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Endocrinology Vol. 144, No. 4 1549-1563
Copyright © 2003 by The Endocrine Society

ARTICLE

Rab8B GTPase and Junction Dynamics in the Testis

Ann S. N. Lau and Dolores D. Mruk

Population Council, Center for Biomedical Research, New York, New York 10021

Address all correspondence and requests for reprints to: Dolores D. Mruk, Ph.D., Population Council, Center for Biomedical Research, 1230 York Avenue, New York, New York 10021. E-mail: mruk{at}popcbr.rockefeller.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Throughout spermatogenesis, germ cells migrate from the basal to the adluminal compartment while remaining attached to Sertoli cells via actin-based adherens and intermediate filament-based anchoring junctions. However, the events that trigger deadhesion and adhesion remain largely unknown. As part of our continued effort in elucidating the mechanism of germ cell movement, we have examined the role of Rab8B, a GTPase probably participating in intracellular trafficking events at the site of the adherens junction. By RT-PCR Rab8B mRNA was found in the brain, testis, heart, kidney, and spleen. Immunohistochemical studies revealed that Rab8B was concentrated predominantly in the basal compartment, localizing to a similar site at which immunoreactive E-cadherin was found. Additional experiments demonstrated that Rab8B associated with the actin, intermediate filament, and microtubule cytoskeletal networks. When Sertoli cells were cultured at high density or germ cells were cocultured with Sertoli cells, Rab8B increased significantly during junction assembly. Moreover, inclusion of germ cell-conditioned medium in Sertoli cell cultures resulted in stimulation of Rab8B expression. Conversely, treatment of adult rats with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide reduced Rab8B mRNA and protein levels, coinciding with the time of germ cell loss from the epithelium. Taken collectively, these studies suggest that Rab8B participates in adherens junction dynamics in the testis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADHERENS JUNCTIONS (zonula adherens), specialized junctions that join adjacent epithelial cells via the actin cytoskeletal network, participate in an array of cellular processes (1, 2, 3). For instance, their functionality, in particular their mechano-adhesive role, is mediated in part by a complex consisting largely of cadherin and catenin molecules. However, substantial evidence has recently suggested the involvement of cadherins and catenins in events other than cell adhesion, such as in signal transduction (4, 5), illustrating that adherens junctions have additional functions and can be regulated by other as yet to be identified molecules. Indeed, after nearly two decades since the first guanosine triphosphatase (GTPase) was discovered, a mechanism regulating adherens junction dynamics in epithelial cells, which involves the participation of GTPases, has recently been proposed. Using surface biotinylation and recycling assays, Stow’s group (6) has shown the internalization of E-cadherin and ß-catenin in Madin-Darby canine kidney (MDCK) cells after cell adhesion was perturbed by depletion of extracellular Ca2+, followed by their relocalization back to the cell membrane once Ca2+ was replenished in the culture medium. They also convincingly demonstrated the colocalization of E-cadherin and Rab5 in early endosomes, implicating Rab GTPases in the dynamics of adherens junctions (6). These findings are comparable with those of earlier studies that described the recycling of integrins by vesicular trafficking (7, 8). These results, taken collectively, may imply that the disassembly and/or reassembly of adherens junctions in other physiological systems, such as during the movement of germ cells from the basal to the adluminal compartment of the seminiferous epithelium, may involve a similar mechanism.

Rab GTPases are proteins of approximately 20 kDa that constitute the largest family of monomeric GTPases (9, 10, 11). Their function is vested primarily in their ability to shuttle between GTP (active form, membrane-associated)- and GDP (inactive form, cytosol-associated)-bound conformations, constraining them to temporally and spatially regulate intracellular events, such as vesicular trafficking, polarized transport of proteins, and cell movement. What is not known, however, is how these events correlate with adherens junction disassembly and/or reassembly in the testis. These intriguing observations have also raised several other questions. For example, what is the stimulus triggering cycling between the two conformations? Could germ cells possibly elicit the stimulus that is responsible for mediating the events of adherens junction disassembly and/or reassembly via Rab GTPases? If they do, then how? We hypothesize that Rab8B participates in adherens junction dynamics by possibly regulating the arrangement of cell adhesion proteins, such as cadherins and catenins, during germ cell translocation across the seminiferous epithelium via the cytoskeleton. Such a postulate is not without precedent, as a previously published report has demonstrated Rab8 (an isoform of Rab8B) to be enriched in the area of the junctional complex in MDCK cells (12). In addition, a survey of several tissues by Northern blot has shown the mRNA level of Rab8B to be highest in the testis (13), suggesting its importance in the biology of the testis. In the present study we have used Sertoli cell cultures and Sertoli-germ cell cocultures in vitro in conjunction with chemical cross-linking and coimmunoprecipitation experiments to determine whether Rab8B participates in adherens junction dynamics. To our knowledge, this is one of the first reports illustrating the participation of Rab8B in the dynamics of adherens junctions, and this study will serve as a framework for future investigations in the field that aim at examining the relationship between GTPases and adherens junctions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley male rats at different ages were purchased (Charles River Laboratories, Inc., Kingston, NY). Rats were killed by CO2 asphyxiation. Organs were removed immediately, frozen in liquid nitrogen, and stored at -80 C until used. For Sertoli and germ cell cultures, testes were removed and used immediately for the isolation of cells. The use of all animals was approved by The Rockefeller University Animal Care and Use Committee (Protocol 00111).

Sertoli cell cultures
To determine whether Rab8B participates in Sertoli cell junction dynamics, an in vitro model was used in which Sertoli cells were cultured at high density to initiate junction assembly (14). Sertoli cells were isolated from 20-d-old testes as previously described (15, 16, 17). For Sertoli cells cultured at low density, where tight junctions could not form, but cell-cell adherens and cell-substratum anchoring junctions were present, cells were plated at a density of 0.05 x 106 cells/cm2 on 100-mm dishes. Sertoli cells were also plated at a substantially lower density (0.025 x 106 cells/cm2) to initiate the assembly of cell-substratum anchoring junctions only. For high density Sertoli cell cultures, cells were plated on Matrigel (BD Biosciences, San Diego, CA)-coated (diluted 1:7 in Ham’s F-12 nutrient mixture and DMEM, 1:1; Sigma-Aldrich, St. Louis, MO) 12-well dishes at cell densities of either 0.75 x 106 or 0.5 x 106 cells/cm2. In these cultures the time of cell plating was designated time 0 (control), and cells were terminated thereafter at specified time points using either RNA STAT-60 (Tel-Test, Friendswood, TX) or cell lysis buffer A [0.125 M Tris (pH 6.8) at 22 C containing 1% sodium dodecyl sulfate (SDS; wt/vol), 20% glycerol (vol/vol), 1.6% 2-mercaptoethanol (vol/vol), 2 mM phenylmethylsulfonylfluoride (PMSF), 2 mM 1,10 phenanthroline, 1 µg/ml pepstatin A, and 2 mM N-ethylmaleimide]. In experiments in which freshly isolated germ cells, germ cell-conditioned medium (GCCM), astrocyte-conditioned medium (ACM), or steroids were added, Sertoli cells (0.5 x 106 cells/cm2) were hypotonically treated (20 mM Tris, pH 7.4, at 22 C) 48 h after plating to remove residual germ cells (18). Thereafter, cells were successively washed and cultured for an additional 3 d to allow the formation of an epithelium with intact tight, anchoring, and communicating gap junctions. Media were replaced every 24 h until the addition of germ cells or factors. For 10-d-old Sertoli cell cultures, cells were isolated as previously described with minor modifications (15, 17). For Sertoli cells isolated from 45-, 60-, and 90-d-old testes, seminiferous tubules were subjected to sequential enzymatic treatments as described previously (19). Residual germ cells were removed from 45-, 60-, and 90-d-old Sertoli cell cultures by consecutive hypotonic treatments on d 2 and 3, whereas 10-d-old Sertoli cells were hypotonically treated on d 2 only. These cells were then allowed to recover for 24 h before termination using either RNA STAT-60 or cell lysis buffer A.

