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Population Council, New York, New York 10021
Address all correspondence and requests for reprints to: C. Yan Cheng, Ph.D., Population Council, Center for Biomedical Research, 1230 York Avenue, New York, New York 10021. E-mail: Y-Cheng{at}popcbr.rockefeller.edu
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Abstract |
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-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-ß and TNF-, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway. |
Introduction |
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Tight junctions (TJs) in the testis are constituted by several TJ-integral membrane proteins, such as occludins, claudins, and junctional adhesion molecules (JAMs), and peripheral proteins, such as zonula occludens (ZO)-1, ZO-2, ZO-3, cingulin, and AF-6 (for a review, Ref. 11). In the testis, TJs between adjacent Sertoli cells at the basal compartment of the seminiferous epithelium create the blood-testis barrier (BTB) (for a review, see Ref. 11), which provides a unique microenvironment for germ cell development by sequestering proteins in the systemic circulation residing in the interstitium from entering the seminiferous epithelium (for review, see Ref. 11). As such, the precise nature of TJ regulation in the testis not only is important to the study of spermatogenesis, a thorough understanding on the biology of TJ dynamics and their regulation will yield new insights in developing novel approaches for male contraception because a disruption of TJ dynamics per se will affect spermatogenesis such as migration of preleptotene and leptotene spermatocytes across the BTB from the basal to adluminal compartment for further development. Recent studies have shown that Sertoli cell TJ dynamics are regulated by an array of molecules, which include proteases, protease inhibitors, cytokines, cAMP, protein phosphorylation and/or dephosphorylation, and intracellular Ca2+ level (for a review, see Ref. 11). Because NO has recently been implicated in the regulation of the junction permeability in the blood-retina TJ barrier and TJ barrier of blood vessels (for a review, see Ref. 13), it is of interest to investigate whether NO plays a role in TJ dynamics in the testis.
Two of the immediate downstream signaling molecules of NO are adenylate cyclase (AC) and soluble guanylate cyclase (sGC), which generate cAMP and cGMP, respectively. These two molecules in turn activate cAMP-dependent protein kinase (protein kinase A, PKA) and cGMP-dependent protein kinase (protein kinase G, PKG) (for reviews, see Refs. 12 and 14). Whereas the precise nature of the NO downstream regulatory pathways via cAMP, PKA, cGMP, and PKG that affect junction dynamics is virtually unknown, emerging evidence from studies of other epithelia have clearly implicated these molecules in TJ dynamics (13, 15). For instance, it is known that cAMP at 4–20 µM or 100–500 µM can facilitate or perturb the Sertoli cell TJ barrier in vitro, suggesting the regulatory effects of cAMP on Sertoli cell TJ dynamics are concentration dependent (16). Herein, we report our findings using primary Sertoli cells cultured in vitro as a model to elucidate the roles of iNOS and eNOS in the TJ assembly in vitro. Collectively, these results illustrate the biological significance of NOS in TJ regulation in the testis.
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Materials and Methods |
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-tubulin (H-300; catalog no. sc-5546, lot D042), vimentin (V9; catalog no. sc-6260, lot B252) and nectin-3 (C-19; catalog no. sc-14806, lot K261). Afadin (catalog no. 610732, lot 1) was purchased from BD Transduction Laboratories, Inc. (San Diego, CA). Bovine antirabbit IgG (catalog no. sc-2370), bovine antimouse IgG (catalog no. sc-2371), and bovine antigoat IgG (catalog no. sc-2350) conjugated to horseradish peroxidase from Santa Cruz Biotechnology, Inc. were used as secondary antibodies. Both the catalog and lot numbers are listed herein because several antibodies from other vendors failed to yield satisfactory results in preliminary experiments.
Primary testicular cell cultures
Sertoli cells were isolated from 20-d-old rat testes as described (17, 18). Freshly isolated Sertoli cells were suspended in serum-free Ham’s F-12 Nutrient Mixture and DMEM (F12/DMEM, 1:1, vol/vol, Sigma, St. Louis, MO) supplemented with 15 mM HEPES, 1.2 g/liter sodium bicarbonate, 10 µg/ml bovine insulin, 5 µg/ml human transferrin, 2.5 ng/ml epidermal growth factor, 20 mg/liter gentamicin, and 10 µg/ml bacitracin. For the isolation of Sertoli cells from 45- and 90-d-old rats, an earlier protocol (19) modified in this laboratory (18) was used. Sertoli cells isolated from 20-d-old rat testes were plated at high cell density at 0.5 x 106 cells/cm2 on Matrigel (Collaborative Research, Inc., Bedford, MA)-coated 12-well dishes (effective surface area, 
3.8 cm2 per well) with 3 ml F12/DMEM per well. Cells were hypotonically treated with 20 mM Tris, pH 7.4, for 2.5 min to lyse the contaminating germ cells (20) on d 2 (36 h after plating), and cultures were washed twice to remove lysed cells and debris. The purity of Sertoli cells isolated from 20-d-old and adult rat testes was approximately 95% and 85%, respectively. For germ cell preparation, cells were isolated from 20-, 45-, 60-, and 90-d-old rats by a mechanical method with sequential filtrations using miracloth (Calbiochem Corp., La Jolla, CA), 100-µm and 20-µm nylon meshes (Spectrum, Houston, TX) and glass wool as detailed elsewhere (21, 22). Germ cell preparations with purity of greater than 90% were routinely obtained. The purity of Sertoli and germ cell preparations were monitored using markers specific for germ, Leydig, Sertoli, and myoid cells, such as c-Kit receptor, 3ß-hydroxysteroid dehydrogenase, testin, and fibronectin, respectively, as detailed elsewhere (18). Seminiferous tubules were isolated from adult rat (300 g body weight) testes as previously described (18). The final tubule preparations were nonresponsive to human chorionic gonadotropin (10 ng/ml) treatment when the levels of testosterone in spent media were quantified, suggesting that they had negligible Leydig cell contamination (18).
