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The Population Council (M.K.Y.S., D.D.M., C.Y.C.), Center for Biomedical Research, New York, New York 10021; and Department of Zoology (W.M.L.), University of Hong Kong, Hong Kong, Special Administrative Region of China
Address all correspondence and requests for reprints to: C. Yan Cheng, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: y-cheng{at}popcbr.rockefeller.edu.
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Introduction |
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Several proteins have been identified at the site of ES in the testis, which include 
6ß1 integrin (8, 9); vinculin (10); -actinin (11); fimbrin (10); espin (12); myosin VIIa (13); c-Src (14); Csk (14); integrin-linked kinase (ILK) (15); gelsolin (16); phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2; Ref. 16); phosphoinositide-specific phospholipase C (16); Fyn, a member of the Src family protein tyrosine kinase (17); Keap1 (18); and testin (19, 20). Yet little is known about these molecules regarding their function and regulation in the testis. In this report, we have investigated the roles of ß1-integrin and vinculin in AJ dynamics using Sertoli-germ cell cocultures in vitro. These proteins were selected because they are known to colocalize to the actin filament bundles at the site of ES in a stage-dependent manner (9, 15, 21) and are putative constituent proteins of ES. Recent studies have shown that 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) is a potential male oral contraceptive that can induce germ cell loss from the seminiferous epithelium possibly by disrupting the Sertoli-germ cell AJs, such as ES, in the rat testis, without affecting the hypothalamus-pituitary-testicular axis (22, 23) and AJ structures in other epithelia (for review, see Ref. 1). Also, this compound is neither nephrotoxic nor hepatotoxic (1, 22, 23) unlike its analog, lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid], which is known to disrupt the stress fibers and microfilament network in Sertoli cells when administered in vivo by gavage but is toxic (for reviews, see Refs. 1 and 24). As such, this AF-2364-induced germ cell loss is being used as a model to study the cascade of events leading the disassembly of Sertoli and germ cell AJs both in vivo and in vitro.
Studies by immunohistochemistry using a specific phospho-Tyr antibody have shown intensive staining at the site of ES (14, 15), indicating that at least some molecules at this site can be tyrosine phosphorylated. There are also reports illustrating focal adhesion kinase (FAK) can become tyrosine phosphorylated in cells upon integrin clustering or during integrin-mediated cell adhesion (for reviews, see Refs. 25, 26, 27, 28). Activation of FAK occurs during its interaction with the cytoplasmic tail of the clustered ß1-integrin (for review, see Ref. 26). On activation, autophosphorylation of FAK at Tyr397 takes place; this in turn creates a binding site for the Src homology 2 (SH2) domain of Src or Fyn (29, 30) and other effector molecules, such as phosphatidylinositol-3-kinase (PI3K; Ref. 31). The coupling of Src family protein tyrosine kinases to this FAK/Src complex can further modify FAK by inducing phosphorylation of FAK at Tyr576 and Tyr577 in the kinase domain activation loop, which enhances its catalystic activity (32), or else at Tyr925, which creates a binding site for the adaptor Grb2 SH2 domain (33). Besides, Src kinases can phosphorylate other focal adhesion-associated substrates, such as paxillin (34) and p130 Cas (35, 36). Treatment of cells with cytochalasin D, also known to disrupt actin filaments in ES (37, 38), that disrupts the actin cytoskeleton can also inhibit the phosphorylation of FAK (39, 40). It is therefore logical to speculate that ß1-integrin at the site of ES activates FAK, leading to changes in Sertoli-germ cell AJ dynamics.
To test this hypothesis, we have investigated: 1) the protein levels of FAK, p-FAK-Tyr397, PI3K p85 paxillin, and p130 Cas during Sertoli-germ cell AJ assembly and disassembly both in vitro and in vivo; 2) the localization of FAK, p-FAK-Tyr397, and p-FAK-Tyr576 in the seminiferous epithelium from normal and AF-2364-treated rats; 3) colocalization of p-FAK-Tyr397 with vinculin, a putative ES-associated protein (10, 15, 21), FAK, or ZO-1, a putative TJ-associated protein (41) in the seminiferous epithelium by immunofluorescent microscopy; and 4) the constituents of the molecular complex that is present at the site of ES by immunoprecipitation using an anti-p-FAK-Tyr397 antibody. These results suggest that the dynamics of ES, a testis-specific, cell-cell actin-based AJ structure, are regulated by some of the same component proteins, such as FAK and ß1-integrin, that are found in focal adhesion complex (FAC) in other epithelia at the site of cell-matrix focal contacts.
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Materials and Methods |
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Preparation of testicular cells and spent media
Sertoli cell cultures.
Sertoli cells were isolated from 20-d-old rats as previously described (19, 42). At low cell density (5 x 104 cells/cm2), inter-Sertoli TJs could not form because of the lack of close proximity between cells rendering them incapable of assuming the columnar shape, which is common in Sertoli cells when they form TJs in vitro (42, 43, 44). Nonetheless, both AJs and communicating gap junctions (GJs) were present (19, 45). 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 and DMEM (F12/DMEM, 1:1, vol/vol; 
4.5 x 106 cells/9 ml/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 (0.5 or 1 x 106 cells/cm2), all three types of junctions (TJs, AJs, and GJs) were formed. In some experiments, cells were plated on Matrigel-coated (Collaborative Biochemical Products, Bedford, MA; diluted 1:7 with F12/DMEM, vol/vol) 12-well dishes (Corning, Inc., Corning, NY) at a density of 1 x 106 cells/cm2 as previously described (19, 45, 46). 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 of incubation, cultures were hypotonically treated with 20 mM Tris (pH 7.4) for 2.5 min to lyse the residual germ cells (47), to be followed by two successive washes with F12/DMEM to remove germ cell debris. Media were replaced every 24 h. For adult Sertoli cell cultures, Sertoli cells were isolated from rats at 45 and 90 d of age with a purity of approximately 95% as previously described (48). The purity of these Sertoli cell cultures were analyzed microscopically (48, 49) and by RT-PCR using primer pairs specific to markers of Leydig cells (3ß-hydroxysteroid dehydrogenase), germ cells (c-kit receptor), and peritubular myoid cells (fibronectin; Ref. 50).
Germ cell cultures.
Total germ cells were isolated from 90-d-old rats by a mechanical procedure as previously described (51). Sequential filtrations were performed to remove cellular debris, spermatozoa, and elongated spermatids. When the final preparation was analyzed by DNA flow cytometry as previously described (51) and direct microscopic examination (51), it consisted largely of spermatogonia (17%), spermatocytes (18%), and round (prestep 8; 57%) and elongating (step 8) spermatids (8%). 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 (51, 52, 53); c-kit receptor; a spermatogonium product (54); 3ß-hydroxysteroid dehydrogenase; a Leydig cell product (55); and fibronectin, a peritubular myoid cell product (56). The cell number of freshly isolated germ cells was determined by a hemocytometer. The desired cell density was obtained by reconstituting cells in F12/DMEM, supplemented with 6 mM sodium lactate, 2 mM sodium pyruvate, 20 mg/liter gentamicin, and 10 µg/ml bacitracin and were used within 1 h for coculture experiments. In selected experiments (see Fig. 7B
, lower panel), elongated spermatids were not removed by omitting the glass wool filtration steps.
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Preparation of germ cell-conditioned medium (GCCM).
For the preparation of GCCM, freshly isolated germ cells were cultured at 0.3 x 106 cells/cm2 in a 100-mm dish (22.5 x 106 cells/9 ml/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, centrifuged at 800 x g for 1 h to remove residual germ cells, followed by 3000 x g for 1 h to remove cellular debris and designed GCCM. GCCM were concentrated by a Minitan tangential ultrafiltration unit (Millipore Corp., Bedford, MA) equipped with eight Minitan plates [relative molecular mass (Mr)cut-off at 10 kDa], and filtered through 0.2-µm filter units. The protein content was estimated by Coomassie blue dye-binding assay using BSA as a standard (57). To study the effects of GCCM protein on Sertoli cell target gene expression, GCCM were incubated with Sertoli cells cultured alone for 5 d at 5 x 104 cells/cm2 in a 100-mm dish (4.5 x 106 cells/9 ml medium per dish) at a concentration of 1, 50, 500, and 1500 µg protein/ml for 2 h before termination for RNA extraction.
Preparation of nonviable germ cells.
