Connexin 43 and plakophilin-2 as a protein complex that regulates blood–testis barrier dynamics

Abstract

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 β-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.

In 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 spermatocytes through the BTB while they differentiate into leptotene and zygotene spermatocytes. However, the immunological barrier must be maintained and remain intact (5). The mechanisms by which this timely BTB restructuring event is regulated have remained obscure for almost half a century (3, 6).

In other epithelia, the TJ barrier that confers the barrier function is supported structurally by discrete adherens junction (AJ) plaques located behind the TJ fibrils, followed by the desmosome, forming the junction complexes (7). Interestingly, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES, an atypical testis-specific AJ), and desmosome-like junction (1). We envisioned that these coexisting junctions must have a physiological significance at the BTB other than their structural role in contributing to the barrier integrity. The desmosome-like junction in the seminiferous epithelium was reported to possess the ultrastructural features of both the desmosome and gap junction (GJ) when examined by electron microscopy (8). There is accumulating evidence that desmosome can serve as a platform for signal transduction (9) and that connexin 43 (Cx43) can mediate cell–cell communication (10). We thus sought to examine the physiological significance of the desmosome-like junction at the BTB. We report herein a protein complex consisting of Cx43 and plakophilin-2 (PKP2) at the desmosome-like junction and describe how it regulates BTB dynamics through its effects on the TJ protein complex occludin/ZO-1.

Results

Stage-Specific Expression of Cx43 at the Blood–Testis Barrier in Rat Testes.

The specificity of the anti-Cx43 antibody is shown in Fig. 1A. A single band of ≈39 kDa was detected by immunoblot analysis using lysates of seminiferous tubules, and an additional phosphorylated isoform of ≈41 kDa was detected using lysates of Sertoli cells (see also supporting information (SI) Fig S1). This antibody was also used to localize Cx43 in the seminiferous epithelium of adult rat testes (Figs. 1 B and C and Table S1). The stage-specific expression of Cx43 is shown in Fig. 1B, compared with the control shown in Fig. 1D. In various stages of the seminiferous epithelial cycle, Cx43 was detected near the apical ES at the Sertoli cell–elongating spermatid interface and predominantly at the BTB (Fig. 1C). Its expression at the apical ES and the BTB was highest from stage V to early stage VIII, but it diminished greatly at both sites at late stage VIII (Fig. 1C). The decline in Cx43 at late stage VIII coincided with spermiation, which involves the disassembly of the apical ES to release the fully developed spermatids into the tubule lumen, and with the restructuring at the BTB that facilitates the transit of preleptotene spermatocytes. These findings implicate the likely involvement of Cx43 in cell adhesion at the apical ES and the BTB.

Fig. 1.

Stage-dependent expression of Cx43 in the seminiferous epithelium of adult rat testes. (A) Immunoblot analysis illustrates the specificity of the rabbit anti-Cx43 antibody using lysates of seminiferous tubules (ST) and Sertoli cells (SC). (B) Immunohistochemical localization of Cx43 in the seminiferous epithelium of adult rat testes. The expression of Cx43, which appears as reddish-brown precipitates, in the epithelium is stage dependent. (C) The localization of Cx43 in the epithelium at different stages of the epithelial cycle. Cx43 is detected at the BTB and the apical ES, but its expression is more predominant at the basal region. The expression of Cx43 at both the BTB and apical ES is highest from stage VI to early stage VIII; it diminishes drastically at both sites at late stage VIII. (D) In the negative control, rabbit IgG was substituted for the primary antibody. (Scale bar, 150 μm in B and D and 30 μm in C.)

The Decline in Cx43 During Adjudin-Induced Anchoring Junction Restructuring in the Seminiferous Epithelium.

