Proteomic Analysis of Changes Induced By ... - ACS Publications

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Chem. Res. Toxicol. 2009, 22, 668–675

Proteomic Analysis of Changes Induced By Nonylphenol in Sprague-Dawley Rat Sertoli Cells Jiang Wu,†,‡,| Fuqiang Wang,§,| Yi Gong,†,‡ Dongmei Li,†,‡ Jiahao Sha,§ Xiaoyan Huang,*,§ and Xiaodong Han*,†,‡ Immunology and ReproductiVe Biology Laboratory, Medical School, Nanjing UniVersity, Nanjing, 210093, P. R. China, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing UniVersity, Nanjing, 210093, P. R. China, and Laboratory of ReproductiVe Medicine, Nanjing Medical UniVersity, Nanjing, 210029, P. R. China ReceiVed October 31, 2008

Nonylphenol (NP) is a common environmental contaminant that is known to disrupt the reproductive system. The testicular Sertoli cells play a pivotal role in the regulation of spermatogenesis and are susceptible to NP-induced reproductive lesions. Our goal was to ascertain whether NP could induce apoptosis in Sertoli cells and to explore the preapoptotic changes in Sertoli cells at low NP concentrations, similar to environmental conditions. In order to survey events that occur at the protein level in Sertoli cells after exposure to NP, we used a proteomic approach with two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify proteins with altered expression in rat Sertoli cells treated with 0.01 and 0.1 µM NP for 24 h. We separated 63 protein spots and identified 41 that were differently expressed in the NP-treated groups and the control. Of these 41 spots, we focused on Raf kinase inhibitor protein (RKIP), Annexin A7 (ANXA7), ERp57, and Peroxiredoxin 6 (PRDX6) for further analysis by Western blot. These proteins are involved in the response of Sertoli cells to programmed cell death. These data help to outline mechanisms by which NP might induce apoptotic tendencies in Sertoli cells. Introduction As one of the most common nonionic surfactants, alkylphenol ethoxylates (APEs) are widely used in the manufacture of agricultural chemicals, detergents, pesticides, plastics, and paper (1). APEs, which consist of approximately 80% nonylphenol ethoxylates (2), enter aquatic ecosystems through sewage treatment, pulp mill effluent, industrial effluent, and urban and agricultural runoff (3). In aquatic ecosystems, APEs are degraded into alkylphenols (APs) by microbes (4). Nonylphenol (NP, Figure 1), the most critical member of the APs, exists in ambient air, water, soil, sediments, and biota. The average concentration of NP in rivers is less than 0.005 µM, but it can be present at much higher levels in the outfall of sewage treatment plants (5). It has been reported that the concentration of NP in the aquatic environment, particularly in sediment, can reach up to 1.5 µM (6). Finally, NP can accumulate within the internal organs of fish and birds, and pass to humans through the food chain because of its high stability and lipid solubility. A number of studies show that NP has weak estrogenic activity; for instance, it induces the expression of the estrogen receptor (ER) and inhibits estrogen binding to ER, causing endocrine disruption (7-9). Other studies show that NP can induce cell death by inhibiting the activity of endoplasmic reticulum Ca2+ pumps (8) and can produce oxidative stress by enhancing reactive oxygen species (ROS) (10). * Corresponding author. (X. Han) Tel: 86-25-83686497. Fax: 86-2583686497. E-mail: [email protected]. (X. Huang) Tel: 86-25-86862038. Fax: 86-25-86862038. E-mail: [email protected]. † Immunology and Reproductive Biology Laboratory, Medical School, Nanjing University. ‡ Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University. § Nanjing Medical University. | These authors contributed equally to this work.

Figure 1. Chemical structures of nonylphenol.

