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The Interplay of Collagen IV, Tumor Necrosis Factor-, Gelatinase B (Matrix Metalloprotease-9), and Tissue Inhibitor of Metalloproteases-1 in the Basal
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     Abstract

    During spermatogenesis, preleptotene and leptotene spermatocytes must translocate across the blood-testis barrier formed by inter-Sertoli cell-tight junctions (TJs) from the basal compartment of the seminiferous epithelium adjacent to the basement membrane to the adluminal compartment at stages VIII–IX for further development. Because of the close proximity between extracellular matrix (ECM) that constitutes the basement membrane and the blood-testis barrier, we sought to investigate the role of ECM in Sertoli cell TJ dynamics. When Sertoli cells were cultured in vitro to initiate the assembly of the Sertoli cell TJ-permeability barrier, the presence of an anticollagen IV antibody indeed perturbed the barrier. Because ECM is known to maintain a pool of cytokines and TNF{alpha} has been shown to regulate TJ dynamics in other epithelia, we investigated whether TNF{alpha} can regulate Sertoli cell TJ function via its effects on collagen {alpha} 3(IV) and other proteins that maintain the homeostasis of ECM. As expected, recombinant TNF{alpha} perturbed the Sertoli cell TJ-barrier assembly in vitro dose dependently. TNF{alpha} also inhibited the timely induction of occludin, which is known to associate with the Sertoli cell TJ-barrier assembly. Furthermore, TNF{alpha} induced the expression of Sertoli cell collagen {alpha} 3(IV), gelatinase B (matrix metalloprotease-9, MMP-9) and tissue inhibitor of metalloproteases-1 but not gelatinase A (matrix metalloprotease-2), and promoted the activation of pro-MMP-9. These results thus suggest that the activated MMP-9 induced by TNF{alpha} is used to cleave the existing collagen network in the ECM, thereby perturbing the TJ-barrier. This in turn creates a negative feedback that causes TNF{alpha} to induce collagen {alpha} 3(IV) and tissue inhibitor of metalloproteases-1 expression so as to replenish the collagen network in the disrupted TJ-barrier and limit the activity of MMP-9. Taken collectively, these observations strengthen the notion that ECM is involved in the regulation of junction dynamics in addition to its structural role in the testis.

    Introduction

    THROUGHOUT SPERMATOGENESIS, preleptotene and leptotene spermatocytes residing at the basal compartment of the seminiferous epithelium must translocate across the Sertoli cell-tight junctions (TJs) that constitute the blood-testis barrier (BTB) near the basal lamina, entering into the adluminal compartment for further development (for reviews, see Refs. 1, 2, 3). However, the mechanism(s) and molecules that regulate the opening and closing of the BTB to facilitate the timely passage of developing germ cells across the BTB are largely unknown (for a review, see Ref. 3). In other epithelia, such as those found in the small intestine and the collecting tubule in the kidney, TJs are located at the apical portion of adjacent cells, farthest away from the extracellular matrix (ECM). Below the TJ structures are the cell-cell actin-based adherens junctions (AJs), followed by cell-cell intermediate filament-based anchoring junctions called desmosomes. These structures in turn constitute the junctional complex, responsible for anchoring cells together forming an impermeable epithelium (for reviews, see Refs. 3 and4). Gap junctions (GJs) are found behind this junctional complex, and the epithelial cells in turn attach to ECM via hemidesmosomes or focal contacts (for reviews, see Refs. 3 and4). In contrast to these epithelia, Sertoli cell TJs that constitute the BTB in the testis are located adjacent to the basement membrane, a modified form of ECM in the testis (for a review, see Ref. 5) and are structurally present side by side with cell-cell actin-based AJs, cell-cell intermediate filament-based desmosomes, cell-matrix actin-based focal contacts, and cell-matrix intermediate filament-based hemidesmosomes (for a review, see Ref. 3). As such, the relative position of TJs and AJs in the testis is different from other epithelia (for reviews, see Refs. 2 and3). Because of such morphological intimacy between TJs and the basal lamina, we sought to examine whether the ECM in the testis takes an active role in regulating TJ dynamics.

    In the testis, the basal lamina is composed of: 1) the basement membrane, which is comprised of thin, sheetlike extracellular structures constituted by ECM proteins, such as type IV collagen, laminin, heparan sulfate proteoglycan (6), entactin (7), and other substances; 2) a thin collagen layer; and 3) a layer of peritubular myoid cells (for a review, see Ref. 5). ECM is known to play an important role in the regulation of Sertoli cell function by affecting its morphology, differentiation, and migration (for a review, see Ref. 5). Modifications of the basement membrane by passive transfer of antibodies raised against seminiferous tubule basement membranes (STBMs; Ref. 8) or noncollagenous fraction of STBMs have been shown to cause focal sloughing of the seminiferous epithelium in the rat (9). Yet it is not known whether ECM plays any role in the regulation of TJ dynamics in the testis. Previous studies have demonstrated the presence of type IV collagen in the rat testis (10). Also, the expression of collagen {alpha} 3(IV) chain peaked at 10–20 d after birth coinciding with the assembly of the BTB in the mouse testis at approximately postnatal d 13 (11). As such, we thought it pertinent to examine whether both Sertoli and germ cells contribute to the pool of collagen {alpha} 3(IV) in the basal lamina and whether collagen {alpha} 3(IV) can affect Sertoli TJ assembly in vitro.

    The homeostasis of ECM proteins are regulated, at least in part, by the coordinated activity between proteases and protease inhibitors (for reviews, see Refs. 12 and13). For instance, matrix metalloproteases (MMPs), such as MMP-9 and MMP-2, and tissue inhibitors of metalloproteases (TIMPs), such as TIMP-1 and TIMP-2, produced by testicular cells under the hormonal and paracrine regulation (14), are believed to take part in these processes because collagen IV and laminin, the major components in STBMs, are putative substrates for MMP-2 and MMP-9 (for reviews, see Refs. 13 and15). Moreover, abnormal thickening and multiple lamination of the STBM can induce infertility, further implicating the importance of ECM homeostasis for normal testis function (for a review, see Ref. 5).

    There is accumulating evidence that the expression and action of MMPs and TIMPs can be regulated by cytokines, such as TNF{alpha} (for reviews, see Refs. 12 and13). Indeed, rats administrated with TNF{alpha} displayed a massive loss of germ cells from the adluminal compartment (16). Other studies have shown that TNF{alpha} affects TJ dynamics in both epithelial and endothelial cells (for a review, see Ref. 17). Furthermore, TNF{alpha} can inhibit the expression of occludin, a TJ-integral membrane protein (for a review, see Ref. 3), in HT-29/B6 cell line (18). As such, we hypothesized that the role of TNF{alpha} on TJ dynamics may rely not only on its effects on TJ proteins, but it can also be mediated, at least in part, via the ECM proteins, such as collagen, MMPs, and TIMPs. To test this hypothesis, we have investigated: 1) the expression of TNF{alpha} by Sertoli cells during the assembly of the Sertoli cell TJ-permeability barrier; 2) the effects of recombinant TNF{alpha} on the Sertoli cell TJ-barrier; 3) its effects on the expression of collagen, MMPs, TIMPs, and TJ-associated proteins during Sertoli cell TJ-barrier assembly; and 4) the downstream signaling pathway mediating these effects.

    Materials and Methods

    Animals

    Male Sprague Dawley rats were obtained from Charles River Laboratories, Inc. (Kingston, MA). The use of animals for this study was approved by the Rockefeller University Animal Care and Use Committee with protocol nos. 97117 and 00111.

    Preparation of testicular cells and spent media

    Sertoli cell cultures.

