Pearl P.Y. Lie

Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel regulator of cell adhesion and the blood-testis barrier integrity in the seminiferous epithelium

In the seminiferous epithelium, Eps8 is localized to actin‐based cell junctions at the blood‐testis barrier (BTB) and the apical ectoplasmic specialization (ES) in stage V—VI tubules but is considerably diminished in stage VIII tubules. Eps8 down‐regulation coincides with the time of BTB restructuring and apical ES disassembly, implicating the role of Eps8 in cell adhesion. Its involvement in Sertoli‐germ cell adhesion was substantiated in studies using an in vivo animal model by treating rats with 1‐(2,4‐dichlorobenzy)‐1H‐indazole‐ 3‐ carbohydrazide (adjudin) to induce anchoring junction restructuring, during which Eps8 disappeared at the apical ES before germ cell departure. In Sertoli cell cultures with established permeability barrier mimicking the BTB in vivo, the knockdown of Eps8 by RNAi led to F‐actin disorganization and the mislocalization of the tight junction proteins occludin and ZO‐1, suggesting the function of Eps8 in maintaining BTB integrity. In vivo knockdown of Eps8 in the testis caused germ cell sloughing and BTB damage, concomitant with occludin mislocalization, further validating that Eps8 is a novel regulator of cell adhesion and BTB integrity in the seminiferous epithelium.— Lie, P. P. Y., Mruk, D. D., Lee, W. M., Cheng, C. Y. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel regulator of cell adhesion and the blood‐testis barrier integrity in the seminiferous epithelium. FASEB J. 23, 2555–2567 (2009)

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

In epithelia, a primary damage of tight junctions (TJ) always leads to a secondary disruption of adherens junction (AJ), and vice versa. This response, if occurring in the testis, would disrupt spermatogenesis because the blood–testis barrier (BTB) must remain intact during the transit of spermatids in the seminiferous epithelium, which is associated with extensive apical ectoplasmic specialization (apical ES, a testis-specific AJ type) restructuring. As such, apical ES restructuring accompanied with the transit of developing spermatids during spermiogenesis must be segregated from the BTB to avoid an immunological barrier breakdown in all stages of the seminiferous epithelial cycle, except at stage VIII when spermiation and BTB restructuring take place concurrently. We report herein a mechanism involving restricted spatial and temporal expression of Arp2/3 complex and N-WASP, whose actin branching activity associated with apical ES and BTB restructuring in the seminiferous epithelium. High expression of Arp3 at the apical ES was shown to correlate with spermatid movement and proper spermatid orientation. Likewise, high Arp3 level at the BTB associated with its restructuring to accommodate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. These findings were validated by in vitro and in vivo studies using wiskostatin, an inhibitor that blocks N-WASP from activating Arp2/3 complex to elicit actin branching. Inhibition of actin branching caused a failure of spermatid transit plus a loss of proper orientation in the epithelium, and a “tightened” Sertoli cell TJ permeability barrier, supporting the role of Arp2/3 complex in segregating the events of AJ and BTB restructuring.

Focal adhesion kinase-Tyr407 and -Tyr397 exhibit antagonistic effects on blood–testis barrier dynamics in the rat

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood–testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin–zonula occludens-1 complex. Herein, we report that p-FAK-Tyr407 and p-FAK-Tyr397 display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction–permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr407 and Tyr397. Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott–Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr407 phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular “switch” to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.

Coordinating cellular events during spermatogenesis: a biochemical model

Throughout spermatogenesis, a select pool of germ cells, the leptotene spermatocytes, must traverse the blood-testis barrier (BTB) to enter the adluminal compartment of the seminiferous epithelium. This event requires extensive restructuring of cell junctions, and it must also coincide with germ cell cycle progression in preparation for primary spermatocyte meiosis. Recent findings show that cell-cycle-associated kinases and phosphatases, including mitogen-activated protein kinases (MAPKs), participate in the pathways that also direct germ cell adhesion and movement. Our new biochemical model explains, in part, how two distinct cellular events, BTB restructuring and spermiation, are coordinated to maintain spermatogenesis and fertility. In this way, MAPKs would synchronize cell cycle progression in primary spermatocytes with junction remodeling and cell migration across the BTB.

