Originally published In Press as doi:10.1074/jbc.M513218200 on April 11, 2006
J. Biol. Chem., Vol. 281, Issue 25, 17286-17303, June 23, 2006
Laminin 
3 Forms a Complex with 
3 and 
3 Chains That Serves as the Ligand for 61-Integrin at the Apical Ectoplasmic Specialization in Adult Rat Testes*
Helen H. N. Yan1 and
C. Yan Cheng2
From the
Center for Biomedical Research, Population Council, New York, New York 10021
Received for publication, December 12, 2005
, and in revised form, April 10, 2006.
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ABSTRACT
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Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin 
3, restricted to elongating spermatids, is a putative binding partner of 
6
1-integrin localized in Sertoli cells at the apical ES. However, the identity of the and chains, which constitute a functional laminin ligand with the 3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that 2, 3, 1, 2, 3, and 3 chains were found in germ cells, whereas 1, 2, 4, 5, 1, 2, 1, 2, and 3 chains were found in Sertoli cells, implying that 3 and 3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of 3. To verify this postulate, recombinant proteins based on domain G of 3 and domain I of 3 and 3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin 3, 3, and 3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with 1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin 3, 3, and 3, as well as 1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin 3 or 3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.
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INTRODUCTION
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During spermatogenesis, preleptotene spermatocytes residing at the basal compartment of the seminiferous tubules must traverse the BTB3 to the apical compartment at late stage VII through early stage VIII of the epithelial cycle in adult rat testes (1). Once in the adluminal compartment, spermatocytes differentiate into round spermatids, and the elongating/elongate spermatids orientate themselves with heads and tails pointing toward the basal lamina and the tubule lumen, respectively. The primary anchoring device that facilitates this process of orientation, while maintaining adhesion at the Sertoli cell/spermatid interface, is the apical ectoplasmic specialization (ES), a cell/cell actin-based adherens junction (AJ) type (2-4). In recent years, numerous reports have been published identifying the adhesion molecules at the apical ES. These include the cadherin·catenin, nectin·afadin, and integrin·laminin protein complexes (5-10), which are the primary adhesion protein complexes at the Sertoli cell/developing spermatid interface. Interestingly, in virtually all other epithelia, the integrin·laminin complex is usually restricted to the basement membrane at the cell/matrix interface (11, 12). Yet studies have shown that integrin is a crucial adhesion molecule at the apical ES (7, 13) and together with laminin is likely the most important functional protein complex at the Sertoli cell/spermatid interface at the apical ES, suggesting that apical ES is a hybrid cell/cell and cell/matrix anchoring junction type (4, 14).
Laminins are glycoproteins and heterotrimers composed of , , and chains. To date, there are five known -subunits, four -subunits, and three -subunits that can give rise to at least 16 different functional laminin ligands that bind to integrins restricted to the cell/matrix anchoring junctions, also known as focal contacts or focal adhesion complexes (15, 16). Laminins are crucial scaffolding proteins that provide structural stability to epithelia/endothelia in the basement membrane (12). More important, laminins and integrins at focal contacts provide adhesion between epithelial cells and basal lamina. This laminin·integrin protein complex also serves as an efficient structure to facilitate cell migration during normal and in pathological conditions, such as tumor invasion. The roles of laminins have been expanded from cell/matrix site to cell/cell interface following the identification of a non-basement membrane-associated laminin 3 chain in mouse testes (5); laminin-423 and laminin-523 (previously known as laminins 14 and 15, respectively) were also found to reside outside the retinal basement membrane (17). Although their functions at the apical cell surface remain to be defined, it has been speculated that laminins expressed at the apical retinal epithelium may play a role in photoreceptor morphogenesis (17).
In adult rat testes, laminin 3 chain has recently been shown to be a putative binding partner of 61-integrin at the Sertoli cell/spermatid interface (18); however, the and chains that constitute a functional laminin remain to be identified. In this report, we sought to identify the laminin and chains that form a functional ligand together with 3 for 61-integrin at the apical ES. Using specific blocking antibodies, we also sought to examine the changes in the status of spermatogenesis, the integrity of the Sertoli-germ cell adhesion, and the BTB when functional laminin chains at the apical ES were perturbed. This is an initial attempt to study cross-talk between apical ES and BTB.
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EXPERIMENTAL PROCEDURES
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Animals—The use of rats and rabbits was approved by The Rockefeller University Laboratory Animal Use and Care Committee with protocol numbers 00111, 03017, 03040, and 06018.
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FIGURE 1.
A study by RT-PCR to assess the distribution of different laminin mRNAs in rat testes. A, testin, a Sertoli cell-specific marker, was amplified only in testes and Sertoli cells but not in germ cells (left panel), whereas c-Kit receptor, a spermatogonium-specific marker, was amplified only in testes and germ cells but not in Sertoli cells (right panel), illustrating that each cell type (used in studies shown in B-D) was contaminated with negligible numbers of other cell types. B, laminins 1-3 were detected in testes. Laminin 2 was expressed in both Sertoli and germ cells, and laminin 3 was expressed predominantly in germ cells (right panel). C, laminin -subunits were also amplified by PCR using primers specific to the different chains (see supplemental Table S1). In testes, all three laminin chains were detected. Laminin 2 was expressed in both Sertoli and germ cells, whereas the expression of laminin 3 was restricted to germ cells. D, laminin 1 was detected in testes and Sertoli cells but not in germ cells, whereas the expression level of laminin 3 was higher in germ cells than in Sertoli cells. The asterisk illustrates the absence of a specific laminin chain. SC, Sertoli cells; GC, germ cells; D, day, indicating the age of animals from which cells or testes were isolated or obtained.
