Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

Blood-testis barrier: a review on regulators in maintaining cell junction integrity between Sertoli cells.

The blood-testis barrier (BTB) is formed adjacent to the seminiferous basement membrane. It is a distinct ultrastructure, partitioning testicular seminiferous epithelium into apical (adluminal) and basal compartments. It plays a vital role in developing and maturing spermatocytes into spermatozoa via reorganizing its structure. This enables the transportation of preleptotene spermatocytes across the BTB, from basal to adluminal compartments in the seminiferous tubules. Several bioactive peptides and biomolecules secreted by testicular cells regulate the BTB function and support spermatogenesis. These peptides activate various downstream signaling proteins and can also be the target themself, which could improve the diffusion of drugs across the BTB. The gap junction (GJ) and its coexisting junctions at the BTB maintain the immunological barrier integrity and can be the "gateway" during spermatocyte transition. These junctions are the possible route for toxicant entry, causing male reproductive dysfunction. Herein, we summarize the detailed mechanism of all the regulators playing an essential role in the maintenance of the BTB, which will help researchers to understand and find targets for drug delivery inside the testis.

The Planar Cell Polarity Protein Fat1 in Sertoli Cell Function.

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 ß-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.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Inhibition of miR-143-3p Restores Blood-Testis Barrier Function and Ameliorates Sertoli Cell Senescence.

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-ß 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.

Cross-talk between Vimentin and autophagy regulates blood-testis barrier disruption induced by cadmium.

The blood-testis barrier (BTB) plays a vital role in mammalian spermatogenesis by separating the seminiferous epithelium into an adluminal and a basal compartment. Cadmium (Cd) is a toxic heavy metal that is widely present in the environment. We observed that Cd can induce BTB disruption, leading to apoptosis of testicular cells. However, the molecular mechanisms contributing to BTB injury induced by Cd have not yet been fully clarified. Vimentin (Vim) is an important desmosome-like junction protein that mediates robust adhesion in the BTB. In this study, we investigated how Vim responds to Cd. We found that Cd treatment led to a significant decrease in Vim expression, accompanied by a marked increase in LC3-II expression and a higer number of autophagosomes. Interestingly, we also observed that Cd-induced autophagy was associated with decreased Vim activity and enhanced apoptosis of testicular cells. To further investigate the role of autophagy in Vim regulation under Cd exposure, we treated cells with an autophagy inhibitor called 3-MA. We found that 3-MA treatment enhanced Vim expression and improved the disruption of the BTB under Cd exposure. Additionally, the inhibition of Vim confirmed the role of autophagy in modulating Vim expression. These results reveal a previously unknown regulatory mechanism of Cd involving the interplay between a heavy metal and a protein.

Rreb1 is a key transcription factor in Sertoli cell maturation and function and spermatogenesis in mouse.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood-testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.

A short-term high-fat diet alters rat testicular activity and blood-testis barrier integrity through the SIRT1/NRF2/MAPKs signaling pathways.

Background: Overweight/obesity are metabolic disorder resulting from behavioral, environmental, and heritable causes. WHO estimates that 50% of adults and 30% of children and adolescents are overweight or obese, and, in parallel, an ongoing decline in sperm quality and male fertility has been described. Numerous studies demonstrated the intimate association between overweight/obesity and reproductive dysfunction due to a highly intricate network of causes not yet completely understood. This study expands the knowledge on the impact of a short-term high-fat diet (st-HFD) on rat testicular activity, specifically on steroidogenesis and spermatogenesis, focusing on the involved molecular mechanisms related to mitochondrial dynamics, blood-testis barrier (BTB) integrity, and SIRT1/NRF2/MAPKs pathways. Methods: Ten adult Male Wistar rats were divided into two groups of five and treated with a standard diet or an HFD for five weeks. At the end of the treatment, rats were anesthetized and sacrificed by decapitation. Blood was collected for serum sex hormone assay; one testis was stored at -80ÅãC for western blot analysis, and the other, was fixed for histological and immunofluorescence analysis. Results: Five weeks of HFD results in reduced steroidogenesis, increased apoptosis of spermatogenic cells, and altered spermatogenesis, as highlighted by reduced protein levels ofmeiotic and post-meiotic markers. Further, we evidenced the compromission of the BTB integrity, as revealed by the downregulation of structural proteins (N-Cadherin, ZO-1, occludin, connexin 43, and VANGL2) other than the phosphorylation of regulative kinases (Src and FAK). At the molecular level, the impairment of mitochondrial dynamics (fission, fusion, andbiogenesis), and the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways, were evidenced. Interestingly, no change was observed in the levels of pro-inflammatory markers (TNFα, NF-kB, and IL-6). Conclusions: The combined data led us to confirm that overweight is a less severe state than obesity. Furthermore, understanding the molecular mechanisms behind the association between metabolic disorders and male fertility could improve the possibility of identifying novel targets to prevent and treat fertility disorders related to overweight/obesity.

