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.published_or_final_versio

Differential interactions between transforming growth factor-β3/ TβR1, 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)-β3) 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-β3 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-β3 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-β3-TβR1 complex with adaptors TAB1 and CD2AP because if TβR1 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-β3, and perhaps other TGF-βs 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 TβRI interacts with adaptors TAB1 and CD2AP. However, TGF-β3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TβRI interacts only with adaptor CD2AP. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.postprin

Disruption of the blood-testis barrier integrity by bisphenol A in vitro: Is this a suitable model for studying blood-testis barrier dynamics?

Bisphenol A, an estrogenic environmental toxicant, has been implicated to have hazardous effects on reproductive health in humans and rodents. However, there are conflicting reports in the literature regarding its effects on male reproductive function. In this study, it was shown that in adult rats treated with acute doses of bisphenol A, a small but statistically insignificant percentage of seminiferous tubules in the testes displayed signs of germ cell loss, consistent with some earlier reports. It also failed to disrupt the blood-testis barrier in vivo. This is possibly due to the low bioavailability of free bisphenol A in the systemic circulation. However, bisphenol A disrupted the blood-testis barrier when administered to immature 20-day-old rats, consistent with earlier reports concerning the higher susceptibility of immature rats towards bisphenol A. This observation was confirmed using primary Sertoli cells cultured in vitro with established tight junction-permeability barrier that mimicked the blood-testis barrier in vivo. The reversible disruption of Sertoli cell tight junction barrier by bisphenol A was associated with an activation of ERK, and a decline in the levels of selected proteins at the tight junction, basal ectoplasmic specialization, and gap junction at the blood-testis barrier. Studies by dual-labeled immunofluorescence analysis and biotinylation techniques also illustrated declining levels of occludin, connexin 43, and N-cadherin at the cell-cell interface following bisphenol A treatment. In summary, bisphenol A reversibly perturbs the integrity of the blood-testis barrier in Sertoli cells in vitro, which can also serve as a suitable model for studying the dynamics of the blood-testis barrier. © 2009 Elsevier Ltd. All rights reserved.postprin

Ectoplasmic specialization: A friend or a foe of spermatogenesis?

The ectoplasmic specialization (ES) is a testis-specific, actin-based hybrid anchoring and tight junction. It is confined to the interface between Sertoli cells at the blood-testis barrier, known as the basal ES, as well as between Sertoli cells and developing spermatids designated the apical ES. The ES shares features of adherens junctions, tight junctions and focal contacts. By adopting the best features of each junction type, this hybrid nature of ES facilitates the extensive junction-restructuring events in the seminiferous epithelium during spermatogenesis. For instance, the α6β1-integrin- laminin 333 complex, which is usually limited to the cell-matrix interface in other epithelia to facilitate cell movement, is a putative apical ES constituent. Furthermore, JAM-C and CAR, two tight junction integral membrane proteins, are also components of apical ES involving in spermatid orientation. We discuss herein the mechanisms that maintain the cross-talk between ES and blood-testis barrier to facilitate cell movement and orientation in the seminiferous epithelium. © 2006 Wiley Periodicals, Inc.postprin

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFalpha treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.postprin

Unraveling the molecular targets pertinent to junction restructuring events during spermatogenesis using the Adjudin-induced germ cell depletion model

During spermatogenesis, extensive restructuring takes place at the Sertoli-Sertoli and Sertoli-germ cell interface, which is regulated via intriguing interactions among cytokines, proteases, protease inhibitors, kinases, phosphatases, and transcription factors. This in turn determines the steady-state levels of integral membrane proteins at the cell junctions. We sought to further expand these observations using the Adjudin model. Adjudin is a potential male contraceptive that targets Sertoli-germ cell adhesion, causing exfoliation of spermatids and spermatocytes, but not spermatogonia, from the seminiferous epithelium. This model thus provides the means to identify crucial regulatory molecules and signaling pathways pertinent to junction restructuring events during spermatogenesis. In this study, genome-wide expression profiling of rat testes after treatment with Adjudin at the time of extensive junction restructuring was performed. Differentially regulated genes, such as cytokines, proteases, protease inhibitors, cell junction-associated proteins, and transcription factors pertinent to junction restructuring were identified. These data were consistent with earlier findings; however, much new information was obtained which has been deposited at the Gene Expression Omnibus data repository website: http://www.ncbi.nih.gov/geo/ with Accession number: GSE5131. The primary signaling events pertinent to junction restructuring in the testis induced by Adjudin were also delineated using bioinformatics. These findings were also consistent with recently published reports. The identified molecular signatures or targets pertinent to junction dynamics in the testis as reported herein, many of which have not been investigated, thus offer a framework upon which the regulation of junction restructuring events at the Sertoli-Sertoli and Sertoli-germ cell interface pertinent to spermatogenesis can be further studied.postprin

Ectoplasmic specialization: A friend or a foe of spermatogenesis?

