[Show abstract][Hide abstract] ABSTRACT: The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in mammals. As such, it poses a challenge to deliver any drugs to the seminiferous epithelium of the testis, such as a nonhormonal male contraceptive. To circumvent this problem, a genetically engineered follicle-stimulating hormone (FSH) mutant protein was produced in Spodoptera furgiperda (Sf)-9 insect cells to serve as a testis-specific carrier. Subsequently, a 22-amino acid peptide corresponding to the second extracellular loop of occludin, which was known to disrupt BTB integrity in vivo, was inserted to the FSH mutant by polymerase chain reaction (PCR), as well as chemical cross-linking. This molecule was found to have negligible hormonal activity but was still capable of binding to FSH receptors, which are restricted to Sertoli cells in mammals. When this FSH mutant-occludin peptide conjugate was administered to adult rats at 40 microg/adult rat (approximately 300 gm b.w.) via intraperitoneally (i.p.) injection, it induced transient and reversible disruption of the BTB, while at 150 microg/rat, it induced partial germ cell loss from the testis, particularly elongating/elongate spermatids. Most importantly, this effect was limited to the BTB without compromising the TJ-barrier integrity or cell adhesion in epithelia of other organs, such as kidney, liver, and small intestine. In summary, the use of an FSH mutant-occludin peptide conjugate is a feasible nanodevice to transiently compromise the BTB.
The FASEB Journal 03/2007; 21(2):438-48. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In adult rat testes, blood-testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient 'opening'. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), beta-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/beta-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with beta-catenin and ZO-1. Beta-catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an 'engaged'state to reinforce the barrier function in normal testes. However, beta-catenin and ZO-1 were 'disengaged' from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the 'disengagement' of beta-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.
Journal of Endocrinology 01/2007; 191(3):571-86. · 3.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Throughout spermatogenesis, developing germ cells remain attached to Sertoli cells via testis-specific anchoring junctions. If adhesion between these cell types is compromised, germ cells detach from the seminiferous epithelium and infertility often results. Previously, we reported that Adjudin is capable of inducing germ cell loss from the epithelium. In a small subset of animals, however, oral administration of Adjudin (50 mg per kg body weight (b.w.) for 29 d) resulted in adverse effects such as liver inflammation and muscle atrophy. Here, we report a novel approach in which Adjudin is specifically targeted to the testis by conjugating Adjudin to a recombinant follicle-stimulating hormone (FSH) mutant, which serves as its 'carrier'. Using this approach, infertility was induced in adult rats when 0.5 microg Adjudin per kg b.w. was administered intraperitoneally, which was similar to results when 50 mg per kg b.w. was given orally. This represents a substantial increase in Adjudin's selectivity and efficacy as a male contraceptive.
Nature Medicine 12/2006; 12(11):1323-8. · 28.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitogen-activated protein kinases (MAPKs) are important regulators of many cellular processes. In mammalian testes, these kinases are involved in controlling cell division, differentiation, survival and death, and are therefore critical to spermatogenesis. Recent studies have also illustrated their involvement in junction restructuring in the seminiferous epithelium, especially at the ectoplasmic specialization (ES), a testis-specific adherens junction (AJ) type. ES contributes to the adhesion between Sertoli cells at the blood-testis barrier, as well as between Sertoli and developing spermatids (step 9 and beyond) at the adluminal compartment. MAPKs regulate AJ dynamics in the testis via their effects on the turnover of junction-associated protein complexes, the production of proteases and protease inhibitors, and the cytoskeleton structure. In this review, roles of the three major MAPK members, namely extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in ES dynamics are critically discussed. An integrated model of how these three MAPKs regulate adhesion function in the seminiferous epithelium is also presented. This model will serve as the framework for future investigation in the field.
