Disruption of Sertoli-germ cell adhesion function in the seminiferous epithelium of the rat testis can be limited to adherens junctions without affecting the blood-testis barrier integrity: an in vivo study using an androgen suppression model

During spermatogenesis, both adherens junctions (AJ) (such as ectoplasmic specialization (ES), a testis-specific AJ type at the Sertoli cell-spermatid interface (apical ES) or Sertoli-Sertoli cell interface (basal ES) in the apical compartment and BTB, respectively) and tight junctions (TJ) undergo extensive restructuring to permit germ cells to move across the blood-testis barrier (BTB) as well as the seminiferous epithelium from the basal compartment to the luminal edge to permit fully developed spermatids (spermatozoa) to be sloughed at spermiation. However, the integrity of the BTB cannot be compromised throughout spermatogenesis so that postmeiotic germ cell-specific antigens can be sequestered from the systemic circulation at all times. We thus hypothesize that AJ disruption in the seminiferous epithelium unlike other epithelia, can occur without compromising the BTB-barrier, even though these junctions, namely TJ and basal ES, co-exist side-by-side in the BTB. Using an intratesticular androgen suppression-induced germ cell loss model, we have shown that the disruption of AJs indeed was limited to the Sertoli-germ cell interface without perturbing the BTB. The testis apparently is using a unique physiological mechanism to induce the production of both TJ- and AJ-integral membrane proteins and their associated adaptors to maintain BTB integrity yet permitting a transient loss of cell adhesion function by dissociating N-cadherin from beta-catenin at the apical and basal ES. The enhanced production of TJ proteins, such as occludin and ZO-1, at the BTB site can supersede the transient loss of cadherin-catenin function at the basal ES. This thus allows germ cell depletion from the epithelium without compromising BTB integrity. It is plausible that the testis is using this novel mechanism to facilitate the movement of preleptotene and leptotene spermatocytes across the BTB at late stage VIII through early stage IX of the epithelial cycle in the rat while maintaining the BTB immunological barrier function.     

Molecular basis of cryptorchidism-induced infertility

Artificial cryptorchidism or local testicular heat treatment can induce reversible oligospermia or azoospermia in monkeys and rats via germ cell apoptosis. Local warming of monkey testes in water at 43°C for 2 consecutive days (30 min per day) decreased the number of sperm in the semen by up to 80% on d 28, and the effect was completely reversed on d 144. Germ cells rely heavily on Sertoli cells for structural and nutritional support. Specialized junctions that play a pivotal role in spermatogenesis occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact in the seminiferous epithelium. We demonstrated that expression of tight junction (TJ)-associated molecules, such as occludin and zonula occludens-1 (ZO-1), were greatly reduced 24–48 h after heat treatment, while the permeability of the blood-testis barrier (BTB) was simultaneously increased, but recovered 10 d later. These results indicate a reversible disruption of the BTB associated with transient inductions of transforming growth factor (TGF) β2 and β3 expression, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase activation, and concomitant loss of occludin and ZO-1. This suggests that expression of TJ-associated molecules and the BTB was reversibly perturbed by mild testicular hyperthermia, and that the heat-induced induction of TGF-β might be involved in downregulating TJ-associated proteins, leading to cell junction reduction. This review discusses the changes in total gene expression patterns after experimental cryptorchidism in adult mouse testes, and the cloning of several novel, physiologically significant spermatogenesis-specific genes.     

Differential interactions between transforming growth factor-beta3/TbetaR1, TAB1, and CD2AP disrupt blood-testis barrier and Sertoli-germ cell adhesion

The biochemical basis that regulates the timely and selective opening of the blood-testis barrier (BTB) to migrating preleptotene/leptotene spermatocytes at stage VIII of the epithelial cycle in adult rat testes is virtually unknown. Recent studies have shown that cytokines (e.g. transforming growth factor (TGF)-beta3) may play a crucial role in this event. However, much of this information relies on the use of toxicants (e.g. CdCl(2)), making it difficult to relay these findings to normal testicular physiology. Here we report that overexpression of TGF-beta3 in primary Sertoli cells cultured in vitro indeed perturbed the tight junction (TJ) barrier with a concomitant decline in the production of BTB constituent proteins as follows: occludin, N-cadherin, and ZO-1. Additionally, local administration of TGF-beta3 to testes in vivo was shown to reversibly perturb the BTB integrity and Sertoli-germ cell adhesion via the p38 MAPK and ERK signaling pathways. Most importantly, the simultaneous activation of p38 and ERK signaling pathways is dependent on the association of the TGF-beta3-TbetaR1 complex with adaptors TAB1 and CD2AP because if TbetaR1 was associated preferentially with CD2AP, only Sertoli-germ cell adhesion was perturbed without compromising the BTB. Collectively, these data illustrate that local production of TGF-beta3, and perhaps other TGF-betas and cytokines, by Sertoli and germ cells into the microenvironment at the BTB during spermatogenesis transiently perturbs the BTB and Sertoli-germ cell adhesion to facilitate germ cell migration when the activated TbetaRI interacts with adaptors TAB1 and CD2AP. However, TGF-beta3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TbetaRI interacts only with adaptor CD2AP.     

c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes

During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminins β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites.     

