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Testis |
Population Council,3 Center for Biomedical Research, New York, New York 10021
Department of Zoology,4 University of Hong Kong, Hong Kong, Special Administrative Region of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least 1–4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (
ho-ass
iated protein 
inase 1, also called ROKß), LIMK1 (LIM kinase 1, also called 
in-11 
sl-1 
ec3 kinase 1), and cofilin but not p140mDia and profilin via phosphorylation. Immunoprecipitation and immunoblots revealed that the induction of LIMK1 was mediated via an increase in its phospho-Ser but not phospho-Tyr content. Furthermore, Y-27632 ([(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide, 2HCl]), a specific ROCK inhibitor, could effectively delay the AF-2364-induced germ cell loss from the seminiferous epithelium in vivo, illustrating that the integrin/RhoB/ROCK/LIMK pathway indeed plays a crucial role in the regulation of Sertoli-germ cell AJ dynamics. The fact that the RhoB pathway in the kidney and brain was not activated suggests that AF-2364 exerts its effects primarily at the testis-specific ES multiprotein complex structures between Sertoli cells and spermatids. In summary, this report illustrates that Sertoli germ cell AJ dynamics are regulated, at least in part, via the integrin/ROCK/LIMK/cofilin signaling pathway.
Sertoli cells, signal transduction, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin [10, 12], protein tyrosine phosphatases such as myotubularin [14, 15], and other proteins such as sertolin, superoxide dismutase, and prostaglandin D2 synthetase [16–18]. Using an in vitro model of Sertoli cell TJ disassembly induced by cadmium chloride, Chung and Cheng [10] found that a transient but significant decrease in occludin mRNA level was associated with induction of uPA when the Sertoli cell TJ barrier was perturbed, suggesting that the dynamics of these junctions are intricately regulated by the interplay of a variety of molecules. Still, the signal transduction pathway(s) that regulates these genes is not known. A more recent study revealed that transforming growth factor ß3 (TGFß3) can perturb the transient induction of occludin and ZO-1 expression during the assembly of Sertoli cell TJs, which in turn leads to a disruption of the Sertoli cell TJ permeability barrier [9]. The effects of TGFß3 on Sertoli cell TJ dynamics are mediated via the p38 mitogen-activated protein kinase pathway [19]. However, the dynamic events of AJs that facilitate germ cell movement during spermatogenesis and the regulation of these events are virtually unknown. Rho GTPases are members of the Ras superfamily. In mammals, the Rho GTPase family consists of 12 distinct proteins: Rho (isoforms A, B, and C), Rac (isoforms 1 and 2), Cdc42 (isoforms Cdc42Hs and G25K), RhoD, RhoG, Rnd1/Rho6, Rnd2/Rho7, Rnd3/Rho8, TC10, and TTF (for reviews, see [20–22]). Like other members of the Ras superfamily, Rho proteins act as molecular switches to control cellular processes by cycling between GTP-bound (active) and GDP-bound (inactive) states (for reviews, see [21, 22]). The activated Rho proteins in turn interact with their corresponding effector proteins to invoke a wide range of cellular responses, such as actin cytoskeleton reorganization and gene transcription (for reviews, see [20, 23]). The effects of Rho on actin reorganization have been well established and extensively characterized in fibroblasts. For instance, activation of Rho in fibroblasts results in the bundling of actin filaments into stress fibers and the clustering of integrins and the integrin-associated proteins into focal adhesion complexes [21]. Rho also has a biphasic effect on regulation of both the assembly and disassembly of AJs and cell adhesion via its effects on the formation of stress fibers [24]. GTPases are also required for the establishment of cadherin-dependent cell-cell adhesion, redistribution of cadherins in cells, and the actin cytoskeleton organization pertinent to AJ dynamics [25].
In the present study, we first sought to examine whether Sertoli and/or germ cells are equipped with RhoB GTPase and the downstream molecular components necessary to transduce signals regulating AJ dynamics. We next determined whether the assembly and/or disassembly of Sertoli-germ cell AJs is associated with an induction of RhoB GTPase. A drug-induced AJ disruption model in the testis that causes germ cell loss from the seminiferous epithelium was used to investigate the downstream signal transduction events of RhoB. These results suggest that the testis may be using a similar molecular mechanism to regulate AJ dynamics associated with germ cell movement in the seminiferous epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats were obtained from Charles River Laboratories (Kingston, MA). The use of animals for these studies was approved by the Rockefeller University Animal Care and Use Committee with protocol nos. 00111 and 95129-R.
Antibodies
The polyclonal antibodies used were obtained commercially. They were raised in rabbits against proteins of either rat (RhoB), human (integrin ß2, LIMK1), or mouse (ROCK1) origin. Antibodies against RhoB (catalog no. sc-180, lot no. F281) and integrin ß2 (catalog no. sc-8420, lot no. K270) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against ROCK1 (catalog no. 611137, lot no. 1) and LIMK1 (catalog no. 611748, lot no. 1) were from BD Transduction Laboratories (San Diego, CA). Antibodies against phosphoserine (catalog no. 61-8100, lot no. 111) and phosphotyrosine (catalog no. 61-5800, lot no. 00861517) were from Zymed Laboratories (South San Francisco, CA). All antibodies used in this study cross-reacted with the corresponding rat protein as indicated by the manufacturers. Bovine anti-rabbit IgG (catalog no. sc-2370, lot no. B051) and goat anti-mouse IgG (catalog no. A-2304, lot no. 51K4849) conjugated to horseradish peroxidase were used as secondary antibodies and were purchased from Santa Cruz Biotechnology and Sigma (St. Louis, MO), respectively. Both the catalog and lot numbers are indicated because selected antibodies from other vendors failed to yield satisfactory results in preliminary experiments.