The ectoplasmic specialization (ES) is a testis-specific, actin-based hybrid anchoring and tight junction. It is confined to the interface between Sertoli cells at the blood-testis barrier, known as the basal ES, as well as between Sertoli cells and developing spermatids designated the apical ES. The ES shares features of adherens junctions, tight junctions and focal contacts. By adopting the best features of each junction type, this hybrid nature of ES facilitates the extensive junction-restructuring events in the seminiferous epithelium during spermatogenesis. For instance, the α6β1-integrin- laminin 333 complex, which is usually limited to the cell-matrix interface in other epithelia to facilitate cell movement, is a putative apical ES constituent. Furthermore, JAM-C and CAR, two tight junction integral membrane proteins, are also components of apical ES involving in spermatid orientation. We discuss herein the mechanisms that maintain the cross-talk between ES and blood-testis barrier to facilitate cell movement and orientation in the seminiferous epithelium. © 2006 Wiley Periodicals, Inc.postprin

Cytokines and junction restructuring during spermatogenesis - A lesson to learn from the testis

In the mammalian testis, preleptotene and leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle during spermatogenesis, entering the adluminal compartment for further development. However, until recently the regulatory mechanisms that regulate BTB dynamics remained largely unknown. We provide a critical review regarding the significance of cytokines in regulating the 'opening' and 'closing' of the BTB. We also discuss how cytokines may be working in concert with adaptors that selectively govern the downstream signaling pathways. This process, in turn, regulates the dynamics of either Sertoli-Sertoli tight junction (TJ), Sertoli-germ cell adherens junction (AJ), or both junction types in the epithelium, thereby permitting TJ opening without compromising AJs, and vice versa. We also discuss how adaptors alter their protein-protein association with the integral membrane proteins at the cell-cell interface via changes in their phosphorylation status, thereby altering adhesion function at AJ. These findings illustrate that the testis is a novel in vivo model to study the biology of junction restructuring. Furthermore, a molecular model is presented regarding how cytokines selectively regulate TJ/AJ restructuring in the epithelium during spermatogenesis. © 2005 Elsevier Ltd. All rights reserved.postprin

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

published_or_final_versio

Regulation of ectoplasmic specialization dynamics in the seminiferous epithelium by focal adhesion-associated proteins in testosterone-suppressed rat testes

Apical ectoplasmic specialization (ES) is a unique testis-specific cell-cell actin-based adherens junction type restricted to the Sertoli-round/elongating/elongate spermatid interface in the seminiferous epithelium. An endogenous testosterone (T) suppression model was used to study the regulation of apical ES dynamics in the testis. By providing sustained releases of T and estradiol using subdermal implants in rats, this treatment reduced endogenous testicular T level. This in turn led to sloughing of spermatids (step 8 and beyond) from the seminiferous epithelium, which can be reversed by removing the implants, or replacing them with a higher dose of T implants. This model thus allows us to study the restructuring events at the apical ES. It was shown that apical ES restructuring involved proteins that were usually restricted to the cell-matrix focal adhesion site in other epithelia. For instance, the protein levels of β1-integrin, Tyr-phosphorylated focal adhesion kinase (p-FAK), and c-Src were induced during the T suppression-induced germ cell loss and recovery, implicating that these proteins are putative regulators of ES dynamics. Indeed, the formation of p-FAK/c-Src protein complex, but not their association with β1-integrin, was stimulated during T suppression-induced germ cell loss. ERK, a MAPK known to regulate focal adhesion turnover, was also activated during the androgen suppression-induced spermatid loss and the early phase of the recovery when germ cells began to repopulate the epithelium. Collectively, these data suggest that the apical ES is a cell-cell adherens junction type with the characteristics of cell-matrix focal contacts. In addition to its role in conferring cell adhesion formation, the p-FAK/c-Src protein complex apparently also regulates apical ES disruption via the ERK signaling pathway.postprin