Molecular Human Reproduction, Vol. 7, No. 8, 715-722,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Mapping of seminal plasma proteins by two-dimensional gel electrophoresis in men with normal and impaired spermatogenesis
1 INSERM EMI 00-09 Groupe de Recherche en Endocrinologie et Reproduction, IFR 50, UFR de Médecine, avenue de Valombrose, 06107 Nice cedex 2 and 2 Laboratoire de Chimie des Proteines, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France
Abstract
The present study analyses differential polypeptide expression of seminal plasma from fertile and infertile men by two-dimensional gel electrophoresis. Optimization of solubilization of seminal plasma was obtained by using [3-(3-(cholamidopropyl) dimethyl-ammonio)-1-propane sulphonate] and chaotropic agent mixture in lysis buffer before separation in immobilized pH gradient for isoelectric focusing. A two-dimensional map of seminal plasma from a fertile man allowed the detection of about 750 spots. Semi-preparative electrofocusing was performed. Analysis of tryptic fragments of two major spots by matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectroscopy resulted in identification of prostatic acid phosphatase and prostate specific antigen. Three groups of spots and seven individual spots of isoelectric point from 4.6 to 6.2 and mol. wt from 41 to 18 kDa disappeared in the two-dimensional maps of seminal plasma of vasectomized men (n = 4) and of a patient with bilateral anorchidy as compared to that of fertile men (n = 5). Some of these polypeptides were also absent in seminal plasma of patients with alterations of seminiferous tubules showing Sertoli cell-only syndrome characteristics (n = 4) and could be potential diagnostic markers of spermatogenesis impairment.
azoospermia/proteome/seminal plasma/spermatogenesis/two-dimensional gel electrophoresis
Introduction
Seminal plasma combines a lot of proteins originating from the testis, the epidiymis and from the accessory glands. Many attempts have been made to establish a link between testicular specific proteins and the events of spermatogenesis. Such a relationship has been suggested for at least three testicular proteins. Lactate dehydrogenase-C4 (LDH-C4) found in seminal plasma originates mainly from damaged or disintegrated postmeiotic germ cells and appears to be a useful marker for assessing the status of the seminiferous epithelium (Virji and Naz, 1995
). Inhibin B is considered as a marker of the functional state of Sertoli cells (Anawalt et al., 1996). Measurement of inhibin B serum levels in association with FSH has been used for diagnosis of infertility in azoospermic patients (Von Eckardstein et al., 1999). More recently, low levels of anti-Müllerian hormone (AMH) have been reported in seminal plasma of men with alteration of sperm motility (Fallat et al., 1996) or with severe spermatogenic failure (Fénichel et al., 1999).
In light of this limited number of molecules leading to only few available assays, an alternative approach was to characterize other proteins associated with fertility by using a high-resolution electrophoresis method. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of seminal plasma has been carried out in different species (Desnoyers et al., 1994; Binette et al., 1996; McDowell et al., 1996; Mortarino et al., 1998; Brandon et al., 1999). For human seminal fluid, isoelectric focusing (IEF) in carrier ampholytes (CA) has been used as the first dimension and resulted in the detection of a few spots on 2-D PAGE (Edwards et al., 1981; Ayyagari et al., 1987). By using CA-IEF and non-equilibrium pH gradient electrophoresis (NEPHGE), more than 300 polypeptides have been separated from human seminal plasma (Naaby-Hansen et al., 1997). Recently, an immobilized pH gradient (IPG) has also been developed as the first dimension for 2-D PAGE analysis of a specific polypeptide present in the human seminal plasma (Charrier et al., 1999).
The aim of the present study was to improve the polypeptide detection in the human seminal plasma (2-D) map of fertile and infertile men. Solubilization was enhanced in the presence of [3-(3-(cholamidopropyl) dimethyl-ammonio)-1 propane sulphonate] (CHAPS) added to chaotropic agent mixtures and in-gel sample application by rehydration of an IPG strip of great length was applied. Separation in the second dimension was performed in highly reproducible ready-to-use gradient second dimensional thin gels.