Preparation of Testicular Cells for Culture Experiments
Sertoli cell cultures
Primary Sertoli cells were isolated from 20-day-old Sprague-Dawley rats as previously described [26, 27]. Freshly isolated Sertoli cells were cultured at high cell density (0.5 x 106 cells/cm2) in 12-well dishes coated with Matrigel (Collaborative Research, Bedford, MA) in serum-free Ham F12 nutrient mixture and Dulbecco modified Eagle medium (F12/DMEM) (1:1, v:v; Sigma) supplemented with gentamicin (20 mg/L), 15 mM Hepes, sodium bicarbonate (1.2 g/L), bovine insulin (10 µg/ml), human transferrin (5 µg/ml), epidermal growth factor (2.5 ng/ml), and bacitracin (5 µg/ml). For the assessment of the TJ barrier, Sertoli cells were cultured on Matrigel-coated bicameral units instead [9]. Cells were then incubated at 35°C in a humidified atmosphere of 95% air and 5% CO2 (v/v) and treated as cultures at time 0. To obtain Sertoli cell cultures with 98% purity, cells were hypotonically treated with 20 mM Tris, pH 7.4, at 22°C for 2.5 min to lyse residual germ cells [28] 36 h after plating. Cells were washed twice with F12/DMEM to remove cellular debris. Medium was replaced every 24 h thereafter. Cells were incubated for an additional 6 days to allow the assembly of the Sertoli TJ permeability barrier, which was monitored by measuring the transepithelial electrical resistance (TER) across the Sertoli cell epithelium and the restricted diffusion of [3H]-inulin or [125I]-BSA from the apical to the basal compartment of the bicameral unit across the Sertoli cell epithelium as detailed elsewhere [9, 29]. Cultures were terminated at specified time points and processed for RNA extraction. Total RNA was extracted from Sertoli cells by using RNA STAT-60 (Tel-Test "B", Inc., Friendswood, TX).
Isolation of Sertoli cells from adult rat testes Sertoli cells from 60- and 90-day-old Sprague-Dawley rat testes were isolated as previously described [30] with modifications as detailed elsewhere [15, 19]. Sertoli cells were plated in 100-mm Petri dishes in F12/DMEM supplemented with 15 mM Hepes, 1.2 g/L sodium bicarbonate, 20 mg/L gentamicin, 10 µg/ml bovine insulin, 5 µg/ml human transferrin, 2.5 ng/ml epidermal growth factor, and 10 µg/ml bacitracin. Cultures were incubated in a humidified atmosphere of 95% air and 5% CO2 (v/v) at 35°C for 72 h. Residual germ cells were lysed by exposing these Sertoli cell cultures to a hypotonic treatment using 20 mM Tris, pH 7.4, at 22°C for 2.5 min [28]. The purity of Sertoli cells was >85% after this hypotonic treatment step. Sertoli cells were allowed to recover for 24 h, and cultures were terminated by RNA STAT-60 for RNA extraction.
Germ cells
Germs cells were isolated from 5-, 10-, 20-, 40-, 60-, and 90-day-old Sprague-Dawley rat testes by a mechanical procedure without the use of trypsin [31] as previously described [18]. These freshly isolated germ cells were used immediately for RNA isolation or for coculture experiments because >90% of germ cells were nonviable (as assessed by erythrosine red-dye exclusion test) after 18 h when cultured alone [32]. These germ cell preparations had negligible somatic cell contamination since reverse transcription polymerse chain reaction (RT-PCR) using a primer pair specific to testin (a Sertoli cell product) failed to detect testin mRNA, consistent with earlier published results [31].
Sertoli-germ cell coculture Primary Sertoli cells isolated as described above were used for Sertoli-germ cell coculture experiments as described previously [12, 27]. Isolated Sertoli cells were cultured on Matrigel-coated dishes at a cell density of 0.5 x 106 cells/cm2. Cultures were hypotonically treated 36 h after their isolation. Medium was replaced every 24 h thereafter, and Sertoli cells were cultured for an additional 4 days to allow the establishment of cell junctions forming an epithelium with intact TJs and AJs as characterized elsewhere by morphological and other pertinent analyses [12, 13, 29]. Freshly isolated germ cells from adult rat testes were added to this Sertoli cell epithelium on Day 6 using a Sertoli:germ cell ratio of 1:1 to initiate Sertoli-germ cell AJ assembly. For coculture experiments, the F12/DMEM was supplemented with 20 mg/L gentamicin, 1.2 g/L sodium bicarbonate, 15 mM Hepes, 2 mM sodium pyruvate, 6 mM sodium DL-lactate, 10 µg/ml bovine insulin, 5 µg/ml human transferrin, 5 µg/ml bacitracin, and 2.5 ng/ml epidermal growth factor. Cocultures were terminated at specified time points at 0, 5, 15, 30, and 45 min and 1, 2, 3, 4, 6, 24, and 48 h. Total RNA was subsequently extracted using RNA STAT-60.
Treatment of Sertoli-germ cell cocultures with AF-2364 Sertoli-germ cell cocultures were prepared as described above. Freshly isolated germ cells were added to the Sertoli cell epithelium on Day 6 after Sertoli cells had been cultured alone for 5 days in 24-well dishes (Corning) using a Sertoli:germ cell ratio of 1:1 with Sertoli cells at 0.5 x 106 cells/cm2. Germ cells were then cocultured with Sertoli cells for an additional 2 days to allow the assembly of Sertoli-germ cell AJs [33, 34]. Old medium was removed, and fresh medium containing AF-2364 (125 ng/ml, 2 ml/dish using 24-well dishes) was added to these cultures. AF-2364 was dissolved in ethanol at 1 mg/ml. Cells were then terminated at specified time points. A set of controls was prepared, which included cells cultured alone without AF-2364 and with vehicle only. Total RNA was extracted using RNA STAT-60.
Disruption of Sertoli-germ cell AJs by hypotonic treatment Sertoli-germ cell cocultures were prepared as described above in 24-well dishes. To disrupt the AJs between Sertoli cells and germ cells, where germ cells had been added to the Sertoli cell epithelium and cocultured for 2 days to allow the assembly of both desmosome-like AJs and ESs [33, 34], these cocultures were exposed to hypotonic treatment using 20 mM Tris, pH 7.4, at 22°C for 2.5 min [28] to lyse germ cells, disrupting the Sertoli-germ cell AJs. After hypotonic treatment, cells were washed with F12/DMEM to remove cellular debris. Thereafter, these cocultures were terminated at 5, 15, and 30 min, 1, 2, 3, and 6 h, and 1 and 2 days. Total RNA was subsequently extracted using RNA STAT-60. In some experiments, whole cell lysates were obtained for subsequent immunoblot analysis.