To study the restructuring of anchoring junctions in the seminiferous epithelium, an established in vivo model was used. Adult rats were fed a single dose of adjudin (50 mg/kg body weight) to induce germ cell loss, most notably elongating spermatids, from the epithelium beginning at ≈1 day posttreatment (3). The BTB remained intact despite the disruption of adhesion between Sertoli and germ cells (3) when assessed by an in vivo integrity assay (11). A time-dependent decline in Cx43 in the testes was detected following the disruption of anchoring junctions induced by adjudin (Fig. 2 A and B). Results from a representative immunohistochemical experiment are shown in Fig. 2C. In a normal stage VII tubule, the staining of Cx43 was very strong near elongating spermatids at the apical ES (Fig. 2Cb). At the time of adjudin-induced depletion of elongating spermatids, Cx43 staining near these areas diminished (compare Fig. 2C b and h). At the BTB, the intensity of Cx43 staining diminished in some damaged tubules by 11 h and 1 day posttreatment (compare Fig. 2C d–i and a–c). Cx43 staining also was more diffuse and was less restricted to the BTB. When the drug was metabolized by 2 to 4 days (12) and more germ cells had left the epithelium, Cx43 staining considerably weakened in the seminiferous epithelium (Fig. 2C j and k). These findings further support the notion that Cx43 is involved in the germ cell adhesion in the seminiferous epithelium.

Fig. 2.

The expression and localization of Cx43 during adjudin-induced restructuring of anchoring junctions in adult rat testes. Adult rats were treated with adjudin, and testes were obtained for immunoblot analysis (using lysates) (A, B) and immunohistochemistry (frozen sections) (C). The steady-state protein level of Cx43 decreased after adjudin treatment (A, Upper Lane), with actin serving as the protein loading control (A, Lower Lane). (B) The bar graph shows the results of 3 experiments. *, P < 0.05; **, P < 0.01. (C) Changes in the localization of Cx43 in the seminiferous epithelium were studied in testes collected 11 h (H) and 1, 2, and 4 days (D) after adjudin treatment. The boxed areas in a, d, and g are magnified and shown in b, c, e, h, and i. At 11 h and 1 day after adjudin treatment, immunoreactive Cx43 at the basal region remained at the BTB but diminished gradually, and its expression at the apical ES also was considerably reduced compared with controls (C d–f, and g–i versus a– c). In the negative control (Cl), normal rabbit IgG replaced the primary antibody. (Scale bar, 200 μm in a, d, g, j, k, and l, 100 μm in f; 50 μm in b, c, e, h, and i.)

Cx43 as an Integral Component of the Blood–Testis Barrier.

Using dual-labeled immunofluorescent analysis, Cx43 was shown to co-localize with both ZO-1, a TJ adaptor (Fig. 3A a–d), and N-cadherin, a basal ES integral membrane protein (Fig. 3A e–h), at the BTB of the seminiferous epithelium. The structural interaction of Cx43 with some selected junction proteins was detected by co-immunoprecipitation. These proteins include N-cadherin and β-catenin from the basal ES, the GJ protein connexin 26 (Cx26), desmosomal protein PKP2, and c-Src, a non-receptor protein kinase that was shown to be a component of the BTB (Fig. 3B) (13). Even though Cx43 co-localized with ZO-1 and N-cadherin at the BTB (Fig. 3A), Cx43 was shown to interact structurally with the N-cadherin/β-catenin protein complex of the basal ES instead of with the TJ-based occludin/ZO-1 protein complex (Fig. 3B).

Fig. 3.

The co-localization and interaction of Cx43 with other known constituent proteins at the BTB. (A) Partial co-localization of Cx43 (FITC) with either ZO-1 (CY3) (A a–d) or N-cadherin (CY3) (A e–h) at the BTB was detected, showing that Cx43 is a component of the BTB. Nuclei were stained with DAPI. (Scale bar, 50 μm.) (B) Interactions of Cx43 with other junction proteins, including β-catenin, c-Src, ZO-1, occludin, and N-cadherin, which were reported to be present at the BTB, were examined by co-immunoprecipitation using lysates of seminiferous tubules from adult rats. β-Catenin, c-Src, N-cadherin, connexin 26, and PKP2, but not ZO-1, occludin, and laminin γ3, interact with Cx43. Rabbit IgG and goat IgG served as negative controls. These findings are representative data from 3 independent experiments.