[Show abstract][Hide abstract] ABSTRACT: Earlier studies have shown that 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (AF-2364) is a potential male contraceptive when administered orally to adult Sprague-Dawley rats. This compound induces reversible germ cell loss from the seminiferous epithelium by disrupting cell adhesion function between Sertoli and germ cells, in particular, elongating/elongate/round spermatids and spermatocytes but not spermatogonia. Thus, this event is accompanied by a transient loss of fertility in treated rats. Once the drug is metabolically cleared, the remaining spermatogonia can begin repopulating the epithelium, and fertility bounces back. In this review, we summarize recent findings regarding the possible use of this drug for male contraception and its mechanism of action in the rat testis. We also provide an update on the efficacy results of using different treatment regimens in adult rats where AF-2364 was administered by gavage vs. intraperitoneal and intramuscular administration. These results have clearly indicated that AF-2364 is indeed a reversible male contraceptive. Furthermore, the tissue distribution in multiple organs and biological fluids using [3H]-AF-2364 is also reviewed. These data have clearly illustrated the low bioavailability of AF-2364 in rats and that this compound is not specifically taken up by any organs including the testis or the epididymis. These summaries are helpful to investigators in the field who seek to understand the molecular mechanism of action of AF-2364 in the rat testis and to explore its possible use for male contraception.
[Show abstract][Hide abstract] ABSTRACT: During spermatogenesis, extensive restructuring of cell junctions takes place in the seminiferous epithelium to facilitate germ cell movement. However, the mechanism that regulates this event remains largely unknown. Recent studies have shown that nitric oxide (NO) likely regulates tight junction (TJ) dynamics in the testis via the cGMP/protein kinase G (cGMP-dependent protein kinase, PRKG) signaling pathway. Due to the proximity of TJ and adherens junctions (AJ) in the testis, in particular at the blood-testis barrier, it is of interest to investigate if NO can affect AJ dynamics. Studies using Sertoli-germ cell cocultures in vitro have shown that the levels of NOS (nitric oxide synthase), cGMP, and PRKG were induced when anchoring junctions were being established. Using an in vivo model in which adult rats were treated with adjudin [a molecule that induces adherens junction disruption, formerly called AF-2364, 1-(2,4-dichlorobenzyl)-IH-indazole-3-carbohydrazide], the event of AJ disruption was also associated with a transient iNOS (inducible nitric oxide synthase, NOS2) induction. Immunohistochemistry has illustrated that NOS2 was intensely accumulated in Sertoli and germ cells in the epithelium during adjudin-induced germ cell loss, with a concomitant accumulation of intracellular cGMP and an induction of PRKG but not cAMP or protein kinase A (cAMP-dependent protein kinase, PRKA). To identify the NOS-mediated downstream signaling partners, coimmunoprecipitation was used to demonstrate that NOS2 and eNOS (endothelial nitric oxide synthase, NOS3) were structurally associated with the N-cadherin (CDH2)/beta-catenin (CATNB)/actin complex but not the nectin-3 (poliovirus receptor-related 3, PVRL 3)/afadin (myeloid/lymphoid or mixed lineage-leukemia tranlocation to 4 homolog, MLLT4) nor the integrin beta1 (ITB1)-mediated protein complexes, illustrating the spatial vicinity of NOS with selected AJ-protein complexes. Interestingly, CDH2 and CATNB were shown to dissociate from NOS during the adjudin-mediated AJ disruption, implicating the CDH2/CATNB protein complex is the likely downstream target of the NO signaling. Furthermore, PRKG, the downstream signaling protein of NOS, was shown to interact with CATNB in the rat testis. Perhaps the most important of all, pretreatment of testes with KT5823, a specific PRKG inhibitor, can indeed delay the adjudin-induced germ cell loss, further validating NOS/NO regulates Sertoli-germ cell AJ dynamics via the cGMP/PRKG pathway. These results illustrate that the CDH2/CATNB-mediated adhesion function in the testis is regulated, at least in part, via the NOS/cGMP/PRKG/CATNB pathway.
Biology of Reproduction 10/2005; 73(3):458-71. · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as beta1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin.laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-beta1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the beta1-integrin/PI3K/PKB/ERK signaling pathway.