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RNA Isolation, RT-PCR, Isolation of Sertoli and Germ Cells, and Primary Cultures and Co-cultures of Testicular Cells—Total RNA from rat testes, testicular cells, Sertoli cell cultures, and Sertoli-germ cell cocultures were extracted using RNA STAT-60TM (Tel-Test "B" Inc., Friendswood, TX) according to the manufacturer's instructions. RT-PCR was performed as described (19) using primer pairs specific to different target genes (in particular to all the known laminin chains (20)) and co-amplified with ribosomal S16 (see supplemental Table S1). Sertoli and germ cells were isolated from testes of 20- and 90-day-old Sprague-Dawley rats, respectively, as described previously (19, 21, 22). The purity of Sertoli and germ cells used in studies reported herein was monitored and characterized by primers specific to markers of Sertoli cells (e.g. testin), spermatogonia (e.g. c-Kit receptor), Leydig cells (e.g. 3-hydroxysteroid dehydrogenase), and myoid cells (e.g. fibronectin), as well as electron microscopy as detailed elsewhere (10, 23, 24). These analyses have shown that the cell preparations (e.g. Sertoli and germ cells) used in our studies had negligible contamination from other cell types. For co-culture, Sertoli cells were plated on Matrigel-coated dishes (Matrigel diluted 1:7 with F12/Dulbecco's modified Eagle's medium) and cultured alone in serum-free F12/Dulbecco's modified Eagle's medium supplemented with epidermal growth factor, insulin, transferrin, and bacitracin as described (19, 25) for 5 days at 35 °C in a humidified atmosphere of 95% air, 5% CO2 with a hypotonic treatment on day 3 to remove residual germ cells. On day 6, total germ cells isolated from adult rat testes were plated onto the Sertoli cell epithelium using a Sertoli:germ cell ratio of 1:1 or 1:2 to initiate Sertoli-germ cell adherens junction assembly, and co-cultures were terminated at 2, 4, 8, 24, and 48 h thereafter. Cultures (in triplicates) were used for each time point, and each experiment was repeated at least three times using different batches of Sertoli and germ cells. The formation of functional tight junctions between Sertoli cells and adherens junctions (e.g. apical ectoplasmic specialization) at the Sertoli/germ cell interface in these cocultures was assessed by electron microscopy as described previously (23).
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FIGURE 2.
A study characterizing the recombinant laminin proteins and their corresponding antibodies. A, proteins obtained from nickel column purified bacterial lysates were eluted from gel slices (about 2-3 µg of protein/lane; see "Experimental Procedures"), resolved onto 15% T SDS-polyacrylamide gels, stained with Coomassie Blue (left panel), illustrating the purity of the laminin chains used for immunization. These proteins were also verified by immunoblotting using an anti-V5 epitope antibody (right panel). B,  50 µg of total protein from each crude recombinant laminin chain from the corresponding bacterial lysates was resolved by SDS-PAGE under reducing conditions. The blot was probed sequentially with rabbit anti-laminin 3, 3, and 3 antibodies to examine cross-reactivity, illustrating that each antibody reacted only with its corresponding laminin chain. C, an illustration of the extent of homology between the three recombinant laminin chains at the levels of amino acid and nucleotide sequences. IB, immunoblotting; Lam, laminin.
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Production of Recombinant Proteins—Recombinant proteins of laminin 3, 3, and 3 chains were expressed and produced using Escherichia coli BL21 StarTM (DE3) cells with the ChampionTM pET Directional TOPO® expression kit (Invitrogen). First, cDNAs corresponding to domain G of laminin 3 and domain I of laminins 3 and 3 were amplified by PCR with AccprimeTM Pfx SuperMix (catalog no. 12344-040, Invitrogen) using total cDNAs from 90-day-old germ cells that served as the template (note that total RNA isolated from these germ cells were reverse-transcribed to cDNAs with oligo(dT)-15-mer and reverse transcriptase) and specific primers shown in supplemental Table S2 and then subcloned into pET101/D-TOPO® expression vectors. The inserted cDNA was in-frame with the expression vector and was confirmed by direct nucleotide sequencing. The expression vectors were transformed into E. coli BL21 StarTM (DE3) cells by the heat shock method at 42 °C. Protein expression in bacterial culture was induced by adding 1 mM isopropyl--D-thiogalactopyranoside when the A600 nm level reached between 0.5 and 0.8. Bacteria were incubated at 37 °C for an additional 6 h. Cells were harvested by centrifugation (3000 x g for 10 min). To extract lysates, bacterial cell pellets were suspended in a lysis buffer (50 mM potassium phosphate, 400 mM NaCl, 100 mM KCl, 10% glycerol (v/v), 0.5% Triton X-100 (v/v), 100 mM imidazole, pH 7.8), frozen in liquid nitrogen, and thawed at 42 °C (repeated three times) followed by sonication (using a Cole-Parmer model CP 130PB-1 ultrasonic processor, Chicago) and centrifugation. The recombinant proteins were produced with V5 and His6 epitope tags at the C terminus, and the authenticity was verified by a specific mouse anti-V5 epitope antibody (catalog no. R960-25, Invitrogen). Recombinant proteins were purified using a nickel column (Amersham Biosciences). The purity was confirmed by SDS-PAGE and silver-stained gels. The identity of the corresponding laminin recombinant proteins was subsequently confirmed using proteins sliced out of the Coomassie Blue-stained polyacrylamide gels by mass spectrometry, performed at The Rockefeller University Proteomics Resource Center. In some experiments (e.g. Fig. 2A), proteins embedded in Coomassie Blue-stained gel slices were micropurified using electroelution by placing 10-20 gel slices (containing 200 - 400 µg of laminin chain protein) in a small dialysis bag (Spectrapor tubing with Mr cut-off at 3500; Spectrum Laboratories Inc., Rancho Dominguez, CA) and suspended in a final volume of 500 - 800 µl of gel running buffer. The dialysis bag was then placed in a Hoefer horizontal gel unit, and electroelution was performed at 80 V for 4 - 8 h at room temperature using SDS-polyacrylamide gel running buffer without SDS as the electroelution buffer.