To analyze the effects of nonviable germ cells on Sertoli cell ß1-integrin and vinculin expression, germ cells isolated from adult rat testes were stored at 4 C for approximately 24 h in F12/DMEM with 200 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM EDTA. These nonviable cells, confirmed by erythrosine red dye exclusion test (58), were washed twice in F12/DMEM by centrifugation at 800 x g, 10 min each, and layered onto the Sertoli cell (4.5 x 106 cells/9ml F12/DMEM per 100-mm dish) monolayer that had been cultured alone for 5 d using a germ:Sertoli cell ratio of 1:1 and terminated at specified time points.
Sertoli-germ cell cocultures.
To assess changes in target gene expression during the assembly of Sertoli-germ cell AJs, freshly isolated Sertoli cells as described above were first plated at high cell density (0.5 x 106 cells/cm2) on Matrigel-coated 12-well dishes. These cells were cultured alone for 5 d to allow the establishment of a cell epithelium with TJ, GJ, AJ, and desmosome-like junctions (45) and the endogenous target gene expression to subside before their use for coculture experiments because cell-substratum structures were also formed in these cultures. Freshly isolated germ cells as described above were added onto this cell epithelium on d 6 and cocultured using a Sertoli:germ cell ratio of either 1:1, 1:3, or 1:5 to initiate Sertoli-germ AJ. [Although the assembly of desmosome-like junctions also take place in these cultures (59, 60), we limited our discussion on only AJ assembly throughout the text because we had not included any desmosome-like junction protein markers in our investigation because none of the desmosome junction proteins found in other epithelia, such as desmocollins, desmogleins, desmoplakin, and plakophilin, have been positively identified in the testis (for reviews, see Refs. 1 and 3, 4, 5).]
Earlier studies by electron microscopy have shown that anchoring junction structures, such as desmosome-like junctions, are found between Sertoli and germ cells (up to step 7 round spermatids) within 24–48 h when these cells were cocultured in serum-free media in vitro (59, 60). The presence of functional AJ structures in these cocultures was subsequently characterized in our laboratory by light and fluorescent microscopy and other biochemical analysis (43, 50, 61). The isolated germ cells used in the studies reported herein contained up to step 8 spermatids as earlier described (51, 58), and ultrastructures similar to ES found in vivo had been detected between Sertoli cells and step 8 spermatids in vitro (60). This is not entirely surprising because ES can be formed between Sertoli cells and steps 7/8 round spermatids in vivo (62, 63). Furthermore, current investigations in our laboratory by immunofluorescent microscopy have also identified espin, a putative ES-specific marker (12, 62), in these cocultures (Mruk, D. D., and C. Y. Cheng, unpublished observations). In addition, recently completed electron microscopy study using Sertoli-germ cell cocultures from our laboratory performed at the Rockefeller University BioImaging Resource Center has conclusively identified functional ES structure at the ultrastructural level consistent with earlier published reports (Refs. 59 and 60 and Sui, M. K. Y., and C. Y. Cheng, unpublished observations). Taken collectively, these results clearly illustrate functional AJ structures, such as actin-based ES, are present in the Sertoli-germ cell cocultures used in our studies as reported herein.
Cocultures were terminated at specific time points by RNA STAT-60TM (Tel-Test) for RNA extraction or lysed by sodium dodecyl sulfate (SDS) 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 PMSF, 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)] under reducing conditions for immunoblotting. For immunoprecipitation experiments, lysates of cocultures were obtained by using an immunoprecipitation (IP) buffer containing Nonidet P-40 (NP-40) instead of SDS and mercaptoethanol (0.125 M Tris, pH 6.8, at 22 C containing 1% NP-40 (vol/vol), 2 mM EDTA, 2 mM N-ethylmaleimide, 2 mM PMSF, 1 mM sodium orthovanadate, and 0.1 µM sodium okadate) to permit antigen-antibody interactions. In selected experiments (see Fig. 7B
, lower panel), total germ cells with elongated spermatids were also used for the Sertoli-germ cell coculture experiments in which the glass wool filtration steps were omitted. These cocultures were then processed for immunoprecipitation on d 2 using an anti-FAK-Tyr397 as described below.
Seminiferous tubule cultures and lysate preparation
Seminiferous tubules were isolated from testes of adult rats (300 g body weight) by enzymatic treatment as earlier described from this laboratory with negligible Leydig cell contamination (64). Tubules virtually freed of interstitial cell contamination were then trimmed into approximately 2-mm fragments and incubated for 36–48 h at 35 C in F12/DMEM with insulin (20 µg/ml), transferrin (20 µg/ml), gentamicin (100 µg/ml), and penicillin (100 IU/ml) in a final volume of tubules from one testis/25 ml F12/DMEM. Thereafter, lysates were obtained from seminiferous tubules by treating tubules with the IP buffer [0.125 M Tris, pH 6.8, at 22 C containing 1% NP-40 (vol/vol), 2 mM EDTA, 2 mM N-ethylmaleimide, 2 mM PMSF, 1 mM sodium orthovanadate, and 0.1 µM sodium okadate] using an IP buffer:tissue ratio of 3:1, vortexed for 30 sec, sonicated (two times, 5 sec each interspaced by 20 sec, with samples incubated in melting mice) at 4 C, centrifuged at 15,000 x g for 20 min to remove pellets. The clear supernatant was used as seminiferous tubule lysates.
Treatment of rats with AF-2364 to perturb Sertoli-germ cell AJs leading to germ cell loss from the seminiferous epithelium
Treatment of rats with AF-2364 by gavage.
AF-2364 was synthesized as previously described from this laboratory with a purity of greater than 99.8% when assessed by elemental analysis, nuclear magnetic resonance, HPLC, and mass spectrometry (23). This compound was shown to perturb Sertoli-germ cell adhesion function in the testis inducing premature loss of germ cells from the seminiferous epithelium in rats, causing reversible infertility in treated animals (22, 23). Yet AF-2364 is neither nephrotoxic nor hepatotoxic (22, 23), and recent mutagenicity and acute toxicity studies in mice and rats were completed conducted by licensed toxicologists and have shown that it is safe for further development (for review, see Ref. 1). The apparent site by which AF-2364 exerts its action is one of the multiprotein complexes composed of at least integrin-testin-cadherin in the testis-specific ES (for review, see Ref. 1). Adult rats weighing between 250 and 300 g were fed with one dose of AF-2364 at 50 mg/kg body weight, which is known to perturb Sertoli-germ cell AJs inducing germ cells loss, in particular round and elongated spermatids from the seminiferous epithelium. The time when rats were administered with AF-2364 was designated time 0 (control). Thereafter, rats were housed separately for 8 d. Testes were removed at a specified time point with a group of three rats used for each time point. For immunoblotting, testes were homogenized using a Tissumizer (Tekmar, Cincinnati, OH) for protein extraction either by SDS lysis buffer (immunoblotting) or by IP buffer. For immunohistochemistry, testes were immediately frozen in liquid nitrogen and stored at -80 C until sectioning in a cryostat.
Treatment of Sertoli-germ cell cocultures with AF-2364.
Sertoli cells (0.5 x 106 cells/cm2) isolated from 20-d-old rats were cultured for 5 d to form an epithelium with TJs and AJs. On d 6, germ cells isolated from 90-d-old rats were added onto this cell epithelium using a Sertoli:germ ratio of 1:1 and were cocultured for an additional 2 d. On d 9, Sertoli-germ cell cocultures were incubated with either vehicle (ethanol) or different doses of AF-2364 (1, 50, and 500 ng/ml) and terminated at specified time points.
Treatment of Sertoli cells with AF-2364 and lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid].
Sertoli cells (0.5 x 106 cells/cm2) isolated from 20-d-old rats as described above were cultured for 8 d alone and then incubated with 1, 50, and 500 ng/ml AF-2364 for 1 h and terminated thereafter. For lonidamine treatment, Sertoli cells were cultured for 2 d and then incubated with 1, 50, and 500 ng/ml lonidamine for 24 h and terminated thereafter.
Treatment of germ cells with AF-2364.
Germ cells (22.5 x 106 cells/9 ml per 100-mm dish) isolated from 90-d-old rats were incubated immediately with 1, 50, and 500 ng/ml AF-2364 for 1 h and terminated thereafter.