    Sertoli cells were isolated from 20-d-old rats as previously described (19, 20, 21). At low cell density (5 x 104 cells/cm2), Sertoli cell TJ barrier could not form, largely because of the lack of proximity between cells rendering them incapable of assuming the columnar shape, which is common in Sertoli cells when they form TJs in vitro (20, 22, 23, 24, 25). Nonetheless, both AJs and GJs were present (20, 21, 26). Isolated cells were plated at 5 x 104 cells/cm2 in 100-mm Petri dishes in 9-ml serum-free Ham’s F12 nutrient mixture (F12) and DMEM (1:1, vol/vol) (~ 4.5 x 106 cells/9 ml per 100-mm dish) 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. At high cell density (0.75 or 1 x 106 cells/cm2), cells were plated onto Matrigel (Collaborative Biochemical Products, Bedford, MA)-coated (diluted 1:7 with F12/DMEM, vol/vol) 12-well dishes (Corning, Inc., Corning, NY) as previously described (21, 26, 27); TJs, AJs, and GJs were formed. These cultures were incubated in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 35 C. Unless specified otherwise, time 0 represents Sertoli cell cultures that were terminated approximately 3 h after plating. After 48 h, cultures were hypotonically treated with 20 mM Tris, pH 7.4, at 22 C for 2.5 min to lyse residual germ cells (28). This was followed by two successive washes with F12/DMEM to remove germ cell debris. Media were replaced every 24 h. Cell cultures were terminated at specific time points by RNA STAT-60 for RNA extraction.

    For the detection of collagen {alpha} 3(IV) protein, Sertoli cells cultured at low cell density were lysed to prepare whole-cell lysates on d 3 (i.e. 24 h after hypotonic treatment) for immunoblotting. For the preparation of Sertoli cell-enriched culture medium (SCCM), cells were cultured for an additional 8 d after hypotonic treatment, spent media were collected on the following d 4 and 8 as previously described (19). For adult Sertoli cell cultures, Sertoli cells were isolated from rats at 45 and 90 d of age with a purity of about approximately 95% as previously described (29). The purity of these Sertoli cell cultures were analyzed microscopically (29, 30) and by RT-PCR using primer pairs specific to markers of Leydig cells [3ß-hydroxysteroid dehydrogenase (HSD)], germ cells (c-Kit receptor), and peritubular myoid cells (fibronectin; Ref. 30) (PBS containing 0.1% BSA, wt/vol).

    Sertoli cells cultured with recombinant human TNF{alpha} protein.

    Sertoli cells (0.75 x 106 cells/cm2), isolated as described above, were cultured on Matrigel-coated 12-well dishes (effective surface area, ~ 3.8 cm2/dish, containing 3 ml F12/DMEM). Recombinant human TNF{alpha} protein (Calbiochem, La Jolla, CA) at concentrations of either 10 ng/ml or 100 ng/ml was added to Sertoli cell cultures at time 0. TNF{alpha} was prepared as a stock solution of 10 µg/ml in PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4, at 22 C) containing 0.1% BSA (wt/vol). Cultures were hypotonically treated 48 h thereafter to remove residual germ cells as described above. Media containing TNF{alpha} was replenished every 24 h. Cultures were terminated at specific time points for RNA extraction and whole-cell lysate preparation, and media were collected as SCCM. Controls included Sertoli cells cultured alone in F12/DMEM without TNF{alpha} or vehicle alone (PBS containing 0.1% BSA, wt/vol).

    Germ cell isolation.

    Total germ cells were isolated from rat testes at 10, 15, 20, 45, 60, 90, and 200 d of age by a mechanical procedure (31). When the final preparation was analyzed by DNA flow cytometry (31), it consisted largely of spermatogonia, spermatocytes, and round spermatids with a relative percentage of 17%, 19%, and 64%, respectively, because elongate spermatids were removed in the glass wool filtration step (31). These germ cells had a purity of greater than 95% with negligible somatic cell contamination when examined microscopically and assessed by other criteria, such as RT-PCR, to amplify testin, a known Sertoli cell product (31, 32, 33); c-Kit receptor, a spermatogonium product (34); HSD, a Leydig cell product (35); and fibronectin, a peritubular myoid cell product (36). Cell number of freshly isolated germ cells was determined by a hemocytometer. Freshly isolated germ cells were terminated for RNA extraction and for cell lysate preparation. To obtain germ cell-conditioned medium (GCCM), freshly isolated germ cells from 90-d-old rat were cultured at 0.3 x 106 cells/cm2 in 100-mm dish (22.5 x 106 cells/9 ml per 100-mm dish) and incubated in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 35 C for 16 h. Spent media were collected as described (37).

    Effects of anticollagen antibody or human recombinant TNF{alpha} protein on the assembly of Sertoli cell TJ-barrier in vitro

    Freshly isolated Sertoli cells were plated on Matrigel-coated bicameral units (Millicell HA-filters, effective surface area, ~ 0.6 cm2, Millipore Corp., Bedford, MA) at a density of 0.75 x 106 cells/cm2 (20) and treated as cultures at d 0. Cells were incubated in a humidified atmosphere of 95% air and 5% CO2 (vol/vol) at 35 C with 0.5 ml F12/DMEM each in the apical and basal chamber of the bicameral unit. Media were replaced every 24 h. The assembly of the Sertoli cell TJ-barrier was assessed by measuring the transepithelial electrical resistance (TER) across the cell epithelium using a Millicell electrical resistance system (Millipore Corp.) every 24 h as described (21, 25, 38, 39). In experiments targeted to assess the effects of anticollagen antibody on the assembly of the Sertoli cell TJ-barrier, 1 or 5 µg anticollagen IV IgG (rabbit antihuman collagen IV, catalog no. YMPS44, Accurate Chemical & Scientific Corp., Westbury, NY) was added to both the apical and basal compartments of bicameral units at the time of cell plating and to the daily replacement media. In control experiments, 0.1 or 1 µg rabbit {gamma} -globulin (Sigma, St. Louis, MO) was used.

    To assess the effects of TNF{alpha} on the Sertoli TJ-barrier, 10 or 100 ng/ml of recombinant human TNF{alpha} were added to the basal compartment of the bicameral unit immediately following cell plating. Preliminary experiments had shown that its inclusion in the apical compartment had no effects on the TJ-barrier, seemingly suggesting that TNF{alpha} receptors were recruited only to the basal region of polarized Sertoli cells after forming an epithelium. TNF{alpha} at desired concentrations was also included in the replacement medium. Sertoli cells cultured alone in F12/DMEM without TNF{alpha} or with vehicle (PBS containing 0.1% BSA, wt/vol) were used as controls. To assess the specificity of TNF{alpha} treatment, TNF{alpha} was removed by two successive washes in selected experiments on d 2 to examine whether the perturbed TJ-barrier was capable of resealing. Each time point had triplicate cultures, and each experiment was repeated three times using different batches of Sertoli cells.

    Semiquantitative RT-PCR

    Total RNA was isolated from tissues or cells by RNA STAT-60 (Tel-Test \|[ldquo ]\|B\|[rdquo ]\| Inc., Friendswood, TX) according to the manufacturer’s protocol. Semiquantitative RT-PCR was performed essentially as earlier described (26, 27, 40). To enhance the detection sensitivity and yield semiquantitative data for analysis, trace amount of [{gamma} -32P]-labeled primers were included in the RT-PCR tubes. Briefly, the sense primers of a target gene and S16 were labeled at the 5'-end with [{gamma} -32P]-dATP (specific activity, 6000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) using T4 polynucleotide kinase (Promega Corp., Madison, WI). Approximately 1 x 106 cpm were used per PCR. The ratio of the [{gamma} -32P]-labeled sense primer of a target gene to [{gamma} -32P]-labeled S16 was the same as the unlabeled primers. To ensure the linearity of the target gene and S16 during their amplification, 10-µl aliquots of PCR products at 18, 20, 22, 24, and 26 cycles were withdrawn and resolved onto 5% T polyacrylamide gels using Tris-borate EDTA buffer (45 mM Tris-borate; 1 mM EDTA, pH 8.0, at 22 C) as a running buffer in preliminary experiments. Also, different concentrations of primer pairs and reverse transcription products and annealing temperatures were used in preliminary experiments to ensure that the production of each target gene and S16 in each PCR experiment reported herein were in linear phase. Because of the disparity between the endogenous levels of a target gene and S16, the linear phase of the housekeeping gene, such as S16, was close to its saturation phase. However, the linearity for the target gene was at its exponential phase in the PCR. Following gel electrophoresis, PCR products were visualized by ethidium bromide staining, and autoradiography was performed using X-OMAT AR film (Eastman Kodak Co., Rochester, NY). The authenticity of the PCR products for collagen {alpha} 3(IV), MMP-9, MMP-2, TNF{alpha} , occludin, ZO-1, TIMP-1, and S-16 (Table 1) was confirmed by direct nucleotide sequencing as described (27, 41).