Preparation of Antiserum and Purification of IgG—Anti-laminins 3, 3, and 3 were prepared in female New Zealand White rabbits with three immunizations using affinity column and gel-purified recombinant proteins emulsified with Freund's complete and incomplete adjuvants essentially as earlier described (26). In short, to prepare highly purified recombinant proteins for immunization, recombinant laminin 3, 3, and 3 proteins isolated from the nickel columns were resolved by SDS-PAGE on 15% T SDS-polyacrylamide gels, stained with Coomassie Blue, and the bands corresponded to the different laminin chains were sliced out. To remove acetic acid and methanol that were used in the Coomassie Blue staining and de-staining steps, gel slices (10-15 µg protein/slice; about 15 gel slices were used for each immunization per rabbit) were immediately suspended in 5 ml of PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4 at 22 °C) and placed in a rotator at 30 rpm. Gel slices containing 200 µg of recombinant protein were washed with PBS five times and homogenized in 400 µl of PBS using a glass homogenizer. Homogenized gel slices were then emulsified with an equal volume of Freund's complete adjuvant with a sonicator and administered subcutaneously to the back of the rabbit at 4 sites (200 µl/site). Prior to the first immunization, about 30 ml of blood was collected from the ear vein of each rabbit to obtain preimmune serum, which was used for corresponding control experiments described herein. Two booster injections were administered 6 and 8 weeks later using similar amounts of gel-purified recombinant proteins but emulsified with Freund's incomplete adjuvant. Rabbits were bled, 10 days after the final booster injection, from the marginal vein in the ear, and at least four more bleedings were collected weekly thereafter. Blood allowed to clot overnight at 4 °C was centrifuged at 2000 x g for 15 min to obtain serum. Anti-laminin 3, 3, and 3 IgG and the IgG from corresponding preimmune sera were isolated from (5 ml) sera by sequential ammonium sulfate precipitation and DEAE (Bio-Rad) affinity chromatography as described (27). The purity of the IgG was confirmed by SDS-PAGE.
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FIGURE 3.
A study to access the changes in the three laminin chains and their likely binding partners in Sertoli-germ cell co-cultures during functional anchoring junctions assembly. A, antibody specificity was analyzed by immunoblotting using 100 µg of protein from lysates of testes, seminiferous tubules (ST), Sertoli cells (SC), and germ cells (GC) and the corresponding antisera. All three laminins were expressed almost exclusively by germ cells, whereas 1-integrin was restricted to Sertoli cells (see left panel). When Sertoli and germ cells were co-cultured to initiate anchoring junction assembly, the protein levels of both laminins and 1-integrin, but not FAK, were induced; actin served as a protein loading control. B and C, protein levels of each target protein shown in A were scanned densitometrically and compared. Each bar is a mean ± S.D. of three samples. The protein level at time 0 was arbitrarily set at 1, against which one-way ANOVA was performed. nd, not detectable; ns, not significantly different; *, p < 0.05; **, p < 0.01.
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Characterization of Anti-laminin 3, 3, and 3 Antibodies—To confirm that the antibodies raised against laminin 3 chain in rabbits did not cross-react with 3 and/or 3 and vice versa, such that the results obtained from co-immunoprecipitation experiments were not artifacts of antibody cross-reactivity among these three laminin chains (namely 3, 3, and 3), the antigens and these antibodies were characterized as follows. First, the identities of the nickel column-purified recombinant laminin 3, 3, and 3 proteins were confirmed by mass spectrometry. Protein sequence analyses and their alignments were examined by using the EMBOSS software (European Bioinformatics Institute). The purity of the recombinant proteins used for immunization was also confirmed by SDS-PAGE and Coomassie Blue staining prior to their use for immunization. Second, the three antibodies were characterized by immunoblotting as follows. In short, 50 µg of crude bacterial protein lysates from bacterial cultures transformed with the corresponding plasmids, containing either 3, 3, or 3 construct, were subjected to immunoblotting. The protein blots were immunostained sequentially with mouse anti-V5 epitope antibody followed by anti-laminin 3, 3, and 3 antibodies to assess cross-reactivity.
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FIGURE 4.
Localization of laminin 3 at the apical ES of adult rat testes and kidneys. A-D, an immunohistochemistry localization study of laminin 3. A, cross-section of an adult rat testis showing the localization of immunoreactive laminin 3 (reddish-brown precipitates) in the epithelium at different stages of the epithelial cycle. Tubules at stages VI-VII (a), stage VIII (b), stages VII-VIII (c), and stages IV-V (d) are boxed, and the magnified views are shown in corresponding panels ad. B, negative control (Ctrl) in which preimmune serum was used to substitute the primary antibody. C, cross-section of an adult rat kidney showing the localization of laminin 3. D, negative control using preimmune serum. E, laminin 3 (red, Cy3) was detected at the Sertoli cell/elongating spermatid interface (blue, DAPI staining) by fluorescent microscopy. F, an immunoblot using lysates of 100 µg of protein of testicular cells and stained with an anti-laminin 3 antibody in a 6% T SDS-polyacrylamide gel. D, dye front. Laminin 3 with an apparent Mr of 165,000 was detected. G, apparent Mr analyses of the laminin chains. The Mr of laminins 3, 3, and 3 was estimated by interpolation of protein standards versus relative mobility (Rf) in multiple SDS-polyacrylamide gels (n = 3). Scale bars: A, 120 µm, applies also to B; A, panel a,60µm, applies also to A, panels b-d, C, and D; A, panel b, inset,20µm, applies also to insets in A, panel c, and in E.
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Electron Microscopy (EM) and Immunogold EM—Electron microscopy and immunogold EM studies were performed at The Rockefeller University Bio-imaging Resource Center essentially as described previously (28, 29) using adult male Sprague-Dawley rats (250 g body weight). In short, rat testes obtained from controls (normal rats and testes treated with preimmune IgG) and rats treated with either antilaminin 3 or 3 IgG at specified time points were cut into small pieces (1-2 mm) and fixed immediately in ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate, pH 7.4, overnight. Thereafter, samples were embedded in 3% agar to keep the tubules together. After solidification, excessive agar was removed, and the specimens were post-fixed in 1% OsO4 in 0.1 M cacodylate, pH 7.4, on ice for 1-2 h. The tubules in agar blocks were treated en bloc with aqueous uranyl acetate for 1 h at room temperature. Specimens were then dehydrated with ascending graded alcohol, propylene oxide and then embedded in Epon 812. The specimens were oriented such that cross-sections could be obtained in multiple tubules. Semi-thin sections (0.5 µm) were cut with a glass knife and stained with 0.25% toluidine blue in 1% borate, allowing visualization of the tubules at light microscopic level for their orientation. Tissue block was retrimmed, and ultra-thin sections were obtained with a DuPont diamond knife. Ultra-thin sections were collected on copper grids and stained with both uranyl acetate and lead citrate before being examined in a JEOL 100 CX electron microscope operated at 80 kV. For immunogold EM, normal testes were fixed in ice-cold 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 3 h. After dehydration with ascending graded alcohol, specimens were embedded in Epon 812. They were incubated with the antiserum (or anti-laminin or IgG, or preimmune serum for control experiments) at room temperature overnight followed by incubation with 10-nm gold-labeled secondary antibody.
