Wing-Yee Lui

Regulation of blood-testis barrier dynamics: an in vivo study

An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl2. It was shown that the CdCl2-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-β2 and TGF-β3 (but not TGF-β1) and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-β2/-β3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl2-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl2 mediates its BTB disruptive effects via the TGF-β3/p38 MAP kinase signaling pathway. Besides, this CdCl2-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of α2-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. α2-Macroglobulin was also shown to associate physically with TGF-β3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-β3/p-38 MAP kinase pathway also prevented the CdCl2-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on α2-macroglobulin. These results demonstrate for the first time that the TGF-β3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-β3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.

Transforming Growth Factor-β3 Perturbs the Inter-Sertoli Tight Junction Permeability Barrier in Vitro Possibly Mediated via Its Effects on Occludin, Zonula Occludens-1, and Claudin-11

Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that create the blood-testis barrier in the rat must be disassembled and reassembled to permit the timely passage of preleptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism(s) and the participating molecules that regulate this event are largely unknown. Although there is no in vitro model to study the event and regulation of inter-Sertoli TJ disassembly, primary cultures of Sertoli cells in vitro can be used to study junction assembly. In this study, we sought to investigate whether cytokines are involved in the inter-Sertoli TJ assembly in vitro. Sertoli cells isolated from 20-day-old rats were cultured at a density of 0.5–1.2 × 106 cells/cm2 on Matrigel-coated dishes or bicameral units for 8–9 days. The steady-state messenger RNA levels of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β2, and TGF-β3 at different time points were assessed by semiq...

Sertoli Cell Tight Junction Dynamics: Their Regulation During Spermatogenesis

Abstract During spermatogenesis, developing preleptotene and leptotene spermatocytes must translocate from the basal to the adluminal compartment of the seminiferous epithelium so that fully developed spermatids (spermatozoa) can be released to the tubular lumen at spermiation. It is conceivable that the opening and closing of the inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier are regulated by an array of intriguingly coordinated signaling pathways and molecules. Several molecules have been shown to regulate Sertoli cell TJ dynamics; they include, for example, transforming growth factor β3 (TGFβ3), occludin, protein kinase A, protein kinase C, and signaling pathways such as the TGFβ3/p38 mitogen-activated protein kinase pathway. Yet the mechanisms that regulate these events are essentially not known. This minireview summarizes some of the recent advances in the study of TJ dynamics in the testis and reviews several models that can be used to study TJ dynamics. It also highlights specific areas for future research toward understanding the precise physiological relationship between junction dynamics and spermatogenesis.

Transforming Growth Factor β3 Regulates the Dynamics of Sertoli Cell Tight Junctions Via the p38 Mitogen-Activated Protein Kinase Pathway

Abstract Earlier studies have implicated the significance of transforming growth factor-β3 (TGFβ3) in the regulation of Sertoli cell tight junction (TJ) dynamics, possibly via its inhibitory effects on the expression of occludin, claudin-11, and zonula occludens-1 (ZO-1). Yet the mechanism by which TGFβ3 regulates the Sertoli cell TJ-permeability barrier is not known. Using techniques of semiquantitative reverse transcription-PCR (RT-PCR), immunoblotting, immunohistochemistry, and inhibitors against different kinases coupled with physiological techniques to assess the Sertoli cell TJ barrier function, it was shown that this TGFβ3-induced effect on Sertoli cell TJ dynamics is mediated via the p38 mitogen-activated protein (MAP) kinase pathway. First, the assembly of the Sertoli cell-TJ barrier was shown to be associated with a transient but significant decline in both the TGFβ3 production and expression by Sertoli cells. Furthermore, addition of TGFβ3 to Sertoli cell cultures during TJ assembly indeed perturbed the TJ barrier with an IC50 at ∼9 pM. Second, the TGFβ3-induced disruption of the TJ barrier was associated with a transient induction in MEKK2 but not the other upstream signaling molecules that mediate TGFβ3 action, such as Smad2, Cdc42, Rac2, and N-Ras, suggesting this effect might be mediated via the p38 MAP kinase pathway. This postulate was confirmed by the observation that TGFβ3 also induced the protein level of the activated and phosphorylated form of p38 MAP kinase at the time the TJ barrier was perturbed. Third, and perhaps the most important of all, this TGFβ3-mediated inhibitory effect on the TJ barrier and the TGFβ3-induced p-p38 MAP kinase production could be blocked by SB202190, a specific p38 MAP kinase inhibitor, but not U0126, a specific MEK1/2 kinase inhibitor. These results thus unequivocally demonstrate that TGFβ3 utilizes the p38 MAP kinase pathway to regulate Sertoli cell TJ dynamics.

