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Endocrinology Vol. 144, No. 5 2141-2163
Copyright © 2003 by The Endocrine Society

Adhering Junction Dynamics in the Testis Are Regulated by an Interplay of ß1-Integrin and Focal Adhesion Complex-Associated Proteins

Michelle K. Y. Siu, Dolores D. Mruk, Will M. Lee and C. Yan Cheng

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During spermatogenesis, the movement of developing germ cells across the seminiferous epithelium associates with extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specialization (ES, a testis-specific AJ junction), between Sertoli and germ cells. Although this event of germ cell movement is essential to the completion of spermatogenesis, the mechanism(s) that regulates AJ restructuring is largely unknown. Using Sertoli-germ cells cocultured in vitro to study the regulation of AJ assembly, it was shown that this event associated with a transient induction of ß1-integrin, vinculin, p-FAK-Tyr397, and phosphatidylinositol 3-kinase (PI3K) but not the nonphosphorylated form of focal adhesion kinase (FAK), paxillin, and p130 Cas. Furthermore, p-FAK-Tyr397 was shown to coimmunoprecipitate with ß1-integrin, vinculin, and c-Src both in vitro and in vivo using Sertoli-germ cell cocultures and seminiferous tubules, respectively. These results seemingly suggest that the testis is using constituent proteins of the focal adhesion complex (FAC) found in other epithelia between cell and extracellular matrix to regulate AJ dynamics. To further confirm that p-FAK, a putative FAC protein in other epithelia, is indeed present at the site of ES, immunohistochemistry and immunofluorescent microscopy were used. The p-FAK-Tyr397 and p-FAK-Tyr576 were found to localize almost exclusively at the site of apical ES with weak staining at the basal ES in the seminiferous epithelium in a stage-specific manner, being highest at stages VI–VIII. In contrast, FAK was largely restricted to the basal compartment but with weak staining at the apical compartment. When rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to perturb Sertoli-germ cell AJs, an induction of ß1-integrin, vinculin, p-FAK-Tyr397, PI3K, and p130 Cas but not the nonphosphorylated form of FAK and paxillin was also detected in the testis, coinciding with the time spermatids began to deplete from the epithelium, indicating their involvement in AJ disassembly. Thereafter, the levels of vinculin, p-FAK-Tyr397, PI3K, and p130 Cas in the testis plunged, coinciding with the declining events of AJ disruption when virtually all spermatids were depleted from the epithelium. Taken collectively, these results suggest a bifunctional role of p-FAK, being involved in the events of Sertoli-germ cell AJ assembly and disassembly. In summary, the events of AJ dynamics in the testis, in particular at the site of ES, are regulated, at least in part, by proteins that are found in the FAC in other epithelia, such as ß1-integrin, vinculin, and FAK utilizing the integrin/pFAK/PI3K/p130 Cas signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING SPERMATOGENESIS, PRELEPTOTENE and leptotene spermatocytes must translocate across the blood-testis barrier (BTB), which is formed by the inter-Sertoli tight junctions (TJs) near the basal lamina, entering into the adluminal from the basal compartment of the seminiferous epithelium for further development (for reviews, see Refs. 1 and 2). This timely movement of developing germ cells is essential for spermatogenesis; it also associates with extensive restructuring of the actin-based cell-cell, intermediate filament-based cell-cell, and actin-based cell-matrix adhering (or anchoring) junctions, also known as adherens junctions (AJs), desmosome junctions, and focal contacts in other epithelia, respectively (for reviews, see Refs. 1, 2, 3). Ectoplasmic specializations (ESs) are specialized actin-based cell-cell AJs unique to the testis. They are found in Sertoli cells at the sites in which developing and mature spermatids attach (apical ES) and between Sertoli cells at the basal compartment (basal ES; Refs. 1, 4 and 5). The turnover of basal and apical ES are essential for the movement of spermatocytes across the seminiferous epithelium (6), and the movement of spermatids and the release of sperm into the tubular lumen at spermiation (7), respectively.

