Biology and regulation of ectoplasmic specialization, an atypical adherens junction type, in the testis

Anchoring junctions are cell adhesion apparatus present in all epithelia and endothelia. They are found at the cell-cell interface (adherens junction (AJ) and desmosome) and cell-matrix interface (focal contact and hemidesmosome). In this review, we focus our discussion on AJ in particular the dynamic changes and regulation of this junction type in normal epithelia using testis as a model. There are extensive restructuring of AJ (e.g., ectoplasmic specialization, ES, a testis-specific AJ) at the Sertoli-Sertoli cell interface (basal ES) and Sertoli-elongating spermatid interface (apical ES) during the seminiferous epithelial cycle of spermatogenesis to facilitate the migration of developing germ cells across the seminiferous epithelium. Furthermore, recent findings have shown that ES also confers cell orientation and polarity in the seminiferous epithelium, illustrating that some of the functions initially ascribed to tight junctions (TJ), such as conferring cell polarity, are also part of the inherent properties of the AJ (e.g., apical ES) in the testis. The biology and regulation based on recent studies in the testis are of interest to cell biologists in the field, in particular their regulation, which perhaps is applicable to tumorigenesis.     

Extracellular matrix: recent advances on its role in junction dynamics in the seminiferous epithelium during spermatogenesis

Spermatogenesis takes place in the seminiferous epithelium of the mammalian testis in which one type A1 spermatogonium (diploid, 2n) gives rise to 256 spermatids (haploid, 1n). To accomplish this, developing germ cells, such as preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) entering into the adluminal compartment for further development into round, elongating, and elongate spermatids. Recent studies have shown that the basement membrane in the testis (a modified form of extracellular matrix, ECM) is important to the event of germ cell movement across the BTB because proteins in the ECM were shown to regulate BTB dynamics via the interactions between collagens, proteases, and protease inhibitors, possibly under the regulation of cytokines. While these findings are intriguing, they are not entirely unexpected. For one, the basement membrane in the testis is intimately associated with the BTB, which represents the basolateral region of Sertoli cells. Also, Sertoli cell tight junctions (TJs) that constitute the BTB are present side-by-side with cell-cell actin-based adherens junctions (AJ, such as basal ectoplasmic specialization [ES]) and intermediate filament-based desmosome-like junctions. As such, the relative morphological layout between TJs, AJs, and desmosome-like junctions in the seminiferous epithelium is in sharp contrast to other epithelia where TJs are located at the apical portion of an epithelium or endothelium, furthest away from ECM, to be followed by AJs and desmosomes, which in turn constitute the junctional complex. For another, anchoring junctions between a cell epithelium and ECM found in multiple tissues, also known as focal contacts (or focal adhesion complex, FAC, an actin-based cell-matrix anchoring junction type), are the most efficient junction type that permits rapid junction restructuring to accommodate cell movement. It is therefore physiologically plausible, and perhaps essential, that the testis is using some components of the focal contacts to regulate rapid restructuring of AJs between Sertoli and germ cells when germ cells traverse the seminiferous epithelium. Indeed, recent findings have shown that the apical ES, a testis-specific AJ type in the seminiferous epithelium, is equipped with proteins of FAC to regulate its restructuring. In this review, we provide a timely update on this exciting yet rapidly developing field regarding how the homeostasis of basement membrane in the tunica propria regulates BTB dynamics and spermatogenesis in the testis, as well as a critical review on the molecular architecture and the regulation of ES in the seminiferous epithelium.     

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.     

Adaptors, junction dynamics, and spermatogenesis

Adaptors are component proteins of junctional complexes in all epithelia, including the seminiferous epithelium of the mammalian testis. They recruit other regulatory and structural proteins to the site of both anchoring junctions (such as cell-cell actin-based adherens junctions [AJs], e.g., ectoplasmic specialization [ES] and tubulobulbar complex, which are both testis-specific cell-cell actin-based AJ types, and cell-cell intermediate filament-based desmosome-like junctions) and tight junctions (TJ). Furthermore, adaptors per se can be substrates and/or activators of kinases or phosphatases. As such, the integrity of cell junctions and the regulation of junction dynamics during spermatogenesis rely on adaptors for their ability to recruit and link different junctional components to the same site and to tether transmembrane proteins at both anchoring and TJ sites to the underlying cytoskeletons, such as the actin filaments, intermediate filaments, and microtubules. These protein-protein interactions are possible because adaptors are composed of conserved protein binding domains, which allow them to link to more than one structural or signaling protein, recruiting multi-protein complexes to the same site. Herein, we provide a timely review of adaptors recently found at the sites of AJ (e.g., ES) and TJ. In addition, several in vivo models that can be used to delineate the function of adaptors in the testis are described, and the role of adaptors in regulating junction dynamics pertinent to spermatogenesis is discussed.     

