Journal of Andrology, Vol. 28, No. 3, May/June 2007 Copyright E American Society of Andrology
Cytoskeleton Vimentin Disruption of Mouse Sertoli Cells Injured by Nitrogen Mustard In Vitro DAWEI HE, DEYING ZHANG, GUANGHUI WEI, TAO LIN, AND XULIANG LI From the Department of Pediatric Urology, Children’s Hospital of Chongqing Medical University, Chongqing, China.
ABSTRACT: Reproductive toxicity is one of the potential side effects of anticancer alkylating agents, with potential effects on vimentin intermediate filaments, one of the main components of the Sertoli cytoskeleton. Research suggests (Aumuller et al, 1988; Aumuller et al, 1992) that the highly organized and active Sertoli cytoskeleton is important in spermatogenesis. The aim of the current study was to investigate the effects of alkylating agents on vimentin filament expression in vitro. Sertoli cells, isolated from 20-day-old mice testes, were cultured for 5 days and then incubated with 0, 50, 100, and 200 mmol/L nitrogen mustard (HN2). Morphologic changes in Sertoli cells were observed per 30-minute interval at 12-hour exposure time points to 100 mmol/L HN2. Vimentin expression was investigated by immunocytochemistry at 6 hours and 24 hours posttreatment and reverse transcriptase polymerase chain reaction and Western blot at 12 hours posttreatment with 50, 100, and
200 mmol/L HN2. Exposure to HN2 resulted in a comparatively small Sertoli cell body with diminished cytoplasm. Sertoli cells were shrunk or detached. Cytoskeletal disruption increased with increasing HN2 concentration. The optical density values of vimentin antibody and expression of vimentin mRNA and protein were significantly decreased with increasing concentration of HN2. Significant treatment dose-dependent and time-dependent differences of vimentin mRNA and protein expression levels were also noted. Our data suggest that the change in the biochemical properties of vimentin may indicate that one of the mechanisms of reproductive toxicity resulting from HN2 is disruption of Sertoli cell vimentin filament structure, accompanied by a down-regulation of vimentin expression. Key words: Testis. J Androl 2007;28:389–396
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Sertoli cells and more advanced germ cells. Previous research has suggested that vimentin intermediate filaments play a role in anchoring germ cells to the seminiferous epithelium (Amlani and Vogl, 1988). Others have found that the collapse of vimentin after xenobiotic treatment correlated with the loss of structural integrity of the seminiferous epithelium, along with germ cell apoptosis (Johnson et al, 1991; Richburg and Boekelheide, 1996; Dalgaard et al, 2001). Reproductive toxicity is a common side effect of anticancer drug chemotherapy, especially in young men of reproductive age (Byrne et al, 1987; Nicholson and Byrne, 1993; Pauwels et al, 1995; Rueffer et al, 2001). However, the mechanisms underlying the cytotoxic effects of alkylating agents and germ cell loss, as well as treatment effects on the vimentin cytoskeleton, are not completely understood. In the current study nitrogen mustard (bis-[b-chloroethyl] methylamine; HN2) was used to investigate the effects of alkylating agents on vimentin filament mRNA and protein expression in vitro.
ertoli cells are the principal structural element of the seminiferous epithelium, and they play a key role in triggering and regulating spermatogenesis (Langford et al, 1993; de Kretser et al, 1998). Research suggests (Aumuller et al, 1988; Aumuller et al, 1992) that the highly organized and active Sertoli cytoskeleton is important in spermatogenesis. Vimentin intermediate filaments, formed by polymerization of 57 kd vimentin monomers, are an important component of the Sertoli cytoskeleton (Franke et al, 1982) and surround the nucleus, giving it the characteristic ‘‘halo’’ appearance (Show et al, 2003) that radiates outward to the cell periphery and terminates near points of contact between the Sertoli cell and adjacent cells. Vimentin intermediate filaments mediate tight junction contact between neighboring Sertoli cells, as well as the desmosome-like junctions located between Sertoli cells and germ cells and the ectoplasmic specialization junctions between This research was supported in part by a grant from the National Natural Science Foundation of China (3037145/C03030310) and the health bureau research Fund of Chongqing. Correspondence to: Dr Dawei He, Children’s Hospital of Chongqing Medical University, Department of Pediatric Urology, Chongqing, No. 136, Zhongshan 2 RD, Yuzhong District, Chongqing, China 400014 (e-mail:
[email protected]). Received for publication May 9, 2006; accepted for publication October 9, 2006. DOI: 10.2164/jandrol.106.000455
Materials and Methods Animals Twenty-day-old male Kunming mice were obtained from the Experimental Animal Center, Third Military Medical Univer-
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sity, Chongqing, China. The study was approved by the Animal Ethical Committee of the Institute of Zoology and the Chongqing Medical University.