Morphological Analysis of Testis
Adult Sprague-Dawley rats (270–300 g body weight [bw]) received a single dose of AF-2364 at either 300 or 50 mg/kg bw by gavage [35]. These two dosings of AF-2364 were selected because preliminary studies from this laboratory using 21 different treatment regimens [35, 36](unpublished observations) at 37.5–300 mg/kg bw yielded similar results regarding the efficacy of AF-2364 on inhibiting fertility in adult Sprague-Dawley rats and the time course of germ cell loss from the epithelium. Recently completed toxicity studies by licensed toxicologists revealed that at dose of up to 1 g/kg bw in adult rats, AF-2364 had no detectable toxicity [2]. AF-2364 was suspended in 0.25% methylcellulose (w/v) at a concentration of 100 mg/ml (w/v). Rats were killed by CO2 asphyxiation at specified time points at 0.5, 1, 2, 4, and 8 h and 1, 2, 4, 8, and 14 days after treatment. Control rats received no treatment or received vehicle alone. Testes were removed and either fixed immediately in 10% formalin or placed in liquid nitrogen and stored at -80°C for subsequent sectioning in a cryostat. Formalin-fixed testes were embedded in paraffin, sectioned at 8-µm thickness with a microtome, stained with hematoxylin and eosin, mounted in poly-mount (Polysciences, Warrington, PA), and examined microscopically. Micrographs were photographed using a BX40 microscope (Olympus, Tokyo, Japan) interfaced to a PM-30 Exposure Control Unit (Olympus). For each treatment group, at least 100 cross sections were examined microscopically.
Immunohistochemistry
Frozen sections (8 µm thick) were obtained in a cryostat (Model HM 500M; Microm Lab GmbH, Walldorf, Germany) at -20°C with a disposable blade. Sections were placed on poly-L-lysine-coated slides, air dried at room temperature, and fixed in Bouin fixative. The endogenous peroxidase activity in tissue sections was blocked by treatment with 1% hydrogen peroxide (v/v) for 20 min. Nonspecific sites were blocked by incubating with 10% normal goat serum for 30 min. Tissue sections were incubated with a RhoB polyclonal antibody at a dilution of 1:50 at 4°C overnight and washed three times with PBS (5 min each), then incubated with biotinylated goat anti-rabbit IgG for 30 min and washed three times with PBS, and then incubated with streptavidin peroxidase for 10 min. The immunoreactive RhoB protein was visualized using an aminoethyl carbazole reagent system (Zymed) for 5–10 min. The slides were then washed in water for 10 min to stop the reaction and counterstained with Mayer hematoxylin. Controls were performed by replacing the RhoB antibody with normal goat and rabbit serum, and replacing the second antibody with normal goat serum.
Detection of mRNAs Encoding for Different Target Proteins in Testicular Cells and Their Changes in the Testis and Sertoli-Germ Cell Cocultures by Semiquantitative RT-PCR
Semiquantitative RT-PCR was performed essentially as previously described [9, 18, 19] to detect changes in the steady-state mRNA levels of RhoB and its downstream signal transducers and of integrin ß2, which were coamplified with S16 using specific primer pairs (see Table 1). Coamplification with S16 was included to ensure that equal amounts of RNA were reverse transcribed and amplified in each reaction tube. To enhance the detection limit and to yield data for densitometric scanning to permit semiquantitative analysis, PCR was performed by including trace amounts of [
-32P]-labeled primers as previously described [9]. The ratio of the [32P]-labeled RhoB sense primer to the [32P]-S16 sense primer was the same as that for the unlabeled primer so that the autoradiograms are replicates of the corresponding ethidium bromide-stained gels. To ensure linearity in the synthesis of each target gene and the S16 primer in each PCR tube, 10-µl aliquots of PCR products at 18, 20, 22, 24, and 26 cycles were withdrawn and resolved onto 5% T polyacrylamide gels using 0.5x TBE (44.5 mM Tris-borate, 1 mM EDTA, pH 8.0) as a running buffer in preliminary experiments. Different concentrations of RT products and primer pairs were used in preliminary experiments to assess the appropriate amplification conditions for production of both RhoB and S16 cDNAs in the linear phase. Because of the disparity between the endogenous levels of S16 and the target gene in the samples being investigated in this study, when the production of the target gene and S16 was in the linear phase in the PCR, the synthesis of S16 (although it was at the rear end of the exponential phase) was near the plateau phase, whereas the target gene was at its exponential phase. As such, results of RT-PCR were confirmed by immunoblotting in virtually all experiments when antibody against a target protein was commercially available. This additional analysis also supports the notion that an induction of a target mRNA level would translate into a functional physiological event mediated by changes in its protein level. Following gel electrophoresis, PCR products were visualized by ethidium bromide staining, and autoradiography was performed using Kodak X-OMAT AR x-ray film (Eastman Kodak, Rochester, NY). The authenticity of each target gene shown in Table 1 was verified by direct nucleotide sequencing following subcloning into a pGEM-T vector as previously described [8, 18]. Autoradiograms from two or three separate experiments were densitometrically scanned at 600 nm using an Ultroscan XL Laser Densitometer (model LKB 2222-020; Amersham Pharmacia Biotech, Piscataway, NJ), normalized against S16, and used for statistical analysis.
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Preparation of Whole Cell Lysates
Sertoli cell cultures and Sertoli-germ cell cocultures were prepared as described above. To obtain whole cell lysates, medium was first removed from culture dishes at specified time points prior to termination. The remaining cells in each dish of a 12-well plate were resuspended in 1 ml of extraction buffer (0.125 M Tris, pH 6.8, 22°C, containing 1% SDS, 1.6% 2-mercaptoethanol, 2 mM PMSF, 1 mM EDTA) and incubated at room temperature for 5 min. For samples subjected to analysis to detect changes in protein phosphorylation by immunoblotting, 1 mM of sodium vanadate (a protein tyrosine phosphatase inhibitor, PTPi) and 0.1 µM sodium okadate (a protein Ser/Thr phosphatase inhibitor, PPi) were included in the extraction buffer. Samples incubated on ice were then sonicated (twice for 15 sec each interspaced by 30 sec on ice) using a sonicator (model 4710; Cole-Palmer Instrument Co., Chicago, IL), vortexed, and centrifuged at 15 000 x g for 10 min at 4°C. The supernatant was collected and used as whole cell lysate. With this extraction buffer, virtually all the cellular and membrane proteins were solubilized; the pellets were negligible in most instances. Protein estimation was performed with a Coomassie blue dye-binding assay [37] using BSA as a standard.