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 alpha6beta1-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.     

Myotubularin phosphoinositide phosphatases, protein phosphatases, and kinases: their roles in junction dynamics and spermatogenesis

Spermatogenesis in the seminiferous epithelium of the mammalian testis is a dynamic cellular event. It involves extensive restructuring at the Sertoli-germ cell interface, permitting germ cells to traverse the epithelium from basal to adluminal compartment. As such, Sertoli-germ cell actin-based adherens junctions (AJ), such as ectoplasmic specializations (ES), must disassemble and reassemble to facilitate this event. Recent studies have shown that AJ dynamics are regulated by intricate interactions between AJ integral membrane proteins (e.g., cadherins, alpha6beta1 integrins and nectins), phosphatases, kinases, adaptors, and the underlying cytoskeleton network. For instance, the myotubularin (MTM) phosphoinositide (PI) phosphatases, such as MTM related protein 2 (MTMR2), can form a functional complex with c-Src (a non-receptor protein tyrosine kinase). In turn, this phosphatase/kinase complex associates with beta-catenin, a constituent of the N-cadherin/beta-catenin functional unit at the AJ site. This MTMR2-c-Src-beta-catenin complex apparently regulates the phosphorylation status of beta-catenin, which determines cell adhesive function conferred by the cadherin-catenin protein complex in the seminiferous epithelium. In this review, we discuss the current status of research on selected phosphatases and kinases, and how these proteins potentially interact with adaptors at AJ in the seminiferous epithelium to regulate cell adhesion in the testis. Specific research areas that are open for further investigation are also highlighted.     

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.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway

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 approximately 4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least approximately 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 ROKbeta), 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.     

An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: a biochemical and molecular perspective

During spermatogenesis in adult rat testes, fully developed spermatids (i.e. spermatozoa) at the luminal edge of the seminiferous epithelium undergo “spermiation” at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood–testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to destabilize the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood–tissue barriers.     

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.     

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 approximately 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 approximately 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 beta-catenin (but not beta1-integrin and nectin-2), as well as with actin and vimentin (but not alpha-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, beta-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 beta-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 beta-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 beta-catenin are crucial to the regulation of Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics in the seminiferous epithelium.     

Protein kinases and adherens junction dynamics in the seminiferous epithelium of the rat testis

Earlier studies in multiple epithelia have shown that cell-cell actin-based adherens junction (AJ) dynamics are regulated, at least in part, by the interplay of kinases and phosphatases that determines the intracellular phosphoprotein content. Yet it is virtually unknown regarding the role of protein kinases in Sertoli-germ cell AJ dynamics in the seminiferous epithelium of the testis. To address this issue, an in vitro coculture system utilizing Sertoli and germ cells was used to study the regulation of several protein kinases, including c-Src (the cellular form of the v-src transforming gene of Rous Sarcoma virus, RSV), carboxyl-terminal Src kinase (Csk), and casein kinase 2 (CK2), during AJ assembly. Both Sertoli and germ cells were shown to express c-Src, Csk, and CK2 with a relative Sertoli:germ cell ratio of approximately 1:1, suggesting both cell types contributed equally to the pool of these kinases in the epithelium. c-Src and Csk were shown to be stage-specific proteins during the epithelial cycle, being highest at stages VII-VIII. Studies using immunoprecipitation have illustrated that these kinases were structurally associated with the N-cadherin/beta-catenin, but not the nectin/afadin, protein complex, implicating that the cadherin/catenin protein complex is their likely putative substrate. An induction in c-Src, Csk, and CK2 were detected during Sertoli-germ cell AJ assembly in vitro but not when Sertoli cells were cultured alone. When adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364), a compound known to induce germ cell loss from the seminiferous epithelium, in particular elongating/elongate and round spermatids, by disrupting Sertoli-germ cell AJs, an induction of c-Src and Csk, but not CK2, was detected. Furthermore, a transient increase in the intrinsic kinase activities of c-Src, but not CK2, was also detected. This event was also associated with a loss of protein-protein association of N-cadherin and beta-catenin from the cadherin/catenin/c-Src/Csk/CK2 protein complex. Administration of PP1, a c-Src inhibitor, into adult rats via the jugular vein could induce the loss of spermatocytes and round spermatids, but not elongating/elongate spermatids, from the seminiferous epithelium. This result thus implicates the importance of c-Src in maintaining the integrity of AJs and possibly desmosome-like junctions between Sertoli cells and spermatocytes/round spermatids. In short, the data reported herein have shown that c-Src, Csk, and CK2 are novel protein kinases in AJ dynamics in the testis.     