In our previous in vivo research (11), testicular histological changes were observed in NP-treated rats. The arrangement of testicular cells was irregular and disordered. Intercellular connections were incompact and cellular vacuoles increased dramatically. As the most important testicular cells, Sertoli cells play a pivotal role in the regulation of spermatogenesis and rate of spermatozoa production (12, 13). Adjacent Sertoli cells form the blood-testis barrier by contacting each other through tight junctions at the base of the seminiferous tubule (14, 15). This barrier provides an isolated immunologic space by separating spermatocytes and spermatids from the immune system and preventing autoimmunity (16). This space ensures germ cell proliferation and differentiation with nutrients and regulatory factors. Sertoli cells are also responsible for phagocytosis of degenerating germ cells and residual bodies, release of spermatids at spermiation, and synthesis of proteins in response to gonadotropic hormones (17). Damage to the Sertoli cells inevitably results in malfunction of male reproduction. The aim of the present study was to ascertain whether NP could induce apoptosis in Sertoli cells, and to explore the changes in Sertoli cells at low NP concentrations that are similar to environmental conditions. Proteomic analysis was used to compare the protein expression profiles of primary Sertoli cells treated with NP before the appearance of apoptotic changes, and to investigate the mechanism by which NP affects Sertoli cells.

10.1021/tx800406z CCC: $40.75  2009 American Chemical Society Published on Web 03/05/2009

Nonylphenol Induced Proteomic Changes

Materials and Methods Chemicals and Reagents. Analytical grade 98% 4-nonylphenol (NP) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Dulbecco’s modified Eagle’s medium-Ham’s F-12 medium (DMEM-F12 medium), penicillin, streptomycin sulfate, trypsin, and collagenase I were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). C8H17N2O4SNa (HEPES sodium salt) and 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Amresco Inc. (Solon, OH, USA). The Annexin V-PE apoptosis detection Kit was obtained from Biovision (USA). Urea, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), immobilized pH gradient (IPG) buffer, iodoacetamide, tris-(hydroxymethyl)aminomethane (Tris), acrylamide, methylene bis-acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), glycerol, and enhanced chemiluminescence kit were purchased from Amersham Bioscience (Uppsala, Sweden). Halt protease inhibitor cocktail, EDTA-free was from Pierce (Rockford, IL, USA). Sequencing grade-modified trypsin was obtained from Promega (Madison, WI, USA). Thiourea, acetonitrile (ACN), ammonium bicarbonate (NH4HCO3), trifluoroacetic acid (TFA), and formaldehyde were from Sigma (St. Louis, MO, USA). Dithiothreitol (DTT) was from Shenggong (Shanghai, China). Peptide calibration standards and matrix material (R-cyano-4-hydroxy cinnamic acid, R-HCCA) were purchased from Bruker Daltonik (Bremen, Germany). Rabbit antiphosphoserine and mouse antiphosphothreonine were from Zymed Laboratories (San Francisco, CA, USA). Rabbit anti-RKIP polyclonal antibody was from Calbiochem (San Diego, CA). Rabbit anti-Annexin VII polyclonal antibody and Rabbit anti-β-Actin antibody were from Santa Cruz (CA, USA). Rabbit anti-ERp57 (Grp58, Carnitine Palmitoyltransferase) polyclonal antibody was from StressGen (Victoria, Canada). Mouse anti-Peroxiredoxin 6 monoclonal antibody was from Abcam (Cambridge, UK). Goat antirabbit and goat antimouse secondary antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology (Beijing, China). Isolation and Culture of Sertoli Cells. Sprague-Dawley Rats were purchased from Nanjing Medical University and kept in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Sertoli cells were isolated from the testes of 30-day-old rats in accordance with the method of Steinberger with some modifications (18). Briefly, testes were decapsulated and washed twice in phosphate-buffered saline (PBS). The settled seminiferous tubules were digested in 0.25% trypsin (Sigma) at 37 °C for 30 min and washed three times in PBS before further digestion in 0.1% collagenase (Sigma, type 1) at 37 °C for 40 min. The digested fragment was filtrated through a 100-mesh stainless steel filter. Cells were collected by centrifugation and washed three times with PBS before being resuspended in DMEM-F12 medium containing 5% fetal bovine serum (FBS, Invitrogen). Finally, resuspended cells were seeded into 6-well plates and incubated in a humidified chamber at 37 °C with 5% CO2. After 48 h of culturing, when Sertoli cells were attached to the bottom of plates, the supernatant containing most of the germ cells was discarded followed by two successive PBS washes. The remaining germ cells were hypotonically lysed with 20 mM Tris-HCl (pH 7.4) at 22 °C for 2.5 min. Two days later, the medium was changed again, and the enriched Sertoli cells grew to a monolayer in the new medium. Cell Viability Assay. Cell viability was evaluated by the MTT test. Briefly, cells were plated in a 96-well culture plate at 2 × 104 cells/well in 100 µL of culture medium. After incubation for 24 h, the wells were divided into five groups

