. Human exposure to BPs is associated with male reproductive health disorders. Some of the main targets of BPs are intercellular junction proteins of the blood‐testis barrier (BTB) in Sertoli cells because BPs alter the expression or induce aberrant localization of these proteins. In this systematic review, we explore the effects of BP exposure on the expression of BTB junction proteins and the characteristics of in vivo studies to identify potential gaps and priorities for future research. To this end, we conducted a systematic review of articles. Thirteen studies met our inclusion criteria. In most studies, animals treated with bisphenol‐A (BPA) showed decreased occludin expression at all tested doses. However, bisphenol‐AF treatment did not alter occludin expression. Cx43, ZO‐1, β‐catenin, nectin‐3, cortactin, paladin, and claudin‐11 expression also decreased in some tested doses of BP, while N‐cadherin and FAK expression increased. BP treatment did not alter the expression of α and γ catenin, E‐cadherin, JAM‐A, and Arp 3. However, the expression of all these proteins was altered when BPA was administered to neonatal rodents in microgram doses. The results show significant heterogeneity between studies. Thus, it is necessary to perform more research to characterize the changes in BTB protein expression induced by BPs in animals to highlight future research directions that can inform the evaluation of risk of toxicity in humans. [ABSTRACT FROM AUTHOR]

One cause of human male infertility is a scarcity of spermatogonial stem cells (SSCs) in testes with Sertoli cells that neither produce adequate amounts of GDNF nor form the Sertoli-Sertoli junctions that form the blood-testis barrier (BTB). These patients raise the issue of whether a pool of SSCs, depleted due to inadequate GDNF stimulation, will expand if normal signaling is restored. Here, we reduce adult mouse SSC numbers by 90% using a chemical-genetic approach that reversibly inhibits GDNF signaling. Signal resumption causes all remaining SSCs to replicate immediately, but they primarily form differentiating progenitor spermatogonia. Subsequently, self-renewing replication restores SSC numbers. Testicular GDNF levels are not increased during restoration. However, SSC replication decreases as numbers of SSCs and progenitors increase, suggesting important regulatory interactions among these cells. Finally, sequential loss of SSCs and then pachytene spermatocytes causes dissolution of the BTB, thereby recapitulating another important characteristic of some infertile men.

Sertoli cells (SCs) are essential to maintaining germ cell development. Metformin, the main pharmacologic treatment for pediatric type 2 diabetes, is administered to children during SC maturation. The present study aimed to analyze whether metformin affects SC energy metabolism and blood–testis barrier (BTB) integrity. Primary SC cultures were used for the in vitro studies. In vivo effects were studied in Sprague–Dawley rats treated with 200 mg/kg metformin from Pnd14 to Pnd30. Metformin decreased fatty acid oxidation and increased 3-hydroxybutyrate production in vitro. Moreover, it decreased the transepithelial electrical resistance across the monolayer and induced ZO-1 redistribution, suggesting an alteration of cell junctions. In vivo, a mild but significant increase in BTB permeability and ZO-1 expression was observed in the metformin group, without changes in testicular histology and meiosis progression. Additionally, adult rats that received metformin treatment during the juvenile period showed no alteration in BTB permeability or daily sperm production. In conclusion, metformin exposure may affect BTB permeability in juvenile rats, but this seems not to influence spermatogenesis progression. Considering the results obtained in adult animals, it is possible to speculate that metformin treatment during the juvenile period does not affect testicular function in adulthood.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4–8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood–testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, β-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, β-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Nickel (Ni) is an essential common environmental contaminant, it is hazardous to male reproduction, but the precise mechanisms are still unknown. Blood-testis barrier (BTB), an important testicular structure consisting of connections between sertoli cells, is the target of reproductive toxicity caused by many environmental toxins. In this study, ultrastructure observation and BTB integrity assay results indicated that NiCl2 induced BTB damage. Meanwhile, BTB-related proteins including the tight junction (TJ), adhesion junction (AJ) and the gap junction (GJ) protein expression in mouse testes as well as in sertoli cells (TM4) were significantly decreased after NiCl2 treatment. Next, the antioxidant N-acetylcysteine (NAC) was co-treated with NiCl2 to study the function of oxidative stress in NiCl2-mediated BTB deterioration. The results showed that NAC attenuated testicular histopathological damage, and the expression of BTB-related proteins were markedly reversed by NAC co-treatment in vitro and vivo. Otherwise, NiCl2 activated the p38 MAPK signaling pathway. And, NAC co-treatment could significantly inhibit p38 activation induced by NiCl2 in TM4 cells. Furthermore, in order to confirm the role of the p38 MAPK signaling pathway in NiCl2-induced BTB impairment, a p38 inhibitor (SB203580) was co-treated with NiCl2 in TM4 cells, and p38 MAPK signaling inhibition significantly restored BTB damage induced by NiCl2 in TM4 cells. These results suggest that NiCl2 treatment destroys the BTB, in which the oxidative stress-mediated p38 MAPK signaling pathway plays a vital role.