RNA extraction
Testes were removed from 10-, 20-, 60-, and 90-d-old rats and homogenized in RNA STAT-60 for RNA isolation using a Tissumizer (Tekmar, Cincinnati, OH). Total RNA was also isolated from cells using RNA STAT-60 according to the manufacturer’s protocol as described (18, 23). RNA concentration was quantified by using a GeneQuant II spectrophotometer (Pharmacia, Uppsala, Sweden) at 260 nm. Two micrograms of RNA from each sample were routinely used for RT reaction.
Electron microscopy
To assess the presence of TJs between Sertoli cells cultured in vitro in addition to monitoring the transepithelial electrical resistance (TER) across the cell epithelium on bicameral units, electron microscopy was performed. In brief, Sertoli cells were isolated from 20-d-old rat testes and cultured at 0.5 x 106 cells/cm2 on Matrigel-coated 60-mm culture dishes. Sertoli cells were subjected to hypotonic treatment approximately 36 h after plating to remove residual germ cells (20). On d 5, these cells were fixed and processed for electron microscopy. Briefly, cells were fixed in 0.1 M cacodylate (pH 7.5 at 22 C) containing 2.5% (vol/vol) glutaraldehyde and 2.5% (wt/vol) paraformaldehyde for 1 h after a brief rinse with F12/DMEM. Cells were post-fixed with 1% OsO4 (vol/vol) in 0.1 M cacodylate for 1 h on ice and stained with 2% uranyl acetate (wt/vol) at room temperature. After dehydration through 70%, 95%, and 100% ethanol, cells were then detached from culture dishes with propylene oxide treatment and embedded in EPON (Electron Microscopy Sciences, Fort Washington, PA) blocks (24). Silver sections were cut using a Reichert Ultracut II ultramicrotome (Reichert Inc., Depew, NY) and examined with a JEOL 100CXII electron microscope (JEOL USA Inc., Peabody, MA) at 80 kV. Electron microscopy was carried out in the Bio-Imaging Resource Center at the Rockefeller University.
Treatment of Sertoli cells with cytokines
For cytokine treatments, recombinant human TGF-ß1 (Calbiochem) at 0.1–3 ng/ml and recombinant TNF- (Calbiochem) at 0.5–20 ng/ml were added to the Sertoli cell cultures (0.5 x 106 cells/cm2) on d 3 (i.e. 24 h after hypotonic treatment). Control experiments included cells receiving no treatment (Ctrl) or cells exposed to vehicle (Ctrl/V). TNF- and TGF-ß1 were dissolved in PBS (10 mM sodium phosphate and 0.15 M NaCl, pH 7.4, at 22 C) with 0.1% BSA (wt/vol), and 4 mM HCl in PBS containing 0.1% BSA (wt/vol), respectively. Cells were terminated after 6 h of incubation, and total RNA was extracted as described above.
Quantitative analysis on the Sertoli cell TJ-permeability barrier by TER measurement across the cell epithelium
Freshly isolated Sertoli cells (1.2 x 106 cells/cm2) from 20-d-old rat testes were plated on Matrigel-coated (1:5 dilution with F12/DMEM, vol/vol) bicameral units (effective surface area, 0.6 cm2) (Millicell HA filters, Millipore Corp., Bedford, MA) as described (23) with 0.5 ml of F12/DMEM each in the apical and basal compartment of the bicameral unit to allow the assembly of the TJ barrier. The TJ barrier was quantified by recording TER across the Sertoli cell epithelium on a daily basis for up to a period of 5 d. Preliminary studies were performed using different cell densities to determine the optimal cell density for TER measurement. Zinc (II) protoporphyrin-IX (ZnPP, BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), an NOS (25) and sGC (26) inhibitor, KT-5823 (C29H25N3O5) (BIOMOL), a specific inhibitor of PKG, 8-bromo-cGMP (BIOMOL), a cell membrane-permeable cGMP analog, 8-bromo-cAMP (BIOMOL), a cell membrane-permeable cAMP analog, 9-(tetrahydro-2'-furyl) adenine (SQ-22536) (BIOMOL), a cell membrane-permeable adenylate cyclase inhibitor, were added to the basal and apical compartments of the bicameral units on d 1 at the desired concentrations as shown in corresponding figure legends. The concentrations of the inhibitors used in our studies were selected as based on previously published reports (16, 27, 28, 29). These compounds, except ZnPP, which was replenished daily, were incubated with Sertoli cells for only 24 h and were removed by rinsing the cell epithelium with F12/DMEM (3x) thereafter. TER readings from triplicate bicameral units in a single experiment were taken daily and each experiment was repeated at least three times using different batches of Sertoli cells. The cytotoxicity of the compounds was tested by DNA assay (30) monitoring total DNA content in replicate cultures before and after treatment with specified inhibitors.