    fig.ommitteed

    Table 1. Primers used for semiquantitative RT-PCR to assess the steady-state mRNA levels of collagen 3 (IV), MMP-9, MMP-2, TNF-, occludin, ZO-1, TIMP-1, and S16

    Immunoblotting analysis

    For immunoblotting, total cell lysates were prepared as follows: 1 ml lysis buffer [0.125 M Tris, pH 6.8, at 22 C containing 1% SDS (wt/vol), 2 mM EDTA, 2 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 1.6% 2-mercaptoethanol (vol/vol), 1 mM sodium orthovanadate (a protein tyrosine phosphatase inhibitor), and 0.1 µM sodium okadate (a protein Ser/Thr phosphatase inhibitor)] was added to the freshly isolated germ cells or to the Sertoli cells in either 100-mm dishes or each well of a 12-well dish. Samples were vortexed, incubated at room temperature for 5–10 min, centrifuged at 15,000 x g at 4 C to remove cellular debris. The clear supernatant was used as whole-cell lysate. SCCM and GCCM were prepared as previously described (19, 37). Protein content was estimated by Coomassie blue dye-binding assay using BSA as a standard (42). Equal amounts of proteins from each sample (100 µg per lane for cell lysates and 40 µg per lane for SCCM and GCCM) were resolved onto 7.5%, 10%, or 12.5% T SDS-polyacrylamide gels by SDS-PAGE under reducing conditions (43) and electroblotted onto nitrocellulose membranes. Membranes were blocked with 6% nonfat dry milk (Nestle, Solon, OH) in PBS-Tris (10 mM sodium phosphate, 0.15 M NaCl, 10 mM Tris, pH 7.4, at 22 C) containing 0.1% (vol/vol) Tween.

    For immunoblottings, the following primary antibodies were used: rabbit anticollagen type IV, which also cross-reacted with {alpha} 1, 2, 3, and 5 chains of type IV collagen (catalog no. sc-11360, lot no. B021), goat anti-TNF{alpha} (catalog no. sc-1351, lot no. K121), goat anti-TIMP-1 (catalog no. sc-6834), rabbit anti-ß1-integrin (catalog no. sc-8978, lot no. B221), goat antiintegrin-linked kinase (ILK) (catalog no. sc-7516, lot no. G111), mouse antiglycogen synthase kinase-3ß (GSK-3ß) (catalog no. sc-7291, lot no. E171), mouse antiphospho-c-Jun (catalog no. sc-822, lot no. G191), rabbit antifocal adhesion kinase (FAK) (catalog no. sc-558, lot no. J051) and mouse anti-c-Src (catalog no. sc-8056, lot no. C051) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-{alpha} 3 chain of collagen IV (catalog no. MAB3, lot no. 97) was from Weislab (Lund, Sweden). Rabbit anti-MMP-9 (catalog no. AB805, lot no. 21100763) was from Chemicon (Temecula, CA). Rabbit antioccludin (catalog no. 71-1500) was from Zymed Laboratories, Inc. (Burlingame, CA). Rabbit antiphospho-GSK-3{alpha} /ß (Ser21/9) (catalog no. 9331, lot no. 2A) and rabbit antiphospho-stress-activated protein kinase/Jun-terminal kinase (SAPK/JNK) pathway kit (catalog no. 9912, lot no. 4), which included phospho-specific anti-SEK1/MKK4 antibody, were from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit antiphospho-FAK (Tyr397) (catalog no. 07-012, lot no. 21019) was from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse anti-p130 Crk-associated substrate (Cas) (catalog no. P27820, lot no. 7) was from Transduction Laboratories, Inc. (Lexington, KY). These antibodies were known to cross-react with the corresponding target proteins in rats as indicated by the manufacturers.

    Depending on the source of the primary antibody, the following secondary antibodies were used. These include goat antirabbit IgG-horseradish peroxidase (HRP) (catalog no. sc-2004), bovine antigoat IgG-HRP (catalog no. sc-2350) and goat antimouse IgG-HRP (catalog no. sc-2005) (Santa Cruz Biotechnology, Inc.). Both the catalog and lot numbers for each antibody used in this report were listed because preliminary experiments had shown that several antibodies from other vendors failed to yield satisfactory results. The immunoreactive target protein in a blot was visualized with an enhanced chemiluminescence system from Amersham Pharmacia Biotech (catalog no. RPN2106) and Biomax MR films (Eastman Kodak Co.).

    Immunohistochemistry

    Immunohistochemistry was performed to localize TNF and MMP-9 in the seminiferous epithelium of normal adult rat testes essentially as previously described (44, 45, 46) using a Histostain SP kit (catalog no. 95-6143, Zymed Laboratories, Inc.). Frozen sections (8 µm thick) obtained with a microtome in a cryostat were air dried, fixed in Bouin’s fixative, and treated with 3% hydrogen peroxide in methanal to block endogenous peroxidases. To minimize nonspecific antibody binding, sections were incubated with serum-blocking solution. Sections were then incubated with primary antibodies in a moist chamber at 35 C overnight. Primary antibodies were used with the following dilutions: goat anti-TNF{alpha} (1:50) and rabbit anti-MMP-9 (1:75). Sections were rinsed and incubated with biotinylated rabbit antigoat IgG or goat antirabbit IgG for 30 min and then with the streptavidin-peroxidase conjugate for 10 min. Sections were then treated with the aminoethylcarbazole mixture (a substrate-chromogen mixture) for 5–10 min, counterstained in hematoxylin, and mounted. Sections were examined and photographed in BX-40 (Olympus Corp., Melville, NY). Controls include sections incubated with normal rabbit serum by substituting the primary antibody, PBS by substituting the primary antibody, the primary antibody preabsorbed with the corresponding peptide used for antibody generation, and normal rabbit or normal goat serum instead of the biotinylated second antibody.

    Statistical analysis

    Multiple comparisons were performed using one-way ANOVA followed by Tukey’s honest significant different test to compare selected pairs of experimental groups using the GB-STAT statistical analysis software package (version 3.0, Dynamic Microsystems, Inc., Silver Spring, MD). In some experiments, t test was also performed to compare a treatment group with its corresponding control.

    Results

    Purity of testicular cells

    The purity of cells used in the studies described in this report was assessed by 1) direct microscopic examination; 2) RT-PCR analysis for testin, a putative Sertoli cell protein not expressed by normal germ cells (31, 32, 33), c-Kit receptor, a spermatogonium specific product (34), HSD, a Leydig cell product (35) and fibronectin, a peritubular myoid cell product (36); and/or 3) DNA flow cytometry (31, 37). For Sertoli cells isolated from testes using rats of different ages, microscopic analysis revealed that the cell purity was routinely greater than 95%. For germ cells, these preparations did not express testin, HSD, or fibronectin. These analyses also illustrate that these cell preparations were contaminated with a negligible number of other cell types. In selected experiments, germ cells were also subjected to DNA flow cytometry as described (31) to assess the relative percentages of spermatogonia, spermatocytes, and spermatids. For instance, germ cells isolated from adult rat testis had a relative percentages of 17%:19%:64% for spermatogonia (2 C, diploid):spermatocytes (S-phase and 4 C, tetraploid):spermatids (1 C, haploid). Elongated spermatids were not detected because they were removed in the glass wool filtration step (31).