Nanoplastics (NPs) frequently cause adverse health effects by transporting organic pollutants such as dibutyl phthalate (DBP) into organisms by utilizing their large specific surface area, large surface charge, and increased hydrophobicity. However, the effects of NPs combined with DBP on the reproductive systems of mammals are still unclear. The present investigation involved the administration of polystyrene NPs (PS-NPs) to BALB/c mice via gavage, with a size of 100 nm and at doses of 5 mg/kg/day or 50 mg/kg/day, along with DBP at a dose of 0.5 mg/kg/day, or a combination of PS-NPs and DBP, for 30 days, to assess their potential for reproductive toxicity. The co-exposure of mice to PS-NPs and DBP resulted in a significant increase in reproductive toxicities compared to exposure to PS-NPs or DBP alone. This was demonstrated by a marked decrease in sperm quality, significant impairment of spermatogenesis, and increased disruption of the blood-testis barrier (BTB). Furthermore, a combination of in vivo and in vitro investigations were conducted to determine that the co-exposure of DBP and PS-NPs resulted in a noteworthy reduction in the expressions of tight junction proteins (ZO-1 and occludin). Moreover, the in vitro findings revealed that monobutyl phthalate (MBP, the active metabolite of DBP, 0.5 μg/mL) and PS-NPs (30 μg/mL or 300 μg/mL) inhibited autophagy in Sertoli cells, thereby increasing the expression of matrix metalloproteinases (MMPs). The study found that PS-NPs and DBP co-exposure caused harmful effects in male reproductive organs by disrupting BTB, which may be alleviated by reactivating autophagy. The paper's conclusions provided innovative perspectives on the collective toxicities of PS-NPs and other emerging pollutants.

The aim of this study was to evaluate the general histological structure of testis in prepubertal and postpubertal stages of quails and determine the presence and location of claudin-1 and zonula occludens-1 proteins. In this study, testicular tissues obtained from 6 prepubertal and postpubertal stage quails were used. Tissue samples were fixed in 10% formaldehyde and processed for paraffin embedding. Crossman's triple staining method was used for general histological evaluation. Immunohistochemistry and immunofluorescent staining were performed for the expression of claudin-1 and zonula occludens-1 proteins, respectively. It has also been concluded that the proteins of claudin-1 and zonula occludens-1 do not show immunoreactivity because an active blood-testis barrier is not formed yet in seminiferous tubule epithelium in the testis in the prepubertal period as a result of immunohistochemical and immunofluorescence stainings but show immunoreactivity in the basal area in seminiferous epithelium with the blood-testis barrier formed in the postpubertal period. Immunoreactivity wasn't observed in prepubertal quail's testis. The immunoreactivity of claudin-1 has been distinguished as cytoplasmic and membranous in Sertoli cells and spermatogonia in postpubertal quails' testis. The immunoreactivity of zonula occludens-1 has not been observed in seminiferous tubules in prepubertal stage quail's testis. Immunoreactivity has been observed in the basal half of seminiferous tubules in postpubertal stage quail's testis. It has also been concluded that the proteins of claudin-1 and zonula occludens-1 do not show immunoreactivity because an active blood-testis barrier is not formed yet in seminiferous tubule epithelium in the testis in the prepubertal period as a result of immunohistochemical and immunofluorescence stainings but show immunoreactivity in the basal area in seminiferous epithelium with the blood-testis barrier formed in the postpubertal period. [ABSTRACT FROM AUTHOR]

The Sertoli cell is a critical somatic cell that initiates the development of testicular morphology and determines important parameters for spermatogenic function. The blood-testis barrier, also known as the Sertoli cell barrier and one of the tightest tissue barriers in the mammalian body, is an immunological barrier to separate post meiotic germ cell antigens from the systemic circulation. Additionally, creating a unique microenvironment for the development of spermatocytes that exceed into the adluminal compartment from the leptotene stage. It restricts the passage of substances such as paracrine factors, electrolytes, hormones, water, and biological molecules to the apical part of the seminiferous tubule. It separates spermatogenic cells from toxic and drug-containing environmentally harmful substances, hormones, and biomolecules in the systemic circulation. This nearly impenetrable barrier prevents proteins, including antibodies, from reaching the spermatogenic cells. It also prevents protein leakage from developing spermatogenic cells and forming an immune response. This review explains Sertoli's functional properties, the testis barrier's molecular structure, the substances involved in the barrier dynamics, and their importance in realizing spermatogenesis. [ABSTRACT FROM AUTHOR]