Several proteins have been identified at the site of ES in the testis, which include {alpha}6ß1 integrin (8, 9); vinculin (10); {alpha}-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{alpha} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats ranging between 1 and 120 d of age were obtained from Charles River Laboratories, Inc. (Kingston, MA). Rats were killed by CO2 asphyxiation, and testes were removed immediately for the isolation of testicular cells or for RNA extraction. The use of animals for studies described herein was approved by the Rockefeller University Animal Care and Use Committee with protocol no. 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, 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. 7BGo, lower panel), elongated spermatids were not removed by omitting the glass wool filtration steps.



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  		Figure 7. A and B, An immunoprecipitation study to assess the tyrosine phosphorylation of FAK (A) and association of p-FAK-Tyr397 with ß1-integrin, vinculin, and c-Src in Sertoli-germ cell cocultures during AJ assembly in vitro, and in adult testes and seminiferous tubules (B). Whole-cell lysates of Sertoli-germ cell cocultures terminated on d 2 with (S/Sp) and without elongated spermatids (S/G), adult testes (T) and seminiferous tubule cultures (ST; see Materials and Methods) were prepared and immunoprecipitated using either an anti-FAK (A) or an anti-p-FAK-Tyr397 (B) antibody. The immunocomplexes were subjected to immunoblotting as described in Materials and Methods and stained with the corresponding antibody as described in A and B to examine the association of different proteins in the immunocomplexes. Whole-cell lysates of S/G without incubation with antibodies or with normal rabbit serum were used as negative controls (data not shown). Normal IgG immunoprecipitates did not react to any detectable band (data not shown), illustrating the specificity of the IP. This figure is the representative results derived from three independent experiments and each time point had triplicate cultures. S/G, Sertoli-germ cell cocultures using a Sertoli:germ cell ratio of 1:1 (Sertoli cells at 0.5 x 106 cells/cm2) in which elongated spermatids were removed by glass wool filtration. T, Testis lysates; S/Sp, Sertoli-germ cell cocultures using a Sertoli:germ cell ratio of 1:1 (Sertoli cells at 0.5 x 106 cells/cm2) in which elongated spermatids were not removed by omitting the glass wool filtration step; ST, lysates from seminiferous tubule cultures.

 
To assess changes in target gene expression in germ cells during maturation, germ cells were also isolated from rat testes at 10, 15, 20, 45, 60, 90, and 200 d of age as described above. Freshly isolated germ cells were terminated by RNA STAT-60 (Tel-Test Inc., Friendswood, TX) for RNA extraction.

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. 7BGo, 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 [{gamma}-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 [{gamma}-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 [{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 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 1Go) was confirmed by direct nucleotide sequencing as previously described (46, 61).


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  		Table 1. Primers used for semiquantitative RT-PCR to assess the steady-state mRNA levels of ß1-integrin, vinculin, and S16

 
Immunoblotting
Total Sertoli-germ cells lysates were obtained from cocultures plated in 12-well dishes at specified time points. Briefly, spent media were removed from each well, and 1 ml SDS lysis buffer was added to the remaining cells. The solubilized cell suspension was transferred to a microfuge tube, vortexed, incubated at room temperature for 5–10 min, and centrifuged at 15,000 x g to remove cellular debris. The clear supernatant was used as whole-cell lysate. Protein content was estimated by Coomassie blue dye-binding assay using BSA as a standard (57). Equal amounts of proteins (~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{alpha} (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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Relative expression of ß1-integrin in Sertoli and germ cells and its developmental regulation in Sertoli and germ cells and the testis
It is noted that most of the ß1-integrin in the seminiferous epithelium is contributed by Sertoli rather than germ cells in all ages examined except in prepubertal rats (Fig. 1Go, A–F). The steady-state mRNA level of ß1-integrin remained relatively steady during Sertoli cell maturation (Fig. 1Go, C and D). Its expression by germ cells plummeted drastically and became almost undetectable after 20 d of age (Fig. 1Go, 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. 1Go, 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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  		Figure 1. A–H, Differential expression of ß1-integrin in Sertoli and germ cells, and changes on its steady-state mRNA level in these cells and testes during maturation. Total RNA was extracted from Sertoli cells, germ cells, or testes. Sertoli cells isolated from rats at specified ages were cultured in vitro for 3 d before termination so that germ cells could be removed by a hypotonic treatment on d 2 to eliminate RNA contributed by germ cells (see Materials and Methods). For germ cell preparations, cells were terminated soon after their isolation because more than 90% of germ cells became nonviable within 16 h of their isolation (51 58 ). These germ cells, however, had negligible somatic cell contamination using various criteria to confirm their purity as described (50 51 ). Semiquantitative RT-PCR was performed to assess changes in the steady-state mRNA level of ß1-integrin using primer pair-specific to this target gene and coamplified with S16. A, An autoradiogram showing the relative steady-state mRNA level of ß1-integrin in Sertoli and germ cells isolated from 20-d-old rat testes. C, E, and G, Autoradiograms showing the changes in steady-state ß1-integrin mRNA level in Sertoli and germ cells and testes during development, respectively. B, D, F, and H, Corresponding densitometrically scanned results using autoradiograms, such as those shown in A, C, E, and G, 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 ß1-integrin in either cells or testes at other ages vs. d 20 (D), 10 (F), or 3 (H), 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.

