Blood–testis barrier dynamics are regulated by an engagement/disengagement mechanism between tight and adherens junctions via peripheral adaptors

Abstract

In the mammalian testis, the blood–testis barrier (BTB), unlike the blood–brain and blood–retina barriers, is composed of coexisting tight junctions (TJs) and adherens junctions (AJs). Yet these junctions must open (or disassemble) to accommodate the migration of preleptotene and leptotene spermatocytes across the BTB during spermatogenesis while maintaining its integrity. In this report, we show that the BTB utilizes a unique “engagement” and “disengagement” mechanism to permit the disruption of AJ that facilitates germ cell movement without compromising the BTB integrity. For instance, both TJ (e.g., occludin and JAM-1) and AJ (e.g., N-cadherin) integral membrane proteins were colocalized to the same site at the BTB. Although these TJ- and AJ-integral membrane proteins did not physically interact with each other, they were structurally linked by means of peripheral adaptors (e.g., ZO-1 and α- and γ-catenins). As such, these proteins are structurally “engaged” under physiological conditions to reinforce the BTB. When rats were exposed to Adjudin to induce AJ restructuring that eventually led to germ cell loss from the epithelium, this structural interaction between occludin and N-cadherin by means of their adaptors became “disengaged” while their protein levels were significantly induced. In short, when the epithelium is under assault, such as by Adjudin or plausibly at the time of germ cell migration across the BTB during spermatogenesis, the TJ- and AJ-integral membrane proteins can be disengaged. Thus, this mechanism is used by the testis to facilitate AJ restructuring to accommodate germ cell migration while maintaining the BTB integrity.

During spermatogenesis, preleptotene and leptotene spermatocytes that are residing outside the blood–testis barrier (BTB) in the basal compartment of the seminiferous epithelium traverse the BTB at stages VIII–IX of the epithelial cycle in mammalian testes, such as those of the rat (1). As such, extensive restructuring of tight junctions (TJs), adherens junctions (AJs), and desmosome-like junctions must take place in the epithelium to facilitate germ cell movement (for a review, see ref. 2). Interestingly, unlike barriers in other mammalian organs, such as the blood–brain, blood–retina, and blood–epididymal barriers, where TJ is restricted to the apical portion of the cell epithelium and/or endothelium, to be followed by AJ (for reviews, see refs. 3–6), the BTB is composed of coexisting TJs, AJs, and desmosome-like junctions (for reviews, see refs. 7–9). One of the main roles of the TJ barrier is to prevent small molecules from passing through the paracellular space. The endothelial TJ barrier in blood vessels, for example, is only opened occasionally to allow the passage of neutrophils and macrophages during inflammation (for a review, see ref. 10). However, in the testis, the BTB is a highly dynamic structure because it must open (or disassemble) periodically to accommodate the migration of germ cells across the barrier. Yet its integrity must not be compromised, so that the microenvironment behind the barrier can be maintained and the postmeiotic germ cell antigens can be sequestered from the immune system. It is therefore logical to speculate that the BTB must be intriguingly regulated, which likely involves a complicated network of signaling cascades and rapid turnover of junction molecules. In recent years, cytokines (e.g., TGF-β3 and TNF-α), nitric oxide, kinases, phosphatases, proteases, and protease inhibitors have been shown to be crucial regulators of BTB dynamics (11–13). Nonetheless, these findings fail to explain the necessity of the coexisting TJs and AJs at the BTB that maintains its integrity during spermatogenesis. In two recent studies, it was shown that AJ disruption in the seminiferous epithelium induced by a suppression of intratesticular androgen (14) or by means of treatment of rats with Adjudin (12) did not disrupt the TJ barrier, unlike all other epithelia, where a disruption of AJ can lead to TJ barrier damage (15, 16). These findings indicate that a mechanism is in place at the BTB that permits AJ restructuring while maintaining TJ integrity. In this report, we present compelling evidence in that the coexistence of TJ and AJ proteins at the BTB in normal conditions is structurally linked by means of peripheral adaptors (“engaged”). However, at the time of BTB restructuring that facilitates germ cell migration, the peripheral adaptors of the TJ- and AJ-integral membrane proteins become “disengaged” by “opening” the BTB. Because these proteins are significantly induced while disengaged, the coexisting AJs at the BTB can supersede the function of TJs transiently, and vice versa, to maintain the barrier integrity.