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receiving different levels of NP at doses of 0, 0.1, 1, 10, and 20 µM, respectively, and incubated for 24 h. After the incubation, 25 µL of MTT solution (5 mg/mL) was added to each well followed by further incubation for 4 h. The supernatants were removed and 100 µL of DMSO was added to the wells to dissolve the formazan crystal at 37 °C. Thirty minutes later, the absorbance was measured on an automated microplate reader (Bio-Rad, Japan) at 570 nm. Flow Cytometry Assay. Annexin V-PI staining combined with flow cytometry was used to quantitate the percentage of cells undergoing apoptosis. Twenty-four hours after seeding 2 × 106 cells into a 6-well plate, cells were treated with the concentrations of NP listed above for 24 h, collected by centrifugation, and washed twice with cold PBS. The cell pellet was resuspended in assay buffer at a concentration of 1 × 106 cells/mL. One-hundred microliters of the solution (1 × 105 cells) was transferred to a 5 mL culture tube. One microliters of Annexin V and 10 µL of PI were added to the cell samples. Samples were incubated for 15 min in the dark, 400 µL assay buffer was added to each tube, followed by immedicate analysis by a FACScan flow cytometer (Becton-Dickson, Immunocytometry System, San Jose, CA). Sample Preparation. Cells cultured in 6-well plates were treated with 0, 0.01, and 0.1 µM NP for 24 h, digested with 0.25% trypsin and collected. After two PBS washes, cells were homogenized with a homogenizer (ULTRA TURRAX, Ika, Malaysia) in lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, 1% protease inhibitor cocktail (v/v), and 2% (v/v) IPG buffer (pH 3-10) at 11,000 IU/min on ice (ten bursts of 10 s, each interspersed with short pauses). The mixture was centrifuged at 40,000 × g for 1 h at 4 °C and the supernatant saved and stored at -70 °C. The protein concentration in each sample was determined by the bicinchoninic acid (BCA) method using PBS as the standard. Two-Dimensional Electrophoresis (2-DE). Isoelectric focusing (IEF) was performed on an Ettan IPGphor II (Amersham Bioscience) with 24-cm immobilized pH gradient strips (pH 3-10; Amersham Bioscience). Samples containing 80 µg of protein were mixed with rehydration solution containing 8 M urea, 2% CHAPS, 20 mM DTT, 0.5% (v/v) IPG buffer (pH 3-10), and 0.001% bromphenol blue. The linear ramping mode of the IEF voltage was applied. Strips were then equilibrated at room temperature for 15 min in 10 mL equilibration solution and incubated for another 15 min in an equilibration solution consisting of the same recipe with DTT replaced with 2.5% (w/v) iodoacetamide. Second-dimension electrophoresis was performed on 12.5% SDS gels in an Ettan DALTsix apparatus (Amersham Bioscience, Uppsala, Sweden) with constant power at 5W per gel for the first 30 min, followed by 12W per gel for 6-7 h until the bromophenol blue line reached the bottom of the gels. Gels were done in triplicate and silver-stained according to published procedures (19). Gels were run and scanned at 300 dpi resolution (Artix Scan 1010 plus, Taiwan, China). Image Analysis. Differential analysis was performed using ImageMasterTM 2D platinum software (Version 5.0, GE Healthcare, San Francisco, CA, USA) for spot detection, quantification, matching, as well as comparative, and statistical analyses. Data were averaged from three independent experiments, and the means and standard deviations were calculated and assessed for statistical significance using the Student’s t-test (ImageMasterTM 2D platinum software). A spot was regarded as significantly differentially expressed between groups if P values were less than 0.05, and the average spot intensity was greater than 1.2-fold (20).