Germ cell cultures and preparation of crude GCCM
The isolation of all germ cells used for experiments in this study was performed as previously described (20). Elongate spermatids and spermatozoa were removed from all of our germ cell preparations by successive passages through glass wool. The somatic cell contamination was virtually negligible when assessed by various criteria as detailed previously (21). Germ cell viability at the end of the 15-h incubation period was greater than 95% when assessed by the 1) erythrosine red dye exclusion test (22), and 2) integrity of genomic DNA. Thereafter, spent media were collected, centrifuged successively at 1,000 and 45,000 x g for 45 min each to remove cellular debris, and concentrated using a Minitan (Millipore Corp., Bedford, MA) tangential ultrafiltration unit equipped with eight Minitan plates with a molecular mass cut-off of 10 kDa. This sample, designated GCCM, was then filtered through a 0.2-µm pore size filter unit and stored at -20 C until its inclusion in Sertoli cell cultures. In experiments in which the Rab8B mRNA level was examined in germ cells isolated from aging testes, germ cells were isolated and terminated immediately using either RNA STAT-60 or cell lysis buffer A.

Incubation of Sertoli cells with germ cells, crude GCCM, or ACM in vitro
Germ cells isolated from 90-d-old rats were cocultured with Sertoli cells, which had previously been cultured for 5 d, at a Sertoli/germ cell ratio of 1:5 (Sertoli cells at 0.5 x 106 cells/cm2) for up to 3 d to allow the assembly of Sertoli-germ cell adherens junctions as previously described (14). In this experiment, time 0 represents cocultures that were terminated immediately after the addition of germ cells to the Sertoli cell epithelium so that the relative percentages of Sertoli and germ cell RNA at this time point were 20% and 80%, respectively. Sertoli-germ cell cocultures were terminated by using either RNA STAT-60 or cell lysis buffer A. To confirm the germ cell-mediated stimulation in Rab8B mRNA and protein levels, additional in vitro experiments were performed in which the effects of crude GCCM and ACM (control) on Sertoli cell Rab8B expression were examined. In brief, Sertoli cells, cultured as described above, were incubated with increasing concentrations of GCCM (20–1000 µg protein/dish) or ACM (5–1000 µg protein/dish) for 4 h and then terminated by using either RNA STAT-60 or cell lysis buffer A. An incubation period of 4 h was selected because germ cells are capable of stimulating the Sertoli cell Rab8B mRNA level from 2–6 h. In experiments in which increasing concentrations of ACM were incubated with Sertoli cells, astrocytes were isolated from 1-d-old male rats as previously described (23, 24).

In vitro germ cell adhesion assay
The germ cell adhesion assay was performed as previously described (25, 26). In brief, isolated germ cells, consisting largely of spermatogonia, primary spermatocytes, and round spermatids, were metabolically labeled with L-[35S]methionine (1 x 106 cells/~400,000 cpm; 1,000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) for approximately 45 min in medium containing 1/100th of the methionine normally present in laboratory stock Ham’s F-12/DMEM. Thereafter, cells were washed three times by centrifugation at 800 x g, and labeled germ cells were diluted to an appropriate concentration and added to Sertoli cells (0.5 x 106 cells/cm2; Sertoli cells were previously cultured for 5 d to allow the formation of an epithelium with intact tight, anchoring, and gap junctions) at a Sertoli/germ cell ratio of 1:3. At specified time points Sertoli-germ cell cocultures were briefly washed twice in Ham’s F-12/DMEM to remove unbound germ cells, and cocultures were terminated by scraping cells with 1 M NaOH. Radioactivity was quantified by a Tri-Carb 2900 TR liquid scintillation counter (Packard Bioscience, Downers Grove, IL). Samples for radioactivity determination were processed simultaneously to eliminate interassay variations. In another experiment in which the adhesion of germ cells to Sertoli cells was assessed microscopically, isolated germ cells were cocultured with Sertoli cells at a Sertoli/germ cell ratio of 1:3 on Matrigel-coated bicameral units as previously described (14). At specified time points, Sertoli-germ cell cocultures were terminated by submerging bicameral units in 0.2 M collidine buffer, pH 7.4, at 22 C containing 5% glutaraldehyde (vol/vol) for 6–12 h. Thereafter, membranes containing cultured cells were carefully removed from plastic supports, briefly rinsed in PBS, and processed for microscopy as previously described (14).

Incubation of Sertoli cells with androgens in vitro
To assess the effects of androgens on Rab8B expression, Sertoli cells were cultured either at high (0.75 x 106 cells/cm2) or low (0.05 x 106 cells/cm2) density. Cultures were hypotonically treated, and cells were incubated for an additional 3 d with daily changes of medium. Thereafter, Sertoli cells were cultured for approximately 12 h in the presence of increasing concentrations of either testosterone (10-11–10-5 M) or dihydrotestosterone (DHT; 10-11–10-5 M; Sigma-Aldrich). In these cultures, the time of addition of testosterone or DHT was designated time zero (control). Control experiments consisted of Sertoli cells cultured alone and in the presence of vehicle (ethanol) under the conditions described above.

Cell and tissue lysates, membrane and cytosol preparations, and immunoprecipitation
Sertoli and germ cell lysates were obtained by scraping cells at specified time points in cell lysis buffer A, whereas tissue lysates were collected in the same buffer using a tissue/buffer ratio of 1:3. In both cases samples were briefly sonicated on ice and centrifuged at 15,000 x g for 15 min at 4 C, and supernatants were collected as cell and tissue lysates. To obtain membrane and cytosol preparations, Sertoli cells (0.75 x 106 cells/cm2) were terminated at specified time points by scraping cells in cell lysis buffer B (20 mM Tris, pH 7.4, at 22 C containing 2 mM PMSF, 2 mM 1,10 phenanthroline, and 1 µg/ml pepstatin A). This was followed by a brief incubation at room temperature on a rotator, five consecutive rounds of snap-freezing and thawing, centrifugation at 45,000 x g, and collection of supernatants as Sertoli cell cytosol. Thereafter, membrane protein solubilization buffer [0.125 M Tris, pH 7.4, at 22 C containing 0.1% Nonidet P-40 (vol/vol), 0.1% Triton X-100 (vol/vol), 20% glycerol (vol/vol), 1.6% 2-mercaptoethanol (vol/vol), 2 mM PMSF, 2 mM 1,10 phenanthroline, and 1 µg/ml pepstatin A] was added to the remaining pellet and incubated for 6–12 h at room temperature. Supernatants were collected after centrifugation at 45,000 x g as membrane proteins. In selected experiments, such as those involving membrane and cytosol preparations, in which the endogenous level of Rab8B protein was below the detection limit of immunoblotting, immunoprecipitation was performed. Approximately 400 µg protein obtained as described above, except that immunoprecipitation buffer [50 mM Tris, pH 7.4, at 22 C containing 0.15 M NaCl, 1% Nonidet P-40 (vol/vol), 10% glycerol (vol/vol), 2 mM PMSF, 2 mM EDTA, and 2 mM N-ethylmaleimide] was used in place of cell lysis buffer A, was pretreated with normal mouse serum (1:100) for 2–4 h on a rotator, followed by the addition of protein A/G plus agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Thereafter, supernatants, collected after a low speed centrifugation, were incubated with anti-Rab8B (1:100; catalog no. R66320-150, lot 2, BD Transduction Laboratories, Inc., San Diego, CA). Immunocomplexes, precipitated by addition of protein A/G plus agarose, were thereafter washed in immunoprecipitation wash buffer (50 mM Tris, pH 7.4, at 22 C containing 0.15 M NaCl, 2 mM PMSF, 2 mM EDTA, and 2 mM N-ethylmaleimide). Proteins were subsequently extracted by heating in SDS sample buffer [0.125 M Tris, pH 6.8, at 22 C containing 1% SDS (wt/vol), 20% glycerol (vol/vol), and 1.6% 2-mercaptoethanol (vol/vol)], with approximately half of the sample volume was electrophoresed by SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH) for immunoblotting.