Semiquantitative RT-PCR
Semiquantitative RT-PCR was performed essentially as previously described (18, 23). Briefly, 2 µg of total RNA were reverse transcribed into cDNAs using 1 µg oligo(deoxythymidine)15 with a Moloney murine leukemia virus RT kit (Promega Corp., Madison, WI) in a final reaction volume of 25 µl. Each PCR was performed by combining 2 µl of RT product, 0.6 µg each of sense and antisense primers of a target gene (coamplified with 0.01 µg each of sense and antisense primers of rat ribosomal S-16) (Table 1
), 5 µl of 10x PCR buffer, 3 µl MgCl2 (25 mM), 8 µl deoxy (d)-nucleotide triphosphates (200 µM each of dATP, dCTP, dGTP, and dTTP), 1.25 U Taq DNA polymerase, and sterile water to a final reaction volume of 50 µl. The cycling parameters were as follows: denaturation at 94 C for 1 min, annealing at 56–61 C for 2 min (see Table 1), and extension at 72 C for 3 min, for a total of 26–29 cycles, which was followed by a final extension of 15 min in a Techgene (Techne, Cambridge, UK) DNA Thermal Cycler. To obtain data suitable for semiquantitative densitometric analysis, radioactive (i.e. hot) PCR was performed by using [
-32P]-labeled sense primers as described (18, 23). The relative ratio of [32P]-sense primer of a target gene to [32P]-sense S-16 was the same as the unlabeled primers. To ensure that the production of a specific target gene, such as eNOS and iNOS, and S-16 were in their linear phase, preliminary experiments were performed using different concentrations of template cDNAs (i.e. RT products) and primer pairs, and at different annealing temperatures. Autoradiography of PCR products was performed using Kodak X-OMAT AR films (Eastman Kodak Co., Rochester, NY). Autoradiograms were densitometrically scanned and normalized against S-16. In most experiments, results of RT-PCR were verified by immunoblotting, which also ensured that any changes in the mRNA levels were indeed transmitted to functional proteins that could eventually affect cellular functions and junction dynamics.
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200-µl sample size) were first precleared by incubating the sample with normal rabbit serum (or normal mouse or goat serum, depending on the source of precipitating antibody to be used in subsequent steps) at 1:150 for 1 h at 4 C, followed by 10 µl protein A/G-PLUS agarose (Santa Cruz Biotechnology, Inc.) for 1 h at 4 C, centrifuged at 1000 x g for 5 min to obtain the clear supernatant. Samples were then incubated with the corresponding antibodies at 1:150 dilution in microfuge tubes and mounted onto a rotator (Glas-Col, Glass Tech Supplies Inc., Fullerton, CA) at room temperature for 3 h at approximately 15–20 rpm. Thereafter, 20 µl protein A/G-PLUS agarose (Santa Cruz Biotechnology, Inc.) was added to the samples to precipitate the immunocomplexes. After 3 h of incubation at room temperature in a rotator, immunocomplexes bound to protein A/G-PLUS agarose were washed twice (IP buffer, 1000 x g, 5 min) and were extracted in SDS-sample buffer [0.625 M Tris, pH 6.8 at 22 C, containing 1.6% 2-mercaptoethanol, 1% SDS (wt/vol), 1 mM EDTA, 10% glycerol (vol/vol)] (32) and resolved by SDS-PAGE under reducing condition as described (18). It is important that the protein A/G-PLUS agarose should be resuspended by gentle agitation without vortexing so as not to damage the agarose beads.