A Knockdown of Cx43 by RNAi Did Not Affect Sertoli Cell Adhesion at the Blood–Testis Barrier.

The significance of Cx43 in Sertoli cell adhesion at the BTB was assessed by RNAi in primary Sertoli cell cultures having an established TJ permeability barrier that mimics the BTB in vivo. A knockdown of Cx43 by ≈50% in Sertoli cells using specific siRNA duplexes versus non-targeting control siRNA duplexes showed that a reduction of Cx43 did not affect the steady-state levels of various integral membrane or regulatory proteins at the BTB (Fig. 4A a and b). Dual-labeled immunofluorescence analysis revealed no observable changes in the distribution of TJ proteins occludin and ZO-1 at the Sertoli–Sertoli cell interface (see later sections). Using a biotinylation technique to assess changes in proteins at the cell surface following RNAi of Cx43 versus controls, we found that a knockdown of Cx43 did not affect the levels of 4 integral membrane proteins at the cell surface (Fig. 4B). These proteins were the TJ proteins occludin and JAM-A, the basal ES-protein N-cadherin, and the hemidesmosome protein β1-integrin. Furthermore, a knockdown of Cx43 failed to perturb the Sertoli cell TJ permeability barrier (see later discussion). Collectively, a knockdown of Cx43 alone, and thus the suppression of connexons comprising Cx43, had an insignificant effect on the BTB integrity. In short, Cx43 alone may not be required for the maintenance of the BTB between Sertoli cells.

Fig. 4.

A functional study to assess the role of Cx43 in BTB function by RNAi in primary Sertoli cell cultures. Sertoli cells cultured at 0.5 × 10⁶ cells/cm² on Matrigel-coated dishes were used on day 4 when the functional TJ permeability barrier was formed. Cells were transfected with 50 nM of either non-targeting control duplexes or specific Cx43 siRNA duplexes, using RiboJuice transfection reagent. Cells were washed 24 h later, and cultures were terminated after 48 h to obtain cell lysates. The steady-state protein levels of selected markers were investigated (A). Besides a knockdown of Cx43 by ≈50% (Ab), none of the proteins examined showed significant changes in their steady-state levels. The levels of selected integral membrane proteins at the cell surface were then examined to see if the knockdown of Cx43 would affect their levels (B). Immunoblots shown in (Aa) and (Ba) are representative results of 3 independent culture experiments (each in triplicate), which are summarized in (Ab) and (Bb). *, P < 0.01 versus control siRNA.

A Knockdown of Cx43 and PKP2 Perturbs TJ Permeability Barrier Function and Cell Adhesion in Sertoli Cells.