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FIGURE 5.
Localization of laminin 3 at the seminiferous epithelium of rat testes and kidneys by immunohistochemistry and immunofluorescent microscopy. A, cross-section of an adult rat testis showing the localization of immunoreactive laminin 3 (reddish-brown precipitates) at different stages of the epithelial cycle. Tubules at stages XII-XIII (a), stage VIII (b), stages V-VI (c), and stages VI-VII (d) are boxed, and the magnified views are shown in corresponding panels a-d. B, negative control (Ctrl) using preimmune serum. C, cross-section of an adult kidney showing the localization of laminin 3. A collecting tubule in the boxed area is magnified in panel e below. Arrowheads in panel e represent immunoreactive laminin 3 that was found at or near the basement membrane. D, negative control using preimmune serum. E, immunoblot using lysates of seminiferous tubules (ST), Sertoli cells (SC), and germ cells (GC) for SDS-PAGE was probed with the anti-laminin 3 antibody. Only one prominent band of 146 kDa was detected in lysates of seminiferous tubules and germ cells but not in Sertoli cells. The same gel was also probed with an actin antibody to assess equal protein loading. F, laminin 3 (green, FITC) was detected at the Sertoli cell/spermatid interface by fluorescent microscopy. Scale bars: A, 120µm, applies also to B; A, panel a,60µm, applies also to A, panels b-d, C, and D; C, panel e,20 µm, applies also to F.
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Conjugation of Anti-laminin 3 and 3 IgG with Alexa Fluor 488 Dye and Immunofluorescent Microscopy—Conjugation of anti-laminin 3 and 3 IgG with Alexa Fluor 488 dye was performed using the Alexa Fluor® 488 Microscale protein labeling kit from Molecular Probes (Eugene, OR). In short, about 11.3 nmol/µl reactive Alexa Fluor 488 dye was used to label 60 µg of either anti-laminin 3 or 3 IgG suspended in PBS. Excessive dye was removed using a spin column. To demonstrate the co-localization of different laminin chains in the seminiferous epithelium of rat testes, immunofluorescent microscopy was performed as described (30). In brief, frozen sections of testes were first incubated with a 1:300 dilution of anti-laminin 3 containing 1% normal goat serum at 35 °C overnight. The sections were then incubated with a 1:100 dilution of Alexa Fluor 488 dye-conjugated anti-laminin 3 IgG at 4 °C for 7 h. Secondary antibodies conjugated with Cy3 (Zymed Laboratories Inc.), diluted in PBS to 1:50, were incubated with the sections for 30 min. Negative controls were preformed using corresponding normal rabbit IgG, and these sections were incubated with Alexa Fluor 488 dye-conjugated anti-laminin 3 IgG followed by secondary antibodies conjugated with Cy3. Sections were then washed and mounted with Vectashield Hardset with 4',6'-diamino-2-phenylindole (DAPI) (a nucleus stain, Vector Laboratories, Burlingame, CA). Fluorescent micrographs were obtained using a BX40 microscope (Olympus Corp., Melville, NY) equipped with Olympus UPlanF1 fluorescent optics and an Olympus DP70 12.5 MPa digital camera.
Immunohistochemistry and Immunocytochemistry—Immunohistochemistry was performed as described previously (30). Control experiments were preformed using corresponding preimmune rabbit serum or IgG instead of the primary antibodies. The sources of antibodies used for all the studies described herein are listed in supplemental Table S3. Immunocytochemistry was performed to study the co-localization of laminin 3/3 and laminin 3\m=.1-integrin in vitro. Sertoli cells were isolated and cultured for 5 days on a Lab-Tek® Chamber SlideTM system (Nalgene Nunc International) coated with MatrigelTM (Collaborative Biochemical Products, Bedford, MA) at a cell density of 5 x 104 cells/well (1.8 cm2). Thereafter, total germ cells were added to Sertoli cells at a Sertoli:germ cell ratio of 1:2 and cultured for an additional 2 days to allow adherens junction formation. Cells were fixed in ice-cold absolute methanol, blocked with 10% goat serum, and incubated with either an anti-laminin 3 (1:300 in PBS containing 1% normal goat serum) or an anti-laminin 3 (1:300 dilution), mouse anti-N-cadherin, mouse anti-1-integrin, or mouse anti-phospho-Src-Tyr426 antibody (working dilution of these antibodies are listed in supplemental Table S3) in 1% normal goat serum. Secondary antibodies conjugated with fluorescein isothiocyanate (FITC) or Cy3 (obtained from Zymed Laboratories Inc.) in a 1:50 dilution of 10% normal goat serum were used.
Co-immunoprecipitation (Co-IP) and Immunoblotting—To study the binding partners of laminin 3 as well as other interacting proteins in rat testes, co-IP was performed using lysates of either seminiferous tubules or germ cells as described previously (31). Seminiferous tubules used for studies reported herein were isolated from adult rat testes and were shown to be contaminated with negligible Leydig cells as reported previously (31). Seminiferous tubule and germ cell lysates were prepared in an IP buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40 (v/v), 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovandate, 1 µg/ml leupeptin and 1 µg/ml aprotinin, pH 7.4) as described (32). For co-IP using different laminin antibodies, lysates were preincubated at 65 °C for 4 min to unfold proteins, cooled to room temperature for 5 min before adding the corresponding precipitating anti-laminin antibodies with incubation at room temperature overnight in a Labnet Mini Labroller (Labnet International, Inc., Woodbridge, NJ) at 24 rpm. After co-IP, immunocomplexes were denatured in SDS-sample buffer, resolved by SDS-PAGE, and subjected to immunoblotting. Negative controls included IgG isolated from either normal rabbit or normal mouse serum by sequential ammonium sulfate precipitation and DEAE anion-exchange chromatography. Each co-IP experiment was repeated at least three times using different sets of samples, and similar results were obtained from each experiment. Lysates of testes, seminiferous tubules, Sertoli cells, and germ cells used for immunoblotting were prepared in the same lysis buffer as described above. About 50 µg of proteins from each sample was resolved by SDS-PAGE using 7.5, 10, or 12% T SDS-polyacrylamide gels under reducing conditions, depending on the apparent Mr of the target proteins to be investigated. Immunoblottings were performed as described (30).