Materials and Methods

Animals. Male Sprague–Dawley rats (≈280–320 g of body weight) were from Charles River Laboratories. The use of animals was approved by the Animal Care and Use Committee at The Rockefeller University (New York) (protocols 00111 and 03017).

In Vivo Induction of Germ Cell Loss from the Seminiferous Epithelium by Adjudin [AF-2364, 1-(2,4-Dichlorobenzyl)-IH-indazole-3-carbohydrazide]. Adult rats (n = 4 rats per time point; ≈300 g of body weight) were fed with a single dose of Adjudin at 50 mg/kg of body weight (17, 18). Control rats received vehicle only [0.5% (wt/vol) methylcellulose in water]. Rats were killed on d 4 and 7 by CO₂ asphyxiation. Testes and liver were removed and frozen in liquid nitrogen.

Immunofluorescent Microscopy. Immunofluorescent microscopy was performed as described in ref. 19 by using different primary antibodies at different dilutions, which were obtained from different vendors as described in refs. 14, 19, and 20. Negative controls were included, using the corresponding IgGs. Secondary antibodies conjugated with FITC or Cy3 were obtained from Zymed.

Preparation of Samples and Immunoblotting Analysis. Testis and liver lysates used for immunoblotting and coimmunoprecipitation (CoIP) were prepared in immunoprecipitation buffer [50 mM Tris·HCl/150 mM NaCl/1% (vol/vol) Nonidet P-40/1 mM EGTA/1 mM PMSF/1 mM sodium orthovandate/1 μg/ml leupeptin/1 μg/ml aprotinin, pH 7.4] as described in ref. 20. Protein concentration was determined by Coomassie blue dye-binding assay (21). About 100 μg of proteins was used for immunoblot analysis (19, 22–24).

CoIP. About 400 μg of proteins from each sample within an experimental group was used. Testis and liver lysates were first precleared with 2 μg of rabbit or mouse IgG (Sigma-Aldrich) for 1 h, depending on the source of primary antibodies (see refs. 14, 19, 20, and 22–25), followed by 1 h of incubation with 10 μl of Protein A/G Plus-agarose (Santa Cruz Biotechnology) to eliminate non-specific interactions between proteins in lysates and IgG or agarose beads. To this supernatant, 2 μg of primary antibodies was added and incubated at room temperature overnight to precipitate the target protein and its associated binding partners. Negative control was included in which the primary antibody was substituted by 2 μg of IgG (rabbit or mouse). The immunocomplexes were subsequently precipitated by incubating with 20 μl of Protein A/G Plus-agarose for ≈6 h at room temperature. Thereafter, the immunocomplexes were washed four times with a washing buffer [50 mM Tris·HCl/150 mM NaCl/1% (vol/vol) Nonidet P-40/1 mM EGTA/1 mM PMSF, pH 7.4]. Proteins in the immunocomplexes were then extracted in SDS sample buffer [0.125 M Tris/1% (wt/vol) SDS/1.6% (vol/vol) β-mercaptoethanol/20% (vol/vol) glycerol, pH 6.8] at 100°C for 10 min. Proteins were resolved by SDS/PAGE and subjected to immunoblottings. Each blot can be used up to four to eight times with different primary and secondary antibody pairs after stripping as described in ref. 19 without detectable loss of proteins.

Actin Assay, EM, and Statistical Analysis. The relative ratio of the filamentous (F) vs. free globular (G) actin content in samples was quantified by using assay kits obtained from Cytoskeleton (Denver) (catalog no. BK037). EM was performed as described in ref. 19. Statistical analyses were performed by ANOVA.