Semiquantitative RT-PCR
Total RNA was isolated from tissues or cells by RNA STAT-60 (Tel-Test), and RNA concentration was quantified by spectrophotometry at 260 nm using an RNA/DNA calculator (model GeneQuant II, Pharmacia Biotech, Uppsala, Sweden). Semiquantitative RT-PCR was performed essentially as previously described (45, 46, 65, 66). To enhance the detection sensitivity and to yield semiquantitative data for analysis and comparison after densitometric scanning of the resultant autoradiograms, a trace amount of [
-32P]-labeled primers were also included in the RT-PCR tubes. Briefly, the sense primers of a target gene and S16 were labeled at the 5'-end with [-32P]-dATP (specific activity, 6000 Ci/mmol, Amersham) using T4 polynucleotide kinase (Promega Corp., Madison, WI). Approximately, 1 x 106 cpm were used per PCR and the ratio of the [-32P]-labeled sense primer of a target gene to [-32P]-labeled S16 was the same as the unlabeled primers.
To ensure the linearity of a 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 0.5x TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) as a running buffer in preliminary experiments. Also different concentrations of primer pairs, reverse transcription products and annealing temperatures were used in preliminary experiments for each target gene to ensure its production and S16 in each PCR experiment 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. For the target gene, its linearity was at its exponential phase in the PCR. It is because of this disparity issue we had made every effort to include results of immunoblot analysis to verify data of RT-PCR. Furthermore, the additional protein analysis ensures that the detected changes in mRNA levels indeed translate into functional proteins, which are the effectors to induce any phenotypic and/or cellular changes. The PCR products were visualized by ethidium bromide staining and autoradiography was performed using X-OMAT AR film (Eastman Kodak Co., Rochester, NY) after gels were dried. The authenticity of the PCR products for ß1 integrin, vinculin, and S16 (Table 1) was confirmed by direct nucleotide sequencing as previously described (46, 61).
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100 µg/lane) were resolved onto 7.5% or 10% T SDS-polyacrylamide gels by SDS-PAGE under reducing conditions (67) and electroblotted onto nitrocellulose papers. 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% Tween 20 (vol/vol). For immunoblotting, the following primary antibodies were used: rabbit anti-ß1-integrin (catalog no. sc-8978, lot no. B221), rabbit anti-FAK (catalog no. sc-558, lot no. J051), and mouse anti-PI3K p85 (catalog no. sc-1637, lot no. J101) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse antivinculin (catalog no. V9131, lot no. 70K4877) was from Sigma (St. Louis, MO). Rabbit anti-p-FAK-Tyr397 (catalog no. 07-012, lot no. 21019) and rabbit anti-p-FAK-Tyr576 (catalog no. 07-157, lot no. 20262) were from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse antipaxillin (catalog no. P13520, lot no. 16) and mouse anti-p130 Cas (catalog no. P27820, lot no. 7) were 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. After the primary antibody incubation, membranes were incubated with either one of the following secondary antibodies depending on the source of the primary antibody. These include goat antirabbit IgG-horseradish peroxidase (catalog no. sc-2004, lot no. G091) and goat antimouse IgG-horseradish peroxidase (catalog no. sc-2005, lot no. H231), which were purchased from Santa Cruz Biotechnology. The blots were then developed with an enhanced chemiluminescence system using a kit from Amersham Pharmacia Biotech (Piscataway, NJ). We have listed both the catalog and lot numbers for each specific antibody used in this report because preliminary experiments have shown that several antibodies from other vendors failed to yield satisfactory results.
IP
About 500 µg protein of whole-cell lysates of Sertoli-germ cell cocultures (in 250–500 µl sample size) terminated on d 2 after addition of germ cells onto the Sertoli cell epithelium and seminiferous tubules after 36–48 h in culture, and lysates of adult rat testes were pretreated by incubating with 5 µl normal rabbit serum for 3 h at room temperature to be followed by 20 µl protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h to eliminate proteins in the lysates that would otherwise nonspecifically bind to rabbit serum proteins and subsequently bound to protein A/G PLUS-agarose. The lysates were then centrifuged at 1000 x g for 5 min to pellet the agarose beads and supernatant was collected. Two microgram of either anti-FAK or anti-p-FAK-Tyr397 antibody were added to the supernatant and incubated overnight. Thereafter, 20 µl protein A/G PLUS-agarose was added to the lysates and incubated for 4 h. The samples were centrifuged at 1000 x g for 5 min to collect pellet. Supernatant was discarded and the immune complexes were washed four times with IP buffer. After the final wash, the immunocomplexes were resuspended in 50 µl SDS-PAGE sample buffer [0.125 M Tris, pH 6.8 at 22 C containing 1% SDS (wt/vol), 20% glycerol, 1.6% 2-mercaptoethanol (vol/vol)] and heated for 10 min at 100 C to extract the proteins. Beads were pelleted by centrifugation and supernatant was collected and resolved by SDS-PAGE using a 7.5% T polyacrylamide gel under reducing conditions and were immunoblotted with mouse anti-p-Tyr (p-Tyr-100; catalog no. 9411) (Cell Signaling Technology, Inc., Beverly, MA) or anti-FAK antibody (for FAK immunoprecipitation experiment); and anti-ß1-integrin, anti-vinculin, anti-c-Src (catalog no. sc-8056, lot no. C051; Santa Cruz Biotechnology), or p-FAK-Tyr397 antibody (for P-FAK-Tyr397 immunoprecipitation experiment). Whole-cell lysates of Sertoli-germ cell cocultures isolated on d 2, and testicular lysates and/or seminiferous tubular lysates without incubation with antibodies or with incubation with normal rabbit serum were used as negative controls.
Immunohistochemistry
Immunohistochemistry was performed to localize FAK and p-FAK in the seminiferous epithelium of normal and AF-2364-treated rat testes essentially as previously described (68, 69, 70) using Histostain SP kits (catalog no. 95-6143) from Zymed Laboratories, Inc. Corp. (Burlingame, CA). Briefly, animals were killed by CO2 asphyxiation. Testes were removed immediately, embedded in O.C.T. compound (Miles Scientific, Elkhart, IN), and frozen in liquid nitrogen. All tissue blocks were stored at -80 C until used. Frozen sections (8 µm thick) were cut at -20 C with a disposable blade in a cryostat (Hacker, Fairfield, NJ) and mounted on poly-L-lysine (Sigma, Mr > 150 kDa)-coated glass slides. Sections were air dried at room temperature, fixed in modified Bouin’s fixative for 5–10 min, and washed thoroughly with PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4 at 22 C). Streptavidin-biotin peroxidase immunostaining was performed as follows. Briefly, fixed sections were treated with 3% hydrogen peroxide in methanol for 5 min to block endogenous peroxidase activity. To minimize the nonspecific antibody binding, sections were incubated with a serum blocking solution (Zymed Laboratories, Inc. Corp.) or 10% nonimmune goat serum. Sections were then incubated with primary antibodies in a moist chamber at 35 C overnight. Primary antibodies were used with the following dilution: rabbit anti-FAK (1:50 to 1:100), rabbit anti-p-FAK-Tyr397 (1:100 to 1:250), and rabbit anti-p-FAK-Tyr576 (1:20 to 1:150). Sections were washed thoroughly with PBS and incubated with biotinylated goat antirabbit IgG for 30 min and then with the streptavidin-peroxidase conjugate for 10 min. Sections were treated with the aminoethylcarbazole mixture (substrate-chromogen mixture) for 5–10 min. Sections were counterstained in hematoxylin and mounted. Sections were examined and photographed in a BX-40 (Olympus Corp., Melville, NY) using planapochromat x10, x20, and x40 objectives and an 82A blue filter.
All micrographs were digitally acquired using a digital imaging camera (Olympus Corp.) interfaced to a Macintosh G4 computer running under Mac OS 9.22 and analyzed by Adobe Photoshop (version 7.0). At least 50–100 sections were examined from each testis and at least three rats were used per time point in each experimental set. Controls consisted of: 1) sections incubated with PBS instead of the specific primary antibody; 2) sections incubated with normal rabbit serum at the same dilution as of the specific primary antibody but omitting the primary antibody incubation; 3) the secondary antibody replaced with normal rabbit serum; and 4) sections incubated with the primary antibody that had been preabsorbed with lysates of seminiferous tubules or control peptides provided by the manufacturer (such as Santa Cruz Biotechnology). Control and experimental slides were immunostained simultaneously in the same experiment session. For preabsorbed controls, approximately 500 µg protein of seminiferous tubule lysate (or 5–10 µg blocking peptide if available from the antibody vendor) in 10 µl immunoprecipitation buffer (see above) was added to 200 µl diluted rabbit anti-p-FAK-Tyr397, rabbit anti-p-FAK-Tyr576, or rabbit anti-FAK and incubated overnight at 4 C with agitation before it was used for immunostaining. In all experiments, control slides yielded nondetectable staining illustrating specificity of the staining results.