Immunoblotting
Approximately 100 µg protein from cell and tissue lysates or immunoprecipitated samples obtained as described above were electrophoresed by SDS-PAGE under reducing conditions (27, 28). After electrophoresis, proteins were electroblotted onto a nitrocellulose membrane, and nonspecific sites were blocked with 6% (wt/vol) nonfat milk. Thereafter, the blot was incubated with primary antibody, followed by submersion in either bovine antirabbit (1:2000; Santa Cruz Biotechnology, Inc.) or bovine antimouse (1:1000; Sigma-Aldrich) IgG conjugated to horseradish peroxidase. The blot was washed extensively, and immunoreactive proteins were detected by enhanced chemiluminescence using a Western blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies used for immunoblotting experiments were as follows: mouse anti-Rab8B (1:400) and mouse antipaxillin (1:100; catalog no. P13520–150, lot 6, BD Transduction Laboratories, Inc.); rabbit anti-N-cadherin (1:100; catalog no. sc-7939, lot C081), rabbit anti-E-cadherin (1:100, catalog no. sc-7870, lot K080), rabbit anti-integrin ß1 (1:100; catalog no. sc-8978, lot E221), rabbit anti-ß-catenin (1:100; catalog no. sc-7199, lot L060), rabbit anti-{alpha}-tubulin (1:100; catalog no. sc-5546, lot D042), rabbit ß-tubulin (1:100; catalog no. sc-9104, lot I1602), rabbit anti-{gamma}-tubulin (1:100; catalog no. sc-10732, lot A311), and rabbit anti-vimentin (1:100; catalog no. sc-5565, lots E012 and B252; Santa Cruz Biotechnology, Inc.); rabbit antioccludin (1:100; catalog no. 71-1500, lot 11067632) and mouse anti-ZO-1 (1:100; catalog no. 33-9100, lot 20269234; Zymed Laboratories, Inc., San Francisco, CA); mouse anti-Rab9 (1:100; catalog no. 552101, lot B36487; Calbiochem-NovaBiochem, La Jolla, CA); mouse antiactin (1:100; catalog no. 1378996, lot 83149734, Roche Molecular Biochemicals, Indianapolis, IN); and rabbit antitestin (29, 30). It must also be noted that the Rab8B antibody used in this study can detect both Rab8 (i.e. Rab8A) and Rab8B in selected cell and tissue lysate samples. The 24-kDa immunoreactive band corresponding to Rab8B found in lysates from testis, Sertoli, and germ cells was consistently 4-fold the intensity of Rab8 with an apparent molecular mass of about 22 kDa when densitometrically scanned.

Chemical cross-linking and coimmunoprecipitation
To identify proteins interacting with Rab8B, chemical cross-linking and coimmunoprecipitation experiments were performed. In brief, Sertoli cells (0.75 x 106 and 0.05 x 106 cells/cm2) were cultured on Matrigel-coated dishes, hypotonically treated on d 2, and allowed to recover 24 h before the addition of dithiobis-(succinimidylpropionate) (DSP; 2 mM/dish), a membrane-permeable, thio-cleavable cross-linker (Pierce Chemical Co., Rockford, IL; Ref 31). Control experiments consisted of Sertoli cells incubated alone and in the presence of vehicle (dimethylsulfoxide) under the same conditions described above. Cells were terminated by scraping in immunoprecipitation buffer and sonicated briefly on ice, and supernatants were collected after centrifugation. Thereafter, immunoprecipitation was performed, followed by electrophoresis by SDS-PAGE under reducing and nonreducing conditions. In these experiments, in particular, the entire sample volume obtained from the immunoprecipitation step was used for SDS-PAGE. Proteins were electroblotted onto a nitrocellulose membrane for immunoblotting. In another series of experiments, seminiferous tubules were isolated from the testis of a 90-d-old rat, as previously described from this laboratory (21, 32), for cross-linking experiments.

Immunohistochemistry
Immunostaining was performed using a Histostain-SP kit as instructed by the manufacturer with minor modifications (Zymed Laboratories, Inc.). In brief, adult rat testes were removed, immediately frozen in CYTO-FREEZE (1,1,1,2-tetrafluoroethane, Control Co., Houston, TX), and embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Inc., Torrance, CA), and 6-µm sections were cut in a Microm HM 500M cryostat at -20 C. Sections, collected on poly-L-lysine-coated slides (Sigma-Aldrich), were fixed in Bouin’s fixative and washed successively with PBS (10 mM sodium phosphate, pH 7.4, at 22 C containing 0.15 M NaCl), and endogenous peroxidase activity was blocked with hydrogen peroxide (3%, vol/vol). Thereafter, nonspecific sites were blocked in nonimmune goat serum (10%, vol/vol), followed by incubation in anti-Rab8B (1:200). Sections were then saturated with biotinylated goat antimouse secondary antibody (1:500; Zymed Laboratories, Inc.), followed by streptavidin-horseradish peroxidase. Immunoreactive Rab8B was visualized by 3-amino-9-ethyl-carbazole, which produced reddish brown precipitates. Sections were counterstained with hematoxylin and mounted for microscopy using a BX 40 system microscope (Olympus Corp., Melville, NY). Images were captured with a PM-30 automatic photomicrographic system and were compiled using Adobe Photoshop (version 7.0, Adobe Systems, Mountain View, CA). Controls included incubation of sections in either PBS or normal mouse serum (1:200) in place of primary and secondary antibodies.

Treatment of rats with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) in vivo
To assess the effects of AF-2364 on testicular Rab8B expression, in particular during the depletion of round and elongate spermatids from the seminiferous epithelium, a single dose of AF-2364 (40 mg/kg body weight, rats at 250–300 g body weight) was administered to adult rats (n = 4 rats/time point) by ip injection (suspended in 0.25% methylcellulose) as previously described (33, 34). Control animals received PBS alone. Thereafter, rats were killed at specified time points, and testes were removed for RT-PCR, immunoblotting, and immunohistochemistry.

Semiquantitative RT-PCR
Semiquantitative RT-PCR was performed as previously described (14, 16). The primers used for the amplification of Rab8B (13) and S-16 (35) were as follows: 5'-TTCTCAGAGGACGCCTTCAACA-3' (Rab8B sense, nucleotides 82–103), 5'-AGGTATTCTGCAGTCTCTCAGAA-3' (Rab8B antisense, nucleotides 637–659), 5'-TCCGCTGCAGTCCGTTCAAGTCTT-3' (S-16 sense, nucleotides 15–38), and 5'-CCAAACTTCTTGGATTCGCAGCG-3' (S-16 antisense, nucleotides 376–399). After PCR, aliquots of 10 µl from PCR products were withdrawn and resolved onto 5% T polyacrylamide gels in TBE buffer (45 mM Tris, 45 mM boric acid, and 1 mM EDTA, pH 8.0, at 22 C), and PCR products were visualized by ethidium bromide staining. To ensure that the amplifications of Rab8B and S-16 were both in the linear range of cDNA production, a series of preliminary studies was performed in which different concentrations of Rab8B and S-16 primers were combined with testis-, Sertoli-, or germ cell-derived cDNA templates as previously described (16, 36). To obtain semiquantitative data, approximately 0.2 µg each of the Rab8B and S-16 sense primers was 5' end labeled with [{gamma}-32P]ATP (specific activity, 6000 Ci/mmol; Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Promega Corp.). Approximately 1 x 106 cpm of the labeled Rab8B and S-16 sense primers were used per PCR reaction tube. PCR products were visualized by autoradiography.