Results

TJs, AJs, and Their Associated Proteins Are Coexisting at the BTB in the Seminiferous Epithelium. The ultrastructural features of the BTB in normal rat testes are shown in Fig. 1A. The BTB is composed of coexisting TJ and cell–cell actin-based basal ectoplasmic specialization (ES) (see boxed area). The boxed area in Aa was magnified in Ab. Basal ES, typified by the presence of actin filament bundles (white arrow) sandwiched between the Sertoli cell membrane (apposing black arrowheads illustrate the two Sertoli cell plasma membranes) and the cisternae of the endoplasmic reticulum (er) (Fig. 1 Ab), is present side by side with TJ (white arrowhead) to constitute the BTB (Fig. 1 A) (2, 6). Using immunofluorescent microscopy, N-cadherin, an AJ-integral membrane protein, appeared as red fluorescence (Fig. 1Ba and e) and was found to colocalize with two TJ-integral membrane proteins, occludin (Fig. 1Bb) and JAM-1 (Fig. 1Bf) (Fig. 1B c and g vs. a and b and e and f) at the BTB. Their corresponding adaptors, γ-catenin (an AJ adaptor) and ZO-1 (a TJ adaptor), were also colocalized to the same site at the BTB (Fig. 1B i–l). Consistent with earlier reports (25, 26), the N-cadherin/γ-catenin protein complex was also detected outside the basal ES, such as at the apical ES in selected stages of the epithelial cycle (e.g., stages V–VI) (see white arrowheads in Fig. 1Ba and i). TJ proteins (e.g., occludin and JAM-1) were confined to the basal compartment at the BTB.

Fig. 1.

A study to assess the relationship between TJ and AJ protein complexes at the BTB in the rat testis. (A) Electron micrographs of normal rat testes. (a) Cross section of a seminiferous tubule from an adult rat testis. The basement membrane (asterisks) and the collagen fibrils (arrowheads) are shown. GC, spermatogonium; SC, Sertoli cell; N, SC nucleus. (b) The boxed area in a (the BTB) is shown magnified, typified by the coexisting TJ and basal ES. Basal ES refers to the ultrastructure between adjacent Sertoli cells typified by the presence of actin bundles sandwiched between the endoplasmic reticulum (er) and the Sertoli cell plasma membranes that are found on both sides of the Sertoli cells. Two Sertoli cell membranes are indicated by the apposing arrowheads. (Scale bars: a, 2.5 μm; b, 0.6 μm.) (B) Representative immunofluorescent micrographs that illustrate the colocalization of AJ and TJ proteins at the BTB in the seminiferous epithelium. (a, e, and i) AJ proteins (N-cadherin and γ-catenin) appeared as red fluorescence (Cy-3). (b, f, and j) TJ proteins (occludin, JAM-1, and ZO-1) appeared as green fluorescence (FITC). (c, g, and k) Merged images of the corresponding immunofluorescent micrographs, in which AJ and TJ proteins were colocalized to the same site at BTB as orange fluorescence. (d, h, and l) The DAPI staining of the corresponding fluorescent micrographs. White arrowheads indicate the localization of N-cadherin and γ-catenin in the epithelium, consistent with their presence at the apical ES. (Scale bar in a, which applies to b–l:80 μm.)

TJ- and AJ-Integral Membrane Proteins Are Structurally Associated by Means of Peripheral Adaptors at the BTB. The study by fluorescent microscopy (see Fig. 1) has illustrated that both AJ and TJ proteins are present at the BTB. As such, it is of interest to investigate whether they have any physical interaction. Using CoIP, it was shown that N-cadherin associated with the adaptors α-, β-, and γ-catenin, p130 Cas, and ponsin but not TJ-integral membrane proteins (e.g., occludin and JAM-1) (Fig. 2 A Left and B). For occludin, it was found to associate with ZO-1 but not JAM-1 or AJ-associated proteins such as N-cadherin, catenins, nectin, and afadin (Fig. 2 A Right and B). This study thus illustrates that AJs and TJs are colocalized to the BTB but that there is no structural interaction between their integral membrane proteins.