Primary Sertoli Cell Culture Sertoli cells were isolated from 20-day-old mice as previously described (Takase et al, 1988), without the use of trypsin digestion. Briefly, 2 decapsulated testes were incubated in 0.5 mg/mL collagenase IV in phosphate-buffered saline (PBS; pH 7.4) at 34uC, shaken for 15 minutes to remove interstitial cells, and then washed 3 times. Sertoli cells and germ cells were separated by incubation with 0.1% collagenase, 0.2% hyaluronidase, and 0.04% deoxyribonuclease I in PBS (pH 7.4) at 34uC with shaking for 20 minutes. Sertoli cells were pelleted by centrifugation (53 6 g for 4 minutes), washed in PBS, repelleted 3 times, resuspended in PBS, and subjected to hypotonic shock in a dilute PBS solution. Cells were collected by centrifugation (130 6 g for 4 minutes), resuspended in PBS, filtered through a 60-mm nylon mesh (Small Parts Inc, Miami Lake, Fla), washed, and resuspended in Ham F12 Nutrient Mixture (F12)/Dulbecco Modified Eagle medium (DMEM) (1:1) tissue culture media (Invitrogen Corp, Carlsbad, Calif). Sertoli cell number and purity were estimated by hemocytometer and light microscopic analyses (Takase et al, 1988), respectively, with an average of 0.5 to 1 6 105 Sertoli cells per testis obtained with approximately 90% purity. Isolated Sertoli cells were cultured (34uC, 95% humidified atmosphere, 5% CO2, vol/vol) at high density (0.5 6 106 cells/ cm2) on Matrigel-coated 12-well dishes in serum-free F12/ DMEM (1:1, vol/vol) as described above. Increased Sertoli cell purity was obtained by hypotonic treatment (20 mM Tris [pH 7.4]) for 2.5 minutes to lyse contaminating germ cells 36 hours after plating (Galdieri and Zani, 1981), after which cells were washed twice to remove cellular debris. Medium was replaced every 24 hours, and cells were incubated for an additional 5 days. Cultures were terminated at specified time points for RNA extraction or for protein lysate preparation.
Sertoli Cell HN2 Treatment Sertoli cells isolated from 20-day-old mice testes were cultured for 5 days and then incubated in DMEM/F12 with 10% fetal bovine serum containing 50, 100, and 200 mmol/L HN2 (34uC, 5% CO2). Control cells were brought to equivalent concentrations of isotonic NaCl solution. Morphologic changes of Sertoli cells were observed per 30-minute intervals at 12-hour exposure time points to 100 mmol/L HN2. Vimentin expression was investigated by immunocytochemistry at 6 hours and 24 hours posttreatment and reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot at 12 hours posttreatment with 50, 100, and 200 mmol/L HN2.
Immunocytochemistry Slides were air-dried and fixed in 4% paraformaldehyde (wt/ vol in PBS; 10 mM sodium phosphate, 0.15 M NaCl [pH 7.4]) at 28uC for 20 minutes. Then slides were incubated at 4uC overnight with the primary antibody diluted in PBS containing
Figure 1. Culture of Sertoli cells (48 hours) resulted in increases in cell body size and spread in the culture flask. While 3 to 4 cell processes were observed in the cells, no grains were noted in the cytoplasm (2006).
10% normal goat serum (NGS). Anti-human vimentin monoclonal primary antibody and normal IgG for negative control (Santa Cruz Biotechnology, Santa Cruz, Calif,) were diluted 1:50. After 2 washes in PBS, sections were incubated with horse anti-mouse biotinylated Ig in 2% NGS (1:200; Santa Cruz Biotechnology) for 30 minutes. Sections were then washed twice in PBS, incubated with avidin-biotin complex at room temperature for 30 minutes, and developed with diaminobenzidine. Slides were counterstained with Mayer hematoxylin, dehydrated, and mounted for microscopic examination.
