Elissa W.P. Wong

An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII-IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl(2) to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics.

Polarity Proteins and Cell–Cell Interactions in the Testis

In mammalian testes, extensive junction restructuring takes place in the seminiferous epithelium at the Sertoli-Sertoli and Sertoli-germ cell interface to facilitate the different cellular events of spermatogenesis, such as mitosis, meiosis, spermiogenesis, and spermiation. Recent studies in the field have shown that Rho GTPases and polarity proteins play significant roles in the events of cell-cell interactions. Furthermore, Rho GTPases, such as Cdc42, are working in concert with polarity proteins in regulating cell polarization and cell adhesion at both the blood-testis barrier (BTB) and apical ectoplasmic specialization (apical ES) in the testis of adult rats. In this chapter, we briefly summarize recent findings on the latest status of research and development regarding Cdc42 and polarity proteins and how they affect cell-cell interactions in the testis and other epithelia. More importantly, we provide a new model in which how Cdc42 and components of the polarity protein complexes work in concert with laminin fragments, cytokines, and testosterone to regulate the events of cell-cell interactions in the seminiferous epithelium via a local autocrine-based regulatory loop known as the apical ES-BTB-basement membrane axis. This new functional axis coordinates various cellular events during different stages of the seminiferous epithelium cycle of spermatogenesis.

Cancer/testis (CT) antigens, carcinogenesis and spermatogenesis

During spermatogenesis, spermatogonial stem cells, undifferentiated and differentiated spermatogonia, spermatocytes, spermatids and spermatozoa all express specific antigens, many of which transiently, yet the functions of many of these antigens remain unexplored. Studies in the past three decades have shown that many of these transiently expressed genes in developing germ cells during spermatogenesis are proto-oncogenes and oncogenes, which are expressed only in the testis and various types of cancers in both humans and rodents. As such, these antigens are designated cancer/testis antigens (CT antigens). Since the early 1980s, about 70 families of CT antigens have been identified with over 140 members are known to date. Due to their restricted expression in the testis and in various tumors in humans, they have been used as the target of immunotherapy, and multiple clinical trials at different phases are being conducted with some promising results. Interestingly, in a significant number of cancer patients, antibodies against some of these CT antigens were detected in their sera. However, antibodies against these CT antigens in humans under normal physiological conditions have yet to be reported even though many of these antigens are residing outside of the blood-testis barrier (BTB), such as in the basal compartment of the seminiferous epithelium and in the stem cell niche in the testis. In this review, we summarize latest findings in the field regarding several selected CT antigens which may be intimately related to spermatogenesis due to their unusual restricted expression during different discrete events of spermatogenesis, such as cell cycle progression, meiosis, and spermiogenesis. This information should be helpful to investigators in the field to study the roles of these oncogenes in spermatogenesis.

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.

Palladin Is a Regulator of Actin Filament Bundles at the Ectoplasmic Specialization in Adult Rat Testes

In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.

Adjudin disrupts spermatogenesis via the action of some unlikely partners

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a potent analog of lonidamine [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] known to disrupt germ cell adhesion, most notably elongating and elongated spermatids, in the seminiferous epithelium of adult rat testes and thus, leads to infertility in rats. Since the population of spermatogonia and spermatogonial stem cells (SSCs) in the seminiferous tubules is not significantly reduced by the treatment of rats with adjudin, adjudin-induced infertility is highly reversible with the use of appropriate regimens, which enables re-initiation of spermatogenesis and germ cell re-population of the voided seminiferous epithelium. Furthermore, adjudin appears to exert its effects at the testis-specific atypical adherens junction (AJ) type known as ectoplasmic specialization (ES), most notably the apical ES at the Sertoli cell-spermatid interface. Thus, the hypothalamic-pituitary-gonadal axis is not unaffected and systemic side-effects are minimal. This also makes adjudin a potential candidate for male contraceptive development. Herein, we critically evaluate recent findings in the field and provide an updated model regarding the mechanism underlying adjudin-induced apical ES disruption. In short, adjudin targets actin filament bundles at the apical ES, the hallmark of this testis-specific junction type not found in any other epithelia/endothelia in mammals, by suppressing the expression of Eps8 (epidermal growth factor receptor pathway substrate 8), an actin capping protein that also plays a role in actin bundling, so that actin filament bundles can no longer be maintained at the apical ES. This is concomitant with a mis-localization of Arp3 (actin-related protein 3, a component of the Arp2/3 complex that induces actin nucleation/branching), causing “unwanted” actin branching, further destabilizing actin filament bundles at the apical ES. Additionally, adjudin blocks the expression of PAR6 (partitioning defective protein 6) and 14–3-3 (also known as PAR5) considerably at the apical ES, disrupting the homeostasis of endocytic vesicle-mediated protein trafficking, which in turn leads to an increase in protein endocytosis. The net result of these changes destabilizes cell adhesion and induces degeneration of the apical ES, causing premature release of spermatids, mimicking spermiation.