Fig. 2.

A study by CoIP to assess the structural interactions between TJ and AJ proteins at the BTB in the rat testis. (A) Protein (400 μg) of normal testis lysates was subjected to CoIP with either an anti-N-cadherin or an anti-occludin antibody. Immunocomplexes were resolved by SDS/PAGE, and the blots were probed with antibodies against different target proteins. Rabbit IgG replaced the primary antibody to serve as negative control. (B) A summary of the CoIP results from three experiments. +, interaction; –, no interaction.

Effects of AJ Restructuring Induced by Adjudin on the Localization Patterns of TJ- and AJ-Protein Complexes at the BTB During Germ Cell Depletion from the Seminiferous Epithelium. To explore the structural relationship between TJ- and AJ-protein complexes at the BTB during extensive restructuring pertinent to spermatogenesis, an in vivo model was used in which germ cells were induced to deplete from the epithelium by treating adult rats with a single dose of Adjudin, because an earlier study had shown that this treatment would cause AJ disruption without compromising the BTB integrity (12). Fig. 3Aa illustrates the cross section of a normal rat testis, showing tubules at different stages of the epithelial cycle. However, after treatment with Adjudin, germ cells, elongating/elongate spermatids in particular, began to deplete from the epithelium, whereas some spermatocytes and round spermatids were found in the tubule lumen by d 4 (Fig. 3Ab). Almost all elongate spermatids were depleted from the epithelium by d 7 (Fig. 3Ac). Because there were extensive junctions restructuring at this time due to germ cell loss, we had examined changes in selected integral membrane proteins and their peripheral adaptors at the BTB. In normal rat testes, N-cadherin and occludin were colocalized at the BTB (Fig. 3B a–d). However, when AJ was perturbed after the Adjudin treatment, there was a surge in the levels of both N-cadherin and occludin. Interestingly, these proteins appeared to diffuse away from the BTB, as illustrated by the increasing width of the white brackets (see Fig. 3B a, e, and i), which showed the thickening of N-cadherin and occludin proteins from their origin at the BTB (Fig. 3Be–l). White arrowheads illustrate germ cells in the lumen, which were being depleted from the epithelium (Fig. 3B h and l). Their adaptors, γ-catenin and ZO-1, displayed a similar pattern of changes during Adjudin-induced germ cell loss (Fig. 3 C vs. B, D, and E). These data illustrate that TJ and AJ proteins are moving away from the BTB site during germ cell depletion.

Fig. 3.

A study using an in vivo model of AJ disruption to assess changes in the localization of TJ and AJ proteins in the epithelium during Adjudin-induced germ cell loss. (A) These micrographs are cross sections of adult rat testes that illustrate the effects of Adjudin to the epithelium. (a) Normal rat testes showing tubules at different stages of the epithelial cycle. (b and c) Testes from rats treated with a single dose of Adjudin on d 0 and killed on d 4 and 7, respectively. (B and C) These fluorescent micrographs illustrate that AJ- and TJ-integral membrane proteins (B) and their corresponding adaptors (C) were diffusing away from the BTB site (dashed white line) during Adjudin-induced germ cell loss as manifested by the appearance of fluorescence that moved away from the dashed white line that marked the relative location of the BTB. (a–l) Fluorescent micrographs of normal rat testes (a–d), testes from rats treated with Adjudin for 4 (e–h) and 7 (i–l) d. N-cadherin and ZO-1 appeared as green fluorescence (FITC), whereas occludin and γ-catenin appeared as red fluorescence (Cy-3). (c, g, and k) Merged images of the corresponding micrographs. (d, h, and l) DAPI staining of the corresponding sections. The white brackets shown in a, e, and i illustrate the relative distance of target protein that was diffused away from the BTB, which is marked by the dashed white line. (D and E) Bar charts represent the relative distance of TJ and AJ proteins that diffused away from the BTB. Data are estimated by measuring the mean distance of the fluorescence that moved away from the BTB from three randomly selected points on a micrograph. Both integral membrane proteins and adaptors resided closely to the BTB, as shown in control testes (a and b). These proteins disengaged by diffusing away from the BTB during Adjudin-induced AJ restructuring (e and f and i and j). The distance of a target protein from the BTB in control testes was arbitrarily set at 1, against which treatment groups were compared. Each data point is the mean ± SD of 60 randomly selected tubules from two rats. *, significantly different by ANOVA; P < 0.05; **, P < 0.01.