 
Relative expression of vinculin in Sertoli and germ cells and its developmental regulation in Sertoli and germ cells and the testis
RT-PCR was performed to study whether Sertoli and germ cells express vinculin. In 20-d-old rats, like ß1-integrin, Sertoli cells contributed most of the vinculin expression in the seminiferous epithelium (Fig. 2Go, A and B). The steady-state mRNA level of vinculin plummeted during Sertoli cell maturation (Fig. 2Go, C and D) in contrast to the pattern of ß1-integrin, which remained relatively stable (Fig. 2Go, C and D vs. Fig. 1Go, 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. 2Go, 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. 2Go, 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. 2Go, G and H, could be the result of increased Sertoli-germ cell interactions, to be investigated in the following section (see Fig. 4Go).



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  		Figure 2. A–H, Differential expression of vinculin in Sertoli and germ cells and the changes of its steady-state mRNA level in these cells and testes during maturation. A, Autoradiogram showing the relative steady-state mRNA level of vinculin in Sertoli and germ cells isolated from 20-d-old rat testes. C, E, and G, Autoradiograms showing changes in the steady-state vinculin mRNA level in Sertoli and germ cells, and in testes during development, respectively. B, D, F, and H, Corresponding densitometrically scanned results using autoradiograms, such as those shown in A, C, E, and G, and normalized against S-16. Each bar represents a mean ± SD of two to three experiments using different batches of cells. Statistical analysis was performed by t test by comparing the steady-state mRNA level of vinculin in either cells or testes at other ages vs. d 20 (D, F) or 5 (H). *, Significantly different, P < 0.05; **, significantly different, P < 0.01; ns, not significantly different.

 


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  		Figure 4. A–H. Changes in the steady-state mRNA and/or protein levels of vinculin when Sertoli cells were cultured with germ cells, GCCM, and nonviable germ cells. Semiquantitative RT-PCR was performed to assess changes in the steady-state mRNA level of vinculin, coamplified with S16. Sertoli (0.5 x 106 cells/cm2) and germ cells were cocultured using a Sertoli:germ cell ratio of 1:1 (A and B) or at different ratios (G, H) to initiate the Sertoli-germ cell AJ assembly, where Sertoli cells were cultured alone for 5 d, forming an epithelium, before germ cells were added to this cell epithelium on d 6. Upper and lower panels in A represent the results of the steady-state mRNA and protein levels of vinculin when Sertoli cells were cocultured with germ cells, respectively. C and D, Effects of GCCM on Sertoli cells vinculin expression. Sertoli cells (5 x 104 cells/cm2) were cultured alone for 5 d; thereafter, different concentrations of GCCM (1, 50, 500, and 1500 µg protein/ml) were added to this cell monolayer and cultures were terminated after 2 h. E and F, Sertoli cells (5 x 104 cells/cm2) cultured with nonviable germ cells using a Sertoli:germ cell ratio of 1:1 and cultures were terminated at specified time points. B, D, F, and H, Corresponding densitometrically scanned results using autoradiograms or immunoblots, such as those shown in A, C, E, and G. For RT-PCR, data were normalized against S16. Each bar represents a mean ± SD of three experiments. Each experiment had replicate cultures. *, Significantly different from cultures at time 0, which was arbitrarily set at 1 (except for H, Sertoli cells:germ cells at 1:1 was set at 1) for results of RT-PCR and immunoblotting, by t test (P < 0.05). **, P < 0.01; ns, not significantly different.