[Role of autophagy in cadmium-induced damage to the blood-testis barrier in mice].

OBJECTIVE: To investigate the role of autophagy in cadmium chloride (CdCl2)-induced damage to the blood-testis barrier (BTB) in mice. METHODS: Twenty four-week-old male C57BL/6 mice were randomly divided into four groups and intraperitoneally injected with CdCl2 at 0 mg/kg/d (the control), 0.5 mg/kg/d (low-dose), 1.0 mg/kg/d (medium-dose) and 2.0 mg/kg/d (high-dose) respectively for 28 consecutive days. Then the morphological changes of the testis tissue was observed by HE staining, the integrity of BTB measured with the biotracer, and the expressions of the BTB components ZO-1 and N-Cadherin proteins detected by Western blot. The TM4 Sertoli cells were treated with CdCl2at 0, 2.5, 5 and 10 µmol/L respectively for 24 hours, followed by determination of the expression levels of ZO-1 and N-Cadherin as well as the autophagy-related proteins LC3II and p62. Then the cells were again treated with CdCl2 in the presence of the autophagy inhibitor chloroquine (CQ) at 5 µmol/L or the autophagy inducer rapamycin (Rap) at 50 nmol/L for 24 hours, followed by measurement of the expressions of LC3II, p62, ZO-1 and N-Cadherin by Western blot. RESULTS: Compared with the control group, the cadmium-exposed mice showed increased interstitial space in the seminiferous tubules, formation of intracellular cavitation in the germ cells with decreased layers and disordered arrangement, and damaged integrity of the BTB. The expressions of the ZO-1 and N-Cadherin proteins were significantly down-regulated in the testis tissue of the mice in the medium- and high-dose CdCl2 groups (P < 0.05), and even more significantly in the CdCl2-exposed cells in comparison with those in the control mice (P < 0.01), while the expressions of the LC3II and p62 proteins were remarkably up-regulated (P < 0.05). The expressions of ZO-1, N-Cadherin, LC3II and p62 were also up-regulated in the cells co-treated with CQ and CdCl2 (P < 0.01), those of ZO-1, N-Cadherin and p62 down-regulated (P< 0.05) and that of LC3II up-regulated (P < 0.05) in the cells co-treated with Rap and CdCl2. CONCLUSION: CdCl2 can damage the integrity of the mouse BTB, which may be attributed to its ability to enhance the autophagy in Sertoli cells and regulate the expressions of BTB proteins.

WIN18,446 enhances spermatogonial stem cell homing and fertility after germ cell transplantation by increasing blood-testis barrier permeability.

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation.

Nickel induces blood-testis barrier damage through ROS-mediated p38 MAPK pathways in mice.

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.

Sertoli cell-derived extracellular vesicles traverse the blood-testis barrier and deliver miR-24-3p inhibitor into germ cells improving sperm mobility.

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 GSK3ß 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.

[Effects of cadmium chloride on testicular autophagy and blood-testis barrier integrity in prepubertal male rats].