α-Catenin, γ-Catenin, and ZO-1 Are Linkers Between TJ- and AJ-Integral Membrane Proteins at the BTB, and They Can Be Disengaged During AJ Restructuring. The above observations imply that there is some sort of coordination between TJ and AJ proteins during extensive AJ restructuring associated with germ cell loss after Adjudin treatment. Because TJ- and AJ-integral membrane proteins were found to have no direct physical association (see Fig. 2), we sought to examine whether these proteins had any interactions by means of their peripheral adaptors by CoIP (Fig. 4). Consistent with results shown in Fig. 3 B–E, the steady-state protein levels of α- and γ-catenin and ZO-1 as well as their corresponding integral membrane proteins were indeed significantly induced during Adjudin-mediated germ cell loss from the epithelium when the levels of these proteins were quantified by immunoblotting (Fig. 4A Left). The same trend was also observed in CoIP experiments when a target protein (e.g., γ-catenin) was visualized (Fig. 4 A–C Right). Furthermore, the use of an anti-γ-catenin antibody could immunoprecipitate AJ- but not TJ-integral membrane proteins, such as occludin and JAM-1, from lysates even though there was an induction in these protein levels (see Figs. 3 and 4A). Perhaps most important of all, it was shown that adaptors of AJ-integral membrane proteins (e.g., γ- and α-catenin) were associated with those of TJ (e.g., ZO-1) in normal testes (Fig. 4 A and B), yet this association was significantly weakened during extensive AJ restructuring. Fig. 4D illustrates the relative change in adaptor–adaptor associations at the BTB site during Adjudin treatment. As much as 6-fold reduction in association between γ-catenin and ZO-1 was detected when spermatids were depleting from the epithelium by d 4 (Fig. 4D vs. Fig. 3A). Interestingly, this weakened association was capable of being “reestablished” when germ cell depletion had almost completed at d 7 (Fig. 4 A and D). Also, a significant reduction between α-catenin and ZO-1 association was also detected at d 4 (Fig. 4 B and E). These results were validated when the CoIP was performed by using an anti-ZO-1 as the precipitating antibody (Fig. 4 C and F). However, the association between α-catenin and ZO-1 in liver in the same animals subjected to the Adjudin treatment remained unchanged (Fig. 4 B and C and E and F), demonstrating that a site-specific disengagement between TJ and AJ at the BTB had occurred when AJ was undergoing extensive restructuring. These data thus imply that in normal testis, these TJ- and AJ-integral membrane proteins were engaged by means of their peripheral adaptors, plausibly to reinforce the BTB integrity. Yet they became disengaged during extensive AJ restructuring so that the TJ barrier integrity could be maintained while facilitating germ cell movement during spermatogenesis.

Fig. 4.