Measurement of intracellular levels of cAMP and cGMP
ZnPP-treated and control Sertoli cell cultures were terminated using an extraction buffer (50 mM Tris, pH 7.5, at 22 C, containing 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, and 1 mM caffeine) by incubating cultures at 100 C for 5 min to coagulate proteins. Cells were then sonicated and centrifuged at 12,000 x g at 4 C for 15 min to remove cellular debris. The amount of cellular cAMP and cGMP in the supernatant was quantified using a [3H]cAMP assay system (with a detection limit of approximately 0.05 pmol/assay tube and the 50% displacement was at 6.6 pmol) (Amersham Pharmacia Biotech, Piscataway, NJ) and a [3H]cGMP assay system (with a detection limit of 0.04 pmol/assay tube and the 50% displacement was at 3.2 pmol) (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The interassay and intraassay coefficient of variation was at 8–10% and 6–8% for both assays. All samples within a given experiment were assayed simultaneously in a single experimental session to eliminate interassay variations.
Statistical analysis
Student’s t test was performed by comparing treatment group at each time point with the corresponding control using the GB-STAT Statistical Analysis Software Package (version 7.0, Dynamic Microsystems, Inc., Silver Spring, MD). Each experiment was repeated at least three times using different batches of cells, and each time point (or treatment group) had duplicate or triplicate cultures.
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Results |
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, A–D). A much more drastic increase in iNOS expression (Fig. 1, A and B) in Sertoli cells during maturation was detected when compared with eNOS (Fig. 1, C and D), 40-fold vs. 7-fold (Fig. 1, A and B vs. C and D). In contrast, the iNOS steady-state mRNA in germ cells plunged steadily during maturation (Fig. 1, E and F), whereas the eNOS mRNA level rose steadily in germ cells during maturation (Fig. 1, G and H). It was noted that the level of eNOS in 20-d-old germ cells was too low to be detected (Fig. 1, G and H). In the testes, iNOS expression level tumbled steadily from 10–90 d of age (Fig. 1, I and J). In contrast, eNOS increased steadily with testicular maturation (Fig. 1, K and L). Taken together, these data illustrate the differential response of both NOSs in Sertoli cells, germ cells, and testes during maturation, suggesting that although these NOSs are performing similar biological function of producing NO, they are regulated differently in the testis.
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, A and E) and eNOS (Fig. 2, B and F) were quantified. The iNOS level stayed high during the initial 1–2 d in culture and tumbled rapidly thereafter (Fig. 2, A and E). This pattern of changes in expression is similar to eNOS (Fig. 2, B and F vs. A and E), suggesting the assembly of junctions between Sertoli cells may require neither NOSs. Yet Sertoli cells cultured at low cell density (2.5 x 104 cells/cm2) where TJ barrier failed to form due to low cell number, the levels of iNOS and eNOS proteins remained relatively stable throughout the entire culture period (Fig. 2C). Figure 2D is the same blot of Fig. 2, A and B (lower panel), but stained with an antiactin antibody illustrating equal protein loading in this SDS-polyacrylamide gel.
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, as denoted by arrowheads. The basal and apical sides of the Sertoli cell epithelium could be distinguished by the presence of the microvilli (Fig. 3B) on the apical side of Sertoli cells, which were not found in the basal side, consistent with results of earlier reports (for a review, see Ref. 35). The magnified inter-Sertoli cell TJ barrier as shown in Fig. 3C (indicated by arrowheads) was found near the basal side of the Sertoli cell epithelium. In parallel to this morphological analysis, the TER across the cell epithelium was quantified to confirm the assembly of an electrical resistance between Sertoli cells in vitro (Fig. 3D). For instance, when Sertoli cells were plated on Matrigel-coated bicameral units at 1.2 x 106 cells/cm2 and TER across the cell epithelium was quantified daily, a steady rise in TER was detected (Fig. 3D), correlating with the formation of a functional TJ barrier. The TER reached its peak by d 3 and remained steady when the TJ-barrier was established (Fig. 3D vs. 3, A–C).
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, a significant decline in both iNOS (Fig. 2, A and E) and eNOS (Fig. 2, B and F) was detected at the time Sertoli cell TJ barrier was assembled in vitro, suggesting the presence of NO might perturb the TJ barrier. Indeed, ZnPP, an NOS inhibitor, at 1 and 10 µM, when added to Sertoli cell cultures during TJ assembly facilitated the TJ barrier assembly dose dependently (Fig. 3D). Earlier studies have shown that the assembly of Sertoli cell TJ barrier in vitro was associated with a transient induction of occludin (36), we had investigated if ZnPP would affect occludin expression by Sertoli cells. Consistent with several earlier reports (23, 36), the assembly of the TJ barrier was shown to associate with a transient occludin induction (Fig. 3, E and G), yet the presence of ZnPP stimulated the expression and protein production of occludin by Sertoli cells (Fig. 3, F and H).
iNOS and eNOS are structurally linked to the occludin-based TJ multiprotein complexes
Because ZnPP, an NOS inhibitor, can facilitate the Sertoli cell TJ assembly, suggesting NOS is a putative regulator of TJ dynamics in vitro, we sought to investigate whether NOS physically interacts with the TJ multi-protein complexes. By immunoprecipitation using seminiferous tubule lysates as the starting material, iNOS and eNOS were found to structurally link to occludin, a TJ-integral membrane protein, but not afadin and nectin-3, both of which are adherens junction (AJ)-associated integral membrane proteins (Fig. 4). iNOS and eNOS also associated with actin, vimentin, and -tubulin (Fig. 4). Taken collectively, these results illustrate iNOS and eNOS interact structurally and functionally with the occludin/actin TJ, the intermediate filament vimentin-based desmosomes and hemidesmosomes, and the tubulin cytoskeleton.