Preparation of Tissue Lysates
About 0.2 g of testis was homogenized with an ultrasonic homogenizer (Cole-Palmer) in 0.8 ml of extraction buffer in the presence of both PTPi and PPi using a tissue:buffer ratio of 1:5. Samples were vortexed and incubated on ice for 5 min to allow tissue solubilization. Samples were then centrifuged at 15 000 x g for 10 min at 4°C to remove tissue debris. The supernatant was collected and used as testis lysate.
Immunoblot Analysis
About 200 µg of protein per sample was resolved onto 7.5%, 10%, or 12.5% T SDS-polyacrylamide gels by SDS-PAGE under reducing conditions as previously described [38]. Following electrophoresis, proteins were electroblotted onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and immunoblotting was performed essentially as previously described [19, 39]. RhoB, LIMK1, and ROCK1 protein were detected by using a rabbit anti-rat RhoB or goat anti-mouse LIMK1 or ROCK1 antibody, respectively. Bovine anti-rabbit and goat anti-mouse IgG conjugated to horseradish peroxidase were used as the second antibody. Immunoreactive proteins were visualized by the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). To detect the chemiluminescent proteins, membranes were exposed to Kodak BioMax film for 1–10 min. To ensure equal loading of proteins between samples within an experimental group, the blot was also reprobed with a tubulin antibody.
Treatment of Adult Rats with Y-27632, a Specific ROCK Inhibitor, to Examine Its Effect on the Kinetics of AF-2364-Induced Germ Cell Loss from the Seminiferous Epithelium and Changes in Tubular Diameter
Adult rats (300 g bw) were anesthetized using ketamine HCl (60–80 mg/kg bw). Thereafter, 200 µl of Y-27632 (Mr 338.3; Calbiochem, San Diego, CA), a ROCK inhibitor [40–42], was injected to the right testis at three sites using a 22-gauge needle with 70 µl per site. (Concentration of the Y-27632 stock solution was 10 µM prepared in saline. As such, 0.676 µg Y-27632/testis was administered. Assuming the testicular volume is 1.6 ml/testis, the final concentration of Y-27632 in each treated testis was 1.25 µM. This concentration was selected based on earlier reports [40–42].) The same volume of saline without Y-27632 was injected to the left testis, which served as a control. Rats in groups of four for each time point treated with Y-27632 received a single dose of AF-2364 at 50 mg/kg bw by gavage or received vehicle only (0.5% methylcellulose, w/v, in PBS) within 2 h. Rats were killed daily between Day 0 and Day 7, and testes were removed, frozen in liquid nitrogen, and stored at -80°C. Sections were obtained with a cryostat as described above and stained with Mayer hematoxylin. Cross sections of testes were randomly selected, and seminiferous tubules consisting of elongating or elongate spermatids were scored. At least 80 tubules selected randomly from each testis (four testes from four rats were examined with a total of 300 tubules per time point) were photographed, digitally stored, and printed using a 785 EPX printer (Epson America, Inc., Long Beach, CA) for scoring. A tubule from rats treated with AF-2364, Y-27632, or AF-2364 plus Y-27632, which contained <50% of the number of elongating or elongate spermatids found in the control tubules was scored as a tubule with significant loss of elongating or elongate spermatids from the epithelium (110 ± 12 elongating spermatids, 115 ± 18 elongate spermatids, and 93 ± 14 elongating spermatids were found in each cross section of a tubule in control rats at stages I–V, stages VII–VIII, and stages XI–XIV, respectively, except for stages IX–X where no elongating or elongate spermatids were found). The percentage of seminiferous tubules (ST) with normal elongating or elongate spermatids after AF-2364, Y-27632, or AF-2364 plus Y-27632 treatment was calculated as follows:
To assess the effects of Y-27632 on the changes in tubular diameter induced by AF-2364, the following formula was used:
STD/Ctrl is the diameter of tubules in control rats and STD/AF-2364 with or without Y-27632 is the diameter of tubules in AF-2364-treated rats with or without pretreatment with Y-27632. Eighty tubule sections per testis were scored by measuring the diameter, and the mean ± SD was computed from four testes of four different rats.
Immunoprecipitation of LIMK Protein
To eliminate interexperimental variations, all samples within a treatment group were processed simultaneously for immunoprecipitation analysis. About 400–500 µg protein per lysate sample, from testes extracted using an IP lysis buffer (see below), was first pretreated with normal mouse serum (1:40 dilution) for 1 h. Thereafter, proteins that would precipitate nonspecifically were removed by incubating each sample with 20 µl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) at 4°C for 3 h. Samples were then centrifuged at 1000 x g for 5 min to remove the nonspecific complexes in the pellet. Supernatant (150 µl) was then incubated with a mouse monoclonal anti-LIMK1 antibody (1:50; Transduction Laboratories, San Diego, CA) at 4°C overnight with agitation. Thereafter, 20 µl of Protein A/G PLUS-Agarose was added to each sample and incubated at 4°C for 4 h. The pellet was washed four times in 300 µl of an IP lysis buffer (50 mM Tris, pH 7.4 at 22°C, containing 0.15 M NaCl, 2 mM PMSF, 2 mM EDTA, 1 mM sodium orthovanadate, 0.1 µM sodium okadate, 2 mM ethylmaleimide, 1% NP-40, 10% glycerol) at 1000 x g for 5 min each time. Immunocomplexes (40 µg protein containing largely IgG and LIMK1) bound to the Protein A/G agarose in the pellet were extracted in 50 µl of SDS sample buffer (0.125 M Tris, pH 6.8 at 22°C, containing 1% SDS, 1.6% ß-mercaptoethanol, 20% glycerol) at 100°C for 10 min. The extracted proteins in each sample were then subjected to immunoblotting. To investigate whether there was any change in the phosphorylation of LIMK1 during AF-2364-induced AJ disruption in the testis, an additional step was taken to normalize against differences in LIMK1 protein between samples prior to immunoblotting using either specific phospho-Ser or phospho-Tyr antibody. The protein concentration among the samples within an experimental group following immunoprecipitation using an anti-LIMK1 antibody was quantified by Coomassie blue dye-binding assay [37]. Thereafter, the same amounts of immunoprecipitated proteins (30 µg) from each sample within an AF-2364 treatment group, which consisted largely of IgG and LIMK1, were then resolved onto 7.5% T SDS-polyacrylamide gels under reducing conditions. Following electrophoresis, proteins were electroblotted onto nitrocellulose membranes. Blots were immunostained with an anti-LIMK antibody to confirm that a similar amount of LIMK was found among samples within a treatment group. In a parallel experiment, the same blot was stained with a rabbit polyclonal anti-phospho-Ser or anti-phospho-Tyr antibody. Any increases in phosphoprotein content could be attributed to an increase in the phosphorylation status of LIMK1. This step of protein normalization is necessary because an increase in LIMK1 protein was detected in the testicular lysates as a result of AF-2364-induced AJ disruption prior to germ cell loss from the epithelium, which would mask any putative changes in the phosphorylation status of LIMK.