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Figure 2. MTT assay for cell viability after Sertoli cells were treated with 0.1, 1, 10, and 20 µM for 24 h. Data are the ratio of cell viability to control and expressed as mean ( SD (n ) 6). The bar with an asterisk is significantly different (P < 0.05) from the control group.

Protein Identification. Protein spots were excised, dehydrated in acetonitrile, and dried at room temperature. Proteins were reduced and alkylated. Gel pieces were thoroughly washed and dried in a Speedvac (Concentrator 5301, Eppendorf, Hamburg, Germany). Dried gel pieces were reswollen with 2-3 µL of trypsin (Promega, Madison, WI, USA) solution (10 ng/ µL in 25 mM ammonium bicarbonate) at 4 °C for 30 min. Excess liquid was discarded, and gel plugs were incubated at 37 °C for 12 h. Trifluoroacetic acid (TFA) was added to a final concentration of 0.1% to stop the reaction. Digests were immediately spotted onto 600 µm anchorchips (Bruker Daltonics, Bremen, Germany). The Bruker Peptide Calibration Mixture was spotted for external calibration. All samples were allowed to air-dry at room temperature, and 0.1% TFA was used for on-target washing. MALDI-TOF mass spectrometry and tandem TOF/TOF mass spectrometry were carried out on a time-of-flight Ultraflex II mass spectrometer (Bruker Daltonics, Bremen, Germany). Peptide mass maps were acquired in positive reflection mode, averaging 800 laser shots per MALDI-TOF spectrum and 800 shots per TOF/TOF

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spectrum. Resolution was 15000-20000. The Bruker calibration mixtures were used to calibrate the spectrum to a mass tolerance within 0.1 Da. Each acquired mass spectrum (m/z range 700-4000) was processed using the software FlexAnalysis v. 2.4 (Bruker Daltonics, Bremen, Germany). The peak detection algorithm was SNAP (Sort Neaten Assign and Place); S/N threshold, 3; quality factor threshold, 50. The tryptic autodigestion ion picks (842.51 Da and 2211.10 Da) were used as internal standards to validate the external calibration procedure. Matrix and/or autoproteolytic trypsin fragments and known contaminant ions, such as keratins, were excluded. The resulting peptide mass lists were used to search the IPI rat sequence database (http:// www.ebi.ac.uk/IPI/, version 3.31; 41251 sequences; 21545744 residues). The following search parameter criteria were used: significant protein MOWSE score at p < 0.05, minimum mass accuracy 100 ppm, trypsin as enzyme, 1 missed cleavage site allowed, cysteine carbamidomethylation, acrylamide modified cysteine, methionine oxidation and similarity of pI and relative molecular mass specified, and minimum sequence coverage of 15%. Western Blotting. Samples containing 50 µg of protein from normal and NP-treated rat Sertoli cells were electrophoresed on a 12% SDS polyacrylamide gel and transferred to a polyvinylidenefluoride (PVDF) membrane (Pall, Ann Arbor, MI, USA). The membranes were blocked in phosphate buffered saline with 0.1% Tween 20 (PBST) containing 5% nonfat milk powder for 2 h at room temperature and then incubated at 4 °C overnight with a 1:1000-diluted anti-ERp57 rabbit polyclonal antibody, 1:1000-diluted anti-Peroxiredoxin 6 mouse monoclonal antibody, 1:800-diluted anti-Annexin VII rabbit polyclonal antibody, 1:4000-diluted anti-RKIP rabbit polyclonal antibody, or 1:10000-diluted anti-β-Actin rabbit polyclonal antibody in PBST containing 5% nonfat milk powder. After four PBST washes, antirabbit or antimouse IgG (dilute 1:2000) with horseradish peroxidase (HRP)-conjugated was applied to the membranes for 2 h at room temperature. The membranes were washed with PBST for 1 h and signal detected with an enhanced chemiluminescence detection kit (Amersham). The intensity of each band on the Western blot was quantified with Bio-Rad Quantity One software.