The present study identified a critical role of Cx43 in LYC maintaining BTB integrity, which is a prerequisite for normal spermatogenesis. This study reveals the role of Cx43 in LYC preventing phthalate-induced spermatogenic disorders, and Cx43 regulation may provide the basis for future therapeutic male infertility. [Display omitted] • Phthalate induced spermatogenic disorders and destroyed BTB integrity. • Phthalate caused cell migration capacity decline and cytoskeletal disorganization. • LYC alleviated DEHP-induced disruption of intercellular junctions in mouse testes. • LYC improved DEHP-induced spermatogenic disorders by regulating BTB integrity. • LYC ameliorated DEHP-induced BTB disruption by modulating Cx43 expression. Male infertility is a multifactorial pathological condition and may be a harbinger of future health. Phthalates are ubiquitous environmental contaminants that have been implicated in the global decline in male fertility. Among them, di-(2-ethylhexyl) phthalate (DEHP) is the most prevalently used. Lycopene (LYC) is a possible preventive and therapeutic agent for male infertility owing to its antioxidant properties. The blood-testis barrier (BTB) is formed between Sertoli cells where it creates a unique microenvironment for spermatogenesis. We hypothesize that phthalate caused male infertility and LYC plays an important role in phthalate-induced male fertility disorders. Hematoxylin-eosin (H&E) staining, ultrastructure observation, and fluorescence microscopy were used to examine the morphological changes. RNA-Seq, and western blotting were conducted to detect gene and protein levels. Routine testing for sperm morphology and sperm-egg binding assay were conducted to examine the morphological structure and function of sperm. Cell scratch assay and transepithelial electrical resistance (TER) were used to detect cell migration capacity and barrier integrity. In vivo experiments, we showed that LYC prevented DEHP-induced impairment of BTB integrity, which provided a guarantee for the smooth progress of spermatogenesis. LYC improved DEHP-induced change in sperm parameters and fertilization ability. Subsequent in vitro experiments, LYC alleviated MEHP-induced disruption of intercellular junctions in mouse Spermatogonia cells (GC-1 cells) and mouse Sertoli cells (TM4 cells). In MEHP-induced BTB impairment models of Sertoli cells, treatment with LYC or overexpressing connexin-43 (Cx43) promoted cell migration capacity and normalized BTB integrity. Cx43 knockdown inhibited cell migration capacity and perturbed BTB reassembly in LYC preventing DEHP-induced BTB impairment. Our study provides evidence for the role of LYC in phthalates-induced spermatogenic disorders and points to Cx43 as a potential target for male fertility. [ABSTRACT FROM AUTHOR]

The adhesion protein nectin-like molecule 2 (NECL2) is involved in spermatogenesis and participates in the connections between Sertoli cells and germ cells. Necl2 deficiency leads to infertility in male mice. We found that NECL2 is relatively highly expressed on the cell membranes of preleptotene spermatocytes. It is known that preleptotene spermatocytes pass through the blood-testis barrier (BTB) from the base of the seminiferous tubules to the lumen to complete meiosis. We hypothesized that the NECL2 protein on the surfaces of preleptotene spermatocytes has an effect on the BTB when crossing the barrier. Our results showed that Necl2 deficiency caused the levels of proteins in the BTB to be abnormal, such as those of Claudin 3, claudin 11, and Connexin43. NECL2 interacted and colocalized with adhesion proteins forming the BTB, such as Connexin43, Occludin, and N-cadherin. NECL2 regulated BTB dynamics when preleptotene spermatocytes passed through the barrier, and Necl2 deficiency caused BTB damage. Necl2 deletion significantly affected the testicular transcriptome, especially the expression of spermatogenesis-related genes. These results suggest that before meiosis and spermatid development occur, BTB dynamics regulated by NECL2 are necessary for spermatogenesis. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disrupts the blood-testis barrier (BTB), resulting in alterations in spermatogenesis. However, whether BTB-related proteins (such as ZO-1, claudin11, N-cadherin, and CX43) are targeted by SARS-CoV-2 remains to be clarified. BTB is a physical barrier between the blood vessels and the seminiferous tubules of the animal testis, and it is one of the tightest blood-tissue barriers in the mammalian body. In this study, we investigated the effects of viral proteins, via ectopic expression of individual viral proteins, on BTB-related proteins, the secretion of immune factors, and the formation and degradation of autophagosomes in human primary Sertoli cells. Our study demonstrated that ectopic expression of viral E (envelope protein) and M (membrane protein) induced the expressions of ZO-1 and claudin11, promoted the formation of autophagosomes, and inhibited autophagy flux. S (spike protein) reduced the expression of ZO-1, N-cadherin, and CX43, induced the expression of claudin11, and inhibited the formation and degradation of autophagosomes. N (nucleocapsid protein) reduced the expression of ZO-1, claudin11, and N-cadherin. All the structural proteins (SPs) E, M, N, and S increased the expression of the FasL gene, and the E protein promoted the expression and secretion of FasL and TGF-β proteins and the expression of IL-1. Blockage of autophagy by specific inhibitors resulted in the suppression of BTB-related proteins by the SPs. Our results indicated that SARS-CoV-2 SPs (E, M, and S) regulate BTB-related proteins through autophagy. [ABSTRACT FROM AUTHOR]