Statistical Analysis
Statistical analysis was performed by ANOVA with Tukey honestly significant difference (HSD) test using the GB-STAT Statistical Analysis Software Package (version 7.0; Dynamic Microsystems, Silver Spring, MD). In selected experiments, a Student t-test was also used to compare results of treated samples with those of the corresponding control within an experimental group. For each study, an experiment was repeated at least two or three times using different batches of cells unless otherwise specified, and each time point had at least triplicate cultures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies by semiquantitative RT-PCR and immunoblotting revealed that both Sertoli and germ cells isolated from 20-day-old rat testes expressed RhoB (Fig. 1, A and B). The steady-state mRNA (Fig. 1A) level of RhoB was higher in Sertoli cells than in germ cells, with a relative Sertoli:germ cell ratio of 2:1 (Fig. 1, A and C). This result was consistent with those obtained by immunoblotting using a RhoB antibody to detect the protein level using cell lysates (Fig. 1, B and C). The same blot was also reprobed with an anti--tubulin antibody to ensure equal protein loading (data not shown). We next investigated whether there was any change in RhoB expression during the maturation of Sertoli cells, germ cells, and the testis. The expression of RhoB in germ cells increased after birth, peaked in 10-day-old rats, and then declined rapidly with age (Fig. 1, D and E). RhoB in 90-day-old germ cells was only 10% of that in 5-day-old rats (Fig. 1, D and E). In Sertoli cells, the steady-state mRNA level of RhoB also declined during maturation (Fig. 1, F and G). In the testis, the pattern of RhoB expression during maturation is somewhat similar to that in germ cells; it began to rise on Day 5 and remained high until 40 days of age, yet it peaked at 10–20 days of age, coinciding with the assembly of the BTB, and plummeted thereafter (Fig. 1, H and J). Results of the RT-PCR assay depicting changes in the steady-state RhoB GTPase mRNA level in testes during maturation are consistent with results of immunoblotting (Fig. 1, I versus H and J) except that the protein level peaked at ages 10–40 (Fig. 1, I and J).
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Immunohistochemical Localization of RhoB in the Seminiferous Epithelium of Adult Rat Testes
RhoB was detected in the seminiferous epithelium in virtually all stages of the epithelial cycle in 90-day-old rats (Fig. 2, A–E) except at stages VII–VIII, for which staining was markedly reduced. The localization (Fig. 2, A–E) appeared to be specific; immunoreactive reddish-brown precipitate corresponding to RhoB was not found in control sections where the primary antibody was replaced with normal rabbit serum (Fig. 2, F versus A–E). RhoB was associated almost exclusively with elongating spermatids (but not elongated spermatids) and Sertoli cells at the periphery of the nucleus near the site of apical ESs (Fig. 2, A–E). Some immunoreactive RhoB staining was found at the periphery of the nucleus in spermatogonia and spermatocytes at stages I–X possibly at the site of basal ESs (Fig. 2, A–D). However, RhoB immunoreactive substance was not detected in elongate spermatids at stages VII–VIII. Its localization appeared to be predominant at stages IX–XIV (Fig. 2, D and E) and became greatly diminished at stage VII (Fig. 2C). Although RhoB appeared to localize near the site of apical ESs based on light microscopy, work is now in progress to investigate the precise localization of RhoB in the epithelium using electron microscopy (EM).
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Changes in Steady-State mRNA and Protein Levels of RhoB in Sertoli-Germ Cell Cocultures During AJ Assembly
Sertoli cells were cultured in Matrigel-coated dishes or bicameral units at 0.5 x 106 cells/cm2 to allow the assembly of TJs in vitro. RT-PCR and immunoblot analysis revealed that the steady-state RhoB mRNA and protein levels remained steady throughout the entire culture period (Fig. 3A) during the assembly of the Sertoli cell TJ barrier. In parallel experiments, the assembly of the Sertoli cell TJ barrier was assessed by quantifying the TER across the Sertoli cell epithelium (Fig. 3B) coinciding with a transient induction of occludin and ZO-1 (data not shown) as previously described [8, 9]. These results suggest that RhoB may not actively participate in the Sertoli cell TJ assembly. However, when germ cells isolated from 90-day-old rat testes were added on Day 6 to Sertoli cells that had been cultured alone for 5 days to form an epithelium with intact TJs and AJs to initiate AJ assembly between Sertoli and germ cells, there was a 3- to 5-fold increase in the steady-state RhoB mRNA level at the time germ cells adhered onto Sertoli cells [12], which was about 30 min–6 h after addition of germ cells to the epithelium (Fig. 3, C and D). This time frame is somewhat earlier than the actual establishment of AJs (such as ESs) and desmosome-like junctions, which is known to be completed within 24–48 h as revealed by EM [33, 34]. However, this time frame is consistent with results of an earlier study that illustrated an induction of proteases at the time germ cells attached to Sertoli cells [12]. This pattern of RhoB induction during Sertoli-germ cell AJ assembly was further confirmed by immunoblotting when whole cell lysates were prepared in parallel coculture experiments, except that an increase in RhoB protein level was detected within 45 min, a delay of 15 min compared with the increase in mRNA level, and persisted until 6 h (Fig. 3, C, lower panel, and D). These observations suggest that RhoB is involved in the early events of AJ assembly between Sertoli and germ cells, such as cell-cell attachment, but not in AJ assembly between Sertoli cells nor between Sertoli cells and the substratum.