Materials and methods
Patients
Samples of seminal plasma from five fertile men, four vasectomized men, one anorchid man and four azoospermic patients with Sertoli cell-only syndrome (SCOS) were collected after informed consent. Each seminal plasma sample was analysed in 2-D PAGE in quadruplicate. Two-dimensional maps of seminal plasma of patients were compared to that of all the fertile men, except for one patient who served as his own control with a map performed before and after vasectomy.
Apparatus
First and second dimensions of 2-D PAGE were carried out horizontally on the Multiphor II apparatus connected to the EPS XL 3500 power supply from Amersham Pharmacia Biotech (Orsay, France). Scans of dried gels carried out on the Sharp Jx330 laser scanner were processed by the 2-D Image master software from Pharmacia Biotech. Mass spectra of the tryptic digests were acquired on a Biflex (Bruker-Franzen Analytik, Bremen, Germany) matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometer equipped with a gridless delayed extraction.
Reagents
Gel Fix for covers were obtained from Behringer Ingelheim (Bioproducts, France). Urea, dithiothreitol (DTT), the isoelectric focusing calibration kit (broad pI kit pH 3–10), carbamylated creatine kinase (CPK) as pI standard proteins, low and high mol. wt standard proteins, carrier ampholytes Pharmalytes 3–10, silicon oil fluid, immobilized pH gradients (IPG) 3–10, polyacrylamide Excel gels 12% T–14% T gradient and buffer strips for Excel gels, and enhanced chemiluminescence (ECL) reagents were from Amersham Pharmacia Biotech. Amberlite AMB-150 was obtained from ICN Pharmaceuticals (Costa Mesa, CA, USA) and sodium dodecyl sulphate (SDS) from Merck (Darmstadt, Germany). CHAPS, thiourea, iodoacetamide, anti-human albumin antiserum, goat anti-rabbit IgG peroxidase conjugate and Kodak Bio-Max Light films were from Sigma (St Louis, MO, USA). Sequencing grade modified porcine trypsin was from Promega (Madison, WI, USA) and bicinchoninic acid (BCA) protein assay reagents from Pierce (Rockford, IL, USA). The protease inhibitors were the CompleteTM mixture from Roche Diagnostics (Mannheim, Germany). Polyvinylidene difluoride (PVDF) blotting membrane was from Millipore corporation (Bedford, MA, USA).
Sample preparation
Semen samples were allowed to liquefy for 30 min, clarified free of spermatozoa by centrifugation, and protease inhibitors were added according to the manufacturer's instructions. Seminal plasma was then aliquoted and frozen at –80°C.
A fraction of each sample was used for microscale protein determination. Samples analysed in 2-D PAGE were diluted in lysis buffer containing a mixture of 2 mol/l thiourea and 7 mol/l urea purified by Amberlite MB-150 mixed bed ion exchange resin (10 mg/ml), 4% CHAPS, 65 mmol/l DTT, 0.8% (w/v) Pharmalyte 3–10, 10% isopropanol and the protease inhibitor cocktail (1 mmol/l EDTA). Samples were routinely applied in lysis buffer during reswelling of the IPG strip as previously described (Rabilloud et al., 1994; Sanchez et al., 1997). Analytical separations were performed with 50 µg of seminal fluid proteins in pH 3–10 IPG strips. In semi-preparative separations, 0.5 mg of proteins were separated in pH 3–10 strips. For practical purposes, Coomassie Brillant Blue R detection was considered as a good quantitative indication for spot excision.
For a comparison at the analytical level, proteins were also solubilized replacing the mixture of 2 mol/l thiourea and 7 mol/l urea by 8.5 mol/l urea. Cup-loading experiments were performed after reswelling of the IPG strip, which was carried out overnight in a rehydration solution composed of 8.33 mol/l urea purified by Amberlite MB-150 mixed bed ion exchange resin (10 mg/ml), 0.5% CHAPS, 12 mmol/l DTT and 0.2% (w/v) Pharmalyte 3–10.