    Relative expression of collagen {alpha} 3(IV) in Sertoli and germ cells

    To determine whether Sertoli and germ cells express collagen {alpha} 3(IV), RT-PCR and immunoblot analysis were performed. The steady-state mRNA level of collagen {alpha} 3(IV) in germ cells was found to be comparable to Sertoli cells (Fig. 1, A and B). To confirm that the collagen mRNA detected in particular in germ cells could indeed be translated to a functional protein, immunoblotting analysis was performed (Fig. 1C using both a rabbit anticollagen IV (Santa Cruz Biotechnology, Inc.) and mouse anti-{alpha} 3 chain of collagen IV (Weislab) antibody. Because the rabbit polyclonal antibody from Santa Cruz is reactive with {alpha} 1, 2, 3, and 5 chains of type IV collagen, two immunoreactive bands of approximately 170 kDa (type IV collagen {alpha} 2 chain) and approximately 180 (type IV collagen {alpha} 1/3 chain) kDa were detected in Sertoli and germ cell lysates (Fig. 1C). Only a single band of approximately 180 kDa was detected in GCCM but not SCCM; this immunoreactive band is likely the {alpha} 1(IV) chain and/or {alpha} 3(IV) chain (Fig. 1C). The approximately 180-kDa protein band was subsequently confirmed to be the {alpha} 3(IV) chain using a mouse monoclonal antibody specific to the {alpha} 3(IV) chain (data not shown). Only GCCM, but not SCCM, was shown to contain the {alpha} 3(IV) chain (Fig. 1C). The lack of collagen {alpha} 3(IV) chain in SCCM could be the result of collection time, which was d 8 vs. 16 h for GCCM because the expression of {alpha} 3(IV) chain greatly diminished in Sertoli cells in vitro by d 5 (Fig. 2A).

    fig.ommitteed

    Figure 1. A–I, Differential expression and protein level of collagen 3(IV) in Sertoli and germ cells, SCCM, and GCCM and changes of its expression in these cells and the testis during maturation. Total RNA and whole-cell lysates were extracted from Sertoli cells, germ cells, or whole testis as described in Materials and Methods. Sertoli cells (5 x 104 cells/cm2) isolated from rats at specified ages were cultured in vitro for 3 d with hypotonic treatment on d 2 to remove contaminating germ cells before termination. Semiquantitative RT-PCR was performed to assess changes in the steady-state mRNA level of collagen 3(IV) using a primer pair specific to this target gene and coamplified with S16 (see Table 1). The purity of cells used in these studies was verified by microscopic and other analyses as described in Materials and Methods. A, An autoradiogram showing the relative steady-state mRNA level of collagen 3(IV) in Sertoli and germ cells isolated from 20-d-old rats. B, Corresponding densitometrically scanned results using autoradiograms, such as the one shown in A. C, Immunoblot analysis of collagen IV protein using whole-cell lysates from Sertoli and germ cells, SCCM, and GCCM with approximately 40 µg protein/lane. This analysis illustrates that the collagen 3(IV) mRNA detected in germ cells can translate into functional protein. D, F, and H, Autoradiograms showing changes in the steady-state collagen 3(IV) mRNA level in Sertoli and germ cells and testes during development, respectively. E, G, and I, Corresponding densitometrically scanned results using autoradiograms, such as those shown in D, F, and H and normalized against S-16. Each bar represents a mean ± SD of two to three experiments using different batches of cells or testes from three rats. Statistical analysis was performed by t test by comparing the steady-state mRNA level of collagen 3(IV) in either cells or testes at other ages vs. d 20 (E), 10 (G), or 3 (I), which was arbitrarily set at 1, except for results shown in B in which Sertoli cells were compared with germ cells and vice versa. *, Significantly different, P < 0.05; **, significantly different, P < 0.01; ns, not significantly different.

    fig.ommitteed

    Figure 2. A–L, Changes in the expression of collagen {alpha} 3(IV), MMP-9, and MMP-2 by Sertoli cells during the assembly of inter-Sertoli cell junctions in vitro. Sertoli cells isolated from 20-d-old rat testes were cultured at either low (5 x 104 cells/cm2) (A–F) or high cell density (1 x 106 cells/cm2) (G–L). Cultures were terminated at specified time points for RNA extraction. Time 0 represents RNA STAT-60 added to Sertoli cells approximately 3 h after plating. Semiquantitative RT-PCR was performed to assess the changes in the steady-state mRNA levels of collagen {alpha} 3(IV) (A and G), MMP-2 (C and I), and MMP-9 (E and K) in Sertoli cells and were coamplified with S16 at the time of TJ assembly. B, D, F, H, J, and L are the corresponding densitometrically scanned results using autoradiograms, such as those shown in A, C, E, G, I, and K, and normalized against S-16 and cultures at time 0, which was arbitrarily set at 1. Each bar represents a mean ± SD of three experiments. Each experiment had replicate cultures. *, Significantly different from cultures at time 0 by t test, P < 0.01; **, significantly different from cultures at time 0 by t test, P < 0.001; ns, not significantly different by t test.

    Developmental regulation of collagen {alpha} 3(IV) in Sertoli and germ cells and the testis

    The steady-state mRNA level of collagen {alpha} 3(IV) reduced significantly during Sertoli cell maturation (Fig. 1, D and E). In contrast, its expression increased significantly and peaked at 45–60 d of age in germ cells during maturation (Fig 1, F and G). During testis maturation, the steady-state mRNA level of collagen {alpha} 3(IV) became detectable at 5 d of age and peaked at 10–20 d of age coinciding with the assembly of the BTB (Fig. 1, H and I). Because a decline of the {alpha} 3(IV) chain in the aging testis at 90 d of age was detected (Fig. 1H) but the level of this collagen in germ cells at 90 d of age was relatively high (Fig. 1F), these data seemingly suggest that the collagen {alpha} 3(IV) chain expression by germ cells can be down-regulated by Sertoli cells in the testis in vivo. Another alternative yet possible explanation for the increases in collagen {alpha} 3(IV) expression by germ cells at ages between 45 and 90 d shown in Fig. 1F may be that the collagen {alpha} 3(IV) chain is up-regulated rapidly during germ germ isolation.

    Changes in the expression of collagen {alpha} 3(IV), MMP-9, and MMP-2 in Sertoli cell cultures during junction assembly in vitro

    When Sertoli cells were cultured at low cell density (0.5 x 104 cells/cm2) on Matrigel-coated dishes in serum-free F12/DMEM to allow the assembly of Sertoli cell AJs, it was associated with a significant but transient induction of collagen {alpha} 3(IV) and MMP-9 (Fig. 2, A–D) and a steady rise in the expression of MMP-2 (Fig. 2, E and F) These results seemingly suggest that their expression correlates with Sertoli cell AJ assembly. At high cell density (1 x 106 cells/cm2), a transient but significant increase in collagen {alpha} 3(IV) and MMP-9 steady-state mRNA levels was detected between d 1 and 2 (Fig. 2, G–J) at the time Sertoli cell TJs were being assembled (see also Fig. 3 vs. Fig. 2), whereas MMP-2 was induced throughout the entire culture period (Fig. 2, K and L), implicating the involvement of collagen {alpha} 3(IV), MMP-9, and MMP-2 in Sertoli TJ assembly and its maintenance.

    fig.ommitteed

    Figure 3. Effects of anticollagen antibody on the assembly of inter-Sertoli tight junction-permeability barrier in vitro. Sertoli cells isolated from 20-d-old rats were cultured at a density of 0.75 x 106 cells/cm2 on Matrigel-coated bicameral units for a period of 4 d to allow for the assembly of the inter-Sertoli tight junction-permeability barrier. The assembly of TJ-barrier was assessed by quantifying the TER across the Sertoli cell epithelium as described in Materials and Methods. Sertoli cell monolayers were exposed to either anticollagen antibody (DEAE affinity-purified IgG) or rabbit-globulin (control) by adding anticollagen IgG or {gamma} -globulin at desired concentrations to both the apical and basal compartments of the bicameral units (see arrow) TER readings across the Sertoli cell epithelium were taken at specified time points. Anticollagen IgG or -globulin was also included in the daily replacement medium. Each time point represents triplicate cultures (mean ± SD) from three experiments, using different batches of Sertoli cells. *, Significantly different by t test, compared with the corresponding controls, P < 0.05; **, significantly different by t test, compared with the corresponding controls, P < 0.01; ns, not significantly different by t test, compared with the corresponding controls.