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Changes in Steady-State RhoB mRNA Level in Sertoli-Germ Cell Cocultures and Sertoli Cell Cultures During AF-2364-Induced AJ Disruption
There is some evidence that Rho can become activated during both AJ assembly and disassembly [43, 44]. We thus used Sertoli-germ cell cocultures as an in vitro model to examine changes in RhoB expression during AJ disruption. Germ cells were isolated from 90-day-old rats and cocultured with Sertoli cells for 2 days after Sertoli cells had been cultured alone for 5 days on Matrigel-coated dishes forming an epithelium, and allowing the assembly of Sertoli-germ cell AJs to complete—which is known to take 24–48 h [33, 34]. This also permitted the endogenous RhoB mRNA level to subside (Fig. 3, C and D). Thereafter, AF-2364 was added to these cocultures to induce AJ disruption. As expected, a transient but significant induction in RhoB was detected (Fig. 4, A, upper panel, and B), but the steady-state mRNA level of RhoB remained unaltered in the Sertoli-germ cocultures without AF-2364 treatment (Fig. 4, A, middle panel versus upper panel, and B) and in Sertoli cells cultured alone without germ cells but exposed to AF-2364 (Fig. 4, A, lower panel, and B).
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Changes in Steady-State mRNA and Protein Levels of RhoB in Sertoli-Germ Cell Cocultures During AJ Disruption Induced by Hypotonic Treatment
To further expand and verify the above observation that induction of RhoB is associated with a disruption of AJs between Sertoli and germ cells and is not just a drug-related side effect, an in vitro model of AJ disruption induced by physical means was used. Sertoli cells at 0.5 x 106 cells/cm2 were cultured alone for 5 days to form an epithelium after germ cells had been removed on Day 2 by hypotonic treatment. On Day 6, freshly isolated germ cells from 90-day-old rat testes were added to this Sertoli cell epithelium with a cell purity >95% to initiate Sertoli-germ cell AJ assembly and were cultured for 2 days [33, 34] to allow AJs to become established. Thereafter, these Sertoli-germ cell cocultures were subjected to a hypotonic treatment to cleave AJs, mimicking the AF-2364-induced AJ disruption but without the presence of a chemical entity. Figure 5A shows the control experiments where Sertoli-germ cell cocultures were not subjected to hypotonic treatment, and no changes in either RhoB expression (Fig. 5A, upper panel) or protein level (Fig. 5A, lower panel) were detected. However, there was a transient but significant induction in RhoB mRNA (Fig. 5B, upper panel) and RhoB protein (Fig. 5B, lower panel, and D and E) at the time Sertoli-germ cell AJs were being disrupted by hypotonic treatment when compared with controls where Sertoli-germ cell cocultures were not subjected to hypotonic treatment (Fig. 5A) or Sertoli cells were cultured alone at all time points (Fig. 5C). These results thus demonstrate unequivocally that the AF-2364-induced RhoB expression is not the result of other damaging effects of AF-2364 on Sertoli cells that trigger other physiological responses, thereby causing a surge in RhoB expression. Instead, the changes in RhoB expression are tightly associated with changes in AJ integrity regardless of whether the AJs are disrupted by chemical or physical treatment. We next sought to investigate the signaling pathway(s) that AF-2364 utilizes to perturb Sertoli-germ cell AJ dynamics in the testis. Figure 6 depicts the three pathways known to be used by RhoB GTPase to exert its signaling function downstream to induce changes in the actin-based cytoskeletal network, causing cells in cell adhesive function (Fig. 6).
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Changes in Steady-State mRNA and Protein Levels of RhoB During AF-2364-Induced AJ Disruption in the Testis Versus Brain and Kidney
AF-2364 disrupts AJs in the testis, causing germ cell loss from the seminiferous epithelium in particular spermatids without any apparent effects on spermatogonia and early spermatocytes in the epithelium [35, 36]. However, AF-2364 does not affect the hypothalamus-pituitary-testicular axis and is neither nephrotoxic nor hepatotoxic [35, 36]. AF-2364 treatment of rats (300 mg/kg bw by gavage) induced a transient and significant induction in RhoB mRNA level within 1–4 h (Fig. 7, A and B). Likewise, a transient but significant increase in the RhoB protein was also detected by immunoblotting in lysates of the testes (Fig. 7C and D) but not in lysates of the brain and kidney (Fig. 7E) from the same animals, consistent with results of the RT-PCR (Fig. 7, C and D versus A and B, and E). A significant increase in RhoB protein level was detected between 4 h and 2 days in the testis, illustrating a 4-h lag between a surge in RhoB mRNA and an increase in RhoB protein level (Fig. 7, C versus A). The lack of response of RhoB to AF-2364 treatment in the brain and kidney (Fig. 7E) clearly illustrates that action of this compound is restricted to the testis via an upstream transducer specific to the testis. We hypothesize that this transducer is the ES structure, which is composed of integrins and testin (for review, see [2]). Induction of both RhoB mRNA and RhoB protein occurred long before any visible germ cell loss from the seminiferous epithelium was detected by histological analysis (Fig. 7F). At least 1 day elapsed before germ cell loss was detected in the testis. The AF-2364-induced AJ disruption and the associated germ cell loss became more widespread when, by 4–14 days posttreatment, virtually all tubules were devoid of germ cells (Fig. 7F), coinciding with the loss of fertility of the treated male rats after the sperm reserve in the epididymis was exhausted [35]. RhoB protein was not induced in the brain and kidney, as determined by examination of tissue extracts from the same animals (Fig. 7E). This finding strongly suggests that although the effects of AF-2364 on AJs are mediated via the ROCK signaling pathways, this compound triggers a unique component(s) in the ES structure between Sertoli cells and elongating/elongate spermatids, possibly testin that associated with the 6ß1 integrin/integrin laminin, cadherin/catenin, or afadin/nectin complex [2, 35, 36]. This finding also explains the lack of nephrotoxicity and hepatotoxicity when rats treated with AF-2364 exhibit reversible infertility, as reported previously [35, 36]. These results suggest that RhoB plays a crucial role in the early cascade of events leading to AJ disruption and germ cell loss from the seminiferous epithelium in addition to its role in AJ assembly in the testis (see Figs. 4 and 5), implicating the physiological significance of RhoB in germ cell movement across the seminiferous epithelium during spermatogenesis. These results also demonstrate the changes in RhoB expression during AJ disruption in vitro (Fig. 4) can be reproduced in vivo (Fig. 7, A–D).