A study to assess the engagement and disengagement between TJ- and AJ-integral membrane proteins and their adaptors at the BTB during Adjudin-induced germ cell loss. (A) Lysates from normal testes and testes from rats treated with Adjudin for 4 and 7 d. Approximately 100 μg of protein from each sample within an experiment group was resolved by SDS/PAGE, and the blot was probed with anti-actin antibody (lowermost blot in Left) to illustrate equal protein loading before CoIP. The same blot was also stripped and reprobed with antibodies against different target proteins (Left). For CoIP, ≈400 μg of protein from each sample was immunoprecipitated by using a mouse anti-γ-catenin antibody. Thereafter, immunocomplexes were resolved by SDS/PAGE, and the blot was probed with an anti-γ-catenin antibody, followed by other antibodies. An increase in γ-catenin level was found during Adjudin-induced germ cell loss in both the immunoblotting (IB) and CoIP experiment. γ-Catenin was found to associate with ZO-1 at the BTB in normal rat testes (engaged); however, these two adaptors became disengaged when germ cells were depleting from the epithelium after Adjudin treatment by d 4, and the γ-catenin–ZO-1 association restored somewhat when virtually all spermatids were devoid from the epithelium by d 7. No direct association was found between γ-catenin and TJ-integral membrane proteins, such as occludin and JAM-1. (B) CoIP was also performed by using an anti-α-catenin antibody. The levels of α-catenin and γ-catenin were induced during germ cell depletion from the epithelium (Left). However, there was a significant loss in α-catenin and ZO-1 association during Adjudin-induced germ cell loss. (C) A reverse CoIP was also performed by using an anti-ZO-1 antibody to pull down ZO-1 and α-catenin. During Adjudin-induced germ cell loss, there was a significant increase in ZO-1 (Left), which was pulled down by the anti-ZO-1 antibody (Right). Consistent with data shown in A and B, there was a drastic loss of ZO-1 and α-catenin association during germ cell loss. However, the association of ZO-1 and α-catenin in the liver of treated rats was unaffected. (D–F) Corresponding histograms of A–C, respectively. Each bar represents the mean ± SD of six experiments. The association between two adaptors in normal rat testes (control) was arbitrarily set at 1. ns, not significantly different by ANOVA; *, P < 0.05; **, P < 0.01.

An Increase in F-actin:G-actin Ratio in the Seminiferous Epithelium During Disengagement Between Adaptors as a Result of Adjudin-Induced AJ Restructuring. We next examined the relative ratio of F-actin to G-actin in the testis at the time of germ cell depletion from the epithelium by using a specific actin assay. In normal testes, almost an equal amount of F- and G-actin was found (Fig. 5). However, when germ cells were induced to deplete from the epithelium and AJ proteins dissociated from TJ proteins at the BTB site by d 4, there was an increase in the F-actin:G-actin ratio, seemingly suggesting that more F-actin proteins were present in the epithelium to reinforce the junction integrity. The F-actin protein level dropped after d 7, and the F-actin:G-actin ratio returned to 1:1, similar to the ratio found in normal testes when most of the germ cells were already depleted from the epithelium by d 15.

Fig. 5.

Changes in F- and G-actin ratio in the testis during Adjudin-induced germ cell loss from the testis. Each bar represents mean ± SD of three experiments. ns, not significantly different by ANOVA; *, P < 0.05; **, P < 0.01.