    Effects of anticollagen antibody on the assembly of Sertoli TJ-permeability barrier in vitro

    Because there was a transient but significant induction of collagen {alpha} 3(IV) at the time Sertoli TJs were being assembled in vitro, it is logical to investigate whether a blockade of the collagen network by using an anticollagen IV antibody could perturb the assembly of Sertoli TJ-barrier. The assembly of the TJ-barrier was monitored by quantifying the TER across the Sertoli cell epithelium. It was noted that the TER across the cell epithelium soon after cells were plated in vitro rose steadily, which reached plateau by d 3–4 with a level of approximately 80 ohms·cm2 (Fig. 3), consistent with results of earlier reports (25, 38, 39). The presence of an anticollagen IV antibody indeed perturbed the assembly of the Sertoli cell TJ-barrier in vitro manifested by a dose-dependent decline in TER, compared with Sertoli cells cultured alone (Fig. 3). These effects appeared to be specific because {gamma} -globulin alone had no effects in perturbing the TER (Fig. 3). These results also suggest the potential significance of collagen IV in Sertoli cell TJ assembly.

    Changes in the endogenous steady-state mRNA and protein levels of TNF{alpha} in Sertoli cell cultures during junction assembly in vitro

    Previous studies have shown that an infusion of TNF to rats iv could perturb spermatogenesis by causing germ cell loss from the seminiferous epithelium (16). TNF{alpha} is also a known regulator of collagen and MMPs (47, 48). We sought to investigate whether the mechanism by which TNFinduced germ cell loss relates to its effects on ECM remodeling in the rat testis. The changes in the steady-state mRNA and protein levels of TNF{alpha} in Sertoli cells cultured at high and low cell density were first monitored (Fig. 4, A–D). However, during Sertoli cell TJ-barrier (Fig. 4, A and B, vs. Fig. 3) or AJ (Fig. 4, C and D) assembly, there was a time-dependent TNF{alpha} decline in Sertoli cell cultures (Fig. 4, A–D). These results seemingly suggest that the presence of TNF{alpha} may have a negative effect on the assembly and maintenance of Sertoli TJs and AJs. This trend of time-dependent plummeting in TNF{alpha} expression was verified by immunoblotting using an anti-TNF{alpha} antibody and 40 µg protein of SCCM from the same experiment shown in Fig. 4A (see inset in Fig. 4B) for analysis, which clearly demonstrated a significant reduction in TNF{alpha} (19 kDa) protein during TJ assembly.

    fig.ommitteed

    Figure 4. A–D, Changes in the expression and protein level of TNF{alpha} during the assembly of inter-Sertoli cell TJs (A and B) and AJs (C and D) in vitro. Sertoli cells were cultured at either high (0.75 x 106 cells/cm2) or low cell density (5 x 104 cells/cm2). Cultures were terminated at specified time points for RNA extraction and the preparation of SCCM. Time 0 represents the lysis of Sertoli cells approximately 3 h after plating except in A and B in which 0H represents freshly isolated Sertoli cells terminated immediately after their isolation. Semiquantitative RT-PCR coamplified with S16 and immunoblotting were performed to assess changes in TNF{alpha} steady-state mRNA and protein levels, respectively. B and D, Corresponding densitometrically scanned results using autoradiograms, such as those shown in A and C, and normalized against S16 where cultures at time 0 were arbitrarily set at 1. Each bar represents a mean ± SD of three experiments. Each experiment had replicate cultures. *, Significantly different from cultures at 0H by t test, P < 0.01; **, significantly different from cultures at 0H by t test, P < 0.001; ns, not significantly different.

    Effects of recombinant TNF protein on the assembly of Sertoli cell TJ-barrier in vitro

    To verify the data shown in Fig. 4, that the changes in TNF{alpha} during TJ assembly indeed relate to the event of junction dynamics instead of an endogenous cellular maintenance event, we sought to investigate whether TNF{alpha} can indeed perturb the assembly of Sertoli cell TJs. As expected, the presence of TNF{alpha} perturbed the assembly of Sertoli cell TJs in vitro dose dependently (Fig. 5). When TNF{alpha} was removed by two successive washes on d 2, the perturbed Sertoli cell TJ-barrier resealed as manifested by a rise in TER, illustrating the effects of TNF{alpha} on the assembly of Sertoli cell TJs were likely not the result of cell cytotoxicity (Fig. 5).

    fig.ommitteed

    Figure 5. Effects of recombinant TNF on the assembly of the inter-Sertoli tight junction-permeability barrier in vitro. Sertoli cells were cultured at a density of 0.75 x 106 cells/cm2 on Matrigel-coated bicameral units to allow the assembly of the TJ-permeability barrier. Different concentrations of TNF were included in the basal compartments of bicameral units throughout the culture period. The effects of TNF on the assembly of Sertoli cell TJs in vitro were assessed by quantifying TER across the cell epithelium at specified time points. In selected experiments, TNFwas removed by two successive washes with F12/DMEM on d 2, and the replacement media also did not contain TNF (see arrow). It was noted that TNF had a dose-dependent effect on perturbing the Sertoli cell TJ-barrier. These TNF effects appear to be specific because removal of TNF permitted resealing of the TJ-barrier. Each time point is a mean ± SD of triplicate cultures from two experiments. *, Significantly different by t test, compared with the corresponding controls, P < 0.05; **, significantly different by t test, compared with the corresponding controls, P < 0.01; ns, not significantly different by t test, compared with the corresponding controls.

    Effects of recombinant TNF on the steady-state mRNA and/or protein levels of occludin and ZO-1 in cultured Sertoli cells during Sertoli cell TJ-barrier assembly in vitro

    Recent studies have shown that there is a transient but significant induction of occludin (a TJ-integral membrane protein; Refs. 38 and39 ; for a review, see Ref. 3) and zonula occludens-1 (ZO-1, a TJ-associated peripheral protein that has a signaling function and can also act as a scaffold protein at the site of TJs; Refs. 25 and26 ; for a review, see Ref. 3) at the time of Sertoli TJ assembly in vitro. We thus sought to investigate whether the TNF{alpha} -induced perturbation on the Sertoli TJ barrier is mediated via its effects on these two TJ-proteins. In control cultures without TNF{alpha} , there was an 8-fold and 15-fold increase in occludin and ZO-1 expression, respectively, coinciding with the Sertoli cell TJ assembly (Fig. 6 vs. Fig. 5). However, TNF{alpha} at 10 and 100 ng/ml inhibited this transient induction of occludin expression, but not ZO-1, when Sertoli TJs were assembled (Fig. 6). These changes in the occludin mRNA level during Sertoli cell TJ assembly in the absence or presence of TNF{alpha} were confirmed by immunoblot analysis (see lower panel in Fig. 6A). These results thus suggest that the perturbing effects of TNF{alpha} on the Sertoli cell TJ assembly may be mediated, at least in part, via its inhibitory effects on occludin, a building block of TJs (for a review, see Ref. 3), but not via ZO-1, which is a TJ-associated signaling protein.

    fig.ommitteed

    Figure 6. A–C, Effects of recombinant TNF{alpha} on occludin and ZO-1 gene expression during Sertoli cell culture in vitro. Sertoli cells (0.75 x 106 cells/cm2) isolated from 20-d-old rats were cultured with 10 or 100 ng/ml TNF{alpha} or without TNF{alpha} (control) immediately after their isolation as described in Materials and Methods. Cell cultures were terminated at specified time points for RNA extraction and protein analysis. Semiquantitative RT-PCR and immunoblot were performed to assess the changes in different target gene expression. A and B, Autoradiograms showing changes in the expression of occludin and ZO-1 in Sertoli cells during the assembly of TJ-permeability barrier, respectively, in the absence (control) or presence of either 10 or 100 ng/ml TNF{alpha} . The lower panel in A is the immunoblot to visualize occludin protein using Sertoli cell lysates treated with or without (control) 100 ng/ml TNF{alpha} (~ 100 µg protein/lane). C, Corresponding densitometrically scanned results using autoradiograms, such as those shown in A and B, and normalized against S16 where the level of the corresponding target gene at OH was arbitrarily set at 1. Each bar represents a mean ± SD using results from three experiments. Each experiment had replicate cultures. *, Significantly different by t test, compared with the corresponding controls, P < 0.05; **, significantly different by t test, compared with the corresponding controls, P < 0.01; ns, not significantly different by t test, compared with the corresponding controls.