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Changes in Steady-State mRNA and Protein Levels of the Rho Signal Transducers During AF-2364-Induced Sertoli-Germ Cell AJ Disruption in Testis
Other studies have shown that RhoB regulates AJ dynamics through its effects on the actin cytoskeletal network via one of the three upstream signal transducers and three possible downstream signal transduction pathways (Fig. 6). Because integrins have been implicated in the regulation of ESs [45] and junction dynamics [2], we sought to examine whether an upstream signal transducer of RhoB, such as integrin ß2, was induced in the testis after AF-2364 treatment. The expression of integrin ß2 at the mRNA and protein level (Fig. 7, A–D) was induced by AF-2364, and the steady-state mRNA and protein levels of ROCK, a downstream RhoB effector protein, increased at 1–8 h, coinciding with the AF-2364-induced RhoB expression (Fig. 7, A–D). The downstream signal transducers of ROCK, such as LIMK and cofilin, also displayed expression patterns similar to those of ROCK during AF-2364-induced AJ disruption (Fig. 7, A–D). Collectively, these results thus illustrate that RhoB exerts its effects via the ROCK/LIM kinase/cofilin signaling pathway (see Fig. 6 versus Fig. 7).
Phosphorylation Status of LIMK after AF-2364 Treatment as Determined by Immunoprecipitation and Immunoblotting
Although the LIMK1 protein level was increased by AF-2364 treatment (Fig. 7, C and D), it is not absolutely certain that this protein is activated. Because LIMK activation is mediated via Ser/Thr phosphorylation by ROCK (LIMK per se is a Ser/Thr protein kinase and a putative substrate of ROCK; for review, see [23]), we investigated the Ser/Thr phosphorylation level of LIMK during AF-2364-induced AJ disruption. Using immunoprecipitation, an increase in LIMK1 was detected during AF-2364-induced AJ disruption in the testis (Fig. 8A). When the immunoprecipitated LIMK protein level was adjusted between samples in an experimental group so that the same amount of immunoreactive LIMK protein was resolved by SDS-PAGE (Fig. 8B versus 8A), the phospho-Ser (Fig. 8C) but not phospho-Tyr (Fig. 8D) level in LIMK1 was stimulated by the AF-2364 treatment (Fig. 8, A–E). These results thus demonstrate beyond refute that RhoB mediates its effects through the ROCK/LIMK signaling pathway via changes in protein phosphorylation.
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Effects of a ROCK Inhibitor on Kinetics of AF-2364-Induced AJ Disruption and Associated Germ Cell Loss from Seminiferous Epithelium
AF-2364-induced AJ disassembly in vivo and in vitro is associated with a surge in integrin ß2 upstream followed by an increase in RhoB and its associated downstream signal transducers, such as ROCK, LIMK, and cofilin (Figs. 6–8). To further assess the physiological linkage between RhoB and the ROCK/LIMK signaling pathway and AJ disassembly, a specific ROCK inhibitor, Y-27632, was used to block the Rho/ROCK/LIMK pathway to examine whether this blockage would perturb the AF-2364-induced germ cell loss from the seminiferous epithelium. In rats treated with AF-2364, it caused germ cell depletion, in particular spermatids, from the seminiferous epithelium (Figs. 7F and 9, E–H versus A–D). The primary site of action of AF-2364 is the apical ES structure between Sertoli cells and the elongate/elongating spermatids; the percentage of tubules with elongating and/or elongate spermatids was reduced significantly on Day 2 after AF-2364 treatment (Fig. 9, E–H versus A–D, and M). However, the population of round spermatids and spermatocytes, in contrast to elongate or elongating spermatids, remained relatively unaltered in AF-2364-treated rats between Days 2 and 3 (Fig. 9, E and F versus A and B). Tubular diameter was reduced significantly after AF-2364 treatment when compared with the control (saline or Y-27632 alone) (Fig. 9, E–H versus A–D, and N), e.g., by 30% in AF-2364-treated rats by Day 7 posttreatment (Fig. 9N). Intratesticular injection of 1.25 µM Y-27632 effectively delayed the AF-2364-induced loss of elongating and/or elongate spermatids from the seminiferous epithelium (Fig. 9, I–K versus A–D, E–H, and M), indicating that the AF-2364-induced Sertoli-germ cell AJ disruption is indeed mediated via the RhoB/ROCK pathway. However, Y-27632 alone had no effects on germ cell adhesion in the seminiferous epithelium (Fig. 9, B–D). Although Y-27632 could delay AF-2364-induced germ cell loss from the epithelium (Fig. 9, M and K versus G), Y-27632 alone failed to block AF-2364-induced germ cell loss; the seminiferous epithelium also displayed damage by Day 7 in the testis pretreated with Y-27632 prior to AF-2364 treatment (Fig. 9, L versus H, M, and N), suggesting that the Sertoli-germ cell AJ dynamics in vivo are regulated by other unidentified pathways.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During spermatogenesis, actin-based cell-cell AJs, such as ESs, along with desmosomes, must undergo extensive restructuring to facilitate the movement of developing germ cells across the seminiferous epithelium. At present, three AJ structures are known to be found at the site of ESs (for reviews, see [2, 6, 46]): the cadherin-catenin complex, the nectin-afadin complex, and the integrin 6ß1-laminin 3 complex. Recent studies have shown that the dynamics of ESs are regulated, at least in part, by integrin-linked kinase (ILK) [45]. In vitro studies using human keratinocytes and Madin-Darby canine kidney (MDCK) cells have also demonstrated that Rho GTPases are important regulators of cadherin-dependent cell-cell contacts and actin organization [25, 47–50]. For instance, exogenously expressed myc-tagged Rho and Rac are localized at the sites of cell-cell adhesion in MDCK cells [50, 51], suggesting that Rho GTPases are present at or near the site of AJs. Other researchers have demonstrated that the establishment of stable cadherin-mediated cell adhesion requires the activation of Rho proteins. For instance, an inhibition of endogenous Rho GTPases by C3 exoenzyme or dominant negative Rac can induce redistribution of E- and P-cadherin in keratinocytes, causing a loss of the cadherin-mediated cell adhesion between neighboring cells [25]. In the present study, a transient but significant increase in RhoB mRNA, RhoB protein, and integrin was detected during Sertoli-germ cell AJ assembly and disassembly, suggesting that the events of AJ dynamics in the testis may be regulated, at least in part, via the integrin-Rho GTPases pathway (see Fig. 6).