Figure 3. Flow cytometry dot plots of the binding of annexin V-FITC (FL-1) and PI uptake (FL-3) by Sertoli cells in medium alone (a), 0.1 (b), 1 (c), 10 (d), and 20 µM NP (e) for 24 h. The number represents the relative percentage (%) of cells present in the lower right quadrant, which represents apoptotic cells. Similar data were obtained in three independent experiments.



Table 1. Protein List of Differentially Expressed Proteins During NP Treatment spot ID

international protein index accession no.

protein name

score

sequence coverage(%)

Mr/pI

81 82 72 85 132 214 219 95 131 102 70 113 140 166 163 248 123 113

71 36 31 34 55 60 69 31 39 29 8 33 38 49 41 51 24 22

17386/6.92 20902/5.48 26024/6.92 22197/6.29 24860/5.64 36569/5.96 37517/6.67 36510/7.08 42052/5.29 54967/8.27 334582/6.45 49890/7.23 47084/6.5 56318/5.19 53481/5.21 57499/5.88 60835/5.86 66824/6.15

94 133 83 116 119 72 71 118 103 93 72 103 106 72 89 79 70 170 102 78 239 338 83

36 29 31 51 35 28 27 31 49 30 26 29 23 33 26 30 21 38 20 18 45 50 16

27310/5.99 29437/5.65 28614/6.23 22936/6.12 31216/6.18 34484/6.84 19565/8.07 32640/5.93 29784/6.15 30952/5.69 32115/5.97 38112/6.16 50483/4.8 38324/6.76 37961/5.84 48020/6.07 44888/6.26 50272/5.91 48701/6.53 47440/6.16 48943/5.59 59794/5.97 54574/7.96

upa 361 463 522 604 611 1041 1104 1147 1446 1566 1582 1596 1698 1823 1897 1978 2030 2093

IPI00325189 IPI00230937 IPI00231757 IPI00392676 IPI00231260 IPI00207390 IPI00211100 IPI00206092 IPI00189819 IPI00370384 IPI00366081 IPI00371236 IPI00566673 IPI00551812 IPI00421517 IPI00324741 IPI00200847 IPI00215349

517 557 660 726 731 824 834 919 925 976 1030 1094 1185 1230 1250 1346 1377 1660 1675 1754 1787 1806 1898

IPI00559001 IPI00191505 IPI00207184 IPI00201586 IPI00208209 IPI00764535 IPI00392956 IPI00831725 IPI00191748 IPI00365283 IPI00231749 IPI00231148 IPI00195673 IPI00325725 IPI00203390 IPI00476295 IPI00191216 IPI00361924 IPI00193279 IPI00464815 IPI00421600 IPI00231637 IPI00365545