RNA Extraction and Semiquantitative RT-PCR Total RNA was extracted from Sertoli cells using Trizol reagent (Invitrogen Corp), and 1 mg of total RNA was reverse transcribed into first-strand cDNA in a reaction primed by oligo(dT)12–18 primer using Superscript II reverse transcriptase (Invitrogen Corp). First-strand cDNA (2 ml) was used as template for PCR reactions using Taq polymerase (GibcoBRL, Grand Island, NY) with the following protocol: 1 cycle (94uC, 2 minutes) followed by 35 cycles (94uC, 30 seconds; 58uC, 45 seconds; 72uC, 45 seconds), and final cycle (72uC, 7 minutes). Sense and antisense vimentin primers were designed using Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, Calif), with resultant PCR primers as follows: vimentin forward 59-CAGCAGTATGAAAGCGTGG-39 and reverse 59-GGAAGAAAAGGTTGGCAGAG-39, product size: 441 bp; b-actin forward 59-TGTTACCAACTGGGACGACA-39 and reverse 59-GGGGTGTTGAAGGTCTCAAA-39, product size: 165 bp. b-actin was used as a positive control, and PCR products were separated by electrophoresis on 1.5% agarose gels.
Western Blot Sertoli cells were homogenized in RIPA buffer (1% Triton X100, 15 mM HEPES-NaOH [pH 7.5], 0.15 mM NaCl, 1%
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Figure 2. Sertoli cytoskeleton disruption following 12-hour nitrogen mustard (HN2) treatment at (A) 0 mmol/L (2006), (B) 50 mmol/L (2006), and (C) 200 mmol/L (4006). (D–F) Arrows indicate cytoskeletal changes observed per 30-minute intervals at 12-hour exposure time points to HN2 (100 mmol/L) (4006).
392 sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM sodium orthovanadate, 10 mM EDTA, and 0.5% protease inhibitor cocktail) and stored at 280uC until analysis. Protein concentrations were determined using the bicinchoninic acid method (Pierce, Rockford, Ill) according to the manufacturer’s specifications. Protein samples were added to an equal volume of 2X loading buffer (100 mM Tris [pH 6.8], 4% SDS, 0.2% bromophenol blue, and 20% glycerol). Supernatants were obtained by centrifugation at 10 000 6 g for 30 minutes, and samples were then reduced with 0.1% bmercaptoethanol and boiled for 3 minutes. Protein (30 mg) was separated on SDS-polyacrylamide (12%) slab mini gels. Separated proteins were transferred (3 hours, 75 V) to Protran nitrocellulose (Schleicher & Schuell, Keene, NH) with a TransBlot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, Calif), and membranes were blocked at room temperature for 1 hour with 5% nonfat dry milk in PBS and incubated with anti-vimentin primary antibody (1:1000) at room temperature overnight in blocking solution. Membranes were washed 3 times with PBS (5 minutes each) and then incubated at room temperature for 30 minutes with secondary anti-mouse horseradish peroxidase-linked IgG (1:3000, NA 931; Amersham Pharmacia, Piscataway, NJ) in PBS. Signal was detected using the SuperSignal WestPico Chemiluminescent kit (Pierce) according to manufacturer’s specifications.
Statistical Analysis Quantitative data are given as mean 6 SEM (n 5 4 independent experiments). Statistical analysis was conducted using the 1-way analysis of variance test. Students–Newman– Keuls test was used, and P , .05 considered statistically significant.
Results Morphologic Changes in Sertoli Cells Treated With HN2 Sertoli cells adhered to the bottom of the culture flask in a monolayer after 48-hour culture. The Sertoli cell body was polygonal, with 3 or more branches (Figure 1). With increased culture time, cells aggregated and their refractive ability weakened with tight conjunction. The cell nucleus was almost central, and nucleoli were clearly visible. Binucleate cells were also observed by the fifth day of culture (Figure 2A). Sertoli cells treated with HN2 displayed comparatively smaller cell bodies, with disruption of the cytoskeleton and decreased cytoplasm. Our data also suggested that there was a nonsignificant increase in cell length, with narrow columnar or fibroblast-like appearance of cells observed following treatment with low concentrations of HN2 (50 mmol/L) (Figure 2B), while a significant decrease in cellular area was observed (P , .01) (Table 1). Increasing concentrations of HN2 had more dramatic morphologic effects, with reduction in cell size and cytoplasm, atrophy of the cell body, cytoskeletal
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Table 1. Maximum length and area prior to apoptosis following 24-hour nitrogen mustard exposure in vitro (mean 6 SEM) Nitrogen Mustard Concentration (mmol/L) 0 50 100 200
Maximum Length (mm) (n 5 50) 72.9 70.2 63.3 44.7
6 6 6 6
5.72*3 6.013 3.80* 3.46*
Area (mm2) (n 5 50) 6344.81 4371.92 3414.74 1723.65
6 6 6 6
420.42* 353.51* 360.40* 363.01*
* Indicates comparison within groups (t test), P , .01. 3 Indicates comparison within groups (t test), P . .05.