General methods
Statistical analyses were performed by either t test (Dunnett’s posttest; all data points were compared against the control) or ANOVA (Tukey’s or Bonferroni’s posttest; all data points were compared against each other) using the InStat software package (version 3.01, GraphPad Software, Inc., San Diego, CA). All experiments performed in this study had triplicate data points, and each experiment was repeated two to four times. Densitometric scanning was performed using an UltroScan XL enhanced laser densitometer (Amersham Pharmacia Biotech) at 600 nm or the SigmaGel computer software package (version 1.0, SPSS, Inc., Chicago, IL). Protein estimation was quantified by Coomassie Blue dye binding assay using BSA as a standard. The authenticity of the Rab8B PCR product was verified by direct nucleotide sequencing using Sequenase (Amersham Pharmacia Biotech) as described previously (37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of Rab8B mRNA and protein in different rat tissues and cell types, its changes in expression during testicular development, and immunolocalization in the adult testis
To determine the relative mRNA and protein levels of Rab8B in different tissues and cell types, RT-PCR and immunoblotting were performed. An ethidium bromide-stained gel from an RT-PCR experiment in which a 578-bp PCR product corresponding to Rab8B was detected in the brain, testis, heart, kidney, spleen, Sertoli, and germ cells (Fig. 1AGo). By comparison, the mRNA level of Rab8B was apparently higher in germ cells (90-d-old) than in Sertoli cells (20-d-old; Fig. 1AGo). To confirm RT-PCR data, the presence of Rab8B protein in selected tissues was detected by immunoblotting when a 24-kDa protein corresponding to Rab8B was found in the brain, testis, heart, and kidney (Fig. 1BGo). A more detailed analysis of Rab8B expression in the testis by semiquantitative RT-PCR revealed a 6-fold increase by 30 d of age (Fig. 1Go, C and D) during the initiation of meiosis when secondary spermatocytes first appear within the seminiferous epithelium. Studies by RT-PCR and immunoblotting were backed by the immunohistochemical localization of Rab8B in the adult rat testis (Fig. 1EGo). Immunoreactive Rab8B was found to localize in the basal compartment associating with spermatocytes (Fig. 1EGo, panel a, inset, arrowheads) in all stages of the spermatogenic cycle (Fig. 1EGo, panels a–e). Moreover, immunoreactive Rab8B was also detected at the site of elongate spermatids in stages XII–XIV in addition to being found in the basal compartment in these stages (Fig. 1EGo, panel e, arrowheads). Immunoreactive precipitate was not detected in sections incubated with PBS (data not shown) or normal mouse serum in place of primary (Fig. 1EGo, panel c, inset) and secondary (data not shown) antibodies.



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  		Figure 1. A–E, A study of Rab8B expression, protein, and localization. Distribution of Rab8B mRNA and protein in different tissues and cell types. A, An ethidium bromide-stained gel from an RT-PCR experiment demonstrating that a 578-bp PCR product corresponding to Rab8B was detected in the brain, testis, heart, kidney, spleen, Sertoli, and germ cells. B, The relative distribution of Rab8B was confirmed by immunoblot when a 24-kDa immunoreactive protein corresponding to Rab8B was detected in the brain, testis, heart, and kidney. Br, Brain; T, testis; H, heart; Ki, kidney; Sp, spleen; SC, Sertoli cells; GC, germ cells. Changes in Rab8B mRNA during development. C, An autoradiogram of an electrophoresed gel from an RT-PCR experiment demonstrating a 6-fold increase in Rab8B expression at 30 d of age. D, Densitometric scanning of autoradiograms from RT-PCR experiments, such as that shown in C, from at least three experiments in which different sets of aging rat testes were used, normalized against S-16 and testis at 10 d of age. ns, Not significantly different by ANOVA; *, significantly different by ANOVA, P < 0.05; **, significantly different by ANOVA, P < 0.01. Immunolocalization of Rab8B in the adult rat testis. E, Micrographs of seminiferous tubules at different stages of the spermatogenic cycle illustrating that the localization of immunoreactive Rab8B was stage specific. From stages I–X, immunoreactive Rab8B was restricted to the basal compartment (panels a–d) at the level of the spermatocyte (panel a, inset, arrowheads). During stages XII–XIV, immunoreactive Rab8B localized to elongate spermatids in addition to being found in the basal compartment (panel e, arrowheads). Immunostaining was not detected in sections incubated with normal mouse serum (panel c, inset). Bar, 50 µm (panels a–e), 20 µm (panel a, inset), and 200 µm (panel c, inset). The stages of the spermatogenic cycle are noted in each panel in the lower left corner.

 
Changes in Rab8B mRNA and protein in Sertoli and germ cells during development
We proceeded to expand the results shown in Fig. 1Go, C and D to determine whether the increase in testicular Rab8B expression during development can be ascribed to an increase in Rab8B in Sertoli and/or germ cells. All of the Sertoli cells used for this experiment were cultured at high density (0.75 x 106 cells/cm2) to initiate the assembly of tight, anchoring, and gap junctions. Cultures were hypotonically treated and were terminated on d 4 when the mRNA level of Rab8B in Sertoli cells cultured at this density had returned to its basal level. On the other hand, germ cells were terminated immediately after isolation because they are incapable of surviving past 24 h. It was shown that Rab8B expression increased during maturation in both Sertoli (Fig. 2AGo) and germ (Fig. 2BGo) cells when autoradiograms from RT-PCR experiments were densitometrically scanned and normalized against S-16 and Sertoli or germ cells at 10 d of age, as shown (Fig. 2Go, C and D). Figure 2EGo is an immunoblot showing the relative distribution of Rab8B protein in 20- and 60-d-old Sertoli and germ cells.



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  		Figure 2. A–E, Changes in Rab8B expression and protein during development. A and B, Autoradiograms of electrophoresed gels from RT-PCR experiments showing an increase in the Rab8B mRNA level in both Sertoli (A) and germ (B) cells from 45 to 90 d and 20 to 90 d of age, respectively. C and D, Densitometric scannings of autoradiograms from RT-PCR experiments normalized against S-16 and Sertoli or germ cells at 10 d of age. E, A film corresponding to an immunoblot showing the relative distribution of Rab8B in 20- and 60-d-old Sertoli and germ cells. ns, Not significantly different from Sertoli or germ cells at 10 d of age (control) by t test; *, significantly different from control by t test, P < 0.01.

 
Changes in Rab8B mRNA and protein during Sertoli cell junction assembly in vitro
To ascertain whether Rab8B participates in junction assembly, we used an in vitro model in which Sertoli cells were cultured at high and low densities. For high density cultures (0.75 x 106 cells/cm2), functional tight, anchoring, and gap junctions were assembled. Using this model, the expression of Rab8B was found to increase approximately 5-fold from 3–18 h after Sertoli cells were plated on Matrigel-coated dishes (Fig. 3Go, A and B). This was followed by a 2-fold decrease at 1–2 d, but still resulted in a statistically significant increase in Rab8B expression compared with the control on d 0 (Fig. 3Go, A and B). This increase in the Rab8B mRNA level during junction assembly was confirmed by immunoblotting, as shown in Fig. 3Go, B and C. In Sertoli cells cultured at 0.05 x 106 cells/cm2 (Fig. 3DGo) and 0.025 x 106 cells/cm2 (Fig. 3EGo), where anchoring junctions, such as adherens junctions, were not extensive and did not assemble, respectively, we did not detect any significant changes in Rab8B expression. Tight junctions were not present in Sertoli cells cultured at low density due to the lack of proximity between cells. It must also be noted that the basal Rab8B mRNA level in Sertoli cells cultured at low density was barely visible by semiquantitative RT-PCR. The failure of Rab8B mRNA and protein to increase transiently during junction assembly in low density cultures is plausible given the observation that only about 20% of Sertoli cells plated at 0.05 x 106 cells/cm2 participate in actual cell-cell contact (Fig. 3FGo). A more detailed study by immunoblotting using membrane and cytosol preparations revealed that Rab8B is distributed differentially within Sertoli cells during junction assembly (Fig. 3Go, G and H). It was demonstrated that membrane-associated Rab8B increased during junction assembly, whereas cytosol-associated Rab8B decreased during this time period (Fig. 3Go, G and H).