Sertoli-Germ Cell Adherens Junction Dynamics in the Testis Are Regulated by RhoB GTPase via the ROCK/LIMK Signaling Pathway

Abstract During spermatogenesis, cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specializations (ESs), between Sertoli and germ cells undergo extensive restructuring in the seminiferous epithelium to facilitate germ cell movement across the epithelium. Although the mechanism(s) that regulates AJ dynamics in the testis is virtually unknown, Rho GTPases have been implicated in the regulation of these events in other epithelia. Studies have shown that the in vitro assembly of the Sertoli-germ cell AJs but not of the Sertoli cell tight junctions (TJs) is associated with a transient but significant induction of RhoB. Immunohistochemistry has shown that the localization of RhoB in the seminiferous epithelium is stage specific, being lowest in stages VII–VIII prior to spermiation, and displays cell-specific association during the epithelial cycle. Throughout the cycle, RhoB was localized near the site of basal and apical ESs but was restricted to the periphery of the nuclei in elongating (but not elongated) spermatids, spermatocytes, and Sertoli cells. However, RhoB was not detected near the site of apical ESs at stages VII–VIII. Furthermore, disruption of AJs in Sertoli-germ cell cocultures either by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) also induced RhoB expression. When adult rats were treated with AF-2364 to perturb Sertoli-germ cell AJs in vivo, a ∼4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least ∼1–4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (Rho-associated protein kinase 1, also called ROKβ), LIMK1 (LIM kinase 1, also called lin-11 isl-1 mec3 kinase 1), and cofilin but not p140mDia and profilin via phosphorylation. Immunoprecipitation and immunoblots revealed that the induction of LIMK1 was mediated via an increase in its phospho-Ser but not phospho-Tyr content. Furthermore, Y-27632 ([(R)−(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide, 2HCl]), a specific ROCK inhibitor, could effectively delay the AF-2364-induced germ cell loss from the seminiferous epithelium in vivo, illustrating that the integrin/RhoB/ROCK/LIMK pathway indeed plays a crucial role in the regulation of Sertoli-germ cell AJ dynamics. The fact that the RhoB pathway in the kidney and brain was not activated suggests that AF-2364 exerts its effects primarily at the testis-specific ES multiprotein complex structures between Sertoli cells and spermatids. In summary, this report illustrates that Sertoli germ cell AJ dynamics are regulated, at least in part, via the integrin/ROCK/LIMK/cofilin signaling pathway.

cAMP perturbs inter-sertoli tight junction permeability barrier in vitro via its effect on proteasome-sensitive ubiquitination of occludin