    Effects of recombinant TNF{alpha} on the steady-state mRNA and/or protein levels of collagen {alpha} 3(IV), MMP-9, MMP-2, and TIMP-1 in cultured Sertoli cells during TNF{alpha} -induced disruption of the Sertoli cell TJ-permeability barrier in vitro

    We next sought to examine whether collagen {alpha} 3(IV), MMP-9, MMP-2, and TIMP-1 were involved during the TNF{alpha} -induced disruption of the Sertoli cell TJ barrier (Fig. 5). It was noted that TNF{alpha} at 10 and 100 ng/ml was capable of inducing the steady-state mRNA of collagen {alpha} 3(IV) and MMP-9; however, it had no effects on MMP-2 (Fig. 7, A, B, C, and E). The protein level of the collagen {alpha} 3(IV) chain in Sertoli cell lysates was also induced by TNF{alpha} at 100 ng/ml using a mouse anticollagen {alpha} 3(IV) (Weislab) antibody consistent with the results of RT-PCR analysis (Fig. 7A, lower panel vs. upper panel). MMP-9 protein was also activated by TNF{alpha} in cultured Sertoli cells and both the pro-MMP-9 (92 kDa) and active-MMP-9 (84 kDa) were detected by immunoblots (Fig. 7B, lower panel). Also, its effects on the induction of steady-state mRNA and protein levels of TIMP-1 was transient (Fig. 7D) vs. its effects on collagen {alpha} 3(IV) and MMP-9 (Fig. 7, D vs. A and B).

    fig.ommitteed

    Figure 7. A–E, Effects of recombinant TNF{alpha} on collagen {alpha} 3(IV), MMP-9, MMP-2, and TIMP-1 gene expression during Sertoli cell culture in vitro. Sertoli cells (0.75 x 106 cells/cm2) isolated from 20-d-old rats were cultured with TNF{alpha} at 10 ng/ml or 100 ng/ml or without TNF{alpha} (control) immediately after their isolation to allow TJ assembly as described in Materials and Methods. Cultures were terminated at specified time points for RNA extraction and protein analysis. Semiquantitative RT-PCR and immunoblot analysis were performed to assess the changes in different target genes expression. The upper panels in A–D are autoradiograms showing the changes in the expression of collagen {alpha} 3(IV), MMP-9, MMP-2, and TIMP-1, respectively, in Sertoli cells during the assembly of the TJ-permeability barrier in the absence (control) or presence of either TNF{alpha} at 10 or 100 ng/ml. The lower panels in A, B, and D are the corresponding immunoblots to visualize collagen {alpha} 3(IV), MMP-9, and TIMP-1 using whole-cell lysates [collagen {alpha} 3(IV)] and the spent medium (MMP-9 and TIMP-1) from Sertoli cells treated with or without (control) 100 ng/ml TNF{alpha} . About 100 µg protein were used per lane for SDS-PAGE. E, Corresponding densitometrically scanned results using autoradiograms, such as those shown in A–D, normalized against S16 where the control cultures without TNF{alpha} at OH were arbitrarily set at 1. Each bar represents a mean ± SD using results from two experiments. Each experiment had replicate cultures. *, Significantly different by t test, compared with the corresponding controls, P < 0.05; **, significantly different by t test, compared with the corresponding controls, P < 0.01; ns, not significantly different by t test, compared with the corresponding controls.

    Immunohistochemistry localization of TNF{alpha} in the seminiferous epithelium of adult rat testis

    In light of the changes of TNF{alpha} detected at the time of Sertoli cell TJ assembly, we next sought to localize TNF{alpha} in the adult rat testis. The localization of TNF{alpha} in the rat testis was stage specific with the highest staining found in the seminiferous epithelium at stages VI–VII, largely around the heads of elongated spermatids adjacent to the seminiferous tubule lumen (Fig. 8, A and D vs. A–C and E–H). Figure 8B is the control cross-section of an adult rat testis in which the primary antibody was substituted by normal rabbit serum (other controls yielded virtually identical results) indicating the reddish brown precipitate of TNF{alpha} shown in Fig. 8A was specific. At stage VIII, this intense TNF{alpha} staining surrounding elongated spermatids virtually disappeared preceding spermiation (Fig. 8E). In stages IX–V (Fig. 8, F–H and A), light staining of TNF{alpha} was detected in pachytene spermatocytes and elongating spermatids. However, in virtually all stages of the cycle, very weak staining of TNF{alpha} was detected at the basal compartment (Fig. 8) confirming the observations (see Figs. 4 and 5) that TNF{alpha} is an important regulator of Sertoli cell TJ dynamics.

    fig.ommitteed

    Figure 8. A–H, Micrographs of cross-section of adult rat testis showing stage-specific localization of immunoreactive TNF{alpha} in the seminiferous epithelium. A, Cross-section of an adult rat testis at low magnification. B, Corresponding control using normal rabbit serum to substitute the primary antibody. Other controls yielded similar results (data not shown). C–H, Cross-sections of tubules at stages V, VI–VII, VIII, IX–X, XI, and XII, respectively. Immunoreactive TNF{alpha} appears as reddish brown precipitate. Insets are selected regions of the seminiferous epithelium at higher magnification illustrating the localization of TNF{alpha} and its cellular association. Bar, 120 µm for A, B; bar, 60 µm for C–H.

    Immunohistochemical localization of MMP-9

    MMP-9 was also localized to the rat testis by immunohistochemistry as shown in Fig. 9, A–F. MMP-9 was found in the seminiferous epithelium virtually in all stages of the cycle and was largely associated with spermatocytes and round spermatids but not elongated spermatids (Fig. 9, A–E; F is a control section in which the primary antibody was substituted by normal rabbit serum, other controls yielded similar results, data not shown). In contrast to TNF{alpha} , which is highest at stages VI–VII (see Fig. 8), MMP-9 was found largely associated with spermatocytes and round spermatids, peaked after spermiation at stage XIV (Fig. 9 vs. Fig. 8).

    fig.ommitteed

    Figure 9. A–F, Immunohistochemical localization of MMP-9 in the rat testis showing stage-specific staining in the seminiferous epithelium. A–E, Corresponding seminiferous epithelium at stages V, VI–VII, VIII, XI, and XIV, respectively. F, Control in which the primary antibody was substituted by normal rabbit serum. Immunoreactive MMP-9 appeared as reddish brown precipitates. Insets in A–E show selected portion of the corresponding staged epithelium at higher magnification illustrating the localization of MMP-9 and its cellular association. Bar, 60 µm.

    TNF{alpha} -induced signaling events at the contact sites of Sertoli cells and basement membrane

    Because occludin, collagen {alpha} 3(IV), MMP-9, and TIMP-1 were shown to be either up- or down-regulated by TNF, we next sought to investigate the possible signaling pathways by which TNF{alpha} mediated its effects. Changes in the protein levels of signal transducers (ß1-integrin, ILK, GSK-3ß, phospho-GSK-3ß, phospho-SEK1/MKK4, and phospho-c-Jun) and components of focal adhesion complex (FAK, phospho-FAK, c-Src, and p130 Cas) in Sertoli cells during TNF-mediated disruption of the Sertoli cell TJ-barrier were detected. It was noted that TNF{alpha} was capable of inducing significant increase in the protein levels of ß1-integrin, ILK, phospho-GSK-3/ß, phospho-SEK1/MKK4, and phospho-c-Jun, phospho-FAK, c-Src, and p130 Cas. However, it had no effects on the protein levels of the nonphosphorylated form of GSK-3{alpha} /ß and FAK during the TNF{alpha} -induced Sertoli cell TJ-barrier disruption (Fig. 10). These results seemingly suggest that TNF{alpha} perturbed the Sertoli cell-TJ barrier via the ß1-integrin-ILK-GSK-3 pathway.