 
Changes in the expression of ß1-integrin in Sertoli germ cell cocultures during AJ assembly in vitro and the effects of GCCM and nonviable germ cells on Sertoli cell ß1-integrin expression
Earlier studies have shown that ß1-integrin was localized at the site of ES (8, 9, 15). We sought to examine its involvement during Sertoli-germ cell AJ assembly in vitro. Sertoli cells isolated from 20-d-old rats were cultured for 5 d alone at 0.5 x 106 cells/cm2 on Matrigel-coated dishes forming an epithelium with TJs, AJs, and GJs to permit the endogenous ß1-integrin mRNA and protein level to subside (see Fig. 5Go, 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. 3AGo, upper panel) and protein levels (Fig. 3AGo, lower panel) of ß1-integrin was detected during AJ assembly (Fig. 3Go, 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. 3Go, 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. 3Go, 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. 3Go, G and H) when germ cells per se contributed very little ß1-integrin to the cocultures (see Fig. 1Go, 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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  		Figure 5. A–H, Changes in the steady-state mRNA and protein levels of ß1-integrin (A, B, E, and F) and vinculin (C, D, G, and H) in 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–D) or high cell density (1 x 106 cells/cm2) (E–H). At low cell density, TJs were not formed when assessed by quantifying the transepithelial electrical resistance across the Sertoli cell epithelium, which is due to a lack of close proximity between cells as described (19 45 ). Cultures were terminated at specified time points for RNA extraction. Time 0 indicated RNA STAT-60 was 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 ß1-integrin (A and E) or vinculin (C and G) in Sertoli cells and were coamplified with S16 at the time of junction assembly. The lower panels in E and G are the corresponding immunoblots that illustrate the protein levels of ß1-integrin and vinculin in Sertoli cells cultured at high cell density, respectively. B, D, F, and H are the corresponding densitometrically scanned results using autoradiograms or immunoblots, such as those shown in A, C, E, and G. For RT-PCR, data were normalized against S-16. Each bar represents a mean ± SD of three experiments. Each experiment had replicate cultures. *, Significantly different by ANOVA, P < 0.05; significantly different by ANOVA, P < 0.01; ns, not significantly different. nd, not determined. The relative level of target gene and/or protein expression was arbitrarily set at 1 for cultures at time 0.

 


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  		Figure 3. A–H, Changes in the steady-state mRNA and/or the protein level of ß1-integrin when Sertoli cells were cultured with germ cells, GCCM, and nonviable germ cells. Semiquantitative RT-PCR was performed to assess changes in the ß1-integrin steady-state mRNA level and coamplified with S16. Sertoli cells isolated from 20-d-old rats were cultured for 5 d alone at 0.5 x 106 cells/cm2 on Matrigel-coated dishes forming an epithelium with TJs, AJs, and GJs and to permit the endogenous ß1-integrin mRNA level to subside (see Fig. 5Go, 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 using a Sertoli:germ cell ratio of 1:1 (A and B) or at different ratios (G and H) to initiate Sertoli-germ cell AJ assembly. Upper and lower panels in A represent results of the steady-state mRNA and protein levels of ß1-integrin when Sertoli cells were cocultured with germ cells, respectively. C and D, Results showing Sertoli cells (5 x 104 cells/cm2) cultured alone for 5 d; thereafter, different concentrations of GCCM (1, 50, and 500 µg protein/ml) were added to the Sertoli cell monolayer and cultures were terminated after 2 h. E and F, Sertoli cells (5 x 104 cells/cm2) cultured alone for 5 d; thereafter nonviable germ cells obtained from 90-d-old rat testes as described in Materials and Methods were added onto the Sertoli cell monolayer using a Sertoli:germ cell ratio of 1:1. Cocultures were terminated at specified time points. B, D, F, and H, Corresponding densitometrically scanned results using autoradiograms or immunoblots, such as those shown in A, C, E, and G. For RT-PCR, data were normalized against S16. For immunoblotting data presented herein and all subsequent experiments in this report, results were normalized against the protein level 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, which was arbitrarily set at 1 for results of RT-PCR and immunoblotting, by t test (P < 0.05). **, P < 0.01; ns, not significantly different.