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  		Figure 3. A–H, A study to examine the participation of Rab8B in Sertoli cell junction assembly in vitro. Changes in Rab8B mRNA and protein during junction assembly. A, An autoradiogram of an electrophoresed gel from an RT-PCR experiment illustrating an increase in Rab8B expression from 3–18 h when Sertoli cells were cultured at high density. B, Densitometric scanning of autoradiograms from RT-PCR experiments normalized against S-16 and Sertoli cells on d 0. C, A film corresponding to an immunoblot illustrating an increase in Rab8B protein during Sertoli cell junction assembly. B, Densitometric scanning of films from immunoblots normalized against d 0. D and E, Densitometric scannings of autoradiograms from RT-PCR experiments normalized against S-16 and Sertoli cells on d 0 illustrating no detectable changes in Rab8B expression when Sertoli cells (D, 0.05 x 106 cells/cm2; E, 0.025 x 106 cells/cm2) were cultured at low density. Sertoli cells were cultured at low density. F, A micrograph not only illustrating that Sertoli cells cultured at this density do not permit the assembly of extensive cell-cell adherens junctions, but also showing the purity of Sertoli cells used in this study. Bar, 40 µm. Cellular distribution of Rab8B protein during junction assembly. G, Films corresponding to immunoblots demonstrating an increase and a decrease in membrane- and cytosol-associated Rab8B protein during junction assembly, respectively. H, Densitometric scanning of films normalized against Sertoli cells on d 0. ns, Not significantly different from Sertoli cells on d 0 (control) by t test; *, significantly different from control by t test, P < 0.05; **, significantly different from control by t test, P < 0.01.

 
Changes in Rab8B mRNA and protein during Sertoli-germ cell adherens junction assembly in vitro
Results presented herein suggest that Rab8B may participate in junction assembly, as changes in its mRNA and protein levels were detected during junction formation in high, but not low, density Sertoli cell cultures (Fig. 3Go, A–E). To determine whether similar changes could be detected during Sertoli-germ cell adherens junction assembly, isolated germ cells were cocultured with Sertoli cells (0.5 x 106 cells/cm2; Sertoli cells were previously cultured for 5 d to allow the formation of an epithelium with intact tight, anchoring, and gap junctions) at a Sertoli/germ cell ratio of 1:5. It was found that Rab8B mRNA (Fig. 4AGo) and protein (Fig. 4BGo) increased during adherens junction assembly. Figure 4CGo represents the same immunoblot as that shown in Fig. 4BGo, but stripped and reprobed with antiactin (42 kDa), which served as a sample loading control. In control cultures, which consisted of Sertoli cells cultured under the same conditions as those reported for Fig. 4Go, A–C, but without the addition of germ cells, we failed to detect any changes in Rab8B expression (data not shown). To assess the assembly of Sertoli-germ cell adherens junctions, two additional studies were performed. First, microscopic analysis of serial cross-sections of a Sertoli-germ cell coculture (Fig. 4DGo, panels a–c) revealed that germ cell adhesion to Sertoli cells was achieved by 24 h (Fig. 4DGo, panel c). Second, when isolated germ cells were metabolically labeled with [35S]methionine, added to the Sertoli cell epithelium, and adhesion was assessed by quantifying radioactivity at specified time points, it was found that germ cell adhesion reached a maximum by 2 h (Fig. 4EGo). This is consistent with previous results from other laboratories (25, 26). These data are also in agreement with another study from this laboratory in which the mRNA and protein levels of several adherens junction molecules, such as N- and E-cadherin, ß-catenin, and p120ctn, were investigated during Sertoli-germ cell adherens junction assembly (21).



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  		Figure 4. A–E, A study to examine the participation of Rab8B in Sertoli-germ cell adherens junction assembly in vitro. Changes in Rab8B mRNA and protein during Sertoli-germ cell adherens junction assembly are shown. A, Densitometric scanning of autoradiograms from RT-PCR experiments normalized against S-16 and Sertoli-germ cell cocultures at 0 h, illustrating an increase in Rab8B expression from 2–6 h. B, A film corresponding to an immunoblot using lysates obtained from Sertoli-germ cell cocultures, demonstrating a transient, but significant, increase in Rab8B protein during Sertoli-germ cell junction assembly. C, The same immunoblot as that shown in B, but stripped and reprobed with antiactin (42 kDa), which served as a sample loading control. Micrographs of Sertoli-germ cell cocultures. D, Micrographs of a Sertoli-germ cell coculture showing germ cells settle onto the Sertoli cell epithelium by 15 min (panel a), followed by adhesion by 1 h (panel b), and junction assembly by 24 h (panel c). Bar, 5 µm. Assessing the assembly of Sertoli-germ cell adherens junctions in vitro by an adhesion assay. E, The adhesion of 35S-labeled germ cells to the Sertoli cell epithelium reached a maximum by 2 h. ns, Not significantly different from Sertoli-germ cell cocultures at 0 h (control) by t test; *, significantly different from control by t test, P < 0.05; **, significantly different from control by t test, P < 0.001. SC, Sertoli cell; GC, germ cell.

 
Effects of GCCM and ACM on the Sertoli cell steady-state Rab8B mRNA level
Results showed an up-regulation of Rab8B during the initiation of junction assembly when freshly isolated germ cells were cocultured with Sertoli cells. However, this experiment failed to indicate whether the stimulus responsible for the increase in Rab8B was initiated by cell-cell contact, a germ cell-soluble factor, or both. To address this in part, we cultured Sertoli cells in the presence of increasing concentrations of GCCM (20–1000 µg protein/dish). It was demonstrated that crude GCCM, when incubated with Sertoli cells for 4 h, stimulated Rab8B expression dose-dependently (Fig. 5Go, A and B). To demonstrate the specificity of the GCCM-mediated effect on Rab8B expression, Sertoli cells were cultured under the same conditions as those detailed in Fig. 5Go, A and B, but in the presence of increasing concentrations of ACM (5–1000 µg protein/dish). ACM failed to affect the Sertoli cell Rab8B protein level (Fig. 5CGo).



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  		Figure 5. A–C, Effects of GCCM and ACM on Rab8B expression. A, An autoradiogram of an electrophoresed gel from an RT-PCR experiment illustrating a dose-dependent increase in Rab8B expression when Sertoli cells were cultured in the presence of increasing concentrations of crude GCCM for 4 h. B, Densitometric scanning of autoradiograms from RT-PCR experiments normalized against S-16 and Sertoli cells at 0 h without the addition of GCCM. C, A film corresponding to an immunoblot illustrating that increasing concentrations of ACM failed to affect the Sertoli cell Rab8B protein level. ns, Not significantly different from Sertoli cells at 0 h without the addition of GCCM (control) by t test; *, significantly different from control by t test, P < 0.05; **, significantly different from control by t test, P < 0.01.

 
Effects of androgens on the Sertoli cell steady-state Rab8B mRNA level
The GCCM-mediated dose-dependent stimulation of Rab8B expression (Fig. 5Go, A and B) cannot be ascribed to androgens, as ultrafiltration removed such factors from our GCCM sample. Being that the level of testosterone in the testis is 100 times more than that found in the systemic circulation (2 nM) (38), we have examined whether testosterone and its metabolite, DHT, can affect Sertoli cell Rab8B expression. The addition of increasing concentrations of testosterone (10-11–10-5 M) failed to affect the Rab8B mRNA level when Sertoli cells were cultured at high density (0.75 x 106 cells/cm2; Fig. 6Go, A and B). On the contrary, testosterone at 10-9–10-5 M was able to increase Rab8B expression in low density (0.05 x 106 cells/cm2) Sertoli cell cultures (Fig. 6Go, C and D). The effect of this androgen on Rab8B expression was apparently restricted to testosterone, as DHT failed to up-regulate Rab8B mRNA in high (Fig. 6EGo) and low (Fig. 6FGo) density cultures.