Throughout spermatogenesis, inter‐Sertoli tight junctions (TJs) that constitute the blood‐testis barrier must be disassembled and reassembled to permit the timely movement of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism and the participating molecules that regulate the bioavailability of TJ proteins are entirely unknown. Using Sertoli cell culture, it was shown that there was an increase in occludin level, concomitant with a reduction of an E3 ubiquitin ligase, Itch, at the time when inter‐Sertoli TJs were assembled. By co‐immunoprecipitation, occludin was shown to associate with Itch at the TJs. A novel interaction between Itch and UBC4 (an ubiquitin‐conjugating enzyme) was identified. When TJs were disrupted by dibutyryl‐cAMP (db‐cAMP), an increase in protein levels of Itch and UBC4 along with a significant reduction in endogenous occludin was detected. These results seemingly suggest that the interaction of Itch and UBC4 on occludin is potentially involved in regulating Sertoli TJ dynamics. Addition of a proteasome inhibitor, MG‐132, into Sertoli cells cultured with db‐cAMP blocked the db‐cAMP‐induced occludin loss in vitro. Accumulations of ubiquitin‐conjugated and Itch‐conjugated occludin were detected in Sertoli cells cultured in the presence of both MG‐132 and db‐cAMP. These results suggest that MG‐132 prevented db‐cAMP‐induced TJ disruption by altering the rate of occludin degradation. Taken collectively, the results reported herein support the notion that db‐cAMP‐induced TJ disruption was mediated by an induction of Itch protein expression, which in turn triggered the ubiquitination of occludin resulting in TJ disruption.

Interactions among IQGAP1, Cdc42, and the cadherin/catenin protein complex regulate Sertoli‐germ cell adherens junction dynamics in the testis

The movement of developing germ cells across the seminiferous epithelium during spermatogenesis involves extensive adherens junction (AJ) restructuring between Sertoli cells, as well as between Sertoli and germ cells. In this report, we show that the intricate interactions between Cdc42 (a Rho family protein of Mr ∼23 kDa originally identified in membranes of human platelets and placenta, and is the homolog of CDC42Sc, which is known to regulate of bud‐site assembly in Saccharomyces cerevisiae) and its effector, IQ motif containing GTPase activating protein (IQGAP1, Mr ∼189 kDa, it is also an actin‐binding protein known to interact with Cdc42 and Rac1 GTPases), regulate Sertoli‐germ cell, but not Sertoli‐Sertoli cell, AJ dynamics. Using testis lysates for immunoprecipitation (IP), IQGAP1 was shown to associate with E‐cadherin, N‐cadherin, and β‐catenin (but not β1‐integrin and nectin‐2), as well as with actin and vimentin (but not α‐tubulin). Moreover, IQGAP1 was found to localize to the periphery of both Sertoli and germ cells in the seminiferous epithelium, at sites of cell–cell contacts. Using fluorescent microscopy with dual fluorescent probes, IQGAP1 was found to co‐localize, at least in part, with N‐cadherin in the seminiferous epithelium consistent with their localization at the basal and apical ES. Using Sertoli‐germ cell cocultures, it was demonstrated that AJ assembly associated with a transient induction of Cdc42 and IQGAP1, which was not found when Sertoli cells were cultured alone. Lastly, a shift in the interactions of Cdc42, IQGAP1, β‐catenin, and N‐cadherin was detected in Sertoli‐germ cell cocultures using an Ca2+‐induced AJ disruption model, which was used to examine AJ disassembly and its reassembly. In the presence of Ca2+, IQGAP1 bound preferentially to Cdc42 rather than to β‐catenin. However, when Ca2+ was depleted from cocultures using EGTA, a Ca2+ chelating agent, IQGAP1 lost its affinity for Cdc42 and became tightly associated with β‐catenin, destabilizing cadherin‐mediated AJs between Sertoli and germ cells. Yet this shift of protein–protein interaction was not detected in Sertoli cells cultured alone. These results illustrate that the interactions among IQGAP1, Cdc42, and β‐catenin are crucial to the regulation of Sertoli‐germ cell, but not Sertoli‐Sertoli cell, AJ dynamics in the seminiferous epithelium.

Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes

Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood-testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.