Interleukin-1α is a regulator of the blood-testis barrier

Throughout spermatogenesis, the Sertoli cell blood‐testis barrier (BTB) is strictly regulated by cytokines, which mediate its timely restructuring, thereby allowing spermatocytes to enter the adluminal compartment of the seminiferous epithelium for development into spermatozoa. The aim herein was to investigate whether germ cells play a role in BTB restructuring via the action of interleukin‐lo (IL‐1α) since germ cells are known to control Sertoli cell production of this cytokine, and if yes, how these effects are mediated. When Sertoli cells were isolated from Sprague‐Dawley rats and plated at high density, IL‐1α (100 pg/ml) was shown to “open” the Sertoli cell barrier when its integrity was assessed by transepithelial electrical resistance measurements. Further investigation of Sertoli cells treated with IL‐1α revealed striking changes in the cellular distribution of actin filaments when compared to untreated cells. These effects at the Sertoli cell barrier were mediated, in part, by epidermal growth factor receptor pathway substrate 8 (Eps8; an actin bundling and barbed‐end capping protein) and actin‐related protein 3 (Arp3; a component of the actin nucleation machinery). As important, an increase in the kinetics of occludin internalization but a decrease in its rate of degradation was noted following IL‐1α treatment. These results indicate that IL‐1α is a critical regulator of BTB dynamics.—Lie, P. P. Y., Cheng, C. Y., Mruk, D. D. Interleukin‐lα is a regulator of the blood‐testis barrier. FASEB J. 25, 1244–1253 (20ll) (2011). www.fasebj.org

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

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

The Biology of the Desmosome-Like Junction: A Versatile Anchoring Junction and Signal Transducer in the Seminiferous Epithelium

Mammalian spermatogenesis, a complex process that involves the movement of developing germ cells across the seminiferous epithelium, entails extensive restructuring of Sertoli-Sertoli and Sertoli-germ cell junctions. Presently, it is not entirely clear how zygotene spermatocytes gain entry into the adluminal compartment of the seminiferous epithelium, which is sealed off from the systemic circulation by the Sertoli cell component of the blood-testis barrier, without compromising barrier integrity. To begin to address this question, it is critical that we first have a good understanding of the biology and the regulation of different types of Sertoli-Sertoli and Sertoli-germ cell junctions in the testis. Supported by recent studies in the field, we discuss how crosstalk between different types of junctions contributes to their restructuring during germ cell movement across the blood-testis barrier. We place special emphasis on the emerging role of desmosome-like junctions as signal transducers during germ cell movement across the seminiferous epithelium.

A peptide derived from laminin-γ3 reversibly impairs spermatogenesis in rats.

Cellular events that occur across the seminiferous epithelium of the mammalian testis during spermatogenesis are tightly coordinated by biologically active peptides released from laminin chains. Laminin-γ3 domain IV (Lam γ3 DIV) is released at the apical ectoplasmic specialization (ES) during spermiation and mediates restructuring of the blood-testis barrier (BTB), which facilitates the transit of preleptotene spermatocytes. Here we determine the biologically active domain in Lam γ3 DIV, which we designate F5-peptide, and show that overexpression of this domain, or the use of a synthetic F5-peptide, in Sertoli cells with an established functional BTB reversibly perturbs BTB integrity in vitro and in rat testis in vivo. This effect is mediated via changes in protein distribution at the Sertoli and Sertoli-germ cell-cell interface and by phosphorylation of focal adhesion kinase at Tyr407. The consequences are perturbed organization of actin filaments in Sertoli cells, disruption of the BTB and spermatid loss. The impairment of spermatogenesis suggests that this laminin peptide fragment may serve as a contraceptive in male rats.

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.