disruption, gaunter cell shape, and increased detachment (Figure 2C), with a significant increase (P , .01) in cell length and decrease in cell area compared with controls (Table 1). Dramatic effects were observed following 12-hour HN2 treatment (100 mmol/L), with Sertoli cells observed to be much narrower and a noted breakdown of the vimentin cytoskeleton and intercellular conjunction complexes (Figure 2D through F).
Effects of HN2 Treatment on Sertoli Cell Vimentin Structure and Expression When a labeled primary antibody to vimentin was used in control Sertoli cells, vimentin intermediate filaments surrounded the nucleus and radiated out from the nucleus to the cell periphery, terminating near points of contact between the Sertoli cell and adjacent cells. The points of contact included the tight junctions found between neighboring Sertoli cells. Exposure to HN2 caused vimentin intermediate filaments to collapse and Sertoli cell conjunctions to detach; vimentin intermediate filament network was distributed between nuclear and cell membranes, gradually notable with increasing concentrations of HN2. Significant differences in vimentin expression were induced by 6-hour and 24hour HN2 treatment (50, 100, and 200 mmol/L) in vitro. Vimentin expression decreased with increased treatment time at all doses, compared with the strong staining noted in controls. Positive staining was observed mostly in the cytoplasm (Figure 3). Significant time-dependent and dose-dependent differences were also noted in optical density values of vimentin antibody (P , .01) (Table 2).
Semiquantitative RT-PCR To further elucidate the mechanisms underlying HN2mediated Sertoli cytoskeleton vimentin intermediate filaments disruption, we examined vimentin mRNA expression using RT-PCR. Our data suggested that vimentin mRNA expression decreased with HN2 concentration, and there were significant dose-dependent differences in PCR products following HN2 treatment (P , .01) (Figure 4).
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Figure 3. Vimentin expression was diminished at 6-hour and 24-hour nitrogen mustard (HN2) treatment times (4006). (A–D) 6-hour exposure to HN2; (E–H) 24-hour exposure to HN2. (A, E) 0 mmol/L; (B, F) 50 mmol/L; (C, G) 100 mmol/L; (D, H) 200 mmol/L.
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Table 2. The optical density value of vimentin antibody following exposure to varying nitrogen mustard concentrations (mean 6 SEM, n 5 100 cells) Nitrogen Mustard Concentration (mmol/L) 0 50 100 200
6-hour Exposure 85.05 63.37 41.86 15.68
6 6 6 6
2.84 4.77* 5.07* 3.64*
24-hour Exposure 86.90 47.79 26.41 10.60
6 6 6 6
4.19 3.97* 4.47* 2.37*
* Indicates comparison among 50 mmol/L, 100 mmol/L, 200 mmol/ L, and 0 mmol/L (t test), P , .01.
Western Blot Characterization of Vimentin Western blot analysis was used to evaluate vimentin protein expression levels in Sertoli cells following HN2 treatment. Vimentin and b-actin were detected as single bands, as expected, at 57 kd and 42 kd, respectively. Our data suggested that vimentin protein expression levels gradually decreased with increasing concentrations of HN2, with significant differences noted between different HN2 concentrations (P , .01) (Figure 5).