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  		Figure 6. A–F, Effects of androgens on Rab8B expression. A, An autoradiogram of an electrophoresed gel from an RT-PCR experiment demonstrating that no significant changes in Rab8B expression were detected when Sertoli cells (0.75 x 106 cells/cm2) were cultured in the presence of increasing concentrations of testosterone (10-11–10-5 M) for 12 h. B, Densitometric scanning of autoradiograms from RT-PCR experiments normalized against S-16 and Sertoli cells at 0 h without the addition of testosterone. C, An autoradiogram and its corresponding densitometric scanning (D) illustrating an up-regulation in the Rab8B mRNA level when Sertoli cells (0.05 x 106 cells/cm2) were cultured in the presence of testosterone. E and F, Autoradiograms illustrating no significant changes in Rab8B expression when Sertoli cells (E, 0.75 x 106 cells/cm2; F, 0.05 x 106 cells/cm2) were cultured in the presence of DHT (10-11–10-5 M). ns, Not significantly different from Sertoli cells at time 0 h without the addition of testosterone by t test; *, significantly different from control by t test, P < 0.05; **, significantly different from control by t test, P < 0.05. CTRL, Control.

 
Identification of Rab8B interacting proteins by chemical cross-linking and coimmunoprecipitation
To determine whether Rab8B associates with components that confer functionality to anchoring junctions, chemical cross-linking experiments were performed using DSP, a membrane-permeable, thio-cleavable cross-linker. When lysates obtained from 3-d Sertoli cell cultures (0.75 x 106 cells/cm2), which were treated with DSP, were immunoprecipitated with anti-Rab8B and electrophoresed by SDS-PAGE under nonreducing (mercaptoethanol -, lane 2) and reducing conditions (mercaptoethanol +, lane 3), one (band a, ~150-kDa native molecular weight of IgG) and two (band b, ~50-kDa IgG heavy chain; band c, ~25-kDa IgG light chain) immunoreactive complexes were observed (Fig. 7AGo). In addition, an immunoreactive band corresponding to Rab8B was detected in lane 3 run under reducing conditions (Fig. 7AGo, arrowhead), but not in lane 2, in which the DSP-treated Sertoli cell lysate sample was electrophoresed under nonreducing conditions. Lane 1 in this immunoblot consists of lysate obtained from 3-d Sertoli cell cultures without DSP treatment (control) and shows a 24-kDa immunoreactive band corresponding to Rab8B. To expand this study, we examined whether proteins that confer functionality to anchoring junctions associate with Rab8B. Occludin and ZO-1 were also included in this experiment as negative controls. The first lane in all immunoblots shown in Fig. 7BGo represents lysates obtained from 3-d Sertoli cell cultures electrophoresed under reducing conditions (control). The second lane in all immunoblots represents lysates obtained from 3-d Sertoli cell cultures treated with DSP and electrophoresed under reducing conditions. Figure 7BGo illustrates that Rab8B associates with actin, vimentin, {alpha}-tubulin, and ß-tubulin, but not with {gamma}-tubulin, as all of these proteins were found to cross-link with Rab8B in Sertoli cells (lane 2). Moreover, Rab8B was found to associate with E-cadherin and {gamma}-catenin (Fig. 7BGo, lane 2). This GTPase did not associate with N-cadherin, testin, ß-catenin, paxillin, integrin ß1, ZO-1, or occludin (Fig. 7BGo, lane 2). Results shown in Fig. 7BGo were identical to those of another experiment in which 3-d Sertoli cells lysates without DSP treatment were simply used for coimmunoprecipitation and immunoblotting (data not shown). To confirm these results, we proceeded to repeat virtually all of the coimmunoprecipitation and immunoblotting experiments using isolated seminiferous tubules on which chemical cross-linking was performed. These results (Fig. 7BGo, lane 3) corroborated those obtained from DSP-treated Sertoli cells (Fig. 7BGo, lane 2). A negative control, which involved immunoprecipitating 3-d DSP-treated Sertoli cell lysates with anti-Rab9 instead of anti-Rab8B, has shown this GTPase to cross-link weakly with actin, but not with E-cadherin (Fig. 7BGo, lane 2). Additional cross-linking and coimmunoprecipitation experiments on low density Sertoli cell cultures (0.05 x 106 cells/cm2) failed to show an association between Rab8B and E-cadherin (data not shown). Lastly, we examined the localization of E-cadherin in the adult rat testis and found that immunoreactive E-cadherin was present in the basal compartment (Fig. 7CGo, arrowheads), localizing to a site similar to that in which Rab8B was found (Fig. 1EGo).



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  		Figure 7. A–C, A study to identify Rab8B interacting proteins by chemical cross-linking and coimmunoprecipitation. A, A film corresponding to an immunoblot in which lysates obtained from 3-d Sertoli cell lysates that were treated without (control; lane 1) and with DSP were electrophoresed by SDS-PAGE under nonreducing (lane 2) and reducing (lane 3) conditions. An immunoreactive band corresponding to Rab8B was detected in the control and DSP-treated lysate (lane 3, arrowhead). a, Native molecular mass of IgG, ~150 kDa; b, molecular mass of IgG, heavy chain, ~50 kDa; c, molecular mass of IgG, light chain, ~25 kDa. CTRL, Control. B, A flow chart of chemical cross-linking and coimmunoprecipitation experiments illustrating that Rab8B in Sertoli cells associates with the actin-, intermediate filament (i.e. vimentin)-, and microtubule (i.e. {alpha}-tubulin and ß-tubulin)-based cytoskeletal networks. Sertoli cell Rab8B was also found to associate with E-cadherin and {gamma}-catenin, but not with {gamma}-tubulin, N-cadherin, testin, ß-catenin, paxillin, integrin ß1, occludin, or ZO-1. Lane 1 in all immunoblots, 3-d Sertoli cell lysates without DSP treatment (control) electrophoresed by SDS-PAGE under reducing conditions; lane 2, 3-d Sertoli cell lysates with DSP treatment electrophoresed under reducing conditions; lane 3, another series of experiments in which seminiferous tubules isolated from the testis of an adult rat were treated with DSP, and lysates were electrophoresed by SDS-PAGE under reducing conditions to confirm previous results. Another GTPase, Rab9 (control), was found to associate only weakly with actin in Sertoli cells and not at all with E-cadherin. Immunolocalization of E-cadherin in the testis. C, A micrograph of a seminiferous tubule from the adult rat testis illustrating that immunoreactive E-cadherin was restricted to the basal compartment. Bar, 50 µm.

 
Changes in Rab8B expression and protein during adherens junction disassembly in vivo
If Rab8B is involved in the assembly of adherens junctions, logically its mRNA and protein levels should decline during the disassembly of these junctions. As such, we used an in vivo model in which the administration of AF-2364 resulted in the depletion of germ cells, in particular round and elongate spermatids, from the seminiferous epithelium, to determine whether this argument can be supported. When rats were administered a single dose of AF-2364 (40 mg/kg body weight), the mRNA level of Rab8B decreased by d 1 before the loss of germ cells (Fig. 8Go, A and B). Rab8B expression remained at this decreased level until germ cells began to repopulate the epithelium by approximately 140 d (Fig. 8Go, A and B). These results were corroborated when the decrease in testicular weight was taken into account (Fig. 8BGo) and by immunoblotting (Fig. 8CGo). When testes from rats treated with AF-2364 were processed for immunohistochemistry (Fig. 8DGo), it was found that immunoreactive Rab8B was barely detectable at 21 d.