Blocking of Laminin-333 Function by Intratesticular Injection of Antilaminin 3 or 3 IgG—To assess the physiological function of laminin-333 as a crucial cell adhesion protein complex at the apical ES, rats (n = 3 or 4/time point) received 75 µg of either anti-laminin 3 or 3 IgG suspended in 200 µl of PBS at three sites per testis (i.e., 70 µl/site) as described (23). Rats were terminated at day 2, day 4, and day 7 (received one injection), day 10 and day 15 (received two injections at day 0 and day 7), and day 20 and day 25 (received three injections at day 0, day 7, and day 15). There were two groups of control animals in this study. The first group of control rats received 75 µg of preimmune rabbit IgG (suspended in 200 µl of PBS) and were terminated on day 7 (received one injection), day 10 and day 15 (received a total of two injections), and day 20 (received a total of three injections). The second group of control rats received 200 µl of PBS and were terminated on day 7 (received one injection) or day 15 (received a total of two injections) and served as vehicle control. Because of space constraints, results obtained from the first control group in which rats received preimmune IgG were shown. Yet it must be noted that the results obtained from both control groups were similar, illustrating that changes in the status of spermatogenesis in the seminiferous epithelium that were detected following anti-laminin 3 or 3 IgG treatment were not artifacts of IgG administration. At the time rats were sacrificed, one testis from each rat was frozen immediately in liquid nitrogen and stored in -80 °C until used. Another testis was fixed in Bouin's fixative and processed for paraffin sections and staining with hematoxylin and eosin. To assess damaged tubules, a total of 450 tubules from at least three testes of different rats were photographed and printed with an Epson RX300 printer, using a 10x objective in an Olympus BX40 microscope with a built-in Olympus DP70 digital camera. This thus permitted random scoring of 450 tubules with 150 tubules/testis. Two parameters were used to determine a damaged tubule. First, a tubule was considered damaged when >10 germ cells (e.g. round spermatids and spermatocytes) were found in the tubule lumen, because in control (i.e. normal rats and rats treated with vehicle control) testes, only elongated spermatids were found in the tubule lumen at stage VIII of the epithelial cycle, and fewer than 2% of the tubules had round spermatids/spermatocytes in the tubule lumen. Second, the thickness of the germ cell layer (i.e. elongating/round spermatids and spermatocytes) in the seminiferous epithelium of a tubule in a treatment group was measured and compared against normal tubules, and a >30% loss in the germ cell layer was considered significant and scored as a damaged tubule. Furthermore, the diameter of the tubules was also scored and compared with control testes to assess tubule damage following blocking antibody treatment.
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FIGURE 7.
A study by immunogold EM to localize laminin 3 chain in the seminiferous epithelium of adult rat testes. A and D are cross-sections of rat testes from an adult rat (300 g body weight) illustrating step 8 and step 19 spermatids, respectively. In A, the entire developing step 8 spermatid (see the developing acrosome (Ac) overlying the nucleus (Nu) of the spermatid) remained attached to the seminiferous epithelium via apical ES. However, laminin 3 chains, which appear as black dots in the EM micrographs, were found to be restricted to the spermatid side of the apical ES, as shown in B and C of a step 8 and a more advanced spermatid (see black arrowheads), respectively. D, this is a step 19 spermatid (see the fully developed acrosome) in which only the head remains attached to the seminiferous epithelium via apical ES. In E, laminin 3 was also found to be restricted to the germ cell side of the apical ES of a spermatid, as shown by the grains at or near the spermatid plasma membrane (see arrowheads). F, laminin 3 was also localized to the apical ES site at or near the germ cell plasma membrane, which appear as black dots. Scale bars: A, 0.3 µm, applies also to D and E; B, 0.8 µm, applies also to C and F.
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Statistical Analysis—Statistical analyses were performed by ANOVA with Tukey's honestly significant difference (HSD) tests or Student's t test using the GB-STAT statistical analysis software package (version 7.0, Dynamic Microsystems, Silver Spring, MD).
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RESULTS
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Identification of the Bona Fide Partners of Laminin 3 in Adult Rat Testes—As an initial step to identify the likely laminin and chains that interact with laminin 3 at the apical ES, RT-PCR was used to survey different laminin chains in Sertoli and germ cells (see Fig. 1 and supplemental Table S1). Virtually all laminin chains were found in adult rat testes (Fig. 1), consistent with several earlier reports (33-36). We next examined Sertoli and germ cells in the seminiferous epithelium that express different laminins. Fig. 1 illustrates the representative results of this survey using Sertoli and germ cells with negligible contamination from other cell types, using markers specific for Sertoli (e.g. testin) and germ (e.g. c-kit receptor) cells (Fig. 1A). It is interesting to note that laminin 3 was expressed predominantly in germ cells isolated from adult rat testes (containing spermatogonia:spermatocytes:elongating/elongate spermatids at a ratio of 16.7:18:65% as determined by DNA flow cytometry as reported previously (37)), whereas laminins 3 and 3 were restricted to germ cells (see Fig. 1, B-D). These data thus provide the first clue to the likely combination of laminin chains at the Sertoli cell/elongating spermatid interface.
Expression of Recombinant Laminin Proteins and Characterization of These Recombinant Proteins and Their Corresponding Specific Antibodies—To verify the speculation that laminins 3, 3, and 3 indeed form a functional protein complex based on the RT-PCR results, several cDNA constructs based on domain G of laminin 3 and domain I of 3 and 3 were expressed in E. coli (supplemental Table S2 and Fig. S1A), thereby obtaining the corresponding recombinant proteins. These laminin recombinant proteins were isolated by affinity chromatography using nickel columns and then further gel-purified for antibody production (see supplemental Fig. S1B). Polyclonal antibodies were raised by immunizing rabbits with gel slices excised from the Coomassie Blue-stained SDS-polyacrylamide gels (see "Experimental Procedures"). Fig. 2A shows the purity of the recombinant proteins, laminin 3, 3, and 3, on a Coomassie Blue-stained gel (Fig. 2A, left panel), which were also verified by immunoblotting using an anti-V5 epitope antibody (Fig. 2A, right panel). Monospecific polyclonal antibodies against laminin 3, 3, and 3 chains were raised in rabbits, and each antibody was shown to react with the corresponding antigen specifically without cross-reactivity to the other antigens (Fig. 2B). The homology of these recombinant proteins at the levels of nucleotide and amino acid sequences is also shown in Fig. 2C, and protein sequence alignment among laminin 3, 3, and 3 chains using the EMBOSS software program is shown in supplemental Fig. S1C. The analyses shown in Fig. 2C and supplemental Fig. S1C illustrate that cross-reactivity between these antibodies and the corresponding laminin chains is highly unlikely, particularly as gel-purified recombinant proteins (see Fig. 2A) were used for antibody production, consistent with the data shown in Fig. 2B. Using immunoblot analyses, laminin 3, 3, and 3 chains were indeed detected in lysates of testes, seminiferous tubules, and germ cells but not in Sertoli cells (Fig. 3A, left panel, and B).