Besides existing by itself in the testis, the GJ is also a known integral component of the desmosome-like junction, which has the ultrastructural features of both the desmosome and GJ (14). Because Cx43 interacted with the desmosomal protein PKP2 (Fig. 3B), we sought to examine if a concurrent knockdown of Cx43 and PKP2 would affect the BTB function. A knockdown of Cx43 and PKP2, by ≈40% at the protein level and by ≈60% at the RNA level, respectively, was detected in primary Sertoli cell culture (Fig. 5A a and b and Fig. 5B a and b). This knockdown of Cx43 and PKP2 also caused a decline in the steady-state level of ZO-1 (Fig. 5A a and b) and a mild but significant decline in the levels of N-cadherin and Coxsackie virus and adenovirus receptor (CAR) at the Sertoli cell surface using the biotinylation technique (Fig. 5C a and b), suggesting a possible increase in endocytosis of N-cadherin and CAR. A surge in the protein level of the TJ protein claudin-11 was observed, but its level at the cell surface remained unchanged (Fig. 5A a and b and Fig. 5C a and b). The findings shown in Fig. 6C are consistent with results of Fig. 6A, wherein the Sertoli cell TJ permeability barrier was disrupted with simultaneous knockdown of both Cx43 and PKP2 but not when the expression of either Cx43 or PKP2 alone was suppressed (Fig. 6A). A redistribution of ZO-1 and occludin at the cell–cell interface was also observed following knockdown of both Cx43 and PKP2 but not with the knockdown of Cx43 alone (Fig. 6 B and C). However, knockdown of PKP2 alone was sufficient to perturb the distribution of ZO-1, but not occludin, at the Sertoli–Sertoli cell interface. These findings show that the desmosome-like junction is functionally constituted by the GJ protein Cx43 and desmosomal protein PKP2. Furthermore, this Cx43/PKP2 complex is linked functionally to the occludin/ZO-1 complex. A disruption of the Cx43/PKP2 can affect the integrity of the TJ permeability barrier via a mislocalization of occludin and ZO-1, perhaps mediated by enhanced endocytosis of occludin at the BTB.

Fig. 5.

A functional study to assess the role of the Cx43/PKP2 complex in the BTB by RNAi in primary Sertoli cell cultures. On day 4, when the Sertoli cell TJ barrier was established, cells were transfected with siRNA duplexes for 24 h, either non-targeting control or specific Cx43 and PKP2 siRNA pool, at a final concentration of 100 nM using RiboJuice siRNA transfection reagent. Cultures were terminated 48 or 72 h thereafter. Lysates were obtained for immunoblotting, and RNA was extracted for RT-PCR. (A) A knockdown of Cx43 by ≈40% on day 2 (2 D) and by ≈30% on day 3 (3 D) after transfection was detected. An increase in TJ integral membrane protein claudin-11 of ≈10% and a decline in the TJ adaptor protein ZO-1 of ≈10% were detected 3 days (3 D) after transfection. Other selected target proteins at the BTB remained unchanged after RNAi of Cx43 and PKP2. (B) On day 2 (2 D) after RNAi of Cx43 and PKP2, there was a decline in PKP2 at the RNA level of ≈60% when assessed by RT-PCR. (C) Cell surface proteins were biotinylated on day 2 (2 D) after transfection with the corresponding siRNA duplexes to assess the effect of RNAi of Cx43 and PKP2 on the steady-state level of integral membrane proteins versus control siRNA duplexes. Results shown here are representative results of 3 experiments. *, P < 0.05; **, P < 0.01.

Fig. 6.

A functional study to assess the role of Cx43 and PKP2 in BTB function by RNAi in primary Sertoli cell cultures. (A) The TJ barrier function was monitored by quantifying TER across the cell epithelium. Sertoli cells were transfected for 24 h with the corresponding siRNA duplexes at a final concentration of 80 nM. *, P < 0.05; **, P < 0.01. (B, C) Fluorescence staining of ZO-1 (CY3) and occludin (CY3) was performed 48 h after transfection to investigate the distribution of the target proteins after the knockdown of Cx43 and/or PKP2. In the transfection step of the experiment, 52.5 nM of control (Ctrl), Cx43, and/or PKP2 siRNA was co-transfected with 17.5 nM of siGLO Green (FITC), acting as a transfection indicator, at a final concentration of 70 nM. The green staining surrounding nuclei indicates positive transfection. After knockdown of both Cx43 and PKP2, there was less ZO-1 and occludin at the Sertoli–Sertoli cell interface than in controls (B a and b and C a and b vs. B g and h and C g and h, respectively). A decline in ZO-1 at the cell–cell interface was observed after the knockdown of PKP2 only, but not of Cx43 (B e and f and c and d vs. a and b). There was no change in the distribution of occludin in the single knockdown of Cx43 or PKP2 (C c– f vs. C a and b). Results shown are the representative data of 3 experiments. (Scale bars, 40 μm.)

