Michelle W.M. Li

Connexin 43 is critical to maintain the homeostasis of the blood–testis barrier via its effects on tight junction reassembly

In mammalian testes, the blood–testis barrier (BTB) or Sertoli cell barrier created by specialized junctions between Sertoli cells near the basement membrane confers an immunological barrier by sequestering the events of meiotic division and postmeiotic germ cell development from the systemic circulation. The BTB is constituted by coexisting tight junctions (TJs), basal ectoplasmic specializations, desmosomes, and gap junctions. Despite being one of the tightest blood–tissue barriers, the BTB has to restructure cyclically during spermatogenesis. A recent study showed that gap junction protein connexin 43 (Cx43) and desmosome protein plakophilin-2 are working synergistically to modulate the BTB integrity by regulating the distribution of TJ-associated proteins at the Sertoli–Sertoli cell interface. However, the precise role of Cx43 in regulating the cyclical restructuring of junctions remains obscure. In this report, the calcium switch and the bisphenol A (BPA) models were used to induce junction restructuring in primary cultures of Sertoli cells isolated from rat testes that formed a TJ-permeability barrier that mimicked the BTB in vivo. The removal of calcium by EGTA perturbed the Sertoli cell tight junction barrier, but calcium repletion allowed the “resealing” of the disrupted barrier. However, a knockdown of Cx43 in Sertoli cells by RNAi significantly reduced the kinetics of TJ-barrier resealing. These observations were confirmed using the bisphenol A model in which the knockdown of Cx43 by RNAi also perturbed the TJ-barrier reassembly following BPA removal. In summary, Cx43 is crucial for TJ reassembly at the BTB during its cyclic restructuring throughout the seminiferous epithelial cycle of spermatogenesis.

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.

Connexin 43 and plakophilin-2 as a protein complex that regulates blood–testis barrier dynamics

The blood–testis barrier (BTB) formed by adjacent Sertoli cells is composed of coexisting tight junction (TJ), basal ectoplasmic specialization (ES), and desmosome-like junction. Desmosome-like junctions display structural features of desmosome and gap junctions, but its function at the BTB remains unknown. Herein, we demonstrate that connexin 43 (Cx43), a gap junction integral membrane protein, structurally interacts with desmosomal protein plakophilin-2 (PKP2), basal ES proteins N-cadherin and β-catenin, and signaling molecule c-Src, but not with the TJ proteins occludin and ZO-1 in the seminiferous epithelium of adult rats. The localization of Cx43 in the seminiferous epithelium during (i) the normal epithelial cycle of spermatogenesis and (ii) anchoring junction restructuring at the Sertoli–spermatid interface induced by adjudin which mimics junction restructuring events during spermatogenesis have suggested that Cx43 is involved in cell adhesion. The knockdown of Cx43 by RNAi technique using specific siRNA duplexes was performed in primary Sertoli cell cultures with an established TJ permeability barrier that mimicked the BTB in vivo. This knockdown of Cx43 affected neither the TJ barrier function nor the steady-state levels of junction proteins of TJ, basal ES, and desmosome-like junction. However, after the knockdown of both Cx43 and PKP2, the Sertoli cell TJ barrier function was perturbed transiently. This perturbation was concomitant with a mislocalization of occludin and ZO-1 from the cell–cell interface. In summary, Cx43 and PKP2 form a protein complex within the desmosome-like junction to regulate cell adhesion at the BTB, partly through its effects on the occludin/ZO-1 complex, so as to facilitate the transit of primary preleptotene spermatocytes.

Mitogen-activated protein kinases in male reproductive function

Recent studies have shown that male reproductive function is modulated via the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is involved in numerous male reproductive processes, including spermatogenesis, sperm maturation and activation, capacitation and acrosome reaction, before fertilization of the oocyte. In this review, we discuss the latest findings in this rapidly developing field regarding the role of MAPK in male reproduction in animal models and in human spermatozoa in vitro. This research will facilitate the design of future studies in humans, although much work is needed before this information can be used to manage male infertility and environmental toxicant-induced testicular injury in men, such as blood-testis-barrier disruption.

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 TNFalpha and TGFbeta3, 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.

14-3-3 Protein regulates cell adhesion in the seminiferous epithelium of rat testes.