Objective: To study the effects of cadmium chloride (CdCl(2)) exposure on testicular autophagy levels and blood-testis barrier integrity in prepubertal male SD rats and testicular sertoli (TM4) cells. Methods: In July 2021, 9 4-week-old male SD rats were randomly divided into 3 groups: control group (normal saline), low dose group (1 mg/kg·bw CdCl(2)) and high dose group (2 mg/kg·bw CdCl(2)), and were exposed with CdCl(2) by intrabitoneal injection. 24 h later, HE staining was used to observe the morphological changes of testis of rats, biological tracer was used to observe the integrity of blood-testis barrier, and the expression levels of microtubule-associated protein light chain 3 (LC3) -Ⅰ and LC3-Ⅱ in testicular tissue were detected. TM4 cells were treated with 0, 2.5, 5.0 and 10.0 µmol/L CdCl(2) for 24 h to detect the toxic effect of cadmium. The cells were divided into blank group (no exposure), exposure group (10.0 µmol/L CdCl(2)), experimental group[10.0 µmol/L CdCl(2)+60.0 µmol/L 3-methyladenine (3-MA) ] and inhibitor group (60.0 µmol/L 3-MA). After 24 h of treatment, Western blot analysis was used to detect the expression levels of LC3-Ⅱ, ubiquitin binding protein p62, tight junction protein ZO-1 and adhesion junction protein N-cadherin. Results: The morphology and structure of testicular tissue in the high dose group were obvious changed, including uneven distribution of seminiferous tubules, irregular shape, thinning of seminiferous epithelium, loose structure, disordered arrangement of cells, abnormal deep staining of nuclei and vacuoles of Sertoli cells. The results of biological tracer method showed that the integrity of blood-testis barrier was damaged in the low and high dose group. Western blot results showed that compared with control group, the expression levels of LC3-Ⅱ in testicular tissue of rats in low and high dose groups were increased, the differences were statistically significant (P<0.05). Compared with the 0 µmol/L, after exposure to 5.0, 10.0 µmol/L CdCl(2), the expression levels of ZO-1 and N-cadherin in TM4 cells were significantly decreased, and the expression level of p62 and LC3-Ⅱ/LC3-Ⅰ were significantly increased, the differences were statistically significant (P<0.05). Compared with the exposure group, the relative expression level of p62 and LC3-Ⅱ/LC3-Ⅰ in TM4 cells of the experimental group were significantly decreased, while the relative expression levels of ZO-1 and N-cadherin were significantly increased, the differences were statistically significant (P<0.05) . Conclusion: The mechanism of the toxic effect of cadmium on the reproductive system of male SD rats may be related to the effect of the autophagy level of testicular tissue and the destruction of the blood-testis barrier integrity.

mTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.

Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.

Bisphenols as promoters of the dysregulation of cellular junction proteins of the blood-testis barrier in experimental animals: A systematic review of the literature.

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, ß-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.

In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.

Arsenic interferes with spermatogenesis involving Rictor/mTORC2-mediated blood-testis barrier disruption in mice.

Ingestion of arsenic interferes with spermatogenesis and increases the risk of male infertility, but the underlying mechanism remines unclear. In this study, we investigated spermatogenic injury with a focus on blood-testis barrier (BTB) disruption by administrating 5 mg/L and 15 mg/L arsenic orally to adult male mice for 60 d. Our results showed that arsenic exposure reduced sperm quality, altered testicular architecture, and impaired Sertoli cell junctions at the BTB. Analysis of BTB junctional proteins revealed that arsenic intake downregulated Claudin-11 expression and increased protein levels of ß-catenin, N-cadherin, and Connexin-43. Aberrant localization of these membrane proteins was also observed in arsenic-treated mice. Meanwhile, arsenic exposure altered the components of Rictor/mTORC2 pathway in mouse testis, including inhibition of Rictor expression, reduced phosphorylation of protein kinase Cα (PKCα) and protein kinase B (PKB), and elevated matrix metalloproteinase-9 (MMP-9) levels. Furthermore, arsenic also induced testicular lipid peroxidative damage, inhibited antioxidant enzyme (T-SOD) activity, and caused glutathione (GSH) depletion. Our findings suggest that disruption of BTB integrity is one of the main factors responsible for the decline in sperm quality caused by arsenic. PKCα-mediated rearrangement of actin filaments and PKB/MMP-9-increased barrier permeability jointly contribute to arsenic-induced BTB disruption.