Polarity proteins and actin regulatory proteins are unlikely partners that regulate cell adhesion in the seminiferous epithelium during spermatogenesis

In mammalian testis, spermatogenesis takes place in the seminiferous epithelium of the seminiferous tubule, which is composed of a series of cellular events. These include: (i) spermatogonial stem cell (SSC) renewal via mitosis and differentiation of SSC to spermatogenia, (ii) meiosis, (iii) spermiogenesis, and (iv) spermiation. Throughout these events, developing germ cells remain adhered to the Sertoli cell in the seminiferous epithelium amidst extensive cellular, biochemical, molecular and morphological changes to obtain structural support and nourishment. These events are coordinated via signal transduction at the cell-cell interface through cell junctions, illustrating the significance of cell junctions and adhesion in spermatogenesis. Additionally, developing germ cells migrate progressively across the seminiferous epithelium from the stem cell niche, which is located in the basal compartment near the basement membrane of the tunica propria adjacent to the interstitium. Recent studies have shown that some apparently unrelated proteins, such as polarity proteins and actin regulatory proteins, are in fact working in concert and synergistically to coordinate the continuous cyclic changes of adhesion at the Sertoli-Sertoli and Sertoli-germ cell interface in the seminiferous epithelium during the epithelial cycle of spermatogenesis, such that developing germ cells remain attached to the Sertoli cell in the epithelium while they alter in cell shape and migrate across the epithelium. In this review, we highlight the physiological significance of endocytic vesicle-mediated protein trafficking events under the influence of polarity and actin regulatory proteins in conferring cyclic events of cell adhesion and de-adhesion. Furthermore, these recent findings have unraveled some unexpected molecules to be targeted for male contraceptive development, which are also targets of toxicant-induced male reproductive dysfunction.     

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.     

Interactions of proteases, protease inhibitors, and the beta1 integrin/laminin gamma3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis

During spermatogenesis, developing germ cells migrate progressively across the seminiferous epithelium. This event requires extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as the ectoplasmic specialization (ES, a testis-specific AJ type), between Sertoli cells and elongating/elongate spermatids. It was postulated that proteases and protease inhibitors worked in a yin-yang relationship to regulate these events. If this is true, then it is anticipated that both proteases and protease inhibitors are found at the ES. Indeed, matrix metalloprotease (MMP)-2, membrane-type 1 (MT1)-MMP and their inhibitor, tissue-inhibitor of metalloproteases (TIMP)-2, were shown to localize at the apical ES. In order to identify the putative MMP substrate as well as the unknown binding ligand for alpha6beta1 integrin in the ES, immunofluorescent microscopy coupled with immunoprecipitation techniques were used to demonstrate that laminin gamma3, largely a germ cell product, was present at the apical ES and could form a bona fide complex with beta1-integrin. Furthermore, the structural interactions of MMP-2 and MT1-MMP with laminin gamma3 and beta1-integrin, but not with N-cadherin or nectin-3, have implicated the crucial role of MMP-2/MT1-MMP in the regulation of integrin/laminin-based ES dynamics. Using an in vivo model to study AJ dynamics where adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to disrupt Sertoli-germ cell adhesive function, an induction of active MMP-2, active MT1-MMP and TIMP-2 but not active MMP-9 was detected between 0.5 and 8 h after AF-2364 treatment. This time frame coincided with the depletion of elongating/elongate spermatids from the epithelium, illustrating the synergistic relationships between MMP-2, MT1-MMP, and TIMP-2 in AJ disassembly. Perhaps the most important of all, the use of a specific MMP-2 and MMP-9 inhibitor, (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid, could effectively delay the AF-2364-induced elongating/elongate spermatid loss from the epithelium, demonstrating the pivotal role of MMP-2 activation in ES disassembly. Collectively, these studies illustrate that the beta1-integrin/laminin gamma3 complex is a putative ES-structural protein complex, which is regulated, at least in part, by the activation of MMP-2 involving MT1-MMP and TIMP-2 at the apical ES. The net result of this interaction likely regulates germ cell movement in the seminiferous epithelium.     

Sertoli-germ cell anchoring junction dynamics in the testis are regulated by an interplay of lipid and protein kinases

When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as beta1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin.laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-beta1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the beta1-integrin/PI3K/PKB/ERK signaling pathway.     

TGF-beta3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.     

TGF-β3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.