Discussion

Cx43 Alone Does Not Confer Cell Adhesion in the Seminiferous Epithelium.

Cx43, a GJ protein, was shown to express stage specificity in the seminiferous epithelium of rat testes. Based on its localization in normal and adjudin-treated testes, it seems to be associated with the restructuring of junctions. At stage VIII of the epithelial cycle, fully developed spermatids leave the epithelium at spermiation. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes, and considerable loss of Cx43 at the apical ES and the BTB was detected immunohistochemically. Similarly, when elongating spermatids are induced to depart from the epithelium prematurely by adjudin in tubules other than those at stage VIII, the staining of Cx43 is reduced drastically at the apical ES. Collectively, these findings suggest that Cx43 is involved in conferring cell adhesion in the seminiferous epithelium. We further investigated the role of Cx43 in cell–cell adhesion using a loss-of-function approach by RNAi using primary Sertoli cells cultured in vitro with an established TJ permeability barrier that mimicked the BTB in vivo. The knockdown of only Cx43 in Sertoli cells did not cause any changes in the integrity of the TJ barrier or in the distribution and levels of junction proteins, such as occludin and ZO-1, at or near the cell surface. These results suggest that the loss of Cx43 function at the GJ and the desmosome-like junction does not affect the BTB integrity and show that Cx43 alone is not necessary for the maintenance of the BTB integrity. It is likely that other connexins (15) can supersede the transient loss of Cx43 at the BTB conferred by Sertoli cells.

Regulation of the Cell Adhesion by the Cx43/PKP2 Protein Complex at the Blood–Testis Barrier.

Because Cx43 was shown to interact structurally with the desmosomal protein PKP2, as demonstrated by co-immunoprecipitation, the functional role of this Cx43/PKP2 complex in the desmosome-like junction was examined. The simultaneous knockdown of both Cx43 and PKP2 by RNAi, but not the single knockdown of either Cx43 or PKP2, was shown to disrupt the Sertoli cell TJ permeability function. The knockdown of both Cx43 and PKP2 also was shown to induce a redistribution of the TJ proteins occludin and ZO-1 from TJ fibrils at the Sertoli cell surface. As compared with controls, a significant decline in the level of biotinylated cell surface proteins, including N-cadherin and CAR, was detected at the BTB following the knockdown of Cx43 and PKP2. These findings show that the Cx43/PKP2 protein complex is a putative functional GJ/desmosome protein complex that regulates Sertoli cell TJ barrier function through its effects on the occludin/ZO-1 protein complex at the BTB. This observation also is in agreement with an emerging concept that the desmosome can serve as the platform for signal transduction (9), so its disruption at the desmosome-like junction can perturb the TJ barrier function at the BTB.

A Possible Mechanism by Which the Cx43/PKP2 Complex Induces Blood–Testis Barrier Restructuring.

The desmosome is an intermediate filament-based anchoring junction, whereas the GJ is a communication junction (3, 7). Thus, the desmosome-like junction is expected to confer cell adhesion (3). In this study, a transient loss of the functional desmosome-like junction via a knockdown of Cx43 and PKP2 was shown to cause a disruption of the Sertoli cell TJ barrier partly via a redistribution of occludin and ZO-1 at the cell–cell interface. The ERK MAPK pathway, which becomes activated during various junction restructuring events (16), is not involved in this event. The non-receptor protein tyrosine kinase c-Src is likely to be involved.