Discussion HN2 is an alkylating agent that results in DNA, RNA, and protein damage (Sanderson and Shield, 1996). HN2 is also currently used as an anticancer drug, in a highdose regimen, prior to stem cell transplantation. Alkylating agents have been demonstrated to destroy seminiferous epithelium and result in germ cell destruction and infertility (Nicholson and Byrne, 1993). However, it remains unclear that the cause of the progressive and selective absence of germ cells is because of the alkylating agent. Previous research has suggested that Sertoli cells, the primary supportive cells of the seminiferous epithelium, play a key role in triggering and regulating spermato-
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genesis (de Kretser et al, 1998) and have a welldeveloped cytoskeleton that is responsible for the formation of the seminiferous epithelium and the development of germ cells (Boekelheide et al, 1989; Russell et al, 1989; Muffly et al, 1993; Tung et al, 1993). Morphologic studies have implicated the Sertoli cytoskeleton in the maintenance of cell shape, localization and transformation of cytoplasmic organoids, formation and stabilization of cell-cell or cell-extracellular matrix contacts, localization, anchoring and migration of developed germ cells, and the release of mature sperm from seminiferous epithelium. Vimentin is an important Sertoli cell cytoskeleton component. Vimentin intermediate filaments are mostly located around the nucleus, resulting in a clear zone free of organelles surrounding the nucleus (Aumuller et al, 1988; Aumuller et al, 1992). Although biologic role of intermediate filaments in Sertoli and other cells has not been fully elucidated, it is well established that they provide mechanical resiliency and strength to cells (Klymkowsky et al, 1989; Fuchs, Cleveland, 1998; Show et al, 2003). We have shown herein that the Sertoli cell cytoskeleton responds to HN2 by exhibiting a marked collapse in vimentin intermediate filaments structure in vitro. Increasing concentrations of HN2 had more dramatic morphologic effects, with reduction in cell size and cytoplasm, atrophy of the cell body, cytoskeletal disruption, gaunter cell shape, and increased detachment. The changes were the result of disruption of vimentin intermediate filament network between nuclear and cell membranes, as seen by immunocytochemistry. Exposure to HN2 causes vimentin intermediate filaments to collapse and Sertoli cell conjunctions to detach, gradually notable with increasing concentrations of HN2. This change was the result of vimentin mRNA and protein expression down-regulation. Similarly, the collapse of the Sertoli intermediate filament cytoskeleton has been observed in cryptorchid testes of immature rats in vivo (Wang et al, 2002); in such testes, immunostaining of vimentin revealed loss of intermedi-
Figure 4. Vimentin mRNA expression following 24-hour exposure to varying nitrogen mustard concentrations in vitro. b1, b2, b3, b4: b-actin mRNA expression after 0, 50, 100, and 200 mmol/L, respectively. V1, V2, V3, V4: vimentin mRNA expression after 0, 50, 100, and 200 mmol/L, respectively. M: marker. The bar graph shows a semiquantitative comparison of the vimentin to b-actin optical density ratio (P , .01).
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Figure 5. Western blot analysis of vimentin in Sertoli cells following treatment with varying concentrations of nitrogen mustard. Lane 1: 0 mmol/ L; Lane 2: 50 mmol/L; Lane 3: 100 mmol/L; Lane 4: 200 mmol/L (30 mg total protein/lane). Vimentin expression was observed at the upper 57-kd band. The bar graph shows the comparison of the vimentin to b-actin optical density ratio (P , .01).
ate filament extensions and filament collapse to a perinuclear localization, coinciding with massive germ cell apoptosis. Cytoskeletal intermediate filament breakdown has also been linked to mono-(2-ethylhexyl) phthalate and colchicine treatment (Lloyd and Foster, 1988; Allard et al, 1993; Richburg and Boekelheide, 1996), resulting in loss of structural integrity of the seminiferous epithelium as well as germ cell apoptosis (Richburg and Boekelheide, 1996). Thus, loss of normal Sertoli intermediate filament dynamics has been shown to occur in concert with the failure of spermatogenesis after increased temperature (cryptorchidism) or toxic compounds. If this is generally the case, our data suggests that dysfunction of spermatogenesis induced by HN2 may be due to the disruption of the intermediate filament cytoskeleton in Sertoli cells. In conclusion, this study demonstrated that exposure of Sertoli cells to HN2 in vitro resulted in intermediate filament cytoskeleton collapse, with a concomitant decrease in vimentin protein expression and downregulation of vimentin mRNA. The change in the biochemical properties of vimentin may indicate that one of the mechanisms of reproductive toxicity resulting from HN2 is disruption of Sertoli cell vimentin filament structure, accompanied by a down-regulation of vimentin expression. Other testicular insults may result as well. However, future research is needed to confirm these effects in vivo and to further elucidate the role of vimentin intermediate filaments in Sertoli cells.
Acknowledgments We thank Zhang Xiaoping, MD, and Song Xiaofeng, MD, for excellent technical assistance.
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