Dolores D. Mruk

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.

The Blood-Testis Barrier and Its Implications for Male Contraception

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium into the basal and the apical (adluminal) compartments. Meiosis I and II, spermiogenesis, and spermiation all take place in a specialized microenvironment behind the BTB in the apical compartment, but spermatogonial renewal and differentiation and cell cycle progression up to the preleptotene spermatocyte stage take place outside of the BTB in the basal compartment of the epithelium. However, the BTB is not a static ultrastructure. Instead, it undergoes extensive restructuring during the seminiferous epithelial cycle of spermatogenesis at stage VIII to allow the transit of preleptotene spermatocytes at the BTB. Yet the immunological barrier conferred by the BTB cannot be compromised, even transiently, during the epithelial cycle to avoid the production of antibodies against meiotic and postmeiotic germ cells. Studies have demonstrated that some unlikely partners, namely adhesion protein complexes (e.g., occludin-ZO-1, N-cadherin-β-catenin, claudin-5-ZO-1), steroids (e.g., testosterone, estradiol-17β), nonreceptor protein kinases (e.g., focal adhesion kinase, c-Src, c-Yes), polarity proteins (e.g., PAR6, Cdc42, 14-3-3), endocytic vesicle proteins (e.g., clathrin, caveolin, dynamin 2), and actin regulatory proteins (e.g., Eps8, Arp2/3 complex), are working together, apparently under the overall influence of cytokines (e.g., transforming growth factor-β3, tumor necrosis factor-α, interleukin-1α). In short, a “new” BTB is created behind spermatocytes in transit while the “old” BTB above transiting cells undergoes timely degeneration, so that the immunological barrier can be maintained while spermatocytes are traversing the BTB. We also discuss recent findings regarding the molecular mechanisms by which environmental toxicants (e.g., cadmium, bisphenol A) induce testicular injury via their initial actions at the BTB to elicit subsequent damage to germ-cell adhesion, thereby leading to germ-cell loss, reduced sperm count, and male infertility or subfertility. Moreover, we also critically evaluate findings in the field regarding studies on drug transporters in the testis and discuss how these influx and efflux pumps regulate the entry of potential nonhormonal male contraceptives to the apical compartment to exert their effects. Collectively, these findings illustrate multiple potential targets are present at the BTB for innovative contraceptive development and for better delivery of drugs to alleviate toxicant-induced reproductive dysfunction in men.

Cadmium-induced testicular injury.

Cadmium (Cd) is an environmental toxicant and an endocrine disruptor in humans and rodents. Several organs (e.g., kidney, liver) are affected by Cd and recent studies have illustrated that the testis is exceedingly sensitive to Cd toxicity. More important, Cd and other toxicants, such as heavy metals (e.g., lead, mercury) and estrogenic-based compounds (e.g., bisphenols) may account for the recent declining fertility in men among developed countries by reducing sperm count and testis function. In this review, we critically discuss recent data in the field that have demonstrated the Cd-induced toxicity to the testis is probably the result of interactions of a complex network of causes. This is likely to involve the disruption of the blood-testis barrier (BTB) via specific signal transduction pathways and signaling molecules, such as p38 mitogen-activated protein kinase (MAPK). We also summarize current studies on factors that confer and/or regulate the testis sensitivity to Cd, such as Cd transporters and metallothioneins, the impact of Cd on the testis as an endocrine disruptor and oxidative stress inducer, and how it may disrupt the Zn(2+) and/or Ca(2+) mediated cellular events. While much work is needed before a unified mechanistic pathway of Cd-induced testicular toxicity emerges, recent studies have helped to identify some of the likely mechanisms and/or events that take place during Cd-induced testis injury. Furthermore, some of the recent studies have shed lights on potential therapeutic or preventive approaches that can be developed in future studies by blocking or minimizing the destructive effects of Cd to testicular function in men.

Regulation of blood-testis barrier dynamics: an in vivo study

An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl2. It was shown that the CdCl2-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-β2 and TGF-β3 (but not TGF-β1) and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-β2/-β3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl2-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl2 mediates its BTB disruptive effects via the TGF-β3/p38 MAP kinase signaling pathway. Besides, this CdCl2-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of α2-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. α2-Macroglobulin was also shown to associate physically with TGF-β3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-β3/p-38 MAP kinase pathway also prevented the CdCl2-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on α2-macroglobulin. These results demonstrate for the first time that the TGF-β3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-β3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.

A local autocrine axis in the testes that regulates spermatogenesis

Spermiation—the release of mature spermatozoa from Sertoli cells into the seminiferous tubule lumen—occurs by the disruption of an anchoring device known as the apical ectoplasmic specialization (apical ES). At the same time, the blood–testis barrier (BTB) undergoes extensive restructuring to facilitate the transit of preleptotene spermatocytes. While these two cellular events take place at opposite ends of the Sertoli cell epithelium, the events are in fact tightly coordinated, as any disruption in either process will lead to infertility. A local regulatory axis exists between the apical ES and the BTB in which biologically active laminin fragments produced at the apical ES by the action of matrix metalloproteinase 2 can regulate BTB restructuring directly or indirectly via the hemidesmosome. Equally important, polarity proteins play a crucial part in coordinating cellular events within this apical ES–BTB–hemidesmosome axis. Additionally, testosterone and cytokines work in concert to facilitate BTB restructuring, which enables the transit of spermatocytes while maintaining immunological barrier function. Herein, we will discuss this important autocrine-based cellular axis that parallels the hormonal-based hypothalamic–pituitary–testicular axis that regulates spermatogenesis. This local regulatory axis is the emerging target for male contraception.

Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells

During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood‐testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) ‐βs], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the “opening” of the BTB above a migrating preleptotene/lepto‐tene spermatocyte and the “resealing” of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB‐associated integral membrane proteins: occludin, JAM‐A, and N‐cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin‐mediated but not the caveolin‐mediated pathway. When T or TGF‐β2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF‐β2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady‐state protein levels at the Sertoli‐Sertoli cell interface at the BTB.— Yan H. H. N., Mruk D. D., Lee W. M., Cheng C. Y. Blood‐testis barrier dynamics are regulated by testosterne and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells. FASEB J. 22, 1945–1959 (2008)

Antioxidant superoxide dismutase: a review: its function, regulation in the testis, and role in male fertility

Extracellular superoxide dismutase (SOD(EX)), an antioxidant enzyme, was found to be present in the testis at a relatively high concentration versus other organs. In a more detailed survey of several rat tissues and cells by reverse transcriptase-polymerase chain reaction, it was shown that germ cells expressed approximately one-third that of Sertoli cells, suggesting both cell types are equipped with the machinery needed to defend themselves from radical-induced damage. When we used an in vitro model in which germ cells were co-cultured with Sertoli cells at a Sertoli:germ cell ratio of 1:1, we failed to detect any changes in the mRNA level of SOD(EX). However, the addition of increasing concentrations of germ cell secretory proteins into Sertoli cell cultures resulted in a decrease in Sertoli cell SOD(EX) expression, illustrating that germ cells can indeed regulate Sertoli cell SOD(EX). On the other hand, Sertoli cell SOD(EX) expression was stimulated when human recombinant interleukin-1alpha (IL-1alpha), a germ cell product, was included into Sertoli cells in vitro. These results, taken collectively, suggest SOD(EX) is an important antioxidant molecule in the testis that is under germ cell regulation.

Sertoli Cell Tight Junction Dynamics: Their Regulation During Spermatogenesis

Abstract During spermatogenesis, developing preleptotene and leptotene spermatocytes must translocate from the basal to the adluminal compartment of the seminiferous epithelium so that fully developed spermatids (spermatozoa) can be released to the tubular lumen at spermiation. It is conceivable that the opening and closing of the inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier are regulated by an array of intriguingly coordinated signaling pathways and molecules. Several molecules have been shown to regulate Sertoli cell TJ dynamics; they include, for example, transforming growth factor β3 (TGFβ3), occludin, protein kinase A, protein kinase C, and signaling pathways such as the TGFβ3/p38 mitogen-activated protein kinase pathway. Yet the mechanisms that regulate these events are essentially not known. This minireview summarizes some of the recent advances in the study of TJ dynamics in the testis and reviews several models that can be used to study TJ dynamics. It also highlights specific areas for future research toward understanding the precise physiological relationship between junction dynamics and spermatogenesis.

Is the Cadherin/Catenin Complex a Functional Unit of Cell-Cell Actin-Based Adherens Junctions in the Rat Testis?

Abstract Much controversy exists regarding the presence of the cadherin/catenin complex and its intracellular attachment site in the testis, which is the functional unit for actin-based cell-cell adherens junctions (AJs) in multiple epithelia. Furthermore, whether germ and Sertoli cells are equipped with the necessary AJ-associated signaling molecules to regulate this cadherin/catenin complex during spermatogenesis is not known. In the present study, it was shown that both Sertoli and germ cells indeed express N-cadherin, E-cadherin, α-catenin, β-catenin, and p120ctn by semiquantitative reverse transcription-polymerase chain reaction and immunoblotting. Furthermore, the assembly of AJs between Sertoli and germ cells was associated with a transient induction in the steady-state mRNA and protein levels of cadherins and catenins. These analyses reveal, to our knowledge for the first time, that the testis may indeed be using the cadherin/catenin complex as one of the functional units to regulate AJ dynamics between Sertoli and germ cells in addition to α6β1 integrin and the nectin/afadin complex. To further confirm the existence of such a complex between Sertoli and germ cells, immunoprecipitation experiments were performed using Sertoli-germ cell lysates during AJ assembly. An anti-N-cadherin antibody can pull out β-catenin, whereas N-cadherin can also be pulled out using an anti-β-catenin antibody. To further expand and validate these in vitro biochemical studies, immunofluorescent histochemistry was performed, which colocalized N-cadherin and β-catenin to the same site of Sertoli-Sertoli and Sertoli-germ cell AJs, possibly ectoplasmic specializations near the basal compartment, at the lower third of the seminiferous epithelium in vivo as well as between Sertoli cells cultured in vitro. Furthermore, studies by cross-linking using dithiobis(succinimidylpropionate) confirmed that the cadherin/catenin complex between Sertoli cells as well as between Sertoli and germ cells indeed structurally linked to actin but not to vimentin (an intermediate filament protein) or to tubulin (a microtubule protein). These results thus unequivocally demonstrate that the cadherin/catenin complex, which can be up-regulated by testosterone, is indeed present between Sertoli and germ cells and is used for the assembly of functional AJs.

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.