Isoelectric focusing in immobilized pH gradient (IPG)
In routine analysis, the entire IPG gel was used for sample application with the protein entering the gel during its rehydration. The rehydration volume was 400 µl of lysis buffer for a gel 3 mm wide, 0.5 mm thick, and 180 mm long, pH 3–10. This in-gel sample application was compared in one experiment to the classical method using rehydration buffer and sample loading. IEF was carried out horizontally under silicon oil using paper electrodes wetted with water, at an optimized temperature of 20°C as reported (Görg et al., 1991). The run was performed for analytical purposes with a preliminary voltage gradient to 3500 V in order to obtain first 3000 Vh, then at 3500 V to reach a total of 20 kVh for a pH 3–10 IPG strip. For semi-micropreparative experiments on Excel gels, IPG strips were loaded with 0.5 mg of the sample and run for 2 h at 150 V, 2 h at 300 V and 3 h at 500 V to reach 40 kVh. After IEF, the IPG strips were covered by Gel fix and stored at –80°C for separation in the second dimension.
In specific experiments, the IPG strips separated in CHAPS with the classical protein loading at a specific point were conserved in order to analyse their contents in terms of solubilization efficiency. In this case, strips were washed twice, incubated in 2% (w/v) glycerol for 10 min and dried in a ventilated place in order to perform complementary IEF experiments.
Equilibration
Equilibration solution for IPG contained 30% (w/v) glycerol, 6 mol/l urea and 0.25% (w/v) Bromophenol Blue in 0.05 mol/l Tris–HCl pH 8.8 as previously reported (Görg et al., 1997). SDS concentration was raised to 3% (w/v) and 0.1 mmol/l EDTA was added as recommended (Westermeier, 1993). The first step of equilibration was carried out with 65 mmol/l DTT. The second portion of equilibration solution contained 260 mmol/l iodoacetamide (Görg et al., 1987). Both equilibration steps usually lasted 15 min at room temperature.
SDS–PAGE
SDS–PAGE used as second dimension was a horizontal procedure performed with the commercially available 0.5 mm thick, 240 mm wide, 180 mm long 12% T–14% T gradient Excel gels. After setting down agarose electrodes wicks interacting on a 4.5x245 mm surface with the second dimensional gel, the equilibrated IPG strips were put down on the 40 mm high stacking gel at 0.5 mm from the wicks. The run was performed at 15°C at 20 mA with voltage limitation set to 1000 V and power limitation to 40 W. IPG strips were removed from the second dimensional gel 45 min after starting. The cathode was then placed at the IPG emplacement and a setting of 30 mA with power supply limitations of 1000 V and 40 W was applied.
Detection of the 2-D pattern
Silver staining of analytical gels was performed using the improved transparent background method involving pretreatment of fixed gels by thiosulphate method (Blum et al., 1987). After the second dimension, the stained gel was immersed before drying in glycerol 10%, covered by a cellophane sheet wetted in the same solution and placed at room temperature for 24 h and overnight at 30°C. The scanned gel was analysed and pI and mol. wt determinations were obtained by computer analysis.
Immunostaining of proteins separated on 2-D PAGE Gel-Bond reinforced gels was previously performed after capillary transfer (Starita-Geribaldi et al., 2000). Non-specific binding was blocked by incubation of the PVDF membrane in PBS buffer containing 0.1% Tween and 5% defatted milk for 2 h at room temperature. Immunodetection was carried out for 1 h with anti-human albumin antiserum (1/8000) or non-immune serum at the same dilution, in parallel experiments. Sheets were then washed for 10 min three times in blocking buffer and incubated with peroxidase-conjugated anti-rabbit IgG (1/10 000) for 1 h. The membrane was then washed for 10 min three times in PBS buffer containing 0.1% Tween. The membrane was allowed to react with ECL reagent and, after protection by a plastic sheet, brought in contact with the autoradiographic film for 5 min before development.