a

nm23-H2 rkip psma2 blvrb Peroxiredoxin 6 annexin A3 3-alpha-hydroxysteroid dehydrogenase aldose reductase-like protein Actin, cytoplasmic 1 actr1a similar to desmoplakin isoform I isoform 2 tu translation elongation factor, mitochondrial 47 KDA protein, gdi2 atp5b dexmin protein disulfide-isomerase A3 precursor (ERp57) T-complex protein 1 subunit alpha wd repeat-containing protein 1 downb 27 KDA Protein, rgd1563391_predicted psmb4 erp29 heat shock protein beta-1 Peroxiredoxin 4 Actin related protein 2/3 complex, subunit 2 20 KDA protein, Peroxiredoxin 6, nitrilase 1 isoform B proteasome subunit alpha type-1 f-Actin-capping protein subunit beta phosphatidylinositol transger protein alpha isoform glycerol-3-phosphate dehydrogenase [NAD+], cytoplasmic tubulin, beta 6 dystonia 1 serine/threonine-protein phosphatase PP1-beta catalytic subunit adenosylhomocysteinase 45 KDA protein, Tardbp annexin A7 ornithine aminotransferase, mitochondrial precursor eno1 psmc2 g6pdx dihydrolipoyl dehydrogenase, mitochondrial precursor

Up-regulated proteins. b Down-regulated proteins.

Results MTT Assay for Cell Viability. Viability of Sertoli cells exposed to NP for 24 h was assessed by the MTT assay (Figure 2). Twenty micromolar NP significantly decreased the cell viability. No significant difference was observed between the control group and the groups exposed to 0.1, 1, or 10 µM NP. NP-Induced Apoptosis of Sertoli Cells. Dot plots were generated to assess apoptosis in Sertoli cells stained with annexin V-PI and analyzed by flow cytometry after 24 h of NP treatment (Figure 3). The percentage of apoptotic Sertoli cells is indicated. A significant difference was observed between the group exposed to 20 µM NP and the control group. The percentage of apoptotic Sertoli cells in medium alone (Figure 3a) and in 20 µM NP (Figure 3e) was 1.16% and 3.09%, respectively. Thus, 20 µM NP significantly increased the percentage of apoptotic cells. Quantitative Comparison and Identification of Protein Spots on 2-DE Gels. Sertoli cell protein samples from control and NP-treated groups were analyzed individually. To detect the proteomic changes following NP exposure, we constructed triplicate 2D maps of protein samples following NP treatment. Figure 4 shows a representative 2-DE gel image of the protein expression pattern in Sertoli cells.

Sixty-three protein spots showed significant differences in expression levels by t-test, between the NP-treated groups and the control (p < 0.05 with average spot intensity greater than 1.2-fold). These spots were subjected to tryptic digestion and MALDI-TOF/MS analysis. Peptide mass fingerprint (PMF) analysis was used to identify the differentially expressed proteins. Of the 63, some could not be identified because of incomplete polypeptide fragments, and some were too low in abundance to obtain useful data. In total, 41 protein entries were identified by MALDI-TOF/MS analyses. An overview of these proteins is presented in Table 1, which also includes accession numbers, protein names, scores, sequence coverage, Mr, and pI of the protein spots from the control and NP-treated Sertoli cells. We focused on four spots that were identified to be RKIP (spot 463), ANXA7 (spot 1660), ERp57 (spot 1978), and PRDX6 (spot 611), which are all involved in either protein kinase signaling cascades or cellular Ca2+ homeostasis and oxidative stress. In all cases, these proteins are involved in cellular pathways leading to apoptosis, an important cellular effect of NP. The mean normalized volumes (% volume) and standard deviations of the four protein spots are shown in Figure 5. Gel spot 463 (RKIP) increased in expression intensity after treatment

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Figure 4. Representative image of 2-DE silver stained polyacrylamide gel. Identified spots are indicated by spot numbers. Proteins were extracted from Sertoli cells treated with 0 (a), 0.01 (b), and 0.1 (c) µM NP for 24 h and subjected to 2-DE.

with 0.01 and 0.1 µM NP. Gel spot 1660 (ANXA7) decreased significantly with 0.01 and 0.1 µM NP. Gel spots 1978 (ERp57) and 611 (PRDX6) increased in expression intensity after treatment with 0.1 µM NP (p < 0.05; Table 1). Western Blotting. We used Western blotting to study the expression of RKIP, ANXA7, ERp57, and PRDX6 in Sertoli cells after treatment with 0, 0.01, and 0.1 µM NP for 24 h. As shown in Figure 6, 0.01 and 0.1 µM NP induced an increase of RKIP and a decrease of ANXA7, while 0.1 µM NP treatment resulted in an increase of ERp57 and PRDX6. The expression trends of these four proteins were consistent with the 2-DE result that is shown in Figure 7.