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FIGURE 8.
In vivo and in vitro co-localization of laminin 3/3 and laminin 3·1-integrin·c-Src by immunofluorescent microscopy. A, laminin 3(green, FITC), in panels a and i, was found to co-localize with 1-integrin (b) and p-Src-Tyr416 (j) at the head of elongating/elongate spermatids in merged images (orange, in c and k) consistent with their localization at the apical ES. DAPI (d, h, and l) shows the nucleus staining. Alexa Fluor 488 dye-conjugated laminin3 in panel e was found to localize at the same site as laminin 3 (red, Cy3) in panel f, which is shown in the merged image (orange, panel g). Scale bars: a, 120 µm, applies also to b-d and i-l; e,12 µm, applies also to f-h. B, laminin 3 (green, FITC), shown in a and i, were found to co-localize with 1-integrin and p-Src-Tyr416 (red, Cy3) in b and j, respectively, and are shown in merged images (orange, in c and k). The orange color was detected at the Sertoli cell/spermatid interface, consistent with their localization at the apical ES. DAPI (d, h, l, and p) stained for nuclei in the merged images. Laminin 3(red, Cy3) in panel e was co-localized to the same site as of laminin 3 (green, Alexa Fluor 488 dye-conjugated) in panel f, and at the Sertoli cell/spermatid interface (g). Panel m, laminin 3 (green, FITC) was restricted to a step 10 spermatid, whereas N-cadherin (red, Cy3 in panel n) was detected in both Sertoli and germ cells. Panel o, co-localization of laminin3 and N-cadherin in germ cells in merged image (orange). Scale bars: panel a, 5 µm, applies also to b-h; panel i, 10 µm, applies also to j-p.
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Induction of Laminin 3, 3, and 3 Chains during Sertoli-Germ Cell Anchoring Junction Assembly—If these laminin chains are indeed the constituent proteins of the apical ES, it is anticipated that their production will be induced during adherens junction assembly. Indeed, the protein levels of these three laminin chains were induced in Sertoligerm cell co-cultures during anchoring junction assembly as manifested by the formation of functional apical ES and desmosome-like junctions when examined by electron microscopy (data not shown, see Refs. 23 and 38) (Fig. 3A, right panel, and C). The protein levels of 1-integrin, the binding partner of laminin residing on Sertoli cells (25), was also induced during AJ assembly in this co-culture experiment, whereas the level of FAK remained relatively constant (Fig. 3, A and C).
Localization of Laminin 3 Chain at the Apical ES in Adult Rat Testes—Localization of laminin 3 chain in the seminiferous epithelium of normal rat testes at different stages of the epithelial cycle versus normal rat kidneys is shown in Fig. 4, A-E. In adult rat testes, laminin 3 was associated mostly with elongating and elongated spermatids (Fig. 4, A, panels a-d, versus control (Ctrl) in B). The strongest signal was detected at stage VIII, predominantly surrounding the heads of elongated spermatids (Fig. 4A, panels b and c); this was consistent with its localization at the apical ES, similar to the results of immunofluorescent microscopy shown in Fig. 4E. In kidneys, laminin 3 was detected at or near the basement membrane in the collecting tubules (Fig. 4C); however, only very weak staining was found in the basement membrane in adult testes (Fig. 4, A versus C). Control experiments (Fig. 4, B and D) were performed in which the primary antibody was replaced by preimmune serum. Specificity of this anti-laminin 3 antibody was shown by immunoblotting using lysates of either germ cells (Fig. 4F) or testes (data not shown) in which an immunoreactive band at 165 kDa was detected. Fig. 4G is the result of an analysis that estimated the apparent molecular weight of the three laminin chains by SDS-PAGE using different protein markers and laminin chains.
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FIGURE 9.
A study by co-IP to access the structural relationship among laminin-333, 1-integrin, and other regulatory proteins at the apical ES. A, co-IP was performed using germ cell lysates and antibodies against laminins 3, 3, and 3, c-Src, and paxillin (see supplemental Table S3). Laminin 3 was shown to associate with laminins 3, 3, and 3 as well as c-Src but not with paxillin (upper panel). c-Src was shown to associate with laminins 3, 3, and 3 in this reverse co-IP experiment (lower panel). B, co-IP was also performed using seminiferous tubule lysates and antibodies against pFAK, c-Src, and 1-integrin, and the resultant blot was probed with an anti-laminin 3 antibody, illustrating that pFAK, c-Src, and 1-integrin were indeed structurally associated with laminin 3. These data were validated in a reverse co-IP experiment using an antibody against c-Src (middle panel). c-Src was associated with laminins 3, 3, and 3 individually or with the laminin-333 complex using a mixture of the three antibodies (lower panel). -ve, negative controls using either rabbit or mouse IgG as the precipitating antibody. IB, immunoblot; Lam, laminin.