In-gel protein digestion
Protein spots were excised from the gel and then digested with trypsin using a previously described procedure (Boussac and Garin, 2000). Briefly, the pieces of gel were immersed in 1 ml of 20 mmol/l ammonium bicarbonate solution for 15 min followed by 50% acetonitrile solution for 15 min. The procedure was repeated twice. Finally, the piece of gel was washed in water, dried in a vacuum centrifuge and conserved at –20°C before trypsin action. The 2-D gel pieces were reswollen with a minimum amount of trypsin solution containing 0.25–0.5 of µg protease, depending on the amount of protein (typically 10 µl of a 0.05 µg trypsin/µl solution, in 25 mmol/l ammonium bicarbonate containing 10% acetonitrile). When necessary, ammonium bicarbonate buffer was further added until the gel piece was completely rehydrated. Digestion was performed at 37°C for 3–5 h.
MALDI-TOF mass spectrometry analysis
The MALDI-TOF mass spectrometer was operated in the reflector mode. The digest solution (0.5 µl) was deposited directly onto the sample probe on a dry thin layer of matrix made of 
-cyano-4-hydroxy-trans-cinnamic acid (CCA) mixed with nitrocellulose as a mixture 4/3 (v/v) of a saturated solution of CCA in acetone and a solution consisting of 5 mg nitrocellulose dissolved in 1 ml isopropanol/acetone 1/1 (v/v). Deposits were washed with 5 µl of 0.1% trifluoroacetic acid before analysis. A mass list of peptides was obtained for each protein digest. This peptide mass fingerprint was then submitted to an appropriate software in order to identify the proteins (MS-FIT: http://prospector.ucsf.edu/ucsfhtml3.2/msfit.htm, or ProFound: http://129.85.19.192/prowl-cgi/ProFound.exe).
Histological procedures
Sections (5 µm thick) from Bouin-fixed, paraffin-embedded testicular biopsies were stained with haematoxylin–eosin. Slides were mounted with mounting medium and examined with a Nikon microscope.
Results
2-D PAGE analysis of seminal plasma from a fertile man
The human seminal plasma 2-D PAGE pattern obtained with the cup-loading method is presented in Figure 1A. Although this pattern displayed a great number of spots, some polypeptides were not detected in the vertical path below the loading zone. This was probably due to the mechanical pressure of the loading apparatus onto the gel. Residual proteins appeared as a diffuse shadow in the acidic part of the IPG strip after the first dimension (data not shown). Using a `stepwise' procedure for IEF, this residual content was analysed, submitting again the IPG strip, dried after a first IEF separation, to rehydration with urea–thiourea and CHAPS in the lysis buffer. This second reswelling of the gel was followed by IEF at identical voltage. We were then able to separate the remaining molecules and use the strip for separation in the second dimension. Specific spots were enhanced by the use of a mixture of 7 mol/l urea and 2 mmol/l thiourea (Figure 1B). The 2-D pattern displayed 18 spot trains of high mol. wt >70 kDa. Other spots of lower mol. wt appeared in the acidic part of the pattern suggesting an important and specific enhancement of the solubilization of seminal plasma proteins. This feature was confirmed by direct IEF analysis of the seminal plasma sample in the lysis buffer, with proteins entering the gel during its rehydration. The resulting 2-D pattern of the fertile man's seminal plasma is presented in Figure 2. At the top of the second dimensional gel, the IPG strip devoid of residual proteins indicates maximal solubilization. The software allowed detection of 757 spots automatically after subtraction of the standard protein spots. The mol. wt range covered was ~170 to ~10 kDa. Resolved proteins spanned a pI range from ~4.0 to ~8.0, a limit enhanced to pI 8.5 in the presence of isopropanol in the lysis buffer. The analysis of internal protein fragments proved to be an efficient strategy for the characterization and identification of electrophoretically separated proteins. Two main spots belonging to two groups of 44.4–46.9 kDa and pI 5.34–5.35 for the first spot and 30.2–31.8 kDa and pI 6.93–6.95 for the second spot were excised from semi-preparative gels and analysed by MALDI-TOF mass spectrometry analysis after enzymatic cleavage (Figure 2, lower panels). These spots were identified as prostatic acid phosphatase (PAP, access number P15309) and prostate specific antigen (PSA, access number P07288).
