Bisphenol A, an estrogenic environmental toxicant, has been implicated to have hazardous effects to reproductive health in humans and rodents. However, there are conflicting reports in the literature regarding its effects to 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, which can also serve as a suitable model for studying the dynamics of the blood-testis barrier.

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 ␤-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.desmosome-like junction ͉ seminiferous epithelial cycle ͉ Sertoli cell ͉ spermatogenesis I n adult mammals, including rodents and humans, the blood-testis barrier (BTB) is a unique and important ultrastructure in the seminiferous epithelium for maintaining spermatogenesis. Unlike other blood-tissue barriers, such as the blood-brain barrier, the BTB is constituted by adjacent Sertoli cells near the basement membrane in the seminiferous epithelium, instead of at the endothelial tight junction (TJ) barrier of microvessels in the interstitium (1, 2). In the rat testis, the BTB confers the 'fence' function that monitors the paracellular influx of water, electrolytes, hormones, and biomolecules between adjacent Sertoli cells and maintains cell polarity (3). Furthermore, the BTB creates a specialized microenvironment in the apical compartment of the seminiferous epithelium and segregates the entire event of postmeiotic germ cell development from the systemic circulation (3). Although the BTB is considered to be one of the tightest blood-tissue barriers in mammals (4), it must undergo extensive restructuring at stages VIII and IX of the seminiferous epithelial cycle in adult rat testes. As such, junction restructuring facilitates the transit of primary preleptotene...

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.bisphenol A | calcium switch | gap junction | seminiferous epithelial cycle | spermatogenesis I n mammalian testes, the blood-testis barrier (BTB) segregates the epithelium of seminiferous tubules into apical and basal compartments (1, 2). Unlike other blood-tissue barriers (e.g., blood-brain barrier, blood-retina barrier) that are constituted by tight junctions (TJs) between endothelial cells of the microvessels at the apical region, the BTB is created by adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. The BTB is constituted by coexisting TJs, basal ectoplasmic specialization (basal ES, a testis-specific adherens junction type), desmosome-like junction (or desmosome-gap junction), and gap junction (GJ) (1-3). Whereas the BTB is one of the tightest blood-tissue barriers, it undergoes cyclical restructuring to accommodate the transit of preleptotene spermatocytes from the basal to the apical compartment during spermatogenesis, so that meiotic divisions and postmeiotic germ cell development (i.e., spermiogenesis) can take place in the apical compartment of the seminiferous epithelium behind this immunological barrier. The immunological barrier conferred by the BTB cannot be compromised, even transiently, during the transit of preleptotene spermatocytes to avoid the product...

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. Male reproductive health and the involvement of the mitogen-activated protein kinase pathwayMale infertility and subfertility have been attributed to poor semen quality and/or sexual dysfunction. Abnormal semen quality includes reduced sperm counts and defects in the morphology, genetic content or motility of spermatozoa, whereas sexual dysfunction refers to impotence or defects in ejaculation [1,2]. The causative agents for abnormal semen quality are numerous and include heat, physical insults to testicles, radiation, alcohol and/or drug abuse, cigarette smoking and environmental toxicants. Heavy metals (e.g. cadmium and lead) and some drugs, such as chemotherapeutic compounds and antibiotics, are also known to impair fertility [1]. Even though the direct association of the mitogen-activated protein kinase (MAPK) pathway and reproductive dysfunction in men remains to be established, studies in animal models have indicated that the MAPK pathway is often involved in disrupting spermatogenesis and other functions of germ cells or Sertoli cells (see Glossary). This, in turn, leads to a decline in semen quality and fertility.In this article, the participation of the MAPK pathway in various processes leading to abnormal semen quality is explored, as well as its role in normal spermatogenesis and germ cell functions. We also suggest that targeting kinases in the MAPK pathway might have potential for treating or managing male reproductive dysfunction. To assist the conception of couples with fertility problems, in vitro fertilization and intrauterine insemination are commonly used. Members of the MAPK pathway might serve as therapeutic targets to reverse the decline in semen quality and fertility as a result of disruption of sperm function or after testicular injury such as a disruption of the blood-testis barrier (BTB) induced by environment toxicants (e.g. cadmium). Furthermore, the MAPK pathway has been shown to be involved in the differentiation, maturation, and function of male germ cells. Kinases in the MAPK pathway might therefore also have potential for investigation as candidate targets for male contraception. Overview of the MAPK signaling pathwayThe MAPK pathway is known to have an important role in the signal transduction of many cellular ...

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.