 
Changes in the expression of vinculin in Sertoli-germ cell cocultures during AJ assembly in vitro and the effects of GCCM and nonviable germ cells on the Sertoli cell vinculin expression
We next investigated the participation of vinculin (another ES protein) (15, 21) during Sertoli-germ cell AJ assembly in vitro. Sertoli cells were cultured for 5 d alone to permit the endogenous vinculin mRNA and protein level to subside (see Fig. 5Go, 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. 4AGo, upper panel) and protein (Fig. 4AGo, lower panel) levels of vinculin was detected during AJ assembly (Fig. 4Go, 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. 4Go, 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. 4Go, 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. 4Go, 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. 5Go, A and B) but not vinculin (Fig. 5Go, 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. 5Go, E and F) at the time Sertoli cell TJs were being assembled. Unlike ß1-integrin, no changes in vinculin were detected (Fig. 5Go, 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{alpha}, paxillin, and p130 Cas in Sertoli germ cell cocultures during AJ assembly
Data shown in Figs. 3Go and 4Go 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{alpha} during Sertoli-germ cell AJ assembly (Fig. 6Go). In contrast, the protein levels of the nonphosphorylated FAK, paxillin, and p130 Cas remained unaltered during Sertoli-germ cell AJ assembly (Fig. 6Go). Taken collectively, these results seemingly suggest that p-FAK-Tyr397 and PI3K p85{alpha} take part in the regulation of Sertoli-germ cell AJ assembly. It might be argued that such changes shown in Fig. 6Go (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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  		Figure 6. Changes in the protein levels of FAK, p-FAK-Tyr397, PI3K p85{alpha}, paxillin, and p130 Cas in Sertoli-germ cell cocultures during the assembly of AJs. Sertoli and germ cells were cocultured as described in Material and Methods. Cocultures were terminated at specified time points to obtain whole-cell lysates for immunoblotting. Equal amounts of Sertoli-germ cell lysates (100 µg) were resolved by SDS-PAGE using 7.5 or 10% T polyacrylamide gels under reducing conditions. Proteins on the gel were transferred to nitrocellulose papers and immunostained sequentially using an anti-FAK, p-FAK-Tyr397, PI3K p85{alpha}, paxillin, and p130 Cas antibodies. Each experiment had triplicate cultures. Only one representative set of experiments were shown herein; another set of experiments using different batches of testicular cells for cocultures yielded virtually identical results. In each experiment, each time point had triplicate cultures. The lower panel shows densitometrically scanned results using immunoblots, such as the one shown in the upper panel, in which the levels of the corresponding target proteins at different time points of the Sertoli-germ cell cocultures were normalized against the level at time 0 (i.e. the time when freshly isolated germ cells were added onto the Sertoli cell epithelium on d 6 in which Sertoli cells were cultured alone for 5 d forming an epithelium with intact TJs and AJs), which was arbitrarily set at 1. M, Minute; H, hour; D, day; ns, not significantly different by t test, compared with cocultures at time 0. *, Significantly different, P < 0.05; **, significantly different, P < 0.01.

 
FAK was tyrosine phosphorylated and the phosphorylated FAK was associated with ß1-integrin, c-Src, and vinculin during Sertoli-germ cell AJ assembly in vitro in adult testes and seminiferous tubule cultures
Because there was a transient induction in the p-FAK-Tyr397 protein level during Sertoli-germ cell AJ assembly in vitro, we sought to verify whether FAK could indeed be tyrosine phosphorylated by first isolating FAK from lysates of Sertoli-germ cell cocultures and testes by immunoprecipitation, which was subsequently examined by immunoblotting using a specific antiphospho-Tyr antibody (Fig. 7AGo). 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. 7AGo, left panel) vs. an anti-FAK antibody (Fig. 7AGo, 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. 7AGo, left panel), which had the same electrophoretic mobility when the same blot was stained by an anti-FAK antibody (Fig. 7AGo, right panel), demonstrating Tyr-phosphorylation of FAK indeed occurred in the samples during AJ assembly (Fig. 7AGo).

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. 7BGo, 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. 7BGo (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. 7BGo, lower panel vs. upper panel), p-FAK-Tyr397 indeed was shown to associate with ß1-integrin, vinculin, and c-Src (Fig. 7BGo, lower panel).