Discussion

Junctions Restructuring at the BTB Are Regulated by an “Engagement and Disengagement” Mechanism. Cytokines (e.g., TGF-β3) can reversibly disrupt BTB and Sertoli–germ cell adhesion in the epithelium via the p38 mitogen-activated protein kinase signaling pathway by reducing the levels of TJ proteins (e.g., occludin) and adaptors (e.g., ZO-1) at the BTB (19, 22, 24) (Fig. 6). Interestingly, TGF-β3 can also limit its disruptive effects exclusively on the Sertoli–germ cell adhesion without compromising the BTB when the downstream Ras/extracellular signal-regulated kinase signaling pathway is activated (27). Based on studies in other epithelia, this selective “shifting” of TGF-β3 signaling function is regulated by adaptors, such as CD2AP (11). Nonetheless, these findings cannot fully explain the need for the coexistence of TJs and AJs at the BTB. Besides, the mechanism that the BTB employs to maintain its integrity during extensive anchoring junction restructuring pertinent to spermatogenesis remains unknown. In this report, an in vivo model using Adjudin was used to induce AJ restructuring in the testis without damaging the BTB (12). It is unlikely that the changes reported here are the result of a drug toxicity effect of Adjudin toward junctions, because many of the biochemical changes in the seminiferous epithelium, such as an induction of protein and lipid kinases, phosphatases, adaptors, and AJ-integral membrane proteins, that are associated with the event of germ cell depletion from the epithelium have been reproduced and validated (14, 23, 28) by using an intratesticular testosterone suppression model (29, 30). Furthermore, a more recent study has illustrated that an AJ restructuring event in the testis can indeed be limited to the apical ES without compromising the BTB (14), consistent with data obtained from the Adjudin model (12). From the data reported here, the protein levels of both TJ- and AJ-integral membrane proteins (e.g., occludin and N-cadherin) as well as their peripheral adaptors (e.g., ZO-1 and catenins) were induced when germ cells were depleting from the epithelium. Yet these increases in the TJ and AJ proteins were shown to associate with a temporary disengagement between their adaptors (namely, ZO-1 and α-/γ-catenin) as detected by CoIP. This observation is also consistent with fluorescent microscopy data that have illustrated that proteins were moving away from the BTB site. It is plausible that the disengagement of TJs and AJs at the BTB by means of the dissociating peripheral adaptors can facilitate the passage of preleptotene and leptotene spermatocytes across the TJ barrier while the “induced” AJ proteins can temporarily supersede the TJ barrier function to maintain the BTB integrity. It is equally plausible that the surge in the TJ protein levels, while disengaged from AJ, was also used to reinforce the TJ barrier at the BTB. Thus, these findings illustrate the physiological significance and the necessity for the coexistence of TJs and AJs at the BTB. In short, the results reported here have illustrated that TJ (e.g., occludin) and AJ (e.g., N-cadherin) proteins at the BTB are engaged by means of ZO-1 and catenins under normal physiological conditions but that they can become disengaged during AJ restructuring to facilitate germ cell movement such that the TJ barrier integrity can be maintained. Because the engagement between TJs and AJs by means of their downstream adaptors, ZO-1 and catenins, apparently is not restricted to the BTB, we sought to investigate whether disengagement can also occur in other barriers. From the results of CoIP, the association between ZO-1 and α-catenin was not perturbed in liver lysates of the treated animals.

Fig. 6.

The engagement and disengagement mechanism that regulates BTB dynamics. Under normal physiological conditions (Left), TJ protein complexes and AJ protein complexes are coexisting side by side at the BTB. They are structurally associated with each other by means of adaptors to reinforce the BTB integrity in an engagement state. However, this engagement between TJ and AJ proteins is abolished when germ cells are depleting from the testis (e.g., in Adjudin treatment, Center) or during germ cell movement across the BTB at spermatogenesis, in which catenins are disengaged from ZO-1 even though the steadystate TJ and AJ protein levels are induced. As such, the induced TJ proteins can supersede the role of the dissociated AJ proteins to maintain BTB integrity. Even though AJ proteins (e.g., cadherin and catenins) are induced in parallel with the TJ proteins (e.g., occludin and ZO-1), the Sertoli–germ cell adhesion function is perturbed because there is a loss of association between N-cadherin and γ-catenin. This unique engagement and disengagement mechanism, unlike other epithelia, can prevent unnecessary damage to TJ at the BTB while permitting AJ restructuring. When this Sertoli–germ cell restructuring begins to subside by d 7 after the Adjudin treatment (Right), at the time when most germ cells are depleted from the epithelium, TJ- and AJ-integral membrane proteins reestablish their interactions with the peripheral adaptors. This mechanism is likely working in concert with cytokines (e.g., TGF-β3 and TNFα, which are products of Sertoli and germ cells), which was shown to induce the loss of TJ proteins (e.g., occludin) from the BTB or Sertoli cell TJ barrier (19, 24, 41) to facilitate germ cell movement across the BTB.