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) and cGMP (Fig. 5B) levels at 1–2 d (see Fig. 5 vs. Fig. 3D) when TJ was being formed, suggesting low levels of these cyclic nucleotides are indeed needed for TJ barrier assembly, consistent with an earlier report (16). Yet the presence of ZnPP failed to induce any changes in intracellular cAMP level (Fig. 5A). However, ZnPP induced a significant reduction in cGMP level (Fig. 5B), which in turn facilitated the TJ barrier assembly (see Fig. 3D). Thus, it is likely that ZnPP facilitates the assembly of the Sertoli cell TJ barrier as shown in Fig. 3D by plunging the intracellular cGMP level. This postulate was further confirmed with the study when Sertoli cells were incubated with 8-bromo-cGMP, a cell membrane-permeable cGMP analog, at 0.1–1 mM for 24 h during TJ assembly. 8-Bromo-cGMP was shown to significantly perturb the Sertoli cell TJ barrier dose dependently (Fig. 6A). Because low concentrations of cAMP at 4–20 µM were shown to facilitate Sertoli cell TJ barrier assembly by enhancing TER across the Sertoli cell epithelium in vitro (16), we sought to examine whether cGMP had a similar biphasic effect on the Sertoli cell TJ barrier. At 4 µM, 8-bromo-cGMP indeed facilitated the TJ barrier assembly, enhancing the TER across the cell epithelium (Fig. 6B). In contrast, the presence of KT-5823, a specific PKG inhibitor (see Fig. 8 for the signaling pathway involving cGMP and PKG), facilitated the Sertoli cell TJ barrier in vitro (Fig. 6C). Taken collectively, these data illustrate that the Sertoli cell TJ dynamics was regulated, at least in part, via the NO/sGC/cGMP/PKG pathway. Even though the effects of ZnPP on TJ dynamics as shown in Fig. 5 are likely mediated via the cGMP pathway because its presence in Sertoli cell cultures had no effects on the cAMP level (Fig. 5, A vs. B), it is of interest to verify and expand results of earlier studies illustrating the biphasic effects of cAMP on the Sertoli cell TJ dynamics (16, 27); in particular, results reported in Fig. 5A have demonstrated the crucial role of cAMP in the assembly of Sertoli cell TJs in vitro. Indeed, 8-bromo-cAMP at high concentration, such as 1 mM (but not 0.1 mM), could significantly perturb the Sertoli-TJ barrier (Fig. 6D). Furthermore, SQ-22536, an adenylate cyclase inhibitor, facilitated the Sertoli cell-TJ barrier assembly (Fig. 6E). Collectively, these findings indicate that high levels of cAMP can perturb the Sertoli cell TJ barrier. Although much work is needed to define how the intricate intracellular levels of cAMP and cGMP are being regulated, which in turn modulate the dynamics of the Sertoli cell TJ, these results have clearly demonstrated that the interplay of cAMP and cGMP plays a crucial role in TJ regulation in the testis.
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(37) can perturb the assembly of Sertoli cell TJ barrier in vitro; we thus sought to examine whether TGF-ß1 can affect iNOS and eNOS expression in primary Sertoli cell cultures in vitro. When TGF-ß1 was added to Sertoli cell cultures (0.5 x 106 cells/cm2) on d 3 at 0.1–3 ng/ml and incubated for 6 h, it significantly and dose dependently induced the expression of iNOS and eNOS (Fig. 7, A and B) by Sertoli cells. Likewise, TNF- at 0.5–20 ng/ml also significantly induced the Sertoli cell steady-state iNOS mRNA level dose dependently, but not eNOS (Fig. 7, C and D).