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2-D PAGE analysis of seminal plasma from infertile patients
Variations in the expression of spots were observed essentially in the area of the pattern in a pI range from 3.4 to 6.5 and in mol. wt range from 41 to the 14 kDa. It was in this area that we observed a maximum number of differences between fertile donors and infertile patients. Figure 3
shows the electrophoretic profile of the seminal plasma of a fertile man in this area before (Figure 3A) and after vasectomy (Figure 3B). Various spots disappeared after vasectomy (Figure 3B). Groups of spots numbered G1, G2 and G3 as well as spots S7, S9, S16 were absent after vasectomy. An elliptic zone was drawn in the map of this man before vasectomy (Figure 3A) and contained six spots from S1 to S6. These spots disappeared after vasectomy (Figure 3B), except S1 and S2 which appeared very faint after surgery. The notion of the presence or absence of a spot illustrated above is difficult to apply to other spots since we only observed a decrease in the expression of spots S10, S11, S12 and S13 after vasectomy. Other spots (S8, S14 and S15) also decreased in intensity after vasectomy and resulted in very faint spots (Figure 3B). The electrophoretic pattern of seminal plasma from three other men after vasectomy when compared to that of fertile men confirmed these observations. Similar variations were observed in a patient with bilateral anorchidy (Figure 3C). Groups G1, G2, G3, spots S3, S4, S5, S6, S7, S9 and S16 were not detected in this patient and S1, S2, S8, S14 and S15 were very faint. The expression of spots S10, S11, S12, S13 was also affected (Figure 3B). Table I summarizes the physico-chemical characteristics of the molecules cited above.
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The 2-D PAGE map of seminal plasma from a patient with a severe alteration of spermatogenesis identified as SCOS (Figure 4A
) is shown in figure 4B. As with the vasectomized men, groups of spots G1 and G2, and the spots S3 to S6, S7, S9 and S16 which were present in fertile men were not observed in this azoospermic patient. Similar results were observed in the three other azoospermic patients when compared to all the fertile men. The group of spots G3, and spots S1, S2, S8, S14, S15 were reduced in the four azoospermic men.
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The modification of a specific spot (S7) which was clearly affected in pathological conditions is shown in Figure 5
. This spot was present in seminal plasma of the five fertile men (F1 to F5), but was not detectable either in the four independent vasectomized patients (V1, V2, V3, V5) or in the four independent azoospermic patients (A1 to A4).
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Discussion
Electrophoretic protein maps have been reported for different human fluids, such as plasma (Anderson and Anderson, 1991; Hughes et al., 1992), cerebrospinal fluid (Yun et al., 1992) or amniotic fluid (Liberatori et al., 1997). The present study shows for the first time the 2-D PAGE map of the human seminal plasma describing 757 spots and reveals the presence of numerous groups or individual polypeptides in the 170–10 kDa range. These results suggest that the seminal plasma can be classified in the highly soluble human fluid groups. They are at odds, however, with previous studies on 2-D PAGE analysis of human seminal plasma performed with CA-IEF as first dimension, which mentioned relatively few proteins of high mol. wt above the 80 kDa range (Edwards et al., 1981; Ayyagari et al., 1987; Naaby-Hansen et al., 1997). In the present study, the optimization of the 2-D PAGE map was obtained by maximum solubilization of the sample after addition to the lysis buffer of CHAPS and urea–thiourea mixture as previously reported (Perdew et al., 1983; Rabilloud et al., 1997). Confirmation of this powerful solubilization is evidenced by the depletion of the IPG strip after coupling with the second dimensional gel. The presence of a high concentration of SDS in the equilibration buffer has been suggested to be efficient in solubilizing the sample for the second dimension (Adessi et al., 1997). The absence of vertical streaking on the 2-D map of the current study is in agreement with such an hypothesis. Beside the presence of isopropanol and EDTA in the equilibration buffer (Westermeier, 1993; Görg et al., 1997), the temperature and the thickness of the gels may also be factors which have enhanced the spot resolution in the present study as compared to previous data on seminal plasma (Edwards et al., 1981; Ayyagari et al., 1987; Naaby-Hansen et al., 1997). Indeed, the use of 0.5 mm thick gels allows an optimal cooling. An increase of temperature of 2°C in the run resulted in an important diffusion of the very high mol. wt trains of spots above 80 kDa (data not shown). Finally, the anodic basic area of our 2-D PAGE map is clear enough, as opposed to an abundance of spots below 20 kDa reported by others in the basic pH range and supposed to be seminal plasma degradation products (Naaby-Hansen et al., 1997).