Actin-binding protein drebrin E is involved in junction dynamics during spermatogenesis

The actin-based cytoskeleton plays a critical role in the seminiferous epithelium during spermatogenesis by conferring cell shape, adhesion, structural support and cell polarity to both Sertoli and developing germ cells, which are essential to cell cycle progression, mitosis, meiosis, spermiogenesis and spermiation. However, few functional studies are found in the literature, which explore the functional significance of actin dynamics in these events. This by and large is due to a lack of information on the proteins that regulate actin dynamics. Herein, we report drebrin E is an integrated component of the apical ectoplasmic specialization (apical ES) and the basal ES at the blood-testis barrier (BTB) in the seminiferous epithelium of the adult rat testis. Using immunohistochemistry and dual-labeled immunofluorescence analysis, drebrin E was found to display a stage-specific localization at the apical ES, as well as at the basal ES at the BTB during the seminiferous epithelial cycle of spermatogenesis. Drebrin E was first detected in stage V tubules at the basal ES with the highest expression at the BTB at stages V-VI, but it diminished by stages VII-VIII and was almost non-detectable until stage IV. At the apical ES, drebrin E was also first detected at stage V, surrounding the entire head of the elongating spermatid, but by stage VI its localization had “shifted” to localize most intensely and almost exclusively at the concave side of the spermatid head. In stage VII tubules, drebrin E co-localized with actin, as well as with two other actin regulatory proteins Eps8 (epidermal growth factor receptor pathway substrate 8, an actin capping and bundling protein) and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to regulate actin nucleation and branching). The localization of drebrin E at the apical ES was compromised following treatment of rats with adjudin, which is known to exert its effects primarily at the apical ES by inducing premature loss of elongating/elongated spermatids from the epithelium, mimicking “spermiation.” Instead of being restricted to the concave side of spermatid heads, drebrin E was found to mis-localize in the seminiferous epithelium of adjudin-treated rats; it was also present on the convex side of elongating spermatids, but these cells were mis-oriented so that their heads no longer pointed toward the basement membrane. The expression of drebrin E by Sertoli cells was also found to be modulated by TGF-β3 and TNFα. Since Arp3, but not Eps8, was found to bind drebrin E; and cytokines were also shown to affect the cellular distribution of drebrin E and enhance the interaction between drebrin E and Arp3, these findings illustrate that cytokines may regulate BTB dynamics by recruiting drebrin E and Arp3 to the BTB to induce changes in the configuration of actin filament bundles at the basal ES. In summary, these findings illustrate drebrin E is working in concert with Arp3 to regulate actin filament bundles at both the apical and the basal ES in the testis, conferring adhesion and cell polarity at both sites during spermatogenesis.

Dual transcriptional control of claudin-11 via an overlapping GATA/NF-Y motif: Positive regulation through the interaction of GATA, NF-YA, and CREB and negative regulation through the interaction of Smad, HDAC1, and mSin3A

The expression of claudin‐11, a key integral tight junction protein, is tightly regulated to ensure that the integrity of the seminiferous epithelium could be maintained during the translocation of spermatocytes at the blood–testis barrier at stages VIII–IX. In this study, we elucidate how the overlapping GATA/NF‐Y motif within the core promoter of claudin‐11 gene is modulated by differential binding of various transcription factors, resulting in dual transcriptional control. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we confirmed that GATA, nuclear factor YA (NF‐YA), and cAMP response element‐binding protein (CREB) form a complex in vivo and bind to the GATA/NF‐Y region to promote claudin‐11 gene transcription. Such gene activations were significantly reduced in the presence of siRNA specific to these transcription factors. GATA and CREB transactivation could be further modulated by the presence of Smad3 and Smad4 proteins. Binding of Smad proteins at the GATA/NF‐Y motif could repress the GATA and CREB transactivation of claudin‐11 gene. Such repression which required the recruitment and physical interactions of histone deacetylase 1 and its co‐repressor, mSin3A with Smad proteins, was abolished by treatment with Trichostatin A, thus suggesting the involvement of histone deacetylation at the site of the promoter region. It is believed that cyclic changes in the ratio of positive regulators (GATA, NF‐Y, and CREB) to negative regulators (Smads) in the seminiferous epithelium during the spermatogenic cycle might provide a precise control in claudin‐11 gene transcription. J. Cell. Physiol. 211: 638–648, 2007.