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). The cGMP level, along with intracellular cAMP and Ca2+ levels, and phosphorylation and dephosphorylation of target proteins, such as occludin and other TJ-associated signaling molecules (for review, see Ref. 11), are likely the major players that modulate the TJ dynamics in the testis (Fig. 8). |
Discussion |
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), protein kinases, and protein phosphatases (for a review, see Ref. 11). Data presented herein report yet another potential regulatory system of TJ dynamics in the testis namely the NOS/NO system via the cGMP and protein kinase G signaling pathway. This conclusion was reached based on several lines of evidence. First, the assembly of Sertoli cell TJs in vitro was shown to associate with a significant reduction on the steady-state mRNA and protein levels of NOS, suggesting that NO, the enzymatic product of NOS, if present at high levels, it can perturb the Sertoli cell TJ barrier. Indeed, the presence of ZnPP, an NOS inhibitor (25), which blocks the production of endogenous NO can facilitate the Sertoli cell TJ barrier in vitro dose dependently. Second, sGC (the intracellular putative NO receptor), cGMP (the second messenger), and PKG (the downstream effector protein of NO) are largely localized to the basal compartment of the seminiferous epithelium, residing mostly in Sertoli cells (38, 39, 40) consistent with their localization at the site of the BTB. Such physical intimacy between the downstream effector molecules of NO and the Sertoli cell TJs has further implicated the significance of NO in TJ dynamics. Third, both iNOS and eNOS were shown to structurally associate with occludin, a putative TJ-integral membrane protein largely confined to Sertoli cells in the testis (note: occludin also confers cell adhesion function at the site of TJs) (for a review, see Ref. 11), and the underlying actin network. Furthermore, an inhibition of NOS by ZnPP can stimulate the steady-state mRNA and protein levels of occludin, suggesting NO produced by Sertoli and germ cells may play a role in regulating the homeostasis of TJ-integral membrane proteins at the site of the BTB, thereby affecting TJ dynamics. Other studies have shown that NO is an important intrinsic regulator of microvessel permeability, which may either increase or decrease the TJ permeability barrier of endothelial cells in blood vessels (for a review, see Ref. 41). For instance, NOS inhibitors, such as Nw-monomethyl-L-arginine and L-Nw-nitro-L-arginine methyl ester, are known to attenuate the increased microvessel permeability in response to ionomycin and ATP, that is, making the TJ barrier tighter (42). Needless to say, our results presented herein using ZnPP are consistent with this earlier report illustrating NO can perturb the TJ barrier in endothelial cells in blood vessel, similar to its effects in Sertoli cells, whose action can be blocked by an NOS inhibitor. Furthermore, using microvessel hydraulic conductivity as the means to assess the TJ barrier integrity between endothelial cells in perifused frog mesenteric venular microvessels, both NOS inhibitors, L-Nw-nitro-L-arginine methyl ester and Nw-monomethyl-L-arginine, could dose dependently increase the tightness of the endothelial TJ barrier via a calcium-independent pathway because no changes of intracellular calcium concentration ([Ca2+]i) in these vessels were detected (43). NO apparently mediates its effects on TJ barrier function by perturbing the cell adhesion function in the microvessel because NO was shown to inhibit leukocyte-endothelial cell-adhesive interactions (44), suggesting cross-talks are present between AJs and TJs. Also, NO was shown to perturb the epithelial TJ barrier function in intestinal cells in vitro (45). Yet 3-morpholino-sydnonimine, an NO donor, was shown to increase TER (i.e. promoting the TJ barrier making the junction tighter) (46) across the confluent retinal pigment epithelial cells isolated from rat retinas and cultured in vitro (note: this TJ barrier confers the blood-ocular barrier function in vivo), suggesting that NO can also facilitate TJ assembly. Taken collectively, these results illustrate that the diverging effects of NO on TJ function, whose effect is possibly cell and tissue specific and may be concentration dependent, similar to the effects of cAMP on Sertoli cell TJ barrier (16). For instance, at 4–20 µM, dibutyryl cAMP facilitates the assembly of Sertoli cell TJ barrier, yet at 100–500 µM, it inhibits the TJ barrier function (16). As reported herein, 8-bromo-cAMP at 0.1 mM can also perturb the Sertoli cell TJ barrier, reducing the TER across the cell epithelium by as much as 80%, consistent with this earlier report (16). In this context, it is noteworthy to mention that cytokines can also have a biphasic effects on TJ barrier in different epithelia, either facilitating or inhibiting TJ barrier in vitro (for review, see Ref. 47). Nonetheless, these data illustrate the pivotal role of the NOS/NO system in the regulation of TJ-permeability barrier. And the fact that both Sertoli and germ cells are actively expressing mRNAs encoding both iNOS and eNOS suggests that, although the BTB is constituted by the inter-Sertoli cell TJs, germ cells likely contribute to the making and maintenance of the BTB integrity in the testis. In this connection, it is of interest to note that, during maturation, an age-dependent increase in the Sertoli steady-state mRNAs of iNOS and eNOS is detected, and iNOS and eNOS display an age-dependent decline and induction in expression by germ cells, respectively. Yet the expression of iNOS and eNOS plummets and increases in the testis, respectively, during maturation. Because an induction of testicular eNOS at 60–90 d of age (note: almost a 10-fold increase vs. immature rat testes at 10–20 d of age) favors TJ disruption, whereas a decline of iNOS at these ages presumably facilitates TJ assembly, coinciding with the timing of rapid spermiogenesis and junction restructuring in the seminiferous epithelium at these ages. Based on these data, it is tempting to speculate that these two NOSs may play a crucial role, alike a yin-yang relationship, in regulating the opening and closing of the BTB with iNOS facilitates the closing and eNOS the opening of the BTB. Needless to say, this concept must be vigorously tested in future experiments because NOS is known to regulate an array of physiological function, including Sertoli and germ cell apoptosis, sperm motility, and sperm maturation (2, 3, 4, 5, 6, 7, 8, 9, 10), and these effects must also be taken into considerations for data interpretation. Nonetheless, these data clearly suggest that these two NOSs are regulated differentially between Sertoli and germ cells. Yet the intimate coexistence of both cell types in the seminiferous epithelium further complicates the issue regarding their roles in TJ dynamics.