Although immunohistochemistry has been used to localize RhoB GTPase to the site near the apical and basal ESs, RhoB GTPase is largely found at the periphery of the nucleus of elongating spermatids, spermatocytes, and Sertoli cells. For several reasons, this finding does not negate the functional significance of RhoB for cell adhesion at the site of ESs and its involvement in AJ dynamics. First, GTPases function as molecular switches in intracellular trafficking to move intracellular proteins such as AJ and TJ proteins from one site to another (E-cadherin can become internalized, moving away from the AJ site by GTPases; for review, see [2]) or to move from cytoplasm to nucleus (for review, see [22]). For instance, Rac and Rho GTPases can induce the movement of E- and P-cadherin away from the site of AJs [25]. GTPases are not necessarily restricted to a specific site, such as the cadherin/catenin, integrin/laminin, or nectin/afadin protein complexes, which are ES-associated integral membrane proteins (for reviews, see [2–6]), and RhoB does not have to localize precisely at the apical ES site to mediate its actin-regulating dynamics. Second, the anti-RhoB antibody used in our study was prepared using a synthetic peptide based on an internal stretch of sequence specific to RhoB GTPase. The active form of GTP-bound GTPases accounts for <0.5–1% of the total cellular GTPases (RhoB GTPase cycles between the active GTP-bound and nonactive GDP-bound form within a cell; for review, see [22]) and cannot be selectively identified by this antibody in the seminiferous epithelium. However, the activated form represents those RhoB forms that can execute the intracellular trafficking function to induce redistribution of ES-associated integral membrane proteins. This issue will be resolved only when a specific RhoB GTPase assay is available, which will require the identification and cloning of the RhoB effector protein(s), and when a specific antibody that can detect the activated form of RhoB is available. Other ongoing immunoprecipitation studies and immunoblot analyses using lysates of seminiferous tubules of adult rats have shown that the anti-RhoB antibody can indeed immunoprecipitate E-cadherin and ß-catenin (unpublished observations), further supporting the hypothesis that RhoB is functionally associated with the AJ structural complexes.
Increasing evidence supports the notion that Rho is crucial to cell movement in different physiological and pathophysiological conditions, such as tumor progression. During metastasis, junctions between neighboring cells must be disassembled to permit invasive cells to migrate across an epithelium. Rho activation is required for oncogenic Ras-triggered morphologic transformation and cell motility [44, 52]. Although the mechanism by which Rho affects cell movement remains to be elucidated, these findings illustrate the significance of Rho GTPases in cell migration. As reported here, testicular RhoB was induced in AF-2364-treated rats within 1–4 h, long before visible germ cell loss from the seminiferous epithelium was detected at 24 h after AF-2364 treatment. This result suggests that RhoB is involved in the early steps of Sertoli-germ cell AJ disruption, which eventually leads to germ cell detachment from the epithelium. This transient but significant induction in RhoB was also reproduced in vitro when Sertoli-germ cell AJs were perturbed by AF-2364. A physical disruption of AJs in Sertoli-germ cell cocultures by hypotonic treatment can also induce RhoB expression. These results clearly illustrate that the transient increase in RhoB expression is correlated with Sertoli-germ cell AJ dynamics.
One interesting avenue of investigation concerns how a single class of proteins, such as Rho GTPases, can regulate both AJ disruption and AJ reassembly, because these two biological events, while intricately related, oppose one another. One possibility is that Rho GTPases have complex roles similar to those of Rac GTPases in regulating AJ dynamics. Studies conducted in human epidermal keratinocytes have revealed that Rac GTPases can modulate AJ disassembly and assembly, depending on junctional maturity and cellular context [43, 53], via different downstream signaling mechanisms. For instance, a dominant active form of Rac1 expressed in MDCK cells can either prevent or promote E-cadherin-mediated cell adhesion using different downstream signaling pathways [54]. Another possibility is that Rho activates different downstream target proteins or effector proteins to exert different physiological functions. Each GTPase can activate a variety of effector proteins, and the coupling of a GTPase to different effector proteins can lead to different biological responses (for review, see [20]). Thus, GTPases can affect the opposing events of AJ dynamics, such as AJ disassembly and reassembly, via different mechanistic pathways.
Is the AF-2364-Induced Germ Cell Loss from the Seminiferous Epithelium Mediated, at Least in Part, by the Integrin/RhoB/ROCK/LIMK Cofilin Signal Pathway?
Actin reorganization is regulated, at least in part, by an elaborate network of Rho effector proteins that interact with Rho GTPases, including Rho-associated kinase (ROCK), p140mDia, and phosphatidylinositol 4-phosphate 5-kinase (PIP-5K) (for reviews, see [20, 22]). Both p140mDia and PIP-5K are target proteins of Rho GTPases, which have a broad spectrum of effects on the actin cytoskeleton after binding to profilin (an actin monomer-binding protein), resulting in the release of actin monomers [55–57]. ROCK is also an important target protein and affects actin cytoskeleton regulators, such as myosin light chain, LIMK1, and adducin [41, 58, 59]. These molecules are also implicated in actin-myosin filament assembly. The presence of RhoB GTPase, ROCK, LIMK1, and cofilin in the testis suggests that testicular cells are equipped with the needed effector proteins to mediate the Rho GTPase downstream actions that affect AJ dynamics. Furthermore, integrin and RhoB and its downstream effector proteins are activated in the testis by AF-2364 before the occurrence of the AF-2364-induced germ cell loss from the seminiferous epithelium, suggesting that the events of AF-2364-induced AJ disruption in the testis are mediated via the integrin/RhoB/ROCK/LIMK/cofilin signaling pathway.
AF-2364 was synthesized based on the core structure of lonidamine (1-(2,4)-dichlorobenzyl-indazole-3-carboxylic acid), which had been investigated as a potential male contraceptive based on its specific ability to disrupt the stress fibers and actin filament network in Sertoli cells, thereby inducing germ cell loss from the seminiferous epithelium (for review, see [2]). However, further investigation of lonidamine was abandoned because of its nephrotoxicity (for review, see [2]). Our initial investigation using an in vivo screening assay based on testin had selected AF-2364 from more than 24 newly synthesized candidate compounds, and recently completed studies have shown that AF-2364 has biological activities similar to those of lonidamine but without the unwanted toxicity [35, 36]. This conclusion was reached based on recently completed acute toxicity studies in rats and mice, micronucleus tests in erythrocytes to assess DNA damage, and mutagenicity tests in two bacterial strains (Salmonella typhimurium and Escherichia coli) conducted by licensed toxicologists; AF-2364 produced negative results in these standard toxicity and genotoxicity tests (unpublished observations). We hypothesize that AF-2364 limits its action by perturbing the specialized AJ structures in the testis, such as the ESs, which are composed of testin, integrins, nectin/afadin, cadherin/catenin, and other component proteins (for review, see [2]). This hypothesis was developed based on the following