Immunofluorescent microscopy for colocalization of p-FAK, vinculin, ZO-1, and FAK to the ES in the seminiferous epithelium of rat testes
To confirm results of the immunoprecipitation experiments that p-FAK, the phosphorylated (activated) form of FAK, indeed associates with the apical ES at the adluminal compartment of the seminiferous epithelium, immunofluorescent microscopy was performed essentially as previously described for testin (20) and cadherin/catenin (50) from this laboratory. Double-fluorescent probes, namely fluorescein isothiocyanate (FITC) and Cy3, were used to colocalize p-FAK with vinculin [a putative ES-associated protein (10, 15, 21), which served as a positive control], ZO-1 [a TJ-associated protein in the testis (41), which served as a negative control), or FAK in the same tissue sections. The colocalization of p-FAK with vinculin to the same site of apical ES in the seminiferous epithelium will strengthen results of the immunoprecipitation and immunohistochemistry experiments, indicating that the phosphorylated form of FAK is indeed associated with the ES structure. Briefly, testes removed from adult rats were embedded in O.C.T. compound (Miles Scientific) and frozen in liquid nitrogen. Frozen sections of testis (8 µm thick) were cut at -20 C in a cryostat and mounted on poly-L-lysine (Mr, 150 kDa)-coated glass slides. Sections on slides were placed at 4 C for 10 min and then air dried at room temperature and fixed in modified Bouin’s solution (4% formaldehyde in picric acid). Sections were then treated with 3% H2O2 in methanol to block endogenous peroxidase activity, followed by 10% nonimmune goat serum to minimize nonspecific antibody binding as earlier described (20, 50, 68, 69).
Sections were subsequently incubated with a rabbit anti-p-FAK-Tyr397 antibody (Upstate Biotechnology, Inc., catalog no. 07-012, lot no. 21019) followed by a goat-antirabbit IgG-FITC (Zymed Laboratories, Inc. Corp., catalog no. 62-6111, lot no. 10665523). Thereafter, sections were washed in PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4, at 22 C), and incubated with a mouse antivinculin antibody (Sigma, catalog no. V9131, lot no. 70K4877) or a mouse anti-FAK antibody (Transduction Laboratories, Inc., catalog no. 610088, lot no. 20) followed by a goat antimouse IgG-Cy3 (Zymed Laboratories, Inc. Corp., catalog no. 81-6515, lot no. 11067429). For colocalization study for p-FAK and ZO-1, the first pair of primary and secondary antibody that was rabbit anti-p-FAK-Tyr397 antibody and goat-antirabbit IgG-Cy3 (Zymed Laboratories, Inc. Corp., catalog no. 81-6115; lot no. 10966881), respectively. And the second antibody was mouse anti-ZO-1-FITC conjugate (Zymed Laboratories, Inc. Corp., catalog no. 33-9111, lot no. 10665662). Sections were then mounted in Vectashield (Vector Laboratories, Burlingame, CA), and fluorescent microscopy was performed using a BX40 microscope (Olympus Corp.) equipped with UPlanF1 fluorescent optics (Olympus Corp.). All images were digitally acquired in Adobe Photoshop (version 7.0) and analyzed with Image-Pro Plus (version 4.5) software (Media Cybernetics, Silver Spring, MD) in a Compaq SP700 workstation running under Windows XP. Controls included: 1) sections incubate with normal rabbit serum instead of the primary antibody, 2) secondary antibody alone without the use of primary antibody, and 3) primary antibody preabsorbed with seminiferous tubule lysates or blocking peptide as described above. For all controls, they failed to yield detectable fluorescent staining, illustrating specificity of the antibody used for immunofluorescent microscopy.
Statistical analysis
Multiple comparisons were performed using one-way ANOVA followed by Tukey’s honestly significant difference test to compare selected pairs of experimental groups so that changes in the expression of a target gene at a selected time point between samples within an experimental group can be compared. In selected experiments, t test was also performed by comparing treatment groups with the corresponding controls. Statistical analysis was performed using the GB-STAT statistical analysis software package (version 7.0, Dynamic Microsystem, Inc., Silver Spring, MD).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A–F). The steady-state mRNA level of ß1-integrin remained relatively steady during Sertoli cell maturation (Fig. 1, C and D). Its expression by germ cells plummeted drastically and became almost undetectable after 20 d of age (Fig. 1, E and F). During testis maturation, the steady-state mRNA level of ß1-integrin became clearly detectable at 5 d of ages coinciding with the initiation of spermatogonial proliferation at 3–6 d after birth and peaked at 10–20 d of age coinciding with the assembly of the BTB, and by 90–120 d of age, it was plunged to a level approximately one third of those rats at 5–60 d of age (Fig. 1, G and H). This lowering in ß1-integrin level in adult rat testes could be the result of an increase in the ratio of germ cells vs. Sertoli cells in the seminiferous epithelium.
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, A and B). The steady-state mRNA level of vinculin plummeted during Sertoli cell maturation (Fig. 2, C and D) in contrast to the pattern of ß1-integrin, which remained relatively stable (Fig. 2, C and D vs. Fig. 1, C and D). Unlike ß1-integrin, its steady-state mRNA level in germ cells, although relatively low, compared with Sertoli cells, increased steadily and peaked at 90 d of age during germ cell maturation (Fig. 2, E and F). During testis maturation, the steady-state mRNA level of vinculin became detectable at 5 d of ages and peaked at 60 d of age (Fig. 2, G and H). Because germ cells contributed more RNA than Sertoli cells in the testis samples being analyzed at 90 d of age vs. 20 d, it is likely that the increase in vinculin expression in the testis during maturation, as shown in Fig. 2, G and H, could be the result of increased Sertoli-germ cell interactions, to be investigated in the following section (see Fig. 4).
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, E and F). On d 6, freshly isolated germ cells from 90-d-old rat testes were added onto this Sertoli cell epithelium and cocultured at a Sertoli:germ cell ratio of 1:1 or at different ratios to initiate the Sertoli-germ cell AJ assembly. Cultures were terminated at specific time points. Semiquantitative RT-PCR and immunoblotting were performed. A transient but significant induction of both mRNA (Fig. 3A, upper panel) and protein levels (Fig. 3A, lower panel) of ß1-integrin was detected during AJ assembly (Fig. 3, A and B), seemingly suggesting that ß1-integrin is involved in the Sertoli-germ cell AJ assembly because Sertoli cells cultured alone without germ cell addition failed to display an increase in ß1-integrin (data not shown). To assess the effects of the soluble factor(s) in GCCM on Sertoli cell ß1-integrin expression, different concentrations of GCCM (1, 50, and 500 µg protein/ml) were added to the Sertoli cell monolayer (5 x 104 cells/cm2) on d 6 and incubated for 2 h; a significant and dose-dependent increase in the ß1-integrin expression was observed (Fig. 3, C and D). To examine the effect of cell-cell contact on Sertoli cell ß1-integrin expression by germ cells, Sertoli cells were cultured with nonviable germ cells. Likewise, germ cells, even nonviable, could induce Sertoli cell ß1-integrin expression (Fig. 3, E and F). To further confirm the effects of germ cells on Sertoli cell ß1-integrin expression, increasing numbers of germ cells were cocultured with Sertoli cells for 0 h and 2 h. Similarly, an increase in germ cells indeed increased the Sertoli cell ß1-integrin expression (Fig. 3, G and H) when germ cells per se contributed very little ß1-integrin to the cocultures (see Fig. 1, E and F). These results clearly illustrate that the Sertoli cell ß1-integrin steady-state mRNA level can be induced by germ cells via the soluble factor(s) secreted by germ cells as well as cell-cell contact.