What is the downstream mechanistic pathway used by NOS/NO to affect Sertoli cell TJ dynamics in the testis?
Among the various downstream signaling pathways of NOS/NO, the two best studied signal transducers of NO are cAMP and cGMP (for reviews, see Refs. 12 and 14). Indeed, recent studies of TJ regulation in multiple epithelia including Sertoli cells have shown that TJ dynamics are regulated by these cyclic nucleotides (for a review, see Ref. 11). For instance, dibutyryl cAMP, an analog of cAMP that is noncleavable by cAMP phosphodiesterase, was shown to have a biphasic effect on Sertoli cell TJ barrier in vitro. Dibutyryl cAMP at 4–20 µM or 100–500 µM can either promote or perturb the assembly and maintenance of the Sertoli cell TJ barrier in vitro, respectively (16). As such, we have investigated if the reported effects of NOS/NO on Sertoli cell TJ dynamics are mediated via these cyclic nucleotides (see Fig. 8). Interestingly, a transient but drastic increase in the intracellular cAMP and cGMP levels in Sertoli cells is detected at the time the TJ barrier is being formed in vitro. Yet once the TJ barrier is formed, the levels of both cAMP and cGMP tumbled rapidly and reduced by as much as 2-fold, making them similar to the basal level. These results seemingly suggest that, although cGMP and cAMP may not be required for the maintenance of the Sertoli cell TJ barrier, they are needed for TJ assembly, supporting the reported biphasic effects of cAMP on Sertoli cell TJ barrier function as earlier reported (16). Furthermore, the presence of ZnPP, an inhibitor of NOS and an inhibitor of NO-dependent sGC (26) that can effectively and significantly inhibit the intracellular cGMP level, can facilitate the Sertoli cell TJ assembly, implicating the significance of cGMP in TJ dynamics. Indeed, when Sertoli cells were exposed to 8-bromo-cGMP at 0.1–1 mM, a cell membrane-permeable cGMP analog, on d 1 for a period of 24 h, it perturbed the Sertoli cell TJ barrier dose dependently. Taken collectively, these data suggest that cGMP at much lower concentrations, such as less than 5 µM (note: assuming cGMP has a similar biphasic effect as of cAMP on TJ dynamics; see Ref. 16), can possibly promote TJ barrier assembly. As reported herein, 8-bromo-cGMP at 4 µM can indeed facilitate the TJ barrier function. Furthermore, the use of KT-5823, a PKG inhibitor, can also modulate the Sertoli cell TJ barrier function. It is of interest to note that the effect of the PKG inhibitor is comparably smaller than a cGMP agonist analog, such as 8-bromo-cGMP, suggesting that cGMP may exert its effects via other effectors other than PKG as shown in Fig. 8. Yet this issue will require further investigation in future studies. In summary, these data clearly illustrate that NO is using the sGC/cGMP/PKG pathway to mediate its effects in the regulation of TJ dynamics (see Fig. 8). Whereas the downstream effector(s) of PKG remains to be investigated, this effect, at least in part, is likely mediated via TJ-integral membrane proteins, such as occludin, because the presence of ZnPP that promotes the Sertoli cell TJ barrier, making it tighter, was shown to induce the production and expression of occludin by Sertoli cells. These results are also supported by a recent report demonstrating NO can induce redistribution of occludin and ZO-1, moving them away from the site of TJs to cell cytoplasm, perturbing the TJ barrier (48). Alternatively, the downstream target molecule of PKG can be actin per se. For instance, a recent study using human cervical epithelial cells cultured in vitro has shown that the NO-induced disruption of the TJ barrier is mediated via changes in cGMP and PKG (49). And 8-bromo-cGMP was shown to perturb the TJ barrier in cervical epithelial cells as well (49). Whereas 8-bromo-cGMP had no effects on the total cellular actin content, yet it significantly induced the intracellular G-actin level disrupting the homeostasis of F- and G-actin levels (49), thereby fragmenting the cytoskeleton. This in turn destabilizes the epithelium and hence increases cell permeability.
What are the upstream signaling transducers that associate with the NOS/NO system in the testis?