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  		Figure 8. A–D, A study to examine the participation of Rab8B in adherens junction disassembly in vivo. Changes in Rab8B mRNA and protein during Sertoli-germ cell adherens junction disassembly. A, An autoradiogram of an electrophoresed gel from an RT-PCR experiment illustrating that Rab8B expression decreased during the depletion of germ cells from the epithelium when rats were administered a single dose of AF-2364 (40 mg/kg body weight). B, Densitometric scanning of autoradiograms from RT-PCR experiments normalized against S-16 and testis on d 0. When the reduction in testicular weight during germ cell depletion was taken into account, a decline in Rab8B expression was also noted (B). ns, Not significantly different by ANOVA; *, significantly different by ANOVA, P < 0.05. C, A film corresponding to an immunoblot demonstrating a decrease in Rab8B protein when tubules were devoid of germ cells as a result of AF-2364 treatment. Immunolocalization of Rab8B in adult testes from rats administered AF-2364. D, Micrographs of seminiferous tubules from adult testes 0, 2, 7, and 21 d post treatment, illustrating that the presence of immunoreactive Rab8B in the basal compartment decreased during germ cell depletion (panels a and b vs. panel c, inset, arrowheads, and panel d). Bar, 40 µm (panel a and panel c, inset); 80 µm (panels b–d).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
For several years the function of Rab GTPases was largely unknown. Today at least 60 members exist, and Rab GTPases are known to function as cellular "traffic cops" by regulating intracellular trafficking events (for reviews, see Refs. 10 and 39). We chose to study Rab8B based on previous studies reporting high expression of Rab8B in the testis (13) and, more importantly, its implication in cell junction dynamics (40, 41, 42). However, an extensive literature search revealed that not a single study had been conducted which would connect Rab8B to junction dynamics. In this study we present results that focus on changes detected in Rab8B expression and protein during adherens junction assembly and disassembly coupled with chemical cross-linking and coimmunoprecipitation experiments to provide evidence for the participation of Rab8B in these events. To our knowledge, this is one of the first reports describing a possible link between Rab8B and regulation of the adherens junction (Fig. 9Go).



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  		Figure 9. A schematic illustration depicting a mechanism likely responsible at least in part for adherens junction disassembly and assembly in the testis mediated by Rab GTPases. During the movement of germ cells across the seminiferous epithelium, adherens junctions must be deassembled and then reassembled. As such, these junctions are not static, but instead are continuously restructuring. This requires that adherens junction proteins, such as cadherins, catenins, nectins, and afadins, be momentarily displaced or removed from the membrane without upsetting the delicate balance that is essential to Sertoli-germ cell communication. In brief, Rab8B transiently cycles between the active GTP-bound (membrane-associated) and inactive GDP-bound (cytosol-associated) conformations, and this is probably triggered by upstream signals. When the adherens junction needs to be disassembled, constituents of the adherens junction, such as E-cadherin, become internalized and confined to an endosome in the cytosol (upper panel) where they await relocalization back to the cell membrane once the junction requires reassembling (lower panel). Rab GTPases, such as Rab8B, are proposed to function in this sequestration event. In the GTP-bound conformation, on the other hand, Rab8B is likely to deliver endosome-bound and de novo synthesized proteins to the site of the adherens junction. It is also free to interact with an effector molecule, in turn eliciting a cascade of downstream signaling events that regulate junction dynamics. Currently, the identity of the Rab8B effector molecule remains unknown. Cycling between the two conformations occurs in conjunction with an array of accessory proteins, such as guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), GDP dissociation inhibitor (GDI), and GDI displacement factor (GDF). This figure, which was prepared based on previous reports (4 40 49 50 51 ), will continue to be modified as the role of Rab GTPases in the testis becomes clearer.

 
Although a detailed survey of several tissues and cell types revealed that Rab8B was ubiquitously expressed, its mRNA level was apparently highest in the brain and germ cells. The reason for the high Rab8B mRNA level in germ cells isolated from testes from 90-d-old rats was not immediately known, because these cells were terminated promptly by RNA STAT-60 after their isolation; they were not cultured in vitro to initiate the assembly of anchoring junctions. However, additional studies using immunoblotting and immunohistochemistry have confirmed initial experiments by RT-PCR and assigned germ cells a high endogenous Rab8B level. We were also able to detect an elevation in Rab8B expression during testicular aging at 30 d of age at the initiation of meiosis II. Whether this increase is a direct result of germ cell differentiation, an up-regulation of the Sertoli and germ cell Rab8B mRNA levels during development, an increase in Sertoli-germ cell interactions, or some other unrelated event is not known.

To determine whether Rab8B participates in Sertoli and Sertoli-germ cell adherens junction assembly, we used two in vitro models in which 1) Sertoli cells were cultured at high density to initiate tight, anchoring, and gap junction assembly; and 2) isolated germ cells were cocultured with Sertoli cells to initiate anchoring and gap junction assembly only. It was found that Rab8B mRNA and protein were up-regulated severalfold during junction assembly in Sertoli cell cultures (0.75 x 106 cells/cm2), whereas no significant changes were detected when Sertoli cells were cultured at low density (0.05 x 106 and 0.025 x 106 cells/cm2). The apparent increase in Rab8B protein during junction assembly was confirmed when Sertoli cell membrane preparations were obtained, corroborating a previous study from another laboratory that suggested a link between this Rab GTPase and the cell membrane (12). Moreover, Rab8B may not function in the maintenance of adherens junctions, because Sertoli cell Rab8B expression declined to its basal level after junctions were assembled. In another series of experiments we were also able to detect an increase in Rab8B when freshly isolated germ cells were cocultured with Sertoli cells, coinciding with the time of junction assembly when this event was monitored microscopically and by an in vitro germ cell adhesion assay.

At this point in our study the stimulus responsible for Sertoli cell Rab8B regulation was not known, albeit germ cells were likely suspects, because cells must be able to communicate with Sertoli cells during their translocation across the epithelium. Moreover, previous studies from our and other laboratories have consistently shown that germ cells can dictate Sertoli cell function (for reviews, see Refs. 43, 44, 45). To determine whether this is also applicable to the regulation of Rab8B, we cultured Sertoli cells with increasing concentrations of crude GCCM for 4 h. The fact that GCCM, when incubated with Sertoli cells for such a short period of time, can stimulate the Rab8B mRNA level suggests the existence of a mechanism in which cell junctions can be regulated at times of germ cell movement. Rab8B may indeed be involved in the movement of target proteins to or away from the site of the adherens junction (Fig. 9Go). It must also be noted that GCCM was just as effective in up-regulating Sertoli cell Rab8B as were freshly isolated germ cells. It is not known, however, whether the removal of crude GCCM from Sertoli cell cultures can result in a decline in Rab8B expression.

Testosterone, but not DHT, can also affect the Rab8B mRNA level in Sertoli cells cultured at low, but not high, density. The fact that Rab8B expression failed to be stimulated by testosterone in high density cultures was not immediately known. It is possible, however, that the Rab8B mRNA level in these cultures cannot be further stimulated by testosterone, as cells cultured at high density (0.75 x 106 cells/cm2) already established an epithelium with intact adherens junctions. On the other hand, adherens junctions in low density cultures (0.05 x 106 cells/cm2) are not extensive (~20% of the Sertoli cells can actually establish cell-cell contact), and Rab8B expression is at its basal level. The inclusion of testosterone into these cultures may stimulate the arrangement of adherens junction proteins, but due to lack of proximity between cells, adherens junctions may still not be able to form. Moreover, a recent study has shown that testosterone can stimulate the expression of Sertoli cell N- and E-cadherin, ß-catenin, and p120ctn (21).

Our results have shown that Rab8B may participate in cell junction dynamics. However, evidence that would link Rab8B to the adherens junction was still lacking. To identify proteins associating with Rab8B, chemical cross-linking in conjunction with coimmunoprecipitation was performed. The association of Rab8B with components of the cytoskeletal network (i.e. actin, vimentin, {alpha}-tubulin, and ß-tubulin) suggests that this GTPase is important in orchestrating the events that take place during junction assembly and/or disassembly. We were also able to show that Rab8B interacts with E-cadherin (the putative adherens junction protein that confers to cell adhesion in the testis) (21, 46, 47, 48) and its partner, {gamma}-catenin, but not with ß-catenin. Immunohistochemistry indicated that E-cadherin localized to a site similar to that at which immunoreactive Rab8B was found, illustrating a genuine interaction between these two molecules.