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, G and H). On d 6, freshly isolated germ cells were added onto this Sertoli cell epithelium to initiate AJ assembly, and these cocultures were terminated at specific time points. Semiquantitative RT-PCR and immunoblotting were then performed. A transient but significant induction of both mRNA (Fig. 4A, upper panel) and protein (Fig. 4A, lower panel) levels of vinculin was detected during AJ assembly (Fig. 4, A and B) but not in Sertoli cells cultured alone without germ cell addition (data not shown), suggesting that vinculin is involved in the Sertoli-germ cell AJ assembly. To assess the effects of the soluble factor(s) in GCCM on Sertoli cell vinculin expression, different concentrations of GCCM (1, 50, 500, and 1500 µg protein/ml) were added to the Sertoli cell monolayer (5 x 104 cells/cm2) on d 6 and incubated for 2 h. In contrast to ß1-integrin, the Sertoli cell vinculin expression was not affected by GCCM (Fig. 4, C and D). To define the effects of cell-cell contacts on Sertoli cell vinculin expression by germ cells, Sertoli cells were cocultured with nonviable germ cells. In contrast to GCCM, cocultures of Sertoli cells with nonviable germ cells induced Sertoli cell vinculin expression, indicating the importance of cell-cell contacts in its regulation (Fig. 4, E and F). To further confirm the effects of germ cells on Sertoli cell vinculin expression, increasing numbers of germ cells were cocultured with Sertoli cells for 0 h and 2 h. A germ cell number-dependent increase in Sertoli cell vinculin expression was observed (Fig. 4, G and H). These results thus illustrate that the Sertoli cell vinculin expression is stimulated by germ cells via cell-cell contacts.
Levels of endogenous steady-state mRNA and protein of ß1-integrin and vinculin in Sertoli cell cultures during junction assembly in vitro
To investigate whether there are changes in ß1-integrin and vinculin during Sertoli cell AJ and TJ assembly, their steady-state mRNA and protein levels in Sertoli cells cultured at high and low cell density were quantified when these junctions were assembled. 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 without TJs because of low proximity between cells, it was associated with a significant induction of ß1-integrin mRNA (Fig. 5, A and B) but not vinculin (Fig. 5, C and D). At high cell density (1 x 106 cells/cm2), a significant increase in ß1-integrin steady-state mRNA and protein levels were detected (Fig. 5, E and F) at the time Sertoli cell TJs were being assembled. Unlike ß1-integrin, no changes in vinculin were detected (Fig. 5, G and H). These results seemingly suggest that ß1-integrin, but not vinculin, was involved in Sertoli cell AJ and TJ assembly.
Changes in the protein levels of FAK, p-FAK-Tyr397, PI3K p85, paxillin, and p130 Cas in Sertoli germ cell cocultures during AJ assembly
Data shown in Figs. 3 and 4 have illustrated a transient but significant induction of both ß1-integrin and vinculin during Sertoli-germ cell AJ assembly. It is possible that such an induction in ß1-integrin will in turn activate the downstream integrin-related proteins that constitute the ES complexes. To explore such a possibility, Sertoli-germ cell cocultures were terminated at specific time points for immunoblotting to examine the protein levels of several ES-associated proteins. Indeed, there were transient but significant inductions in the protein levels of p-FAK-Tyr397 and PI3K p85 during Sertoli-germ cell AJ assembly (Fig. 6). In contrast, the protein levels of the nonphosphorylated FAK, paxillin, and p130 Cas remained unaltered during Sertoli-germ cell AJ assembly (Fig. 6). Taken collectively, these results seemingly suggest that p-FAK-Tyr397 and PI3K p85 take part in the regulation of Sertoli-germ cell AJ assembly. It might be argued that such changes shown in Fig. 6 (upper and lower panels) could be the results of changes in Sertoli-substratum structures when germ cells were layered onto the Sertoli cell epithelium. Nonetheless, this possibility is highly unlikely. First, the Sertoli cells used for the coculture experiment had been cultured for 5 d, forming an epithelium with intact cell-substratum structure (see Materials and Methods). Second, it was noted that in the same experiment when germ cells were not added to the Sertoli cell epithelium, these changes in the expression of ES-associated proteins were not detected (data not shown).
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). Sertoli-germ cell cocultures were terminated on d 2 following addition of germ cells onto the Sertoli cell epithelium where Sertoli cells had been cultured alone for 5 d and whole-cell lysates were extracted from these cocultures. In parallel experiments, lysates were also obtained from adult rat testes. Thereafter, immunoprecipitation was performed using an anti-FAK antibody and the immunoblot was stained with an antiphospho-Tyr antibody (Fig. 7A, left panel) vs. an anti-FAK antibody (Fig. 7A, right panel). A 125-kDa band was detected in both Sertoli-germ cell cocultures and extracts of adult rat testes with an antiphospho-Tyr antibody (Fig. 7A, left panel), which had the same electrophoretic mobility when the same blot was stained by an anti-FAK antibody (Fig. 7A, right panel), demonstrating Tyr-phosphorylation of FAK indeed occurred in the samples during AJ assembly (Fig. 7A).
We next investigated whether the phosphorylated FAK associated tightly with ß1-integrin, c-Src, and vinculin forming a complex stable enough to be extracted by immunoprecipitation. Briefly, Sertoli-germ cell, seminiferous tubule, or testicular lysates were immunoprecipitated by using an anti-p-FAK-Tyr397 antibody. The immunocomplexes were then extracted in SDS sample buffer, resolved by SDS-PAGE, electroblotted onto nitrocellulose membrane, and stained sequentially by using antibodies against ß1-integrin, vinculin, c-Src, and p-FAK-Tyr397. It was noted that p-FAK-Tyr397 antibody pulled out ß1-integrin, vinculin, and c-Src (Fig. 7B, upper panel), demonstrating the stable interactions among p-FAK-Tyr397, ß1-integrin, vinculin, and c-Src. It is of note that in the coculture experiments described above, most of the elongated spermatids (poststep 8 spermatids) were removed from total germ cells isolated from testes in the glass wool filtration step and testicular lysates might contain other FAC proteins derived from Leydig or peritubular myoid cells (or even small blood vessels in the interstitium). As such, the association of different proteins with pFAK-Tyr397 shown in Fig. 7B (upper panel) could possibly be the result of other cellular contamination. To verify that this biochemical observation is beyond refute, we had also used Sertoli-germ cell cocultures in which the glass wool filtration step was omitted in germ cell isolation, thereby retaining elongated spermatids in the preparation for lysate preparation. Furthermore, we also included seminiferous tubule lysates for immunoprecipitation, which were shown to have negligible Leydig cell and myoid cell contamination (64). For instance, these tubules failed to respond to human chorionic gonadotropin treatment, illustrating the number of Leydig cells in the tubule cultures, if any, is negligible (50, 64). When similar amounts of samples (all of the immunocomplexes derived from 500 µg total proteins used as starting materials for IP with anti-p-FAK-Tyr397 were analyzed (Fig. 7B, lower panel vs. upper panel), p-FAK-Tyr397 indeed was shown to associate with ß1-integrin, vinculin, and c-Src (Fig. 7B, lower panel).
It is ironic that the results shown in Fig. 7B (both panels) are not precisely quantitative, even though the same amounts of total proteins (500 µg) were used for IP with anti-p-FAK-Tyr397 and all of the recovered immunocomplexes were used for SDS-PAGE. Yet the total immunoprecipitated p-FAK-Tyr397 recovered from Sertoli-germ cell cocultures with elongated spermatids (S/Sp, Fig. 7B, lower panel, last column) is at least approximately 3-fold higher than that recovered from Sertoli-germ cell cocultures in which most, if not all, of the elongated spermatids (poststep 8 spermatids) were removed by the glass wool filtration step (S/G, Fig. 7B, upper panel, last column). This result is thus consistent with the notion that p-FAK is largely associated with apical ES at the site between Sertoli cells and elongating/elongated spermatids. Taking these data and results of immunohistochemistry and immunofluorescent microscopy (Figs. 8 and 9, see below) collectively, which coupled with the fact that the Sertoli and germ cells used for the studies presented herein were contaminated with negligible number of other cell types (see Ref. 50 and Materials and Methods), p-FAK-Tyr397 is structurally and functionally associated with ß1-integrin, vinculin, and c-Src in the ES.