Abstract Background RNA N 6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood–testis barrier (BTB) through cell junctions between adjacent Sertoli cells. Methods ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation–quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation–qPCR, western blot, coimmunoprecipitation, and polysome fractionation–qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure. Results ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice. Conclusions Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.

Di(n‐butyl) phthalate (DBP) is considered a substance of serious concern because of its reproductive toxicity and endocrine‐disrupting properties. Exposure to DBP causes morphological and functional changes in the male reproductive system of birds and mammals. However, there are no detailed reports on the effects of DBP on the Sertoli cell and junctional complexes of the blood–testis barrier (BTB) in birds. The present study investigated dose‐related ultrastructural changes in Sertoli cells and junctional complexes of the BTB in adult Japanese quail (Coturnix coturnix japonica) exposed to DBP prior to puberty. A total of 25 Japanese quail were used for the study. Exposure to DBP doses of 50, 200 and 400 mg DBP/kg/d caused dose‐related ultrastructural changes in junctional complexes including dilation and separation, while disruption of cytoplasmic membranes and mitochondria was observed in Sertoli cells. There was a significant difference in the sum of vacuoles, vacuole diameter, nuclear width, nuclear length, nuclear area, sum of damaged spherical mitochondria, width of elongated mitochondria and the sum of damaged elongated mitochondria among the five treatment groups (p ˂ 0.05). Prepubertal exposure to DBP at doses of 50, 200 and 400 mg DBP/kg/d for 30 days led to adverse effects in the adult male Japanese quail reproductive system by inducing structural changes in the Sertoli cells and junctional complexes. Such changes might disrupt the BTB and potentially interfere with spermatogenesis. Results indicated that the Sertoli cell is sensitive to DBP exposure and might be an important cellular target for DBP‐induced testicular toxicity. [ABSTRACT FROM AUTHOR]

In some special parts of the mammalian body, the movement of molecules between blood and tissues is controlled by structures called "blood-tissue barriers." These barriers are mainly the blood-brain, -placenta, -retina, - thymus, -testis, and -epididymis barriers. The blood-testis barrier (BTB) and the blood-epididymis barrier (BEB) are the two important cellular barriers in the male reproductive system. BTB is localized between adjacent Sertoli cells in the seminiferous epithelium of the testis and is formed by tight junctions, GAP junctions, desmosomes (macula adherens), and adherens junctions (ectoplasmic specialization-a testis-specific adhesion junction). The BTB creates a biochemical and immunological microenvironment that protects developing germ cells, especially post-meiotic spermatids, from harmful agents (such as drugs, toxic chemicals, and mutagens) carried there by blood and lymph and that prevents autoimmune responses against differentiated germ cells. The BTB divides the seminiferous tubule epithelium into basal and adluminal compartments, ensuring cell polarity and helping to maintain the chemical composition of the fluid in the tubule lumen. BTB undergoes remodeling during spermatogenesis, but its integrity remains intact. Thus, thanks to this unique structure, the germ cells are transported across the seminiferous epithelium. Any disruption in the components that make up the barrier can adversely affect Sertoli-germ cell interactions, preventing germ cells from completing their development and leading to male infertility. In addition, female fertility may be indirectly affected as immature germ cells cannot fertilize the secondary oocyte. In summary, this barrier is critical for germ cell survival and maintenance of normal spermatogenesis. This review aims to provide information about the molecular components of the junction complexes that form the blood-testis barrier, which plays an important role in male infertility in mammals. [ABSTRACT FROM AUTHOR]