    fig.ommitteed

    Figure 10. A study to assess the possible signal pathway by which TNF uses to regulate the Sertoli cell TJ-barrier in vitro. Studies were conducted by immunoblotting to examine changes in ß1-integrin, ILK, GSK-3ß, phospho-GSK-3ß, FAK, phospho-FAK, c-Src, p130 Cas, phospho-SEK1/MKK4, and phospho-c-Jun in Sertoli cells cultured in vitro during TJ assembly. Sertoli cells (0.75 x 106 cells/cm2) isolated from 20-d-old rats were cultured with TNF at 100 ng/ml or without TNF (controls) immediately after their isolation as described in Materials and Methods. Cell cultures were terminated at specified time points to obtain whole-cell lysates for immunoblotting. Equal amounts of cell lysates (100 µg protein) per lane from different samples within an experimental group were resolved by SDS-PAGE using 10% T polyacrylamide gels under reducing conditions. Proteins on gels were transferred onto nitrocellulose papers and immunostained sequentially using the corresponding antibody against ß1-integrin, ILK, GSK-3ß, phospho-GSK-3ß, FAK, phospho-FAK, c-Src, p130 Cas, phospho-SEK1/MKK4, and phospho-c-Jun. After probing the blot with a primary antibody specific to a target protein and following its visualization by a corresponding second antibody, the blot was incubated with a stripping buffer to remove these antibodies. It was then blocked and reprobed with a different primary antibody against another target protein. As such, the same blot was used for the analysis of up to four to six target proteins with minimal interexperimental variations. Each experiment had replicate cultures, and each experiment was repeated at least three times using different batches of cells. Only a representative set of blots were shown herein because two other experiments yielded virtually identical results.

    Discussion

    Germ cells contribute to the pool of collagen {alpha} 3(IV) chains in the basal lamina of the testis

    Type IV collagen is a triple helical molecule composed of three {alpha} chains, which in turn constitutes a monomer. These monomers in turn become the building blocks of thetype IV collagen network in the basal lamina (for reviews, see Refs. 49 and50). To date, six genetically distinct chains, designated {alpha} 1 to {alpha} 6, have been identified. Each monomer is characterized by a 7S domain near its N terminus, a middle collagenous domain and a carboxyl-terminal noncollagenous (NC1) domain (for reviews, see Refs. 49 and50). The distribution of {alpha} 1(IV) and {alpha} 2(IV) chains is ubiquitous in tissues, whereas {alpha} 3(IV), {alpha} 4(IV), and {alpha} 5(IV) chains have a more restricted tissue distribution (for a review, see Ref. 49). Studies by immunohistochemistry have localized {alpha} 3(IV) and {alpha} 4(IV) chains in the rat basement membrane of the testicular cords, seminiferous epithelium, myoid cell layer in the basal lamina, and tunica albuginea (51). In this report, we have demonstrated that germ cells also contribute significantly to the pool of the {alpha} 3(IV) chain in the seminiferous epithelium behind the BTB, which in turn maintains the homeostasis of the ECM in the rat STBM. These results may also account for the high level of {alpha} 3(IV) chain found in the bovine STBM (52). An increase in {alpha} 3(IV) chain mRNA level during testis maturation between 5 and 60 d of age as reported herein is in agreement with an earlier immunohistochemistry study, which illustrates the strongest {alpha} 3(IV) chain staining in 15-d-old rat testes (51), coinciding with the assembly of BTB. Likewise, these results support the notion that 3(IV) chain is involved in spermatogenesis because the appearance of {alpha} 3(IV) chain mRNA coincides with the initiation of spermatogonial proliferation at 3–6 d after birth (11).

    ECM remodeling by MMPs and TIMPs plays a crucial role in the regulation of Sertoli cell TJ dynamics

    Collagen has been shown to play a role in TJ assembly in brain endothelial cells and A6 cells, a kidney epithelial cell line in vitro (53, 54). Data presented herein demonstrated that ECM, such as collagen IV, can affect Sertoli cell TJ dynamics because an anticollagen IV IgG can perturb the assembly of the TJ-barrier in vitro. Furthermore, there is a transient but significant induction of collagen 3(IV) at the time of Sertoli TJ assembly.

    MMPs are synthesized as inactive zymogens (pro-MMPs), an initial proteolytic cleavage step is required to activate these proteases, and this activation process is largely regulated by TIMPs. The interplay of these molecules determines the rate and extent of ECM remodeling (for a review, see Ref. 13). In this report, a significant but transient induction of MMP-9 is detected at the time of Sertoli cell TJ assembly, suggesting that MMP-9 may play a crucial role in TJ formation by regulating the ECM dynamics. Because plasmin activated by urokinase plasminogen activator [uPA; the assembly of Sertoli cell TJ barrier in vitro is also associated with an induction of uPA (25)] can in turn activate pro-MMPs in vivo (55); thus, the induction of MMP-9 during Sertoli cell TJ assembly may be regulated, at least in part, by uPA via a yet-to-be-defined pathway or mechanism. In contrast to MMP-9, MMP-2 was induced throughout the entire culture period. The fact that the expression patterns of MMP-9 and MMP-2 in Sertoli cells cultured at both low and high cell density were somewhat similar seemingly implicate that they may take part in both TJ and AJ assembly. It is interesting to note that there are two different patterns of MMP and TIMP expression during Sertoli cell TJ assembly. First, MMP-9 and TIMP-1 were induced during Sertoli cell TJ assembly, which plummeted rapidly soon after TJs were assembled. Second, MMP-2 and TIMP-2 were induced throughout the entire culture period during TJ assembly (56).

    TNF{alpha} perturbs the Sertoli cell TJ-permeability barrier in vitro possibly mediated via its effects on occludin and claudin-11

    Studies to date have shown that germ cells and testicular macrophages are the major source of TNF{alpha} in the testis (57). In this report, freshly isolated Sertoli cells were also shown to express TNF{alpha} ; however, its level tumbled rapidly during the assembly of Sertoli TJs, and by d 8 the level of TNF{alpha} was less than 10% of that detected in freshly isolated Sertoli cells. These observations seemingly suggest a regulatory role of TNF{alpha} in Sertoli cell TJ dynamics. Indeed, addition of recombinant TNF{alpha} to Sertoli cell cultures can perturb the Sertoli cell TJ-barrier assembly. Furthermore, TNF{alpha} also inhibits the timely induction of occludin, a TJ-integral membrane protein in Sertoli cells (58), known to be induced at the time of TJ assembly (38, 39). A recent study has also shown that the expression of claudin-11, also a TJ-integral membrane protein, can be inhibited by TNF{alpha} with an ED50 of 4.5 ng/ml in mouse Sertoli cells in vitro (59). Taken collectively, these results suggest that TNF{alpha} perturbs the Sertoli cell TJ-barrier possible via its effects on TJ-integral membrane proteins, such as occludin.

    Unexpectedly, TNF{alpha} failed to inhibit ZO-1 (60), whose expression was induced during TJ assembly (25, 26, 39). It is possible that TNF{alpha} affects only the redistribution of ZO-1 in cultured Sertoli cells, but not its expression, which is supported by the findings that TNF{alpha} and IFN-{gamma} when used together can induce redistribution of the junctional adhesion molecule, another TJ-integral membrane protein, in human endothelial cells (61). Furthermore, in occludin-deficient embryonic stem cells (62) and oligodendrocyte-specific protein/claudin-11 null mice testes (63), ZO-1 was found at the site of TJs regardless whether occludin or claudin was present. Also, the CdCl2-induced Sertoli cell TJ disruption was associated with a loss of occludin induction but not ZO-1 (38, 64).

    TNF{alpha} is known to induce dissociation of epithelial cells by perturbing the expression of cadherin and ß-catenin (65). These observations further support the notion that a reduced TNF{alpha} expression in Sertoli cells cultured in vitro during AJ assembly is needed, permitting the timely expression of cadherin and ß-catenin during junction assembly. Likewise, studies by immunohistochemistry have detected TNF{alpha} at the basal and adluminal compartment of the seminiferous epithelium throughout the epithelial cycle further strengthening the postulate that TNF{alpha} is actively involved in junction dynamics. The fact that there is a surge in TNF accumulation surrounding elongated spermatids at the site of ectoplasmic specialization (ES) at stages VI–VII seemingly suggest that this cytokine may also be functionally significant at spermiation and an important player of ES dynamics.