It is ironic that the results shown in Fig. 7BGo (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. 7BGo, 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. 7BGo, 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. 8Go and 9Go, 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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  		Figure 8. A–K, Micrographs of cross-sections of adult rat testes showing immunoreactive p-FAK-Tyr397, p-FAK-Tyr576, and FAK in the seminiferous epithelium at different stages of the spermatogenic cycle. A–C are the cross-section of an adult rat testis showing immunoreactive p-FAK-Tyr397, p-FAK-Tyr576, and FAK at low magnification, respectively. D, Corresponding control using normal rabbit serum to substitute the primary antibody at the same dilution (1:100). E–J, Cross-sections of tubules at stages V, VI–VII, VIII (early), VIII (late), XI–XII, and XIV–I, respectively. The left, middle, and right panels in E–J represent the immunostaining of p-FAK-Tyr397, p-FAK-Tyr576, and FAK, respectively. Immunoreactive substances appear as reddish-brown precipitate. Insets are selected regions of the seminiferous epithelium at higher magnification to illustrate the detailed cellular association. Bar, 120 µm for A–D; bar, 50 µm for E–J. K, Immunoblot using adult testis lysates (100 µg protein) for SDS-PAGE, and the blot was immunostained with an anti-FAK, anti-p-FAK-Tyr397, or anti-p-FAK-Tyr576 antibody. All three antibodies reacted specifically with a single band at approximately 125 kDa corresponding to the putative Mr of FAK using testicular lysates for SDS-PAGE and immunoblotting illustrating their specificity for the corresponding proteins, which were used for subsequent immunohistochemistry and immunofluorescent microscopy studies.

 


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  		Figure 9. A–I, Immunofluorescent microscopy to colocalize p-FAK-Tyr397 with vinculin (A–C), FAK (D–F), and ZO-1 (G–I) in the seminiferous epithelium of adult rat testes. A-I, Immunofluorescent micrographs using cross-sections of the seminiferous epithelium from normal Sprague Dawley rats (~300 g body weight) at stages VI–VII. C, F, and I, Merged images of the corresponding immunofluorescent micrographs shown in A and B, D and E, and G and H, for p-FAK-Tyr397/vinculin, pFAK-Tyr397/FAK, and p-FAK-Tyr397/ZO-1, respectively. It is noted that p-FAK-Tyr397 colocalized with vinculin, a putative ES protein largely restricted to apical ES in the rat testis (10 15 21 ) (A–C). Similar to results of the immunohistochemistry analysis shown in Fig. 8Go, the localization of p-FAK-Tyr397 largely restricted to the apical ES, whereas FAK largely confined to the basal ES (D–F). Also, p-FAK-Tyr397 failed to colocalize with ZO-1, which is a TJ-associated peripheral protein at the basal compartment consistent with its presence at the BTB (41 ). Bar, 20 µm.

 
Immunohistochemical localization of p-FAK-Tyr397, p-FAK-Tyr576, and FAK in the seminiferous epithelium of adult rat testis
In light of the changes of p-FAK-Tyr397, but not FAK, detected at the time of Sertoli-germ cell AJ assembly in vitro reported herein and the fact that autophosphorylation of FAK at Tyr397 recruits Src family kinases, which in turn further activates FAK via phosphorylation on Tyr576 and Tyr577 (for reviews, see25, 26, 27, 28), we next sought to localize p-FAK-Tyr397, p-FAK-Tyr576 and FAK in the adult rat testis to examine their cellular localization and stage specificity, if any (Fig. 8Go, 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. 8Go, 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. 8GGo), 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. 6Go). In stages IX to V (left and middle panels, Fig. 8Go, 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. 8Go) 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. 8Go, C, and E–I, right panels) with very weak FAK staining being detected near the apical compartment (Fig. 8Go, C, and E–I, right panels). Figure 8DGo 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. 8DGo), indicating the reddish-brown precipitate of p-FAK and FAK shown in Fig. 8Go 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. 8Go) 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 9Go is the result of colocalization of p-FAK-Tyr397 and vinculin (Fig. 9Go, A–C), p-FAK-Tyr397 and FAK (Fig. 9Go, D–F), and p-FAK-Tyr397 and ZO-1 (Fig. 9Go