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, A–C and E–J). The p-FAK-Tyr397 and p-FAK-Tyr576 were colocalized in the seminiferous epithelium virtually to the same sites showing stage specificity with the highest staining found at stages VI–VIII largely surrounding the heads of elongated spermatids adjacent to the seminiferous tubule lumen and at the site of cell-cell contacts between Sertoli cells and round spermatids at the adluminal compartment (Fig. 8, A and B, left and middle panels of F–H vs. A and B, left and middle panels of E, I, and J). In stage VIII (left and middle panels, Fig. 8G), p-FAK-Tyr397 and p-FAK-Tyr576 were detected at the sites between Sertoli cells and step 8 round spermatids consistent with their localization at the ES, suggesting their functional role in ES dynamics. This result may also account for the induction of p-FAK-Tyr397 in Sertoli germ cell cocultures during AJ assembly (see Fig. 6). In stages IX to V (left and middle panels, Fig. 8, I, J, and E and A and B), staining of p-FAK-Tyr397 and p-FAK-Tyr576 was also detected at the site of cell-cell contacts in apical ES between Sertoli cells and elongating spermatids. Results of the immunohistochemical localization of FAK reported herein (Fig. 8) were consistent with a previous study (15) showing FAK to be largely restricted to the basal compartment in the seminiferous epithelium between Sertoli and germ cells in all stages of the cycle (Fig. 8, C, and E–I, right panels) with very weak FAK staining being detected near the apical compartment (Fig. 8, C, and E–I, right panels). Figure 8D is the control cross-section of an adult rat testis in which the primary antibody was substituted by the same dilution of normal rabbit serum (other controls using PBS or primary antibody preabsorbed with seminiferous tubule lysates or blocking peptide yielded similar results, such as the one shown in Fig. 8D), indicating the reddish-brown precipitate of p-FAK and FAK shown in Fig. 8 was specific staining.
Colocalization of p-FAK-Tyr397 with vinculin, a putative ES constituent protein, to the site of apical ES in the seminiferous epithelium
To further validate that the activated FAK, p-FAK, is indeed localized at the site of ES at the adluminal compartment at stages VI–VIII (see Fig. 8) and to complement results of the IP and immunohistochemistry experiments, immunofluorescent microscopy was used to investigate whether p-FAK-Tyr397 could colocalize with vinculin, a putative ES component protein in the testis (10, 15, 21), to the same site in the seminiferous epithelium. A parallel experiment was performed using ZO-1, a TJ-associated protein (41), which served as a negative control. Figure 9 is the result of colocalization of p-FAK-Tyr397 and vinculin (Fig. 9, A–C), p-FAK-Tyr397 and FAK (Fig. 9, D–F), and p-FAK-Tyr397 and ZO-1 (Fig. 9, G–I) at stage VI–VII tubules of adult rat testes using dual fluorescent probes. The immunofluorescent staining pattern of p-FAK-Tyr397 and vinculin alone in the seminiferous epithelium was shown in Fig. 9, A and B, respectively. It was noted that p-FAK-Tyr397 was associated with the apical ES (Fig. 9A), consistent with results shown in Fig. 8F and the reported results of vinculin in the literature (10, 15, 21) (Fig. 9B). More important, both p-FAK-Tyr397 and vinculin were localized to the same site in the ES (Fig. 9C, merged images, vs. A and B). For FAK, it was localized largely to the basal compartment with very weak immunoreactive FAK being detected near the apical compartment (Fig. 9E), and results of the immunofluorescent staining on p-FAK-Tyr397 and FAK (Fig. 9, D–F) were consistent with immunohistochemistry data shown in Fig. 8. FAK was colocalized with p-FAK-Tyr397 to the same site at the apical ES (Fig. 9, F, merged images, vs. D and E). Furthermore, ZO-1, a TJ-associated peripheral protein (41), was found largely restricted to the site of the BTB in the basal compartment in staged VI–VII tubule (Fig. 9H), consistent with results of an early report (41), and failed to colocalize with p-FAK-Tyr397 (Fig. 9, G–I). Taken collectively, the results shown in Figs. 7–9 have proven beyond refute that FAK found in the adluminal compartment at the site of apical ES is almost exclusively phosphorylated and those detected in the basal compartment were largely nonphosphorylated. It is also clear that ß1-integrin, phosphorylated FAK, c-Src, and vinculin are the putative constituent proteins of the apical ES in the rat testis.
Effects on the expression and/or protein levels of ß1-integrin during AJ disruption in the testis in vivo and Sertoli-germ cell cocultures in vitro induced by AF-2364, a compound known to induce germ cell loss from the seminiferous epithelium by perturbing AJ function
Results presented above have suggested that ß1-integrin and components of the FAC play an important role in Sertoli-germ cell AJ assembly. It is of interest to study their expression during AJ disruption induced by AF-2364, a potential male contraceptive being actively investigated in our laboratory (for review, see Ref. 1). When adult rats were treated with a single dose of AF-2364 at 50 mg/kg body weight by gavage and testes were removed at specified time points for RNA and protein extraction, an induction in the mRNA and protein level of ß1-integrin (Fig. 10, A and B) in the testis by as much as 3.5-fold was detected within 24 h after AF-2364 treatment. And this induction of ß1-integrin was detected up to 8 d after treatment. In vitro studies were also performed to assess treatment using Sertoli-germ cell cocultures (Fig. 10, C and D), Sertoli cell cultures (Fig. 10, E and F), and germ cell cultures (data not shown) following incubation with different doses of AF-2364 (1, 50, and 500 ng/ml). Sertoli cell cultures treated with different doses of lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid] (1, 50, and 500 ng/ml) (Fig. 10, G and H) were also assessed, which is a molecule sharing similar structure with AF-2364 (for reviews, see Refs. 1 and 24 ; also see Refs. 22 and 23), and is known to affect Sertoli cell cytoskeleton filament network (71). Using semiquantitative RT-PCR, it was noted that ß1-integrin mRNA was induced dose dependently by AF-2364 in Sertoli-germ cell cocultures (Fig. 10, C and D) and Sertoli cells cultured alone (Fig. 10, E and F). Furthermore, Sertoli cell ß1-integrin was also induced by lonidamine (Fig. 10, G and H) similar to AF-2364 (Fig. 10, E and F). However, ß1-integrin mRNA could not be detected in adult germ cells with or without treatment with AF-2364 (data not shown) (see also Fig. 1E), which seemingly suggests that ß1-integrin is expressed exclusively by Sertoli cells, but not germ cells, in adult rat testes.
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, A and B) in the testis by as much as 7-fold was detected 8 h after AF-2364 treatment. Its expression plummeted rapidly thereafter (Fig. 11, A and B). In in vitro studies, vinculin mRNA was also induced dose dependently by AF-2364 in Sertoli-germ cell cocultures (Fig. 11, C and D) and Sertoli cells cultured alone (Fig. 11, E and F) but not in germ cells cultured alone (Fig. 11, E and F). Furthermore, Sertoli cell vinculin was also induced by lonidamine (Fig. 11, G and H) similar to AF-2364 (Fig. 11, E and F). Taken collectively, these results suggest that the induction of vinculin in the testis by AF-2364 is mediated via its effects on Sertoli cells.
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, paxillin, and p130 Cas in the testis during AF-2364-induced AJ disruption, and p130 Cas were detected in the testis between 4 and 24 h after AF-2364 treatment (Fig. 12, upper and lower panels). Thereafter, this induction plunged rapidly within 2–8 d after treatment. Although the protein levels of FAK and paxillin were unaltered after AF-2364 treatment (Fig. 12), it is of interest to note that this pattern of induction for p-FAK-Tyr397, PI3K p85, and p130 Cas is similar to that of ß1-integrin and vinculin shown in Figs. 10, A and B, and 11, A and B, suggesting they are part of a functional multiprotein complex, which can be activated and induced simultaneously.