Drug transporter,P-glycoprotein (MDR1), is an integrated component of the mammalian blood–testis barrier

Throughout spermatogenesis, leptotene spermatocytes traverse the blood–testis barrier (BTB) to enter the adluminal compartment of the seminiferous epithelium for continued development. At the same time, the integrity of the BTB, which is constituted by co-existing tight junctions (TJ), basal ectoplasmic specializations (basal ES) and desmosome-like junctions, must be maintained since a breach in barrier function can result in spermatogenic arrest and even infertility. There is evidence to suggest that drug transporters may function at the BTB, but little is known about how they contribute to spermatogenesis. In this study, we investigate the role of P-glycoprotein (P-gp), a drug efflux pump, in BTB dynamics. A survey by RT-PCR revealed several transporter genes to be expressed by the testis, including Mdr1 (gene symbol for P-gp), Mrp1, Abcc5 and Slc15a1. It was also demonstrated that P-gp localizes to the BTB in all stages of the seminiferous epithelial cycle in the adult rat testis, as well as to the Sertoli cell–elongated spermatid interface in stages VII and VIII. We continued our study by examining the levels of several transporters in the testis following oral administration of Adjudin, a compound known to affect Sertoli–germ cell adhesion. In this experiment, the steady-state levels of P-gp, MRP1, ABCG1 and SLC15A1 were all found to increase by several-fold within hours of Adjudin treatment during junction restructuring. More importantly, an increase in P-gp association with TJ proteins (e.g., occludin, claudin-11 and JAM-A) was noted when testis lysates from Adjudin-treated rats were used for co-immunoprecipitation experiments, suggesting that P-gp may enhance BTB function during Sertoli–germ cell junction restructuring.     

Differential effects of testosterone and TGF-β3 on endocytic vesicle-mediated protein trafficking events at the blood-testis barrier

The intricate interaction between protein endocytosis, transcytosis, recycling and endosome- or ubiquitin-mediated protein degradation determines the junction integrity in epithelial cells including Sertoli cells at the blood-testis barrier (BTB). Studies have shown that androgens and cytokines (e.g., TGF-β3) that are known to promote and disrupt BTB integrity, respectively, accelerate protein endocytosis at the BTB. We hypothesized that testosterone-induced endocytosed proteins are transcytosed and recycled back to the Sertoli cell surface, whereas cytokine-induced endocytosed proteins are degraded so that androgens and cytokines have opposing effects on BTB integrity. Herein, we report that both testosterone and TGF-β3 induced the steady-state level of clathrin, an endocytic vesicle protein. Testosterone and TGF-β3 also induced the association between internalized occludin (a BTB integral membrane protein) and clathrin, as well as early endosome antigen-1 (EEA-1). Interestingly, testosterone, but not TGF-β3, also induced the levels of proteins that regulate protein transcytosis (e.g., caveolin-1) and recycling (e.g., Rab11), and their association with internalized occludin and N-cadherin from the cell surface. In contrast, TGF-β3, but not testosterone, induced the level of ubiquitin-conjugating enzyme E2 J1 (Ube2j1), a protein crucial to the intracellular protein degradation pathway, and its association with internalized occludin. Based on these findings and recent reports in the field, we hypothesize that the concerted effects of testosterone and TGF-β3 likely facilitate the transit of preleptotene spermatocytes at the BTB while maintaining the immunological barrier in that testosterone induces the assembly of “new” tight junction (TJ)-fibrils below migrating spermatocytes via protein transcytosis and recycling before cytokines induce the disassembly of “old” TJ-fibrils above spermatocytes via endocytic vesicle-mediated degradation of internalized proteins. This thus provides a unique mechanism in the testis to facilitate the transit of preleptotene spermatocytes, many of which are connected in "clones" via cytoplasmic bridges, at the BTB while maintaining the immunological barrier during stage VIII of the seminiferous epithelial cycle of spermatogenesis.     