The putative signaling molecules that regulate the NOS function in the testis are not entirely known. Yet studies in other epithelia, such as the blood-ocular barrier, and the pathogenesis of ocular inflammation, such as uveitis, have implicated the significance of cytokines in TJ regulation (for a review, see Ref. 50). Furthermore, recent studies have shown that the presence of either TGF-ß3 or TNF- in Sertoli cells cultured in vitro, each cytokine can dose dependently perturb the TJ barrier function (36, 37). It is therefore logical to speculate that cytokines may mediate their effects, at least in part, via the NOS/NO pathway. Indeed, TNF- and TGF-ß1 were shown to activate the steady-state mRNA levels of iNOS and eNOS as reported herein, suggesting that the subsequent increase in NO level was used to perturb the TJ barrier. This argument is consistent with the findings that the assembly of the Sertoli cell TJ barrier in vitro associates with a plunge in NOS, illustrating an inverse relationship between the assembly of Sertoli cell TJ barrier and the endogenous levels of NOS. Taken collectively, these data suggest that cytokines, such as TNF, that promote the expression of NOS will likely reduce the production of occludin, the building block of the TJ fibrils. Indeed, a recent report has shown that TNF can significantly inhibit Sertoli cell occludin production at the time the Sertoli cell TJ barrier is disrupted (37). This conclusion is also supported by results of studies using ZnPP (an NOS inhibitor), illustrating this inhibitor can stimulate Sertoli cell occludin expression and production. These data are also consistent with a recent paper reporting that the disruptive effects of interferon- and lipopolysaccharide on the TJ barrier in rat retinal pigment epithelial cells in vitro could be modulated by using 3-morpholino-sydnonimine, an NO donor (46), confirming the functional relationship between cytokines and the NOS/NO system.
Does NOS/NO play a role in other junction dynamics other than TJ?
Earlier studies by immunohistochemistry have localized both eNOS and iNOS to the human and rat seminiferous epithelium (2, 3, 9). For eNOS, most of the immunostaining is associated with Leydig and Sertoli cells, but not normal germ cells except those undergoing degeneration and apoptosis, and is not a stage-specific protein (3, 9). Yet the pattern of eNOS staining is consistent with its localization at the inter-Sertoli TJ at the basal compartment of the epithelium (3, 9). For iNOS, it is a stage-specific protein in the rat testis, being highest at stages IX–XII (2). iNOS associates with elongating spermatids (but not elongate spermatids), pachytene spermatocytes, Leydig cells, Sertoli cells, and peritubular myoid cells; and its pattern of localization in the seminiferous epithelium of the testes is also consistent with its localization at the inter-Sertoli TJ (2, 9). At present, the precise functional significance regarding the intimate physical association of NOS with TJ proteins, such as occludin, is not known. But this does implicate the role of NOS in TJ function. Furthermore, these immunohistochemical data also illustrate the association of NOS with the site of desmosome-like junctions (cell-cell intermediate filament-based anchoring junction type) and adherens junctions (cell-cell actin-based anchoring junction type) between Sertoli cells as well as between Sertoli and spermatids (also known as ectoplasmic specialization, ES) (2, 3, 9). The demonstration of the association of NOS with vimentin, an intermediate filament constituent protein (for a review, see Ref. 11), by immunoprecipitation as reported herein seemingly suggests that NO can also regulate anchoring junction dynamics, such as desmosome. Indeed, PKG, one of the downstream effectors of NOS/NO (for reviews, see Refs. 12 and 14) was shown to colocalize with vimentin (also a putative substrate of PKG) (51) in human neutrophils cultured in vitro with functional anchoring junctions such as desmosomes, implicating the role of NO in desmosome function (and possibly hemidesmosome function). A more recent study by subcellular fractionation of cellular organelles and immunofluorescent microcopy has shown that eNOS was associated with platelet endothelial cell adhesion molecule 1, a putative AJ-associated cell adhesion molecule, but not vascular endothelial-cadherin and plakoglobin (52). Taken collectively, these data have strengthened the notion that NOS and its downstream effector proteins are also found at the site of desmosomes and ES implicating their role in cell adhesion function and AJ dynamics.
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Abbreviations: AC, Adenylate cyclase; AF-6, s-afadin; AJ, adherens junction; BTB, blood-testis barrier; Ca2+, calcium ions; COX-2, cyclooxygenase 2; d, deoxy; eNOS, endothelial NOS; HO-1, heme-oxygenase 1; iNOS, inducible NOS; IP, immunoprecipitation; JAM, junctional adhesion molecule; NO, nitric oxide; NOS, NO synthase; nNOS, neuronal NOS; PKA, cAMP-dependent protein kinase (protein kinase A); PKG, cGMP-dependent protein kinase (protein kinase G); SDS, sodium dodecyl sulfate; sGC, soluble guanylate cyclase; TJ, tight junction; TER, transepithelial electrical resistance; ZnPP, zinc (II) protoporphyrin IX; ZO, zonula occludens.
Received December 18, 2002.
Accepted for publication March 17, 2003.
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2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells. Mol Cell Endocrinol 89:127–140[CrossRef][Medline]
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