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Localization of Laminin 3 Chain at the Apical ES in Adult Rat Testes—Similar to laminin 3, the 3 chain was associated predominantly with elongating and elongated spermatids at the apical ES (Fig. 5A, panels b-d). The strongest signal was also detected at stage VIII, largely surrounding the heads of elongated spermatids at the apical ES site (Fig. 5A, panel b). The immunohistochemical results are consistent with immunofluorescent microscopy (Fig. 5, F versus A), illustrating that the laminin 3 chain was restricted almost exclusively to the apical ES in adult rat testes. Fig. 5B is a control experiment in which the primary antibody was replaced with preimmune serum. A weak signal was also detected at the apical ES in spermatids at steps 8, 9, and later (Fig. 5A, panels a and c). Similar to laminin 3, the 3 chain was also detected mostly at the basement membrane in the collecting tubules of kidney (Fig. 5C; Fig. 5D is the corresponding control using preimmune serum in place of the anti-laminin 3 antibody in sections of kidney). The specificity of this anti-laminin 3 antibody was illustrated by immunoblotting, as shown in Fig. 5E in which a prominent immunoreactive band of 146 kDa was detected in lysates of seminiferous tubules and germ cells but not Sertoli cells. The distribution of laminin 3 in the seminiferous epithelium of rat testes during maturation was examined next. Interestingly, it was noted that in 16-day-old rats at the time the BTB was being established, laminin 3 was largely confined to the basal compartment of the seminiferous epithelium, consistent with its localization at the BTB (Fig. 6, A-C). Also, at this age, the apical ES was not found because no elongating/elongate spermatids were present at this age (Fig. 6, A-C). However, the expression of laminin 3 was detected at both the basal and apical compartments in 30-day-old rat testes when step 8 and 9 spermatids were found in the epithelium with functional apical ES (Fig. 6, D-F). In adult rats at 60 days of age, when well developed apical ES was found in the epithelium, laminin 3 became mostly restricted to the apical ES (Fig. 6, G-I). These results illustrate a shift in the localization of laminin 3 chain in the seminiferous epithelium during testicular maturation.
Ultrastructural Localization of Laminin 3 and 3 Chains to the Apical ES by Immunogold EM—To further validate results of the immunohistochemistry and immunofluorescent microscopy studies showing that 3 and 3 chains were confined to the non-basement membrane site in adult rat testes, immunogold EM was used. Consistent with the above findings (see Figs. 4, 5, 6), immunogold EM has laminin 3 (see black dots in Fig. 7, B, C, and E versus A and D) and 3 (Fig. 7, F versus A and D) chains localized almost exclusively to the apical ES in adult rat testes. Fig. 7A is a cross-section of a seminiferous tubule showing a step 8 spermatid in which the entire sperm head (see the developing acrosome (Ac) above the condensed nucleus (Nu)) was invaginated into a Sertoli cell and attached to the seminiferous epithelium via the apical ES. Apical ES was typified by the presence of actin filament bundles (Fig. 7A, white arrowheads) sandwiched between the cisternae of the endoplasmic reticulum (ER) and the Sertoli cell plasma membrane (apposing arrowheads represent the apposing Sertoli and germ cell plasma membranes). Fig. 7D is another tubule of a normal rat testis showing a step 19 spermatid in which the entire sperm head is attached to the epithelium via apical ES having the same ultrastructure features as shown in Fig. 7A. Laminin 3, which appeared as black grains in immunogold EM, was localized almost exclusively to the apical ES site of a step 8 spermatid (Fig. 7B; 120 grains were found at the apical ES surrounding the head of a step 8 spermatid) and more developed spermatids (Fig. 7, see arrowheads in C and E). Fig. 7F is the result of an immunogold EM study that also illustrates the localization of laminin 3 chain at the apical ES in an elongated spermatid in the rat testis. More important, both laminin 3 and 3 chains were detected near or adjacent to the germ cell membrane (see Fig. 7, B, C, E, and F), confirming the results of the immunoblotting that they are exclusive germ cell products (see also Fig. 3, A and B).
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FIGURE 10.
A study to assess laminin-333 function by investigating the status of spermatogenesis and the kinetics of tubule damage following intratesticular administration of a blocking antibody using either anti-laminin 3 or 3 IgG. A-H, micrographs of cross-sections of testes from normal rats (A), rats that received normal rabbit IgG and were terminated after 15 days of injection (B), rats that received 75 µg/testis anti-laminin 3 IgG and were terminated on day 4 (C), 7 (D), or 15 (C), and rats that received 75 µg/testis anti-laminin 3 IgG and were terminated on day 4 (F), 7 (G), or 15 (H). The boxed areas in C-H were magnified, respectively, in C:i, D:ii, E:iii, E:iv, F:v, G:vi, and H:vii and H:viii. Damaged tubules (annotated with an asterisk) with germ cell sloughing were found after blocking IgG treatments on day 4; this was not detected in rats that received either preimmune IgG treatment (B) or PBS (data not shown). Multinucleated germ cells were also found in E:iii, E:iv, G:vi, and H:viii, whereas the microvessels in the interstitium remained intact, as shown in F:v. To assess the percentage of damaged tubules and shrinkage in tubule diameter, cross-sections from three different areas of testes were obtained (see broken black lines on the testis illustrated in I, inset), and 3-4 rats for each time point were analyzed. Blue circles in I (inset) illustrate the approximate sites of IgG administration via direct intratesticular injection. About 150 tubules were scored in different cross-sections from at least three rat testes, and the results are plotted in I and J. The diameter of the tubules in control testes (Ctrl) was arbitrarily set at 100%, whereas the diameter of tubules from treatment groups was calculated as % of control. The effects on testis weight of administration of both anti-laminin 3 and3 IgG are shown in K. Each point is the mean ± S.D. of four testes. ns, not significantly different by ANOVA and Student's t test; *, p < 0.05; **, p < 0.01. Scale bars: A, 120µm, applies also to C-H; B, 60 µm; C:i, 40 µm, applies also to panels ii-viii.
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Co-localization of Laminin 3·3-Integrin and Laminin 3·1-Integrin·c-Src in the Seminiferous Epithelium in Vivo and Sertoli-Germ Cell Co-cultures in Vitro—Based on the results of immunogold EM, laminin 3 was localized to the similar site as of 3 chain, near the germ cell membrane in the epithelium. Immunofluorescent microscopy was used to confirm the co-localization of laminin 3 and 1-integrin (Fig. 8, A and B, panels a-d). Furthermore, laminin 3 and laminin 3 were also co-localized to the same site at the apical ES, consistent with their presence in germ cells in the seminiferous epithelium as well as in the co-culture system (Fig. 8, A, panels e-h, and B, panels e-h). We next sought to examine whether the laminin-333·61-integrin·c-Src is a possible regulatory protein unit at the Sertoli/germ cell interface. It was shown that laminin 3 co-localized with phospho-Src-Tyr416 in vivo (Fig. 8A, panels i-l) and in vitro (Fig. 8B, panels i-l). In Sertoli-germ cell co-cultures, laminin 3 was detected exclusively in the germ cells, whereas N-cadherin was found mostly at the Sertoli/Sertoli interface. However, cadherin was also a component of Sertoli-spermatid apical ES, and Fig. 8B, panels m-p, thus illustrating the co-localization of laminin 3 and N-cadherin to the Sertoli/germ cell interface in the cocultures with functional apical ES.