In studies using AF-2364, a compound known to induce germ cell loss from the seminiferous epithelium, apparently by disrupting adhesion between Sertoli and germ cells (33, 34), it was shown that Rab8B protein remained relatively unchanged from d 3–12, despite the fact that this is the time that germ cells begin to deplete the epithelium. For instance, by d 7 most of the tubules were devoid of round and elongate spermatids, and by d 21 only spermatogonia and spermatocytes were present. If germ cells contribute approximately half of the Rab8B in the seminiferous epithelium, then there was a significant induction in Rab8B production by Sertoli cells and/or the remaining germ cells in the seminiferous epithelium from d 3–12 post-AF-2364 treatment. Although the reason for such an induction is not immediately known, Rab8B may be needed to ensure that the remaining adherens junctions between Sertoli and germ cells are not damaged any further. Once the pharmacological effects of AF-2364 are lost between d 35–76, Rab8B protein plummets, but then bounces back by d 150–170 when germ cells repopulate the epithelium (33, 34). Although the biological significance of the decrease in Rab8B at d 21–91 post-AF-2364 treatment is not immediately known, it may be the result of a reduction in intracellular trafficking events in Sertoli and germ cells when tubules are devoid of more advanced germ cells.


   Acknowledgments
 
The authors sincerely thank Dr. C. Yan Cheng for several helpful discussions throughout this study and a critical reading of this manuscript. We also thank Miss Nikki P. Y. Lee and Miss Anne M. Conway for assistance with cross-linking and coimmunoprecipitation experiments, and Dr. Meng-yun Mo for nucleotide sequencing of the Rab8B cDNA.


   Footnotes
 
This work was supported in part by a grant from the CONRAD Program (CICCR, CIG-01-74, to D.D.M.).

Abbreviations: ACM, Astrocyte-conditioned medium; AF-2364, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide; DHT, dihydrotestosterone; DSP, dithiobis-(succinimidylpropionate); GCCM, germ cell-conditioned medium; GTPase, guanosine triphosphatase; MDCK, Madin-Darby canine kidney; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate.

Received August 26, 2002.

Accepted for publication January 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cheng CY, Mruk DD 2002 Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development. Physiol Rev 82:825–874[Abstract/Free Full Text]
  2. Gumbiner BM 2000 Regulation of cadherin adhesive activity. J Cell Biol 148:399–403[Abstract/Free Full Text]
  3. Yap AS, Brieher WM, Gumbiner BM 1997 Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 13:119–146[CrossRef][Medline]
  4. Aplin AE, Howe A, Alahari SK, Juliano RL 1998 Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev 50:197–263[Abstract/Free Full Text]
  5. Vleminckx K, Kemler R 1999 Cadherins and tissue formation: integrating adhesion and signaling. BioEssays 21:211–220[CrossRef][Medline]
  6. Le TL, Yap AS, Stow JL 1999 Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J Cell Biol 146:219–232[Abstract/Free Full Text]
  7. Martenson C, Stone K, Reedy M, Sheetz M 1993 Fast axonal transport is required for growth cone advance. Nature 366:66–69[CrossRef][Medline]
  8. Lawson MA, Maxfield FR 1995 Ca2+- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. Nature 377:75–79[CrossRef][Medline]
  9. Stenmark H, Olkkonen VM 2001 The Rab GTPase family. Genome Biol 2:3007.1–3007.7
  10. Takai Y, Sasaki T, Matozaki T 2001 Small GTP-binding proteins. Physiol Rev 81:153–208[Abstract/Free Full Text]
  11. Zerial M, McBride H 2001 Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117[CrossRef][Medline]
  12. Huber LA, Pimplikar S, Parton RG, Virta H, Zerial M, Simons K 1993 Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol 123:35–45[Abstract/Free Full Text]
  13. Armstrong J, Thompson N, Squire JH, Smith J, Hayes B, Solari R 1996 Identification of a novel member of the Rab8 family from the rat basophilic leukaemia cell line, RBL. 2H3. J Cell Sci 109:1265–1274[Abstract]
  14. Mruk D, Zhu LJ, Silvestrini B, Lee WM, Cheng CY 1997 Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro. J Androl 18: 612–622
  15. Cheng CY, Mather JP, Byer AL, Bardin CW 1986 Identification of hormonally responsive proteins in primary Sertoli cell culture medium by anion-exchange high performance liquid chromatography. Endocrinology 118:480–488[Abstract]
  16. Mruk D, Cheng CY 1999 Sertolin is a novel gene marker of cell-cell interactions in the rat testis. J Biol Chem 274:27056–27068[Abstract/Free Full Text]
  17. Mruk DD, Siu MKY, Conway AM, Lee NPY, Lau ASN, Cheng CY, Role of tissue inhibitor of metalloproteases-1 in junction dynamics in the testis. J Androl, in press
  18. Galdieri M, Ziparo E, Palombi F, Russo MA, Stefanini M 1981 Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions. J Androl 5:249–259
  19. Li JC, Lee WM, Mruk DD, Cheng CY 2001 Regulation of Sertoli cell myotubularin (rMTM) expression by germ cells in vitro. J Androl 22:266–277[Abstract]
  20. Aravindan GR, Mruk D, Lee WM, Cheng CY 1997 Identification, isolation, and characterization of a 41-kilodalton protein from rat germ cell-conditioned medium exhibiting concentration-dependent dual biological activities. Endocrinology 138:3259–3268[Abstract/Free Full Text]
  21. Lee NPY, Mruk D, Lee WM, Cheng CY 2003 Is the cadherin/catenin complex a functional unit of cell-cell actin-based adherens junctions in the rat testis? Biol Reprod 68:489–508[Abstract/Free Full Text]
  22. Pineau C, Syed V, Bardin CW, Jegou B, Cheng CY 1993 Germ cell conditioned medium contains multiple factors that modulate the secretion of testin, clusterin, and transferrin by Sertoli cells. J Androl 14:87–98[Abstract/Free Full Text]
  23. Zwain IH, Grima J, Cheng CY 1994 Regulation of clusterin secretion and mRNA expression in astrocytes by cytokines. Mol Cell Neurosci 5:229–237[CrossRef][Medline]
  24. Zwain IH, Yen SS, Cheng CY 1997 Astrocytes cultured in vitro produce estradiol-17ß and express aromatase cytochrome P-450 (P-450AROM) mRNA. Biochim Biophys Acta 1334:338–348[Medline]
  25. Enders GC, Millette CF 1988 Pachytene spermatocytes and round spermatid binding to Sertoli cells in vitro. J Cell Sci 90:105–114[Abstract/Free Full Text]
  26. Cameron DF, Muffly KE 1991 Hormonal regulation of spermatid binding. J Cell Sci 100:623–633[Abstract/Free Full Text]
  27. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  28. Cheng CY, Musto NA, Gunsalus GL, Frick J, Bardin CW 1985 There are two forms of androgen binding protein in human testis. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin. J Biol Chem 260:5631–5640[Abstract/Free Full Text]
  29. Cheng CY, Grima J, Stahler MS, Lockshin RA, Bardin CW 1989 Testins are two structurally related Sertoli cell proteins whose secretion is tightly coupled to the presence of germ cells. J Biol Chem 264:21386–21393[Abstract/Free Full Text]
  30. Grima J, Wong CCS, Zhu LJ, Zong SD, Cheng CY 1998 Testin secreted by Sertoli cells is associated with the cell surface, and its expression correlates with the disruption of Sertoli-germ cell junctions but not the inter-Sertoli tight junction. J Biol Chem 273:21040–21053[Abstract/Free Full Text]
  31. Joshi S, Burrows R 1990 ATP synthase complex from bovine heart mitochondria. Subunit arrangement as revealed by nearest neighbor analysis and susceptibility to trypsin. J Biol Chem 265:14518–14525[Abstract/Free Full Text]
  32. Zwain IH, Cheng CY 1994 Rat seminiferous tubular culture medium contains a biological factor that inhibits Leydig cell steroidogenesis: its purification and mechanism of action. Mol Cell Endocrinol 104:213–227[CrossRef][Medline]
  33. Cheng CY, Silvestrini B, Grima J, Mo MY, Zhu LJ, Johansson E, Saso L, Leone MG, Palmery M, Mruk D 2001 Two new male contraceptives exert their effects by depleting germ cells prematurely from the testis. Biol Reprod 65:449–461[Abstract/Free Full Text]
  34. Grima J, Silvestrini B, Cheng CY 2001 Reversible inhibition of spermatogenesis in rats using a new male contraceptive, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide. Biol Reprod 64:1500–1508[Abstract/Free Full