Polarity proteins have been implicated in regulating and maintaining tight junction (TJ) and cell polarity in epithelia. Here we report 14-3-3theta, the homolog of Caenorhabditis elegans Par5 in mammalian cells, which is known to confer cell polarity at TJ, is found at the apical ectoplasmic specialization (ES), a testis-specific adherens junction type restricted to the Sertoli cell-elongating spermatid interface, in which TJ is absent. 14-3-3theta was shown to play a critical role in conferring cell adhesion at the apical ES. A loss of 14-3-3theta expression at the apical ES was detected in the seminiferous epithelium before spermiation. Involvement of 14-3-3theta in Sertoli cell adhesion was confirmed by its knockdown by RNA interference in Sertoli cells cultured in vitro with established TJ permeability barrier that mimicked the blood-testis barrier (BTB) in vivo. Mislocalization of N-cadherin and zonula occludens-1, but not alpha- and beta-catenins, was observed after 14-3-3theta knockdown in Sertoli cells, moving from the cell-cell interface to cytosol, indicating a disruption of cell adhesion. Studies by endocytosis assay illustrated that this loss of cell adhesion was mediated by an increase in the kinetics of endocytosis of N-cadherin and junctional adhesion molecule-A at the BTB, which may represent a general mechanism by which polarity proteins regulate cell adhesion. In summary, the testis is using 14-3-3theta to regulate cell adhesion at the apical ES to facilitate spermiation and at the BTB to facilitate the transit of preleptotene spermatocytes at stages VIII-IX of the epithelial cycle. 14-3-3theta may act as a molecular switch that coordinates these two cellular events in the seminiferous epithelium during spermatogenesis.

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.

Cell junctions in the testis as targets for toxicants

Recent declines in male fertility, as evidenced by decreased sperm counts, in industrialized nations have been attributed partly to the exposure of men to environmental toxicants, such as cadmium, bisphenol A, and others. These environmental toxicants are found in drinking water, food, dairy products, and many utensils (e.g., plastics and glass bottles). It is of interest to note that while animal models clearly show effects of environmental toxicants on male reproductive function, linking specific environmental exposures in humans to adverse effects on the male reproductive system is very tenuous for the vast majority of environmental chemicals since human exposure is often orders of magnitude lower than dose levels used in animal studies. Nonetheless, environment toxicants have become an integrated part of our day-to-day routine and food/water intakes. This thus sparks interest in the field to assess if acute and chronic exposure of these toxicants to laboratory animals would cause reproductive damage, and whether such damage can be reversed and/or rescued. In this review, we summarize recent findings in the field regarding damage that are caused by these toxicants to the testis via their actions at the cell–cell interface, thereby inducing premature loss of germ cells from the seminiferous epithelium which leads to reduced sperm counts in semen. Some of these studies have identified specific signaling pathways that are used by these toxicants to induce disruption at the Sertoli–Sertoli and/or Sertoli–germ cell interface, perturbing the blood–testis barrier (BTB) function and germ cell adhesion. This information should be helpful in future studies to design compounds that can ‘reverse’ and/or ‘reduce’ toxicant toxicity to the testis.

Intercellular adhesion molecule 1: recent findings and new concepts involved in mammalian spermatogenesis.

Spermatogenesis, the process of spermatozoa production, is regulated by several endocrine factors, including testosterone, follicle stimulating hormone, luteinizing hormone and estradiol 17β. For spermatogenesis to reach completion, developing germ cells must traverse the seminiferous epithelium while remaining transiently attached to Sertoli cells. If germ cell adhesion were to be compromised for a period of time longer than usual, germ cells would slough from the seminiferous epithelium and infertility would result. Presently, Sertoli-germ cell adhesion is known to be mediated largely by classical and desmosomal cadherins. More recent studies, however, have begun to expand long-standing concepts and to examine the roles of other proteins such as intercellular adhesion molecules. In this review, we focus on the biology of intercellular adhesion molecules in the mammalian testis, hoping that this information is useful in the design of future studies.

Expression of Itch in Sertoli cells is controlled via the interaction of E2F1/DP1 complex with E2F and GATA motifs.

Itch, a ubiquitin E3 ligase, has been implicated in the regulation of the permeability of tight junction (TJ) barrier in Sertoli cells. It is involved in cAMP-mediated TJ disruption by targeting occludin for proteasomal degradation in the testis. However, the molecular mechanisms governing its transcription remain enigmatic. By transient transfection of Itch promoter luciferase construct in TM4 cells, we showed that the minimal Itch promoter was located between nucleotides -151 and -1 (relative to the translation start site). One E2F motifs and two each of GATA and Nkx motifs were identified within the core promoter region. Mutation and overexpression analyses have showed that the E2F and GATA-a motifs are involved in Itch gene transcription, but play different roles. The E2F motif is the crucial cis-acting element that drives the basal gene transcription, while the GATA-a motif functionally co-operate with E2F motif. By electromobility shift assays, we confirmed that E2F1 and DP1 form heterodimers and binds to E2F and GATA-a motifs. Taken together, the GATA-a motif assists/strengthens the binding of E2F1/DP1 complex to the E2F motif, resulting in efficient looping of promoter region of Itch gene for transcription.