Structural Interactions of Laminin 3, 3, and 3 Chains, Which Form a Functional "Laminin-333" Complex at the Apical ES and Its Association with 1-Integrin·pFAK·c-Src—To elucidate whether laminin-333 and integrin are a putative protein complex at the apical ES, a biochemical study was performed using lysates of either germ cells (Fig. 9A) or seminiferous tubules (Fig. 9B) from 90-day-old rats. Using antibodies against laminin 3, 3, and 3 chains, with c-Src or paxillin for co-IP (see supplemental Table S3), laminin 3 was found to associate with 3 and 3 chains as well as c-Src, but not with paxillin, in germ cells (Fig. 9A, upper panel). Interestingly, c-Src was also found to interact structurally with laminin 3, 3, and 3 chains in both lysates of germ cells (Fig. 9A, lower panel) and seminiferous tubules (Fig. 9B, bottom panel; note that c-Src was pulled out by anti-laminin 3, 3, or 3 antibody alone as well as a combination of these three antibodies). Additionally, c-Src was shown to associate with pFAK and 1-integrin (Fig. 9B, middle panel). This implies that c-Src may play a crucial role in signal transduction between Sertoli cells and elongate/elongating spermatids pertinent to spermatogenesis, consistent with two recent reports (30, 32). pFAK, c-Src, and 1-integrin were also shown to associate with laminin 3 (Fig. 9B). Negative controls were performed using either rabbit or mouse IgG instead of the precipitating antibodies. Taking these results collectively, it is seen that the laminin 3, 3, and 3 chains indeed form a functional laminin-333 protein complex that may be the ligand of 1-integrin at the apical ES. This, in turn, forms a functional adhesion complex with pFAK and c-Src.
A Disruption of Laminin-333 at the Apical ES by Blocking Antibodies Perturbs Both AJ and TJ Functions in the Seminiferous Epithelium of Rat Testes—To assess the physiological function of the laminin-333 complex at the Sertoli cell/elongating spermatid interface, laminin-333 was blocked by intratesticular injections of either an anti-laminin 3 or 3 antibody. Following administration of either anti-laminin 3 or 3IgG versus preimmune rabbit IgG, sloughing of spermatids from the epithelium was observed beginning on days 4-15 (Fig. 10, C-H versus A, normal rat testis; or see Fig. 10B, where the testis received preimmune IgG). 40 - 60% of the tubules were damaged when a total of 450 tubules from different testes were scored (Fig. 10I), whereas the blood vessel remained intact (see Fig. 10F:v). Multinucleated giant germ cells (see Fig. 10, E:iii and iv, G:vi, and H:viii) were found in some tubules of rat testes treated with anti-laminin IgG from day 7 onward, an indication of germ cells undergoing necrosis. A drastic reduction was seen in the tubular diameter by up to 40% versus control rats treated with preimmune IgG for 20 days (Fig. 10J). There was no statistical difference in testes weight throughout the whole treatment (Fig. 10K). To further study the changes in cell adhesion function in both the basal and apical compartments of the seminiferous epithelium when the laminin-333 function was perturbed, immunoblot analyses were performed using testes lysates (Fig. 11A). The protein levels of both occludin (a TJ-integral membrane protein in the testis) and ZO-1 (a TJ adaptor) were reduced by 3-fold at day 15 after anti-laminin 3 IgG administration; a more significant reduction in ZO-1 protein level was detected by day 10 (Fig. 11, A and B). Interestingly, a recovery of TJ protein levels was detected by day 20, apparently because the IgGs were metabolized and new laminin-333 was being synthesized. However, JAM-1 (another TJ-integral membrane protein also known as JAM-A) seems to be less susceptible to the blocking antibody. For AJ proteins that are also found at the BTB, such as N-cadherin and -catenin, their protein levels remained relatively steady up to day 4 but were reduced gradually; unlike the TJ proteins, this protein complex failed to bounce back, as shown by immunoblot results (Fig. 11, A and C). Although the protein levels of several TJ and AJ protein markers were found to decrease during germ cell loss induced by the blocking antibodies of the laminin-333 complex, the expression of laminin 3 and 3 were stimulated after treatment, and the steadystate laminin 3 protein level was mildly induced (Fig. 11, A and D). For some downstream signal proteins known to be activated by the laminin·integrin complex, the protein level of FAK remained relatively unchanged, whereas c-Src level displayed a significant reduction by days 15-20 after anti-laminin 3 IgG treatment (Fig. 11, A and C). To further confirm that the disruption of BTB by the blocking antibodies is transient and reversible, immunofluorescent microscopy was performed to monitor the BTB integrity. In testes with or without (normal testes) rabbit IgG (isolated from preimmune serum) administered, an almost continuous belt of fluorescent staining of occludin and ZO-1 was detected near the basement membrane of the seminiferous epithelium consistent with their localization at the BTB (Fig. 12, A-D). Earlier studies have shown that this is a reliable indication that the BTB was intact when the study was performed in conjugation with a micropuncture technique, where rats were administered 125I-labeled bovine serum albumin via the jugular vein prior to fluid collection at the rete testis and seminiferous tubule compartment (22). A weaker signal was detected by day 4 after administration of anti-laminin 3 IgG (Fig. 12, E-H). Staining of both proteins became hardly visible by day 10 after the treatment (Fig. 12, I-L). The BTB damage caused by these blocking antibodies seems to be transient, consistent with results of immunoblot analysis (Fig. 11A), because the fluorescent signals of occludin and ZO-1 bounced back by day 20 (Fig. 12, M-P). This pattern is similar to the pattern obtained by fluorescent microscopy in which the co-localization of N-cadherin·-catenin at the BTB site was examined (data not shown), except that this protein complex failed to bounce back by day 20, which is also consistent with the immunoblot data shown in Fig. 11, A and C.
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