c-Src interacts with Cx43, and it has been reported to be a component of the BTB in the rat testis (13). Studies from other epithelia have shown that occludins localized at TJ fibrils (17) are phosphorylated at both Ser/Thr and Tyr residues (18, 19). A minor pool of less phosphorylated occludins is not assembled into TJ fibrils and is restricted to the basolateral region (18, 20). The modulation of the phosphorylation status of occludins thus provides a unique mechanism to assemble and disassemble TJ fibrils in epithelia rapidly in response to changes in the cellular environment, such as during the transit of preleptotene spermatocytes at the BTB. The c-Src in the Cx43/PKP2/c-Src complex is in proximity to the TJ-based occludin and ZO-1, as demonstrated by their co-localization with Cx43. Following a transient knockdown of Cx43 and PKP2, c-Src may fail to maintain the phosphorylation status of occludin at the TJ fibrils at the BTB, leading to a decline in occludin at the Sertoli cell interface. This change in distribution of occludin, such as moving from the Sertoli–Sertoli cell interface to cell cytosol, thus perturbs the Sertoli cell TJ barrier.

Is the Desmosome-Like Junction Involved in the Preleptotene Spermatocyte Transit at the Blood–Testis Barrier?

It was postulated that junction proteins, such as CAR, residing on primary spermatocytes may interact with those on Sertoli cells to facilitate cell movement at the BTB while maintaining the immunological barrier during spermatogenesis (21). There is a unidirectional movement of primary spermatocytes from the basal to the apical compartment of the epithelium while differentiating into late-stage spermatocytes (3). Desmosome-like junctions, as well as those formed between Sertoli cells, also are formed between Sertoli and spermatocytes/round spermatids but not elongating and elongated spermatids (8). We propose herein that primary preleptotene spermatocytes in transit at the BTB probably use the desmosome-like junction to facilitate cell movement while maintaining the immunological barrier.

At stages VIII and IX of the epithelial cycle, the interaction of a desmosome-like junction protein on the Sertoli cell with one on a primary spermatocyte in transit, instead of with one on another Sertoli cell, may cause a localized reduction in the number of functional desmosome-like junctions between Sertoli cells. It has been reported that GJ communication from germ cells to Sertoli cells differs from that between Sertoli cells (15, 22). A change of Cx43–Cx43 association from 2 Sertoli cells to a Sertoli cell–spermatocyte may destabilize the occludin/ZO-1-based TJ fibrils at the BTB, possibly at the apical region of the spermatocyte, facilitating cell migration at the BTB. Other studies have shown that the formation of an N-cadherin-based AJ is a prerequisite for the assembly of GJ (23, 24) and desmosome (25). Because the knockdown of Cx43 and PKP2 led to a loss of the basal ES protein N-cadherin at the cell surface (Fig. 7), the disruption of basal ES might prohibit the assembly of a desmosome-like junction at the apical region of a spermatocyte in transit. There probably is a feedback loop that promotes the assembly of TJ, basal ES, and/or desmosome-like junction in the basal region of a spermatocyte in transit to assemble new TJ fibrils to maintain the immunological barrier. This feed-back loop might be mediated by paracrine factors produced locally at the BTB, which are known to regulate BTB dynamics (3, 11, 26).

Fig. 7.

A schematic drawing illustrating the regulation of BTB dynamics by desmosome-like junction. (A) A simplified overview of different junctions at the Sertoli–Sertoli interface at the BTB in normal adult rat testes. (B) Following the knockdown of Cx43 alone, there is a decline in the number of connexons constituted by Cx43, possibly in both the GJ and desmosome-like junction, but the cell adhesion conferred by TJ and AJ is unaffected, perhaps because connexons constituted by other GJ proteins maintain the GJ communication and the BTB integrity. As such, the BTB integrity still remains intact. (C) The knockdown of both Cx43 and PKP2 leads to a decline in the levels of TJ proteins occludin, CAR, and ZO-1 and basal ES protein N-cadherin through a yet-to-be defined signaling pathway(s). Nonetheless, this leads to BTB disruption. This mechanism, as depicted here, is likely utilized by the testis at stage VIII of the seminiferous epithelial cycle of spermatogenesis to induce BTB restructuring to facilitate the transit of primary preleptotene spermatocytes at the BTB. AJ refers to the basal ES at the BTB.