The Scribble/Lgl/Dlg polarity protein complex is a regulator of blood-testis barrier dynamics and spermatid polarity during spermatogenesis.

During spermatogenesis, spermiogenesis that releases sperm into the tubule lumen and restructuring of the blood-testis barrier (BTB) that accommodates the transit of preleptotene spermatocytes take place simultaneously, but at the opposite ends of the seminiferous epithelium. These events are tightly regulated and coordinated; however, neither the underlying mechanism(s) nor the involving molecules are known. Herein, the Scribble/Lgl (Lethal giant larvae)/Dlg (Discs large) polarity complex was shown to regulate spermatid polarity during spermiogenesis and tight junction (TJ)-permeability barrier via changes in protein distribution at the apical ectoplasmic specialization and the BTB during the epithelial cycle, respectively. Scribble, Lgl2, and Dlg1 were found to be expressed by Sertoli and germ cells. Scribble also displayed stage-specific expression at the BTB, being highest at stages VII-VIII, colocalizing with TJ proteins occludin and ZO-1. Unlike components of other polarity complex modules, such as partitioning-defective 6, the knockdown of which by RNA interference was found to impede Sertoli cell TJ barrier, a knockdown of the Scribble complex (i.e. simultaneous knockdown of Scribble, Lgl and Dlg or Lgl alone; but not Scribble or Dlg alone) both in vitro and in vivo promoted the TJ integrity. This was mediated by reorganizing actin filament network at the Sertoli cell-cell interface, which, in turn, affected changes in the localization and/or distribution of occludin and/or β-catenin at the BTB. These knockdowns also perturbed F-actin organization at the Sertoli cell-spermatid interface, thereby modulating spermatid adhesion and polarity at the apical ectoplasmic specialization. In summary, the Scribble/Lgl/Dlg complex participates in the regulation of BTB dynamics and spermatid adhesion/polarity in the testis.

Actin Binding Proteins, Spermatid Transport And Spermiation

The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.

Polarity Protein Complex Scribble/Lgl/Dlg And Epithelial Cell Barriers

The Scribble polarity complex or module is one of the three polarity modules that regulate cell polarity in multiple epithelia including blood-tissue barriers. This protein complex is composed of Scribble, Lethal giant larvae (Lgl) and Discs large (Dlg), which are well conserved across species from fruitflies and worms to mammals. Originally identified in Drosophila and C. elegans where the Scribble complex was found to work with the Par-based and Crumbs-based polarity modules to regulate apicobasal polarity and asymmetry in cells and tissues during embryogenesis, their mammalian homologs have all been identified in recent years. Components of the Scribble complex are known to regulate multiple cellular functions besides cell polarity, which include cell proliferation, assembly and maintenance of adherens junction (AJ) and tight junction (TJ), and they are also tumor suppressors. Herein, we provide an update on the Scribble polarity complex and how this protein complex modulates cell adhesion with some emphasis on its role in Sertoli cell blood-testis barrier (BTB) function. It should be noted that this is a rapidly developing field, in particular the role of this protein module in blood-tissue barriers, and this short chapter attempts to provide the information necessary for investigators studying reproductive biology and blood-tissue barriers to design future studies. We also include results of recent studies from flies and worms since this information will be helpful in planning experiments for future functional studies in the testis to understand how Scribble-based proteins regulate BTB dynamics and 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.