Issues on Changes in the Localization Patterns and Levels of Target Proteins in the Seminiferous Epithelium During Germ Cell Loss. One can argue that the altered localization of TJ and AJ proteins during Adjudin-induced germ cell loss as reported here is the result of widening of interstitial space between adjacent tubules that have extensive germ cell loss and/or accumulation of interstitial fluid in the interstitium. These possibilities are supported in part by the histological findings consistent with several earlier reports (17, 25, 31). Furthermore, this significant loss of germ cells from the epithelium can increase the relative contribution of proteins by Sertoli cells and/or other somatic cells in the samples being analyzed by immunoblottings that can artificially account for protein up-regulation. We offer the following explanations to address these interesting issues. First, N-cadherin and occludin are Sertoli cell integral membrane proteins restricted mostly to the BTB site (2), rather than secretory products. Even though the tubule diameter shrinks by as much as 25–30% by d 4 and 7 after Adjudin treatment (31) with a concomitant enlargement of the interstitial space (Fig. 3 Bi–l and Ci–l), they are still restricted to the BTB near the basement membrane and are “diffusing” toward the tubule lumen (Fig. 3B), making it unlikely that those diffusing proteins are integral membrane proteins suspended in the interstitial fluid. Also, studies by EM after Adjudin treatment have illustrated the integrity of Sertoli cells near the basement membrane (25), and cellular hypertrophy was not detected (25, 32). Perhaps the most important proof that supports such protein disengagement comes from the biochemical studies using CoIP. Equally important, recent studies using an androgen suppression model by means of steroid implants to perturb apical ES adhesion have also demonstrated a loss of protein–protein interactions of the apical ES protein complexes (e.g., the cadherin–catenin complex) (14, 28). This finding further strengthens the notion of this disengagement mechanism. Second, many of the proteins that were included in the studies reported here, such as cadherins (33, 34), catenins (33), and JAMs (27, 35), are also products of germ cells including spermatids. As such, an induction of any of these proteins during germ cell loss as detected by immunoblotting is likely an “underestimate” instead of an artifact of “overestimate” for up-regulation, because the testis lysates that were being analyzed had been contributed by Sertoli and other somatic cells (e.g., Leydig and myoid cells). Furthermore, many of these germ cells were likely included in our analysis because these cells would possibly still be in the tubule lumen in particular by d 4 before emptying into the epididymis because lysates of whole testes were used for immunoblot analysis. The results of earlier studies using inhibitors provide another argument against this possibility. For instance, it was shown that the use of Y-27632 (a specific ROCK inhibitor) (36) or 2R-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid (a specific matrix metalloproteinase type 2 inhibitor) (31) could indeed delay Adjudin-induced germ cell loss and the associated up-regulation of several target proteins.

Summary and Concluding Remarks. As reported here, the transient disengagement between TJ- and AJ-associated adaptors, together with a surge of their protein levels and the focal and reversible TJ barrier disruption induced by cytokines (e.g., a loss of occludins induced by TGF-β3) (19, 22, 24), can facilitate the migration of preleptotene/leptotene spermatocytes across the barrier without compromising the barrier integrity (see Fig. 6). However, the mechanism(s) by which the cytokine-induced TJ fibril disassembly that coordinates with TJ and AJ disengagement to facilitate germ cell migration across the BTB remains to be investigated. Nonetheless, this unique junction architecture of having coexisting TJs and AJs also poses a unique problem to the testis. For instance, the testis is extremely sensitive to environmental toxicants (e.g., cadmium) (37–39). This unusual vulnerability is likely the result of the unique junction layouts in the seminiferous epithelium. For example, E-cadherin is the primary target of cadmium (40). Yet in all epithelia present in many organs, such as the small intestine, TJ is located at the apical portion of the cell epithelium, followed by AJ. As such, TJ seals off environmental toxicants from reaching AJ to induce damage there by means of its effects on E-cadherin, which is an AJ component. However, in the testis, both AJ and TJ are adjacent to the extracellular matrix, closest to the interstitium where environmental toxicants can have immediate access to the AJ (e.g., E-cadherin) that present side by side with TJ; they become the molecular target of toxicants (e.g., cadmium).