    TNF{alpha} -induced changes in Sertoli cell TJ dynamics are mediated via its effects on collagen {alpha} 3(IV), MMP-9, and TIMP-1

    The interactions of cytokines with ECM proteins are known to regulate the dynamics of ECM (for a review, see Ref. 66). As reported herein, TNF induces collagen {alpha} 3(IV), MMP-9, and TIMP-1 but not MMP-2 when it perturbs the Sertoli cell TJ-barrier. Unexpectedly, the assembly of Sertoli cell TJs in vitro was associated with an induction of collagen {alpha} 3(IV), MMP-9, and TIMP-1. These results seemingly suggest that these molecules may have a biphasic effect on the Sertoli cell TJ assembly and disassembly. We offer the following rationale to explain the observation that an induction of collagen {alpha} 3(IV), MMP-9, and TIMP-1 is associated with the events of Sertoli cell TJ disassembly. First, the assembly of type IV collagen networks depends not only on the chain availability, but also the NC1 domain in an intact collagen chain also plays a crucial role in chain selection and dimerization for network assembly (50, 67). As such, both the TNF{alpha} -induced expression and activation of MMP-9 [MMP-9 is known to cleave type IV collagen (68)] may break down the collagen network by cleaving the central triple helix of collagen IV, leaving the N-terminal 7S domains and COOH-terminal NC1 domains. Furthermore, this cleavage process may also induce a loss of other basement membrane proteins (such as laminin and entactin) and growth factors (such as TGFß) because type IV collagen network has a scaffolding function for these proteins and growth factors (for reviews, see Refs. 13 and49). This somewhat extensive proteolytic breakdown of ECM proteins may contribute to the TNF{alpha} -mediated disruption of the Sertoli cell TJ-barrier, possibly because cells can no longer attach themselves tightly to an intact ECM (Fig. 11).

    fig.ommitteed

    Figure 11. A schematic drawing that illustrates Sertoli cell TJ dynamics are regulated by the intricate interactions among TNF, MMP-9, TIMP-1, and collagen IV. This diagram was prepared based on earlier reports and reviews (3 13 49 ), illustrating the molecular architecture of tight junctions between adjacent Sertoli cells in the testis. The 6ß1 integrin is localized at the site of ES with the proposed binding ligand, laminin 3 chains, which is a novel, nonbasement membrane-associated laminin chain in the adluminal compartment of the seminiferous epithelium (78 ). The 6ß4 integrin is suggested to localized at base of Sertoli cells (76 ). Based on the results presented herein, it is possible that TNF{alpha} perturbs the Sertoli cell-TJ barrier via its effects on MMP-9, TIMP-1, and collagen IV, which in turn affects the composition and homeostasis of ECM. For instance, the activation of MMP-9 induced by TNF may facilitate the opening of the TJ-barrier permitting the preleptotene/leptotene spermatocytes to translocate across the BTB. Furthermore, cleavage of the collagen network in the basal lamina by the activated MMP-9 can also damage the integrity of the ECM affecting the TJ barrier. The role of TIMP-1 is to limit the action of MMP-9, maintaining the integrity of the BTB.

    Second, the NC1 domains cleaved from the collagen network by MMP-9 during its proteolytic breakdown may set off a chain reaction, further perturbing the type IV collagen network and ECM composition because of the fact that NC1 domains isolated from type IV collagen can bind to the central triple helical domain, which in turn perturbs the lateral association of native type IV collagen, inhibiting the assembly of intact type IV collagen network (69). This hypothesis is further strengthened by a recent study reporting that exogenous addition of NC1 domains can perturb ECM formation in Hydra vulgaris (70).

    Third, proteolysis of ECM proteins by MMPs can also result in the release of biologically active ECM fragments (for reviews, see Refs. 12 and13). For instance, type I collagen fragments are known to induce a rapid disassembly of smooth muscle focal adhesion complex, which is mediated via the integrin-dependent cleavage of FAK, paxillin, and talin (71). Also, these biological active fragments can have a negative feedback effect that inhibits the expression of collagenase and degradation of collagen (72). Thus, an induction of collagen {alpha} 3(IV) and TIMP-1 at the time TNF{alpha} perturbs the Sertoli TJ-barrier may be a negative feedback mediated by the biologically active ECM fragments, which are the cleavage products of the TNF{alpha} -induced MMP-9.

    Furthermore, studies by immunohistochemistry to localize MMP-9 in the seminiferous epithelium have shown that MMP-9 is detected in virtually all stages of the epithelial cycle and is associated with spermatids and spermatocytes largely in the basal and part of the adluminal compartment. Yet MMP-9 peaks at stage XIV and remains associated largely with spermatocytes, spermatids, and possibly spermatogonia in the seminiferous epithelium. In this context, it is of interest to note that the movement of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment occurs in late stage VIII and early IX of the cycle (73) when MMP-9 staining is detected in the seminiferous epithelium near the basal compartment. This observation apparently is in agreement with an earlier report, which also demonstrated an induction of proteolytic activity during junction restructuring, such as the assembly of AJs between Sertoli and germ cells in vitro (24).

    The TNF{alpha} -induced effects on Sertoli cell TJ dynamics are possibly mediated via the ß1-integrin-ILK-GSK-3ß-dependent signaling pathways

    A recent study has shown that MMP-9 in intestinal epithelial cells is regulated by ILK via GSK-3 and activator protein-1 (AP-1) transcription factor (74). ILK also plays a crucial role in cell adhesion because its overexpression in normal epithelial cells can reduce E-cadherin expression as well as induce a redistribution of ß-catenin, moving ß-catenin from the cytoplasm to the nucleus (for a review, see Ref. 75). Interestingly, TNF{alpha} -induced reduction of cadherin and ß-catenin expression also causes the loss of cell adhesiveness in epithelial cells (65). In the testis, ILK has been shown to colocalize with ß1 integrin at the site of both apical and basal ES (76). Thus, the TNF-induced effects on junction dynamics may be mediated via integrin ß-subunit and ILK because the protein levels of these two signal transducers were shown to be stimulated by TNF{alpha} as reported herein. These changes are also coupled with an increase in phosphorylation and inhibition of GSK-3ß activity induced by TNF{alpha} . These observations thus implicate the TNF{alpha} -induced up-regulation of MMP-9 and TJ dynamics is mediated via the ß1-integrin-ILK-GSK-3ß-dependent pathway.

    Besides, several cytokines, including TNF, are known to regulate the expression of MMPs via an induction and/or activation of c-Jun, which binds to the activator protein-1 site in the promoter of MMP genes (for a review, see Ref. 13). Based on the data presented herein, TNF{alpha} apparently activates SEK1/MKK4 and c-Jun, the signal transducers of the SAPK/JNK pathway, in Sertoli cells. And the SAPK/JNK pathway can be regulated, at least in part, by the autophosphorylation of FAK, which recruits c-Src to FAK (for a review, see Ref. 75). Other studies have shown that the TNF{alpha} -induced phosphorylation of proteins in focal adhesion complexes, such as paxillin and FAK, are involved in actin cytoskeleton reorganization (77). In this study, TNF{alpha} was shown to induce phosphorylation of FAK as well as inducing both p130 Cas and c-Src in Sertoli cells. These results further support the notion that the SAPK/JNK pathway is involved in the regulation of MMPs by TNF{alpha} . These data also strongly suggest that TNF{alpha} perturbs the Sertoli cell TJ-barrier by disrupting the actin cytoskeleton via changes in phosphorylation of selected proteins in the focal adhesion complex.

    Acknowledgments

    We want to thank Dr. M. Y. Mo for his excellent assistance in the nucleotide sequence analysis of all the PCR products to confirm their identities used in this studies presented in this article. We also thank Dr. Dolores Mruk for her helpful and critical discussion during the course of this work.

    Received July 31, 2002.

    Accepted for publication September 26, 2002.

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