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), we examined whether there were any changes in the pattern of localization of p-FAK by immunohistochemistry. Adult rats were treated with a single dose of AF-2364 at 50 mg/kg body weight by gavage, and testes were removed at specified time points for immunohistochemical localization. Indeed, AF-2364 induced an increase in p-FAK-Tyr397 in the seminiferous epithelium associated with the heads of elongating spermatids at the cell-cell contact sites between elongating spermatids and Sertoli cells (it is somewhat difficult to discern stages of the epithelial cycle as early as 8 h post treatment because of the loss of elongated spermatids from the epithelium), consistent with its localization at the site of apical ES by 8 h to 2 d (Fig. 13, B–D). And by d 4 post AF-2364 treatment, intense p-FAK-Tyr397 staining was also found to associate with round spermatids and some spermatocytes in most tubules when virtually all elongated and elongating spermatids were depleted from the epithelium (Fig. 13, E vs. A). Yet in control testes (see Figs. 13A and 8), p-FAK-Tyr397 was largely associated with elongated spermatids at stages VI–VII cycle (Fig. 13, B–F vs. A, and Fig. 8). By d 8 post treatment, tubules were virtually devoid of elongated and round spermatids, and the number of spermatocytes was significantly reduced, immunoreactive p-FAK-Tyr397 was detected occasionally within large multinucleated cells (Fig. 13F), and its overall level was also reduced. This pattern of changes in steady-state mRNA level was also consistent with immunoblot data shown in Fig. 12. Based on the results presented in this report, it is apparent that the dynamics of ES are regulated by an array of FA-associated molecules that are functionally and structurally associated with 6ß1 integrins as depicted in Fig. 14.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß1-Integrin and vinculin participate in the assembly of Sertoli-germ cell AJs in vitro
When germ cells were cocultured with Sertoli cells in vitro to initiate AJ assembly, it was associated with transient but significant induction of both mRNA and protein levels of ß1-integrin and vinculin. These results, coupled with the observation that Sertoli cell ß1-integrin and vinculin can be induced by germ cells via cell-cell contacts, have unequivocally demonstrated that ß1-integrin and vinculin are involved in the assembly of AJs between Sertoli and germ cells, which is consistent with previous studies that immunoreactive ß1-integrin and vinculin are found at the contact sites between Sertoli and round and elongating spermatids (8, 9, 15, 21). Other studies have demonstrated the presence of immunoreactive ß1-integrin and vinculin at the basal adhesion site between adjacent Sertoli cells (5, 8, 9, 10, 15), suggesting they may participate in AJ and/or TJ assembly between Sertoli cells. Unexpectedly, only ß1-integrin, but not vinculin, was induced at the time of Sertoli cell AJ and TJ assembly, seemingly suggests that the nature AJ structures between Sertoli cells might be different from those found between Sertoli and germ cells. Indeed, there are other specialized AJ structures, such as tubulobulbar complexes (for reviews, see Refs. 1, 3, 5 and 7), present between Sertoli and germ cells besides ES. Furthermore, vinculin may be involved in other physiological processes.
Ironically, the functional nature of ß1-integrin in Sertoli AJ and TJ assembly remains to be characterized. In this connection, it is noteworthy to mention that these coculture studies should be expanded using purified germ cell types to assess whether the reported effects are mediated by spermatocytes, round spermatids, or elongating and/or elongate spermatids. Furthermore, one can argue that the induction of ß1-integrin and vinculin during Sertoli-germ cell AJ assembly may be unrelated to this event; rather, the addition of germ cells onto the Sertoli cell epithelium may induce the production and/or formation of FA complexes between Sertoli-substratrum (i.e. cell-matrix anchoring junctions). Yet such a possibility is unlikely because the FA-associated proteins that constitute the apical ES, such as ß1-integrin and vinculin (8, 9, 15, 21) and p-FAK as reported herein, were localized to the site of apical ES in the seminiferous epithelium in vivo instead of to the site of FA complexes at the basal compartment. Obviously, this issue can only be settled by using immunogold electron microscopy to precisely localize these FA-associated proteins during AJ assembly in vitro.
Tyrosine phosphorylation of FAK is crucial for the Sertoli-germ cell AJ assembly, and the ß1 integrin/p-FAK-Tyr397/c-Src/vinculin complex is found in the Sertoli-germ cell coculture in vitro and adult rat testis in vivo
It is known that clustering of integrins or integrin-mediated cell adhesion can induce autophosphorylation of FAK at Tyr397 (for reviews, see Refs. 25, 26, 27, 28). Herein, we have reported that tyrosine-phosphorylated FAK at Tyr397 was induced at the time germ cells attached to Sertoli cells in vitro to initiate AJ assembly, indicating tyrosine phosphorylation of FAK indeed plays a crucial role on AJ assembly. Although the binding ligand(s) for the 6ß1 integrin at the site of apical ES in the testis remains to be identified, a recent study has demonstrated the presence of a nonbasement membrane-associated laminin 3-chain in the seminiferous epithelium in the adluminal compartment consistent with its localization at the apical ES (72). In the same study, 1, ß1, and 1 laminin chains were shown to be confined to the basal lamina in the testis by immunohistochemistry (72), suggesting laminin can indeed exist at the adluminal compartment without confining to the extracellular matrix. Taking these results collectively, it is increasingly clear that laminin chains, such as 3, could be the binding partner of 6ß1-integrin. Yet it remains to be determined whether germ cells indeed express and produce laminin 3. Furthermore, the other two chains required to constitute the functional laminin ligand are also not known. The fact that studies by immunoprecipitation using a phospho-specific anti-p-FAK Tyr397 antibody can pull out the ß1-integrin/p-FAK-Tyr397/c-Src/vinculin complex from whole-cell lysates of Sertoli-germ cell cocultures and tissue extracts of the testes and lysates of seminiferous tubule cultures has confirmed the possibility that an activation of FAK is likely a response to the clustering of ß1-integrin in the testis. To the best of our knowledge, this is likely the first article reporting the in vivo interactions of ß1-integrin with p-FAK-Tyr397 in the testis.
p-FAK-Tyr397 and p-FAK-Tyr576 was localized at the site of apical ES
The detection of immunoreactive p-FAK-Tyr397 and p-FAK-Tyr576 at the site of apical ES by immunohistochemistry, and the colocalization of p-FAK-Tyr397 with vinculin, a putative ES-associated protein in the rat testis (10, 15, 21), have provided a strong argument for its colocalization with ß1-integrin forming the ß1-integrin/p-FAK-Tyr397 and Tyr576/c-Src/vinculin complex at the apical ES because ß1-integrin (8, 9, 15), c-Src (14), and vinculin (10, 15, 21) are the putative ES constituent proteins at this site. However, our data were somewhat in contrast to a previous study (15), which failed to detect phosphorylated FAK using rat testis lysates and an anti-FAK antibody for immunoprecipitation. The explanation is not immediately known. Nonetheless, this discrepancy could be due to the fact that the majority of FAK found at the basal compartment of the seminiferous tubules were not phosphorylated, whereas tyrosine-phosphorylated FAK was present exclusively at the site of apical ES as demonstrated in our immunohistochemistry/immunofluorescence analysis. Also, we have clearly illustrated the presence of tyrosine-phosphorylated FAK in lysates of Sertoli-germ cell cocultures and adult rat testes by IP using an anti-FAK antibody.
Constituent proteins of the apical ES are similar to the FAC at the basal lamina: tyrosine-phosphorylated FAK is a potential linker for ß1-integrin, recruiting ES components to the site of apical ES
Recent studies have identified several proteins that constitute the apical ES; many of them can also be found in the FAC at the basal lamina, which are the functional unit of the cell-matrix focal contacts. These include ß1-integrin (8, 9, 15), vinculin (10, 15, 21), c-Src (14), Csk (14), ILK (15), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] (16), phosphoinositide-specific phospholipase C (16), Fyn (17), and Keap1 (18). Because ß1-integrin is a transmembrane receptor and signal transducer, regulating cell adhesion, cell migration, growth, differentiation, and programmed cell death (for review, see Ref. 28), this molecule must associate with several downstream signaling molecules linking the cytoplasmic ES components to exert its signaling function. ILK (15) is one of the putative kinases functionally linking ß1-integrin and other downstream effector proteins in the ES. However, to the best of our knowledge, none of the above-mentioned cytoplasmic ES components are known to interact with ILK either structurally or functionally. Results reported herein strongly suggest that, besides ILK, the tyrosine-phosphorylated FAK may be one of the crucial downstream kinases of ß1-integrin in the apical ES that regulates the integrin/laminin multiprotein complex’s cell adhesion function.