Identification of spots detected in the 2-D map has been well documented for human serum (Hughes et al., 1992). However, relatively few proteins have been identified in the seminal plasma. Some proteins which are present in the serum such as albumin, transferrin, lactoferrin and 1-antitrypsin have also been characterized in seminal plasma (Edwards et al., 1981; present study). In the current study, mass spectrometry analysis of tryptic digests was carried out and allowed us to identify two major proteins of the seminal plasma: PAP and PSA. Thus such an approach can be considered as a new clue for seminal plasma proteome analysis.
Identification of plasma/serum protein alterations by 2-D PAGE has allowed some disease diagnosis on the basis of protein map modifications (Tissot et al., 1991). In the present study, comparison of 2-D maps of clinically normal and abnormal seminal plasma has been performed. Our data clearly show that groups of spots and individual spots which were present in the 2-D map of seminal plasma from fertile men were undetectable in patients undergoing vasectomy or in a man with bilateral anorchidy. The optimized solubilization of proteins, the observation that the absence of spots is not associated with an enhancement of spots of low mol. wt and the reproducibility of our results in different patient groups supported the substractive procedure comparing two different states. Proteins that disappeared are likely to be of epididymal and/or testicular origin. In the latter case, their cellular origin can be related to either the germinal or the Sertoli cells (Sharpe, 1992). 2-D PAGE analysis of conditioned medium of human seminiferous tubules has allowed detection of Sertoli cell products and proteins which originate from germ cells (McKinnell et al., 1995). Comparison of these mapped proteins and spots of interest with those detected in the present study, however, is difficult since noticeable differences in the profile were observed with different techniques of electrophoresis. Among the polypeptides listed from the 2-D PAGE analysis of the seminal plasma of men undergoing vasectomy or of a patient with a bilateral anorchidy, two groups of spots and seven individual spots were absent in the seminal plasma of patients with severe alteration of spermatogenesis identified by histological analysis of the biopsies as SCOS. These results suggest that the presence of these proteins in the seminal plasma reflect functional spermatogenesis and confirm the testicular origin of these products. Thus, these proteins could be powerful candidates as diagnostic markers for spermatogenesis impairment. However, our data do not give information on the testicular cell type responsible for their production within the seminiferous tubules. Since germ cells are absent in the testis with SCOS, it is possible that these proteins originate from this cell-type. Separation by 2-D PAGE of seminal plasma of azoospermic patients with specific arrest of spermatogenesis, identified by histological analysis of the testis, could inform us about the precise nature of germ cells able to secrete these products. Another possibility is that these proteins are produced by Sertoli cells under the control of specific germ cells. Thus it is likely that in the absence of germ cells these proteins are not expressed. Such a regulation of Sertoli cell products by germ cells has been well documented (Jégou, 1992).
In conclusion, the present study provides the first detailed mapping of human seminal plasma and demonstrates that 2-D PAGE may be useful for identification of seminal markers of secretory azoospermia. The present data have indicated several possible candidates. Further investigation will be necessary to identify these markers. The high resolving power of 2-D PAGE associated with the highly soluble proteins of seminal plasma will be key factors for loading higher amounts of proteins for sequencing.
Acknowledgements
We are grateful to Dr M.Donzeau for providing seminal plasma samples and to Mrs L.Nakash for technical assistance. Dr F.Brucker-Davis is kindly acknowledged for revising the English.
Notes
3 To whom correspondence should be addressed. E-mail: mgeribal{at}unice.fr 
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Submitted on March 13, 2001; accepted on May 22, 2001.
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K. L. Pixton, E. D. Deeks, F. M. Flesch, F. L.C. Moseley, L. Bjorndahl, P. R. Ashton, C. L.R. Barratt, and I. A. Brewis Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: Case report Hum. Reprod., June 1, 2004; 19(6): 1438 - 1447. [Abstract] [Full Text] [PDF]
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