Journal of Biological Chemistry 08/2005; 280(26):25029-47. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The blood-testis barrier (BTB), in contrast to the blood-brain and blood-retina barriers, is composed of coexisting tight junctions, gap junctions, and basal ectoplasmic specializations, a testis-specific type of adherens junction. Recent studies showed that BTB restructuring that facilitates germ cell migration during spermatogenesis involves proteolysis, an event that is usually restricted to the cell-matrix interface in other epithelia. For instance, a surge in alpha(2)-macroglobulin (alpha(2)-MG), a protease inhibitor produced by Sertoli cells, was detected at the Sertoli-Sertoli and Sertoli-germ cell interface in the epithelium during cadmium chloride-induced BTB disruption in adult rats. It is thus proposed that the increase in alpha(2)-MG is crucial for protecting the epithelium from unwanted proteolysis as well as regulating the availability of cytokines that affect junction turnover. Although both tight junction and adherens junction dynamics at the BTB are regulated via the p38 MAPK signaling pathway, the mechanism(s) that regulates alpha(2)-MG is entirely unknown. In this study, we have shown that by administering dimethylaminopurine, a c-Jun N-terminal protein kinase (JNK) inhibitor, to the testis, JNK activity was blocked specifically and alpha(2)-MG production was inhibited, worsening the cadmium chloride-induced damage to the epithelium. Studies coupled with inhibitors, immunoblottings, and immunofluorescent and electron microscopy have unequivocally demonstrated that the JNK signaling pathway is a putative regulatory pathway for alpha(2)-MG production in the testis. This finding illustrates for the first time that a cell-matrix restructuring event occurs in normal cell physiology at the cell-cell interface in the testis, highlighting the significance of alpha(2)-MG in the regulation of BTB function.
[Show abstract][Hide abstract] ABSTRACT: Using a well characterized model of cell-cell actin-based adherens junction (AJ) disruption by suppressing the intratesticular testosterone level in adult rats with testosterone-estradiol implants, we have confirmed earlier findings that Sertoli-germ cell AJ dynamics are regulated by the activation of kinases via putative signaling pathways but with some unexpected findings as follows. First, the loss of germ cells from the seminiferous epithelium during androgen suppression was associated with a surge in myotubularin-related protein 2 (MTMR2, a lipid phosphatase, in which adult MTMR2-/- mice were recently shown to be azoospermic because of the loss of cell adhesion function between germ and Sertoli cells); kinases: phosphatidylinositol 3-kinase, c-Src, and C-terminal Src kinase; adaptors: alpha-actinin, vinculin, afadin, and p130 Crk-associated protein; and AJ-integral membrane proteins at the ectoplasmic specialization (ES, a testis-specific cell-cell actin-based AJ type) site: N-cadherin, beta-catenin, integrin beta1, and nectin 3. Second, MTMR2, instead of structurally interacting with phosphatidylinositol 3-kinase, a protein and lipid kinase, was shown to associate only with c-Src, a nonreceptor protein tyrosine kinase, as demonstrated by both coimmunoprecipitation and fluorescent microscopy at the site of apical ES, but none of the kinases, adaptors, and AJ-integral proteins that were examined. Collectively, these results suggest that the MTMR2/c-Src is an important phosphatase/kinase protein pair in AJ dynamics in the testis. Because c-Src is known to associate with the cadherin/catenin protein complex at the ES in the testis, we next sought to investigate any changes in the protein-protein interactions of this protein complex during androgen suppression-induced germ cell loss. Indeed, there was a loss of N-cadherin and beta-catenin association, accompanied by a surge in Tyr phosphorylation of beta-catenin, during germ cell loss from the epithelium. Third, and perhaps the most important of all, during natural recovery of the epithelium after removal of testosterone-estradiol implants when spermatids were reattaching to Sertoli cells, an increase in N-cadherin and beta-catenin association was detected with a concomitant loss in the increased Tyr phosphorylation in beta-catenin. In summary, these results illustrate that the cadherin/catenin is a crucial cell adhesion complex that regulates AJ dynamics in the testis, and its functionality is likely modulated by the MTMR2/c-Src protein complex.