We postulate that the ES dynamics are regulated, at least in part, by a cascade of events with initial autophosphorylation of FAK at the apical ES at Tyr397 on integrin clustering (Fig. 14). This in turn creates a high affinity-binding site for c-Src; the recruitment of c-Src induces further phosphorylation of FAK at Tyr576. This complex associates with vinculin to regulate AJ assembly between Sertoli and germ cells. Because vinculin does not link to p-FAK-Tyr397 through direct interaction, it is postulated that -actinin [also a known ES component (11)] or paxillin may act as a bridge for these proteins (for reviews, see Refs. 25 and 26). Furthermore, the binding of PtdIns(4,5)P2 to the vinculin tail is an essential step known to induce vinculin conformational change from a close (inactive) to an open (active) state (for reviews, see Refs. 73 and 74). As such, the presence of PtdIns(4,5)P2 in the ES (16) seemingly suggests that it has a potential role to activate vinculin, which in turn facilitates the apical ES restructuring during spermatogenesis.
PI3K p85, a subunit of the PI3K, is known to act as an adaptor for coupling the p110 subunit to the activated protein tyrosine kinase (for review, see Ref. 75). It was shown to be induced during Sertoli-germ cell cocultures as reported herein, indicating its participation in Sertoli-germ cell AJ assembly possibly via its effects on the actin cytoskeleton (for review, see Ref. 75). PI3K, also a focal adhesion component (for review, see Refs. 25 and 74), is also an effector molecule that binds to the SH2 domain of p-FAK-Tyr397 (31). Yet their interaction in the testis remains to be elucidated. Although the expression of paxillin and p130 Cas remains relatively steady during the assembly of AJs in Sertoli-germ cell cocultures, work is now in progress to determine whether they can be tyrosine phosphorylated because both molecules are known substrates of kinases during the assembly of the FAK/Src complex (for reviews, see Refs. 25, 26, 27). Taking these results collectively, it is likely that the tyrosine-phosphorylated FAK in the apical ES is a potential linker for ß1-integrin, recruiting ES components to the site of apical ES to regulate ES dynamics.
Sertoli-germ cell AJs disruption induced by AF-2364 is regulated by the interplay of ß1-integrin, vinculin, p-FAK-Tyr397, PI3K p85, and p130 Cas in the ES, proteins that are usually associated with the FAC in other epithelia
AF-2364 is a potential male contraceptive that exerts its effects by inducing germ cell loss from the seminiferous epithelium possibly disrupting the AJ structures, such as ES, between spermatids and Sertoli cells (22, 23). AF-2364 is also a new analog of lonidamine [1-(2,4-dichlorobenyzl)-indazole-3-carboxylic acid] sharing a similar structural formula (for review, see Ref. 1), which was identified from a panel of more than 20 new analogs by screening their ability to perturb the expression of testin (22, 23), a novel AJ signal protein in the testis (19, 20, 65). And earlier studies have shown that lonidamine can disrupt the actin filament network in Sertoli cells when rats were treated with this compound by gavage (71), which is why this compound could induce germ cell loss from the seminiferous epithelium. Its use for male contraceptive, however, was limited because of its nephrotoxicity and hepatotoxicity (for reviews, see Refs. 1 and 24).
The original goal of this laboratory was to synthesize and identify a new compound, which maintains the biological activity of lonidamine by inducing germ cell loss from the seminiferous epithelium without lonidamine’s toxicity (23), and AF-2364 apparently fits into this profile (22, 23). Although the precise mechanism by which AF-2364 perturbs the AJ dynamics is not completely known, several lines of evidence suggest that it limits its action in the specialized AJ structures in the testis, such as ES. First, AF-2364 activates testin in the testis, which is a known AJ-signaling molecule largely restricted to ES (19, 65). Indeed, AF-2364 was selected from more than two dozen candidate compounds based on its ability to induce testin expression in the testis from rats treated with a single dose of this compound by gavage (23). Second, results of serum microchemistry analysis have shown that AF-2364 fails to induce liver and kidney damage (22). Furthermore, histological analysis of tissue sections of liver and kidney also fails to detect any cellular damages (22), suggesting the AJ structures in these organs remain intact. Taking these results collectively, it is likely that AF-2364 perturbs the AJ dynamics in the testis by activating a unique ES structural component complex, which somehow links to testin, and is likely the putative receptor for AF-2364 and lonidamine. In this study, an induction of ß1-integrin, vinculin, p-FAK-Tyr397, PI3K p85, and p130 Cas, but not the nonphosphorylated form of FAK and paxillin, was detected after AF-2364 treatment within 4–24 h. This induction also coincides with the time that elongating and elongate spermatids begin to deplete from the epithelium, followed by the loss of round spermatids, indicating their involvement in Sertoli-germ cell AJ disassembly induced by AF-2364. Furthermore, a transient induction of the immunoreactive p-FAK-Tyr397 was also detected between the heads of elongated spermatids and Sertoli cells and also at the cell-cell contacts between round spermatids and Sertoli cells as shown by immunohistochemistry after AF-2364 treatment before germ cell loss from the epithelium.
In this connection, it is important to note that this pattern of protein activation was also detected in Sertoli-germ cell AJ assembly, suggesting the activation of FAK can take part in both the events of junction assembly and disassembly. Such a potential bifunctional role of FAK signaling in regulating junction disassembly and assembly is not unprecedented. For instance, earlier studies have demonstrated FAK signaling indeed plays a crucial role in both focal adhesion assembly and disassembly (27, 76, 77, 78) via a yet-to-be-defined mechanism that is reminiscent of the observations reported herein. Needless to say, results presented herein have provided compelling evidence implicating the autophosphorylation of FAK at Tyr397 at apical ES likely plays a crucial role for the AF-2364-induced disassembly of Sertoli-germ cell AJs. This postulate is also consistent with a previous study suggesting that tyrosine phosphorylation is important in regulating spermiation (79). Although the precise functional nature of the ß1-integrin induction following AF-2364 treatment before AJ disruption remains obscure, it is possible that such induction relates to the signal transduction event necessary for the integrin-mediated AJ disruption, such as rearranging the microfilament network. For instance, lonidamine is known to induce rearrangement of the stress fibers and cytoskeleton network in Sertoli cells (80), thereby causing germ cell loss from the seminiferous epithelium. In view of the fact that both AF-2364 and lonidamine share similar structural formulas (22, 23), both molecules may exert their effects via similar mechanistic pathway.
Interestingly, the levels of vinculin, p-FAK-Tyr397, PI3K p85, and p130 Cas were also plunged in the testis within 2–8 d after the AF-2364 treatment, coinciding with the declining events of AJ disruption when virtually all elongating and elongate spermatids were depleted from the seminiferous epithelium in more than 98% of tubules examined, followed by the loss of round spermatids and most spermatocytes from the epithelium. These results further support the notion that these proteins are involved in AJ disassembly. In contrast, the protein level of ß1-integrin is still up-regulated within 2–8 d after treatment, indicating ß1-integrin may regulate some other function(s) at the basal ES, such as the maintenance of AJs and TJs between adjacent Sertoli cells and also the AJs between Sertoli cells and spermatogonia/primary spermatocytes at the time of AF-2364-mediated testis damage (see Ref. 50).
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Abbreviations: AF-2364, 1-(2,4-Dichlorobenzyl)-indazole-3-carbohydrazide; AJ, adherens junction; BTB, blood-testis barrier; Csk, protein tyrosine kinase that phosphorylates src family kinases; ES, ectoplasmic specialization; F12, Ham’s F12 nutrient mixture; FAC, focal adhesion complex; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; GCCM, germ cell-conditioned medium; GJ, gap junction; ILK, integrin-linked kinase; IP, immunoprecipitation; Mr, relative molecular mass; NP-40, Nonidet P-40; PI3K, phosphatidylinositol-3-kinase; p130 Cas, protein encoded by Crkas gene also called Crk-associated protein; PMSF, phenylmethylsulfonyl fluoride; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; SH2, Src homology 2 domain; Src, a protein tryosine kinase of the transforming gene of Rous sarcoma virus (besides Src, this family includes Fyn, Yes, Fgr, Lyn, Hck, Lck, Blk, and Yrk proteins); TJ, tight junction.
Received October 8, 2002.
Accepted for publication January 22, 2003.
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6ß1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium. Biol Reprod 52:79–87[Abstract]
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, gelatinase B (matrix metalloprotease-9) and tissue inhibitor of metalloproteases-1 in the basal lamina regulates Sertoli cell tight junction dynamics in the rat testis. Endocrinology 144:371–3872-macroglobulin in rat testis is consistent with its role in germ cell movement and spermiation. J Androl 15:575–5823 chain: a novel, non-basement membrane-associated, laminin chain. J Cell Biol 145:605–617This article has been cited by other articles:
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