Regulation of Sertoli cell function by planar cell polarity (PCP) protein Fjx1.

Four-jointed box kinase 1 (Fjx1) is a planar cell polarity (PCP) protein and a member of the Fat (FAT atypical cadherin 1)/Dchs (Dachsous cadherin-related protein)/Fjx1 PCP complex. Fjx1 is also a non-receptor Ser/Thr protein kinase capable of phosphorylating Fat1 at is extracellular cadherin domains when it is being transported across the Golgi system. As such, Fjx1 is a Golgi-based regulator of Fat1 function by determining its extracellular deposition. Herein, Fjx1 was found to localize across the Sertoli cell cytoplasm, partially co-localized with the microtubules (MTs) across the seminiferous epithelium. It was most notable at the apical ES (ectoplasmic specialization) and basal ES, displaying distinctive stage-specific expression. The apical ES and basal ES are the corresponding testis-specific cell adhesion ultrastructures at the Sertoli-elongated spermatid interface and the Sertoli cell-cell interface, respectively, consistent with the role of Fjx1 as a Golgi-associated Ser/Thr kinase that modulates the Fat (and/or Dchs) integral membrane proteins. Its knockdown (KD) by RNAi using specific Fjx1 siRNA duplexes versus non-targeting negative control siRNA duplexes was found to perturb the Sertoli cell tight junction function, as well as perturbing the function and organization of MT and actin. While Fjx1 KD did not affect the steady-state levels of almost two dozens of BTB-associated Sertoli cell proteins, including structural and regulatory proteins, its KD was found to down-regulate Fat1 (but not Fat2, 3, and 4) and to up-regulate Dchs1 (but not Dchs2) expression. Based on results of biochemical analysis, Fjx1 KD was found to be capable of abolishing phosphorylation of its putative substrate Fat1 at its Ser/Thr sites, but not at its Tyr site, illustrating an intimate functional relationship of Fjx1 and Fat1 in Sertoli cells.

NECL2 regulates blood-testis barrier dynamics in mouse testes.

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.

Cadmium-induced Sertoli Cell Injury Through p38-MAPK and Related Signaling Proteins-A Study by RNA Sequencing.

Environmental toxicants, such as cadmium, found in foods, water, and consumer products are known to induce male reproductive dysfunction. However, the underlying molecular mechanism(s) by which cadmium-induced Sertoli cell injury as manifested by a disruption of the blood-testis barrier (BTB) remains unknown. Interestingly, one of the primary targets of cadmium toxicity in the testis is the cytoskeletons of the Sertoli cells, which, in turn, impedes cell junctions in the seminiferous epithelium. In order to expand these earlier observations and to provide a roadmap for future studies, we embarked a study using RNA sequencing to identify the pertinent genes involved in cadmium-induced Sertoli cell injury. Using bioinformatics analyses, multiple gene sets that regulated actin and microtubule (MT) cytoskeletons were identified along with components of the mitogen-activated protein kinase (MAPK) signaling protein and several signaling pathways. More important, we have also discovered that while the gene expression of p38-MAPK (also JNK or c-Jun) was considerably up- or downregulated during cadmium-induced Sertoli cell injury, the activated (phosphorylated) form was upregulated. Importantly, doramapimod (also known as BIRB 796), a specific p38-MARK inhibitor, that was shown to selectively block cadmium-induced p-p38 MAPK activation via phosphorylation in Sertoli cells, was indeed capable of blocking cadmium-induced Sertoli cell injury including disruption of the Sertoli cell-permeability barrier function, disruptive distribution of BTB-associated proteins, and disruptive organization of the actin and MT cytoskeletons. These data provide a helpful source of information for investigators to probe the role of signaling proteins and/or their signaling cascades, besides MAPKs, that likely utilized by cadmium to induce reproductive dysfunction.