Materials and Methods

Animals.

Sprague-Dawley rats were obtained from Charles River Laboratory. The use of animals was approved by the Animal Use and Care Committee of the Rockefeller University (protocol number 06018).

Primary Sertoli Cell Culture.

Sertoli cells were isolated from testes of 20-day-old Sprague-Dawley rats and were cultured in F12/DMEM supplemented with growth factors and bacitracin (27). The functional TJ permeability barrier, which mimicked the BTB in vivo morphologically and functionally, was established on day 3 or 4 when assessed by transepithelial electrical resistance (TER) across the cell epithelium (27) and by electron microscopy (28). Ultrastructures of TJ, ES, and desmosome-like junction were observed. In this in vitro system, virtually no apical ES was present, because the cultured Sertoli cells were contaminated with negligible germ cells, including elongating and elongated spermatids.

Transient Transfection of siRNA Duplexes and TJ Permeability Barrier Assessment.

Sertoli cells were cultured on Matrigel (BD Biosciences)-coated culture plates at 0.5 × 10⁶ cells/cm², Millicell bicameral units at 1.0 × 10⁶ cells/cm², or coverglasses at 0.08–0.1 × 10⁶ cells/cm². Cells were transfected with siRNA duplexes (Dharmacon, Thermo Fisher Scientific), namely the ON-TARGET plus non-targeting siRNA control pool (D-001810–10), the siRNA pool specifically targeting Cx43 (J-100614–09, -10, -11) or PKP2 (J-087525–09 -10, -11, -12), and siGLO green transfection indicator (D-001630–01), at a final concentration of 70–100 nM using RiboJuice siRNA transfection reagent (Novagen, EMD Biosciences) on day 3 or 4 after an intact cell epithelium was established. In the group transfected with both Cx43 and PKP2 siRNA, equal amounts of each siRNA duplex pool were used so that the final concentration was equivalent to the non-targeting control siRNA duplex. For treatment groups transfected with either Cx43 or PKP2 siRNA alone, an equal amount of Ctrl siRNA was used together with Cx43 or PKP2 siRNA to obtain the same final siRNA concentration. Cells then were incubated in the transfection mixture for 24 h and replenished with fresh DMEM/F12 thereafter. In selected experiments where Sertoli cells were plated on bicameral units, the TJ barrier across the cell epithelium was monitored by TER as described (27).

Estimation of the Cell Surface Protein Levels Following RNAi by the Biotinylation Technique.

For Sertoli cell cultures transfected with non-targeting control siRNA, specific siRNA duplexes targeting Cx43, PKP2, or Cx43 and PKP2 were used to assess whether there were any changes in the level of junction proteins at the cell surface following RNAi. In short, proteins on the Sertoli cell surface following transfection were biotinylated using sulfo-NHS-SS-biotin (Pierce/Thermo-Fisher Scientific) at 4 °C for 30 min, and non-bound biotins were quenched with 50 mM ammonium chloride solution (26, 29). At this temperature, endocytosis and endosome-mediated protein degradation were inhibited. Thus, the level of cell surface proteins following RNAi in treatment versus control groups could be assessed quantitatively. Cell lysates were prepared in Nonidet P-40 lysis buffer (26). Biotinylated cell surface proteins were isolated from cell lysates using UltraLink Immobilized NeutrAvidin Protein Plus (Pierce/Thermo-Fisher Scientific) and were resolved in SDS/PAGE for immunoblot analysis using the corresponding specific antibodies.

Statistical Analyses.

GB-STAT statistical analysis software package (Version 7.0, Dynamic Microsystems) was used to perform Student's t test and 1-way ANOVA, followed by the Tukey/Kramer procedure.

General Methods.

Information on the lysate preparation, RT-PCR, immunoblot analysis, immunohistochemistry, and dual-labeled immunofluorescence analysis can be found in the SI Text.