Discussion NP has been intensively studied on the basis of its wide use in manufacturing. Investigations have demonstrated that NP can induce apoptosis in neuronal cells (21), human embryonic stem cells (22), and other cells (23, 24). Our present study showed that incubation in 20 µM NP for 24 h resulted in reduced cellular viability and apoptosis of Sertoli cells. No significant changes in cellular viability or apoptosis were observed when Sertoli cells were incubated with NP at lower concentrations. As the average concentration of NP in rivers is about 0.005 µM (5), our experiment used 0.01 and 0.1 µM NP to explore the proteomic alterations of Sertoli cells at close to environmental levels. Proteomic studies are sensitive tools for investigating the toxic response of many proteins. Consequently, toxicoproteomics gives us new, comprehensive patterns of effects at the molecular level (25).

Figure 5. Mean normalized volumes (% volume) and standard deviations of apoptosis-associated proteins. To reflect quantitative variations in the volume of protein spots, spot volumes were normalized as a percentage of the total volume of all spots present in a gel. Data are presented as the ratio of spot intensity to control, and expressed as mean ( SEM. The bars with an asterisk are significantly different (P < 0.05 and average spot intensity greater than 1.2-fold of the control) from the control group.

RKIP, a member of the phosphatidylethanolamine-binding protein (PEBP) family, is also called PEBP-1 and PBP. RKIP is expressed in numerous tissues and cell types with the highest protein levels detectable in the testes and brain (26, 27). RKIP plays a vital regulative role in several protein kinase signaling


Figure 6. Western blot analysis of RKIP, ANXA7, ERp57, and PRDX6 protein expression in Sertoli cells after treatment with 0, 0.01, and 0.1 µM NP for 24 h. The intensity of each band obtained by Western blotting was quantified by Bio-Rad Quantity One software. The expression of β-Actin in corresponding Sertoli cells is displayed in the bottom panel as a loading control. The experiment was performed three times with similar results.

Figure 7. 2-DE images of representative up- or down-regulated proteins induced by NP at concentrations of 0 (a), 0.01 (b), and 0.1 (c) µM for 24 h.

cascades. RKIP binds to Raf-1, inhibiting Raf-1-mediated phosphorylation of mitogen-activated protein kinase and extracellular signal-regulated kinase (28, 29). G-protein-coupled receptor kinase was inhibited when bound to phosphorylated RKIP. Because of its role in cell signaling, RKIP has the potential to modulate many processes. RKIP has been identified in the testis and epididymis, where it affects spermatogenesis and reproductive behavior (30, 31). The collective evidence indicates that RKIP inhibits cell proliferation and sensitizes the cell to apoptosis (32, 33). The high RKIP expression following NP exposure observed in the present study may suggest the Sertolicellsaremoresensitivetoapoptosis,affectingspermatogenesis. ANXA7, also known as synexin, was first described by Creutz et al. in 1978 (34). As Pollard et al. explained, the remarkable