双酚A 干扰大鼠生精过程时TJ 渗透性屏障的体外研究

目的:研究体外大鼠睾丸支持细胞紧密连接蛋白(SCJP)在类雌激素-双酚A(BPA)干扰下的损伤机制。方法:对Wistar大鼠睾丸支持细胞(Sertoli细胞)离体原代培养4-5d,通过双室培养模型建立体外紧密连接(TJ)渗透性屏障,并测量其跨上皮电阻值(TER)反应紧密连接结构的形成及BPA对紧密连接的损害程度。设溶剂(DMSO)做阴性对照,以终浓度为25μM、100μM的BPA作用于支持细胞24h,MTT法测不同浓度BPA作用的Sertoli细胞增殖活性。Western bloting观察occludin、ZO-1、Cx43表达的变化。结果:成功分离并培养Wistar大鼠睾丸支持细胞,并建立良好的体外TJ屏障模型。双室培养支持细胞上皮TER值在培养的d4达到顶峰,然后在d4-9维持相对较稳定的状态,d4以200μM,100μM,25μM BPA染毒,分别于染毒后24,48,72,96和120h测TER:与DMSO溶剂对照组相比,200μM,100μM的BPA组TER值明显下降(P<0.05),而25μM的BPA组在染毒后TER值无明显变化(P>0.05)。MTT结果显示:经不同浓度BPA作用24h后,Sertoli细胞的吸光度(OD值)随着染毒剂量的增加而逐渐降低。102、103μM浓度组与溶剂对照组有显著性差异(P<0.05),而10-2、10-1、100、101μM组和溶剂对照组无显著性差异(P>0.05)。Western blot结果显示:occludin、ZO-1、Cx43在各剂量组均有表达,与溶剂对照组相比,occludin、ZO-1表达均分别随作用剂量的增加而降低:25μM组、100μM组与溶剂对照组相比,差异均存在显著性(P<0.05);100μM组与25μM组相比,差异亦存在显著性(P<0.05)。Cx43的表达却随染毒剂量的增加而增加,与溶剂对照组相比,25μM组表达无明显增加(P>0.05),而100μM组则明显增加(P<0.05);与25μM组相比,100μM组表达明显增加(P<0.05)。结论:双酚A可通过损伤支持细胞连接蛋白正常表达,破坏了TJ屏障渗透性,从而影响正常的精子形成过程。     

TGF-β3 and TNFα perturb blood-testis barrier (BTB) dynamics by accelerating the clathrin-mediated endocytosis of integral membrane proteins: A new concept of BTB regulation during spermatogenesis

In adult mammals such as rats, the blood-testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood-tissue barriers. Yet it must “open” transiently at stages VIII to IX of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-β3 (TGF-β3) and tumor necrosis factor α (TNFα) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-β3 (200 ng/testis) to the testis was shown to reversibly perturb the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-β3 (3 ng/ml) and TNFα (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 μM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-β3-induced protein internalization. This inhibition of TGF-β3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-β3 on protein internalization was further confirmed by RNAi using specific TGF-β receptor I (TβR1) siRNA duplexes. When TβR1 was knocked down, the TGF-β3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-β3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-β3 and TNFα via an enhancement of protein endocytosis at the BTB.     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*

Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.     

A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH(2)-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209-230 in the second extracellular loop of rat occludin, at 0.2-4 microM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH(2)-TKVNERYELCDTYPALLAVPAN-COOH (residues 156-177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5-10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8-16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [(125)I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [(125)I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.     

Dynamic cross-talk between cells and the extracellular matrix in the testis

In the seminiferous tubule of the mammalian testis, one type A1 spermatogonium (diploid, 2n) divides and differentiates into 256 spermatozoa (haploid, n) during spermatogenesis. To complete spermatogenesis and produce approximately 150 x 10(6) spermatozoa each day in a healthy man, germ cells must migrate progressively across the seminiferous epithelium yet remain attach to the nourishing Sertoli cells. This active cell migration process involves precisely controlled restructuring events at the tight (TJ) and anchoring junctions at the cell-cell interface. While the hormonal events that regulate spermatogenesis by follicle-stimulating hormone and testosterone from the pituitary gland and Leydig cells, respectively, are known, less is known about the mechanism(s) that regulates junction restructuring during germ cell movement in the seminiferous epithelium. The relative position of tight (TJs) and anchoring junctions in the testis is of interest. Sertoli cell TJs that constitute the blood-testis barrier (BTB) are present side by side with anchoring junctions and are adjacent to the basement membrane. This intimate physical association with the TJs, the anchoring junctions and the basement membrane (a modified form of extracellular matrix, ECM) suggests a role for the ECM in the junction dynamics of the testis. Indeed, evidence is accumulating that ECM proteins are crucial to Sertoli cell TJ dynamics. In this review, we discuss the pivotal role of tumor necrosis factor alpha (TNFalpha) on BTB dynamics via its effects on the homeostasis of ECM proteins. In addition, discussion will also be focused on the novel findings regarding the role of non-basement-membrane-associated ECM proteins and components of focal adhesion (a cell-matrix anchoring junction type) in the regulation of junction dynamics in the testis.     

Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.     

Claudin-11 is over-expressed and dislocated from the blood–testis barrier in Sertoli cells associated with testicular intraepithelial neoplasia in men

In mouse testis, claudin-11 is responsible for the formation of specific parallel TJ strands of the blood–testis barrier (BTB). Concerning the human BTB, there is no information about the transmembrane TJ proteins. We recently demonstrated the loss of functional integrity of the BTB in testicular intraepithelial neoplasia (TIN), associated with a dislocation of the peripheral TJ proteins ZO-1 and ZO-2. Here, we determined the expression and distribution of claudin-11 at the human BTB in seminiferous tubules with normal spermatogenesis (NSP) and TIN. Immunostaining of claudin-11 revealed intense signals at the basal BTB region in seminiferous epithelium with NSP. Within TIN tubules, claudin-11 immunostaining became diffuse and cytoplasmic. Double immunogold labeling demonstrated a co-localization of claudin-11 and ZO-1 at the inter-Sertoli cell junctions. Real-time RT-PCR of laser microdissected tubules showed an up-regulation of claudin-11 mRNA in TIN. Additionally, increased claudin-11 protein was observed by Western blot. We conclude that claudin-11 constitutes a TJ protein at the human BTB. In TIN tubules, claudin-11 is up-regulated and dislocated from the BTB. Therefore, the disruption of the BTB is related to a dysfunction of claudin-11 and not to a failure of its expression.     

Regulation of blood-testis barrier dynamics by focal adhesion kinase (FAK): An unexpected turn of events

The blood-testis barrier (BTB) is conferred by co-existing tight junctions (TJs), basal ectoplasmic specialization (basal ES), desmosome-like junctions and gap junctions (GJs) between adjacent Sertoli cells near the basement membrane in the seminiferous epithelium. While the concept of the BTB has been known for more than a century and its significance to spermatogenesis discerned for more than five decades, its regulation has remained largely unknown. Recent studies, however, have demonstrated that focal adhesion kinase (FAK), a modulator of the integrin-based signaling that plays a crucial role on cell movement, apoptosis, cell survival and gene expression at the focal adhesion complex (FAC, also known as focal contact, a cell-matrix anchoring junction type), is an integrated component of the BTB, associated with the TJ-integral membrane protein occludin and its adaptor zonula occludens-1 (ZO-1). Herein, we summarize recent findings in the field regarding the significance of FAK in conferring BTB integrity based on some unexpected observations. We also critically discuss the role of FAK in regulating the timely "opening" and "closing" of the BTB to facilitate the transit of primary preleptotene spermatocytes across the BTB at stage VIII of the seminiferous epithelial cycle of spermatogenesis. Lastly, we propose a working model, which can be used to design future functional experiments to explore the involvement of FAK in BTB dynamics by investigators in the field.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton

Multiple isoforms of the red cell protein 4.1R are expressed in nonerythroid cells, including novel 135-kDa isoforms. Using a yeast two-hybrid system, immunocolocalization, immunoprecipitation, and in vitro binding studies, we found that two 4.1R isoforms of 135 and 150 kDa specifically interact with the protein ZO-2 (zonula occludens-2). 4.1R is colocalized with ZO-2 and occludin at Madin-Darby canine kidney (MDCK) cell tight junctions. Both isoforms of 4.1R coprecipitated with proteins that organize tight junctions such as ZO-2, ZO-1, and occludin. Western blot analysis also revealed the presence of actin and alpha-spectrin in these immunoprecipitates. Association of 4.1R isoforms with these tight junction and cytoskeletal proteins was found to be specific for the tight junction and was not seen in nonconfluent MDCK cells. The amino acid residues that sustain the interaction between 4.1R and ZO-2 reside within the amino acids encoded by exons 19-21 of 4.1R and residues 1054-1118 of ZO-2. Exogenously expressed 4.1R containing the spectrin/actin- and ZO-2-binding domains was recruited to tight junctions in confluent MDCK cells. Taken together, our results suggest that 4.1R might play an important role in organization and function of the tight junction by establishing a link between the tight junction and the actin cytoskeleton.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Glutamine regulates Caco-2 cell tight junction proteins

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.