[Show abstract][Hide abstract] ABSTRACT: The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the "opening" and "closing" of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing "old" BTB above preleptotene spermatocytes in transit in "clones" at the BTB, but also contribute to the assembly of "new" BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the "gateway" to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin β3 or γ3 chains) derived from the α6β1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on β1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local functional axis in the seminiferous epithelium in spermatogenesis. We also provide molecular modeling information on the interactions between biologically active laminin fragments and β1-integrin, which will be important to assist in the design of more potent laminin-based peptides to disrupt this axis, thereby perturbing spermatogenesis for male contraception and to understand the underlying biology that coordinates spermiation and BTB restructuring during spermatogenesis.
[Show abstract][Hide abstract] ABSTRACT: 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, which enables reinitiation 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 ultrastructure 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) recruited by drebrin E, 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.