function of ANXA7 is to regulate the calcium-dependent membrane fusion and its voltage-dependent calcium-channel activity (35). Clemen’s results suggested an involvement of ANXA7 in Ca2+-dependent signaling or Ca2+-homeostasis in astrocytes (36). ANXA7 removes Ca2+ from the endoplasmic reticulum, which can enhance IP3-mediated Ca2+ release, thus contributing to proper Ca2+ homeostasis in the cell (37). In the current investigation, the decrease of ANXA7 would affect the proper Ca2+ homeostasis in Sertoli cells. The imbalance of intracellular Ca2+ influences a number of calcium sensors, resulting in calcium-dependent disorders and finally initiating a tendency to cell death (38). In other research, Yu et al. (39) reported that following various apoptotic stimuli, galectin-3, a multifunctional oncogenic protein found in the nucleus and cytoplasm, translocates to the perinuclear membrane and exerts antiapoptotic function. The down-regulation of ANXA7 would prevent galectin-3 from translocating to the perinuclear membranes. This may imply another role ANXA7 plays in NPinduced apoptosis of Sertoli cells. Oxidative stress acts as a causative factor in programmed cell death (40). Our previous work indicated that direct exposure to NP could trigger oxidative stress in rat Sertoli cells, but the molecular details remain unexplored. ERp57, a multifunctional thiol-disulfide oxidoreductase, is a member of the protein disulphide isomerase family of oxidoreductases localized mainly in the endoplasmic reticulum, but also existing in the cytosol and nucleus. Associated with both calnexin and calreticulin, ERp57 helps fold nascent polypeptides with glycans (41). Evidence showed that overexpression of ERp57 is an early adaptive response of the cell in toxin-mediated oxidative stress (42, 43). Knockdown of ERp57 by RNA interference increased the cellular apoptotic response and suggested that decline of homeostatic endoplasmic reticulum stress responses would enhance oxidative stress-induced apoptosis (44). In addition, changes in endoplasmic reticulum lumenal Ca2+ concentration are responsible for the regulation of protein-protein interactions (45). Li and Camacho found that ERp57 modulates the redox state of the endoplasmic reticulum in a Ca2+-dependent fashion and maintains the endoplasmic reticulum Ca2+ homeostasis microenvironment. We found down-regulation of ANXA7, which may lead to an imbalance of cellular Ca2+. Increased ERp57 was observed to resist the deregulation of Ca2+ homeostasis and protect Sertoli cells from oxidative stress insult as a adaptive response. In addition to the up-regulation of ERp57, oxidative stress also stimulates other protective reactions such as PRDX6 (46), which is the only mammalian 1-cysteine member of the peroxiredoxin superfamily expressed in all major organs (47). In contrast to other peroxiredoxins, PRDX6 uses glutathione rather than thioredoxin as an electron donor to reduce the oxidized peroxidatic cysteine, consequently reducing H2O2 and other organic hydroperoxides including phospholipid hydroperoxides. As a result of its function in removing reactive oxygen species (48), PRDX6 acts as a cytoprotective enzyme to enhance cell resistance to oxidative stress-induced apoptosis caused by various agents. Kumin A observed that a strong increase in expression of PRDX6 reduced the number of apoptotic cells after UV irradiation and demonstrated that PRDX6 protects keratinocytes from cell death induced by reactive oxygen species, suggesting that activation of this enzyme could be a novel response to stress conditions (49). In the current study, increased expression of PRDX6 after incubation with 0.1 µM NP for 24 h suggests a response to protect Sertoli cells from oxidative stress-induced apoptosis (50).

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In summary, 20 µM NP can reduce cellular viability and induce apoptosis of Sertoli cells. There were no morphological changes to Sertoli cells when incubated at lower concentrations. With the method of differential proteomics, a total of 41 proteins were identified as expressed differently in Sertoli cells treated with 0.01 and 0.1 µM NP. Among these proteins, RKIP, ANXA7, ERp57, and PRDX6 were correlative with programmed cell death and chosen for further study. The upregulation of RKIP and down-regulation of ANXA7 implied the subtle apoptotic trend induced by environmentally relevant levels of NP. At the same time, ERp57 and PRDX6 were upregulated to protect Sertoli cells. Oxidative stress and change of Ca2+ homeostasis may play important roles in the process. Acknowledgment. We are grateful to the foundation of the program: National Natural Science Foundation of China (20577019), 973 program (2009CB941703), National Natural Science Foundation of China (30800115), and Key Laboratory of Yangtze River Water Environment, Ministry of Education, China (YRWEF07006).

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