[Show abstract][Hide abstract] ABSTRACT: Apical ectoplasmic specialization (ES) is a unique testis-specific cell-cell actin-based adherens junction type restricted to the Sertoli-round/elongating/elongate spermatid interface in the seminiferous epithelium. An endogenous testosterone (T) suppression model was used to study the regulation of apical ES dynamics in the testis. By providing sustained releases of T and estradiol using subdermal implants in rats, this treatment reduced endogenous testicular T level. This in turn led to sloughing of spermatids (step 8 and beyond) from the seminiferous epithelium, which can be reversed by removing the implants, or replacing them with a higher dose of T implants. This model thus allows us to study the restructuring events at the apical ES. It was shown that apical ES restructuring involved proteins that were usually restricted to the cell-matrix focal adhesion site in other epithelia. For instance, the protein levels of beta1-integrin, Tyr-phosphorylated focal adhesion kinase (p-FAK), and c-Src were induced during the T suppression-induced germ cell loss and recovery, implicating that these proteins are putative regulators of ES dynamics. Indeed, the formation of p-FAK/c-Src protein complex, but not their association with beta1-integrin, was stimulated during T suppression-induced germ cell loss. ERK, a MAPK known to regulate focal adhesion turnover, was also activated during the androgen suppression-induced spermatid loss and the early phase of the recovery when germ cells began to repopulate the epithelium. Collectively, these data suggest that the apical ES is a cell-cell adherens junction type with the characteristics of cell-matrix focal contacts. In addition to its role in conferring cell adhesion formation, the p-FAK/c-Src protein complex apparently also regulates apical ES disruption via the ERK signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: The blood-testis barrier (BTB) in mammals, such as rats, is composed of the tight junction (TJ), the basal ectoplasmic specialization (basal ES), the basal tubulobulbar complex (basal TBC) (both are testis-specific actin-based adherens junction [AJ] types), and the desmosome-like junction that are present side-by-side in the seminiferous epithelium. The BTB physically divides the seminiferous epithelium into basal and apical (or adluminal) compartments, and is pivotal to spermatogenesis. Besides its function as an immunological barrier to segregate the postmeiotic germ-cell antigens from the systemic circulation, it creates a unique microenvironment for germ-cell development and confers cell polarity. During spermatogenesis, the BTB in rodents must physically disassemble to permit the passage of preleptotene and leptotene spermatocytes. This occurs at late stage VII through early stage VIII of the epithelial cycle. Studies have shown that this dynamic BTB restructuring to facilitate germ-cell migration is regulated by two cytokines, namely transforming growth factor-beta3 (TGF-beta3) and tumor necrosis factor-alpha (TNFalpha), via downstream mitogen-activated protein kinases. These cytokines determine the homeostasis of TJ- and basal ES-structural proteins, proteases, protease inhibitors, and other extracellular matrix (ECM) proteins (e.g., collagen) in the seminiferous epithelium. Some of these molecules are known regulators of focal contacts between the ECM and other actively migrating cells, such as macrophages, fibroblasts, or malignant cells. These findings also illustrate that cell-cell junction restructuring at the BTB is regulated by mechanisms involved in the junction turnover at the cell-matrix interface. This review critically discusses these latest findings in the field in light of their significance in the biology and regulation of the BTB pertinent to spermatogenesis.
Current Topics in Developmental Biology 02/2005; 71:263-96. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl(2). It was shown that the CdCl(2)-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-beta2 and TGF-beta3 (but not TGF-beta1) 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-beta2/-beta3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl(2)-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl(2) mediates its BTB disruptive effects via the TGF-beta3/p38 MAP kinase signaling pathway. Besides, this CdCl(2)-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 alpha(2)-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. alpha(2)-Macroglobulin was also shown to associate physically with TGF-beta3, 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-beta3/p-38 MAP kinase pathway also prevented the CdCl(2)-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on alpha(2)-macroglobulin. These results demonstrate for the first time that the TGF-beta3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-beta3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Recent studies using Sertoli cells cultured in vitro to permit tight junction (TJ) assembly have shown that TJ dynamics are regulated, at least in part, by TGF-beta3 via the p38 mitogen activated protein (MAP) kinase pathway. This in turn regulates the production of occludin, a TJ-integral membrane protein, by Sertoli cells. Yet it is not known if this pathways is used by Sertoli cells to regulate the blood-testis barrier (BTB) function in vivo. Using an in vivo model for studying BTB dynamics, we report herein the CdCl(2)-induced BTB damage in rats was associated with a significant reduction in testicular occludin along with a loss of immunoreactive occludin in the seminiferous epithelium at the site of the BTB. Also, this CdCl(2)-induced occludin loss from the BTB coincided with a surge in testicular TGF-beta3, as well as p-p38 MAP kinase (the phosphorylated/activated form of p38), but not p38 MAP kinase and neither extracellular signal-regulated kinase nor its phosphorylated form (ERK/p-ERK), consistent with results of in vitro studies. More important, intratesticular administration of SB202190, a specific p38 MAP kinase inhibitor, could block the CdCl(2)-induced occludin loss from the BTB. These results illustrate that BTB dynamics in vivo are regulated by the TGF-beta3/p38 MAP kinase pathway, which in turn determines the level of occludin at the site of Sertoli cells TJs.