Molecular Human Reproduction, Vol. 8, No. 7, 606-611,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Prolactin receptor expression in human testis and accessory tissues: localization and function
MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK
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Abstract |
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Experimental studies in animals have established prolactin (PRL) as a progonadal hormone that promotes the function of the testis and reproductive accessory glands. The present study investigated the localization of PRL receptor (PRL-R) expression in the human testis and accessory tissues. Expression of PRL-R was identified in human testis and vas deferens by RT–PCR, and further localized by immunohistochemistry to the Leydig cells and differentiating germ cells of the testis (developmental stages extending from pachytene spermatocytes to elongating spermatids). Positive staining for PRL-R was also clearly evident in the epithelium of vas deferens, epididymis, prostate and seminal vesicles. Functional activation of PRL-R was demonstrated in fresh samples of vas deferens collected at vasectomy by examination of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAP (mitogen-activated protein) kinase ERK (extracellular signal-regulated kinase) signalling pathways. Within the vas deferens, PRL induced rapid tyrosine phosphorylation of JAK 2 and STAT 5 (after 10 and 20 min respectively), and tyrosine and threonine phosphorylation of ERK 1 and 2 (after 5 min). The demonstration of function and localization of PRL-R presented here suggests multiple roles for PRL in the human male reproductive tract.
male reproductive tract/prolactin/prolactin receptor/testis
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Introduction |
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Experimental studies in animals have established that prolactin acts in concert with the classical gonadotrophins, LH and FSH, to stimulate full testicular function in the adult male rat, mouse and hamster (Bartke, 1971
; Bartke et al., 1975; Zipf et al., 1978; Dombrowicz et al., 1992). In the ram, a functional role for prolactin (PRL) in the testis is indicated by the observation that hypothalamo-pituitary disconnected rams, that permanently lack gonadotrophin due to a blockade of GnRH secretion, continue to express cycles in testicular size in response to photoperiod-induced changes in PRL secretion. These gonadal changes are minor and occur with a long latency, but they indicate that PRL is a weak gonadotrophin in the absence of LH and FSH (Lincoln et al., 1996).
Prolactin receptor (PRL-R) gene expression in the testis has been demonstrated in different species including rat, ram and red deer (Ouhtit et al., 1993; Hondo et al., 1995; Jabbour et al., 1998a; Jabbour and Lincoln, 1999). These studies show PRL-R to be localized to the Leydig cells in the interstitium and germ cells within the seminiferous tubules (Jabbour and Lincoln, 1999). The addition of PRL to testicular explants of ruminant species induces phosphorylation of JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signalling proteins, consistent with a functional PRL-R in the testis (Jabbour et al., 1998b). PRL is believed to stimulate testicular steroidogenesis by regulating LH receptors (Bex and Bartke, 1977; Takase et al., 1990), or androgen/estrogen biosynthesis through the control of rate-limiting enzymes in the Leydig cells (Takeyama et al., 1986; Chandrashekar and Bartke, 1988). However, the mechanism of action of PRL on spermatogenesis remains to be clarified.
Expression of PRL-R has also been demonstrated in the rat dorsal and lateral prostate, and seminal vesicles (Ouhtit et al., 1993; Nevalainen et al., 1996). In cultured prostatic cells, androgens and estrogens stimulate the expression of PRL-R (Nevalainen et al., 1996), and both gonadal steroids and PRL, induce the secretion of prostate-specific proteins (Costello and Franklin, 1994). In transgenic mice engineered to over-express the PRL gene, the prostate gland becomes grossly enlarged illustrating the importance of PRL in the control of accessory gland function (Wennbo et al., 1997). Chronic suppression of blood concentrations of PRL secretion in the ram produces a decrease in size and fructose content of the seminal vesicles, with no change in testosterone secretion (Ravault et al., 1977), and manipulations of PRL and androgens in the macaque monkey affect seminal vesicular enzymes (Arunakaran et al., 1988); thus, PRL may promote the function of various androgen-dependant male accessory structures.
Clinical observations also support a role for PRL in the regulation of the testis and accessory glands in man. For example, the restoration of normal PRL levels in a cohort of subfertile, hypoprolactinaemic men caused an increase in sperm density and quality, and restored fertility (Ufearoet al., 1995). In another study, suppression of gonadotrophins and PRL secretion in eugonadal men treated for prostatic carcinoma caused a more marked reduction in testicular weight and spermatogenesis than suppression of gonadotrophin secretion alone (Huhtaniemi et al., 1991). Both observations are consistent with a progonadal role of PRL in the testis, although early studies using I125-iodo PRL failed to demonstrate the presence of PRL binding in the human testis, in contrast to the situation in the rat (Wahlstrom et al., 1983). PRL binding has been demonstrated in the human prostate (Leake et al., 1983), and other studies suggest that PRL may play a role in the aetiology of benign prostatic hyperplasia and cancer (Kadar et al., 1988; Nevalainen et al., 1997). The synchronous reduction in both PRL and androgen improves the efficacy of the treatment of prostatic carcinoma (Rana et al., 1995).
The purpose of the present study was to provide direct evidence for a role of PRL in the regulation of the testis and reproductive tract in man. To this end, the expression of the PRL-R gene was investigated by RT–PCR using RNA extracted from human testis and vas deferens. The localization of expression of the PRL-R protein was further studied using immunohistochemistry in sections prepared from the human testis, epididymis, vas deferens, prostate and seminal vesicles. Lastly, a functional PRL-R was identified in the vas deferens by investigating activation of the JAK/STAT and MAP (mitogen activated protein) kinase ERK (extracellular signal-regulated kinase) specific intracellular signalling pathways. Activation of JAK/STAT and ERK proteins, following binding of PRL to its receptor, mediates both proliferative and differentiating effects in target cells (Findiori and Kelly, 1995; Lewis et al., 1998).
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Subjects and tissues
Testicular tissue (n = 6), exhibiting normal morphology, was obtained by biopsy from men exhibiting unexplained infertility. Vas deferens tissue (n = 10) was obtained from normal men undergoing vasectomy. Left and right vas deferens were used for comparison between treatment and control. Ethical approval was obtained from Lothian Paediatric and Reproductive Medicine Research Ethics Subcommittee, and written informed consent was obtained from each subject. Prostate and bladder tissues were obtained from archival surgical re-section specimens stored by the Department of Pathology, Western General Hospital, Edinburgh. Epididymis and seminal vesicles were obtained from a commercially available human cadaver tissue library.
Tissue culture
Following collection, vas deferens tissue was washed in phosphate-buffered saline (PBS) twice and subsequently minced thoroughly with fine scissors. Four aliquots of each tissue (~0.17 g each) were then incubated overnight in 2 ml serum-free RPMI 1640 medium (Sigma Chemical Co., Dorset, UK) containing 100 IU/ml penicillin and 100 mg/ml streptomycin in a 37°C incubator with 5% CO2:95% air. The following day, samples were treated with 100 ng/ml human PRL (hPRL-SIAFP-B2; donated by NIDDK, NIH) for 0, 5, 10 and 20 min. The tissue was stored at –70°C prior to analysis by immunoprecipitation and/or Western blotting.
RNA extraction and RT–PCR
Total RNA was extracted from testis and vas deferens using the guanidinium thiocyanate method as previously described (Chomczynski and Sacchi, 1987). Polyadenylated RNA (poly A+) was purified on oligo(deoxythymidine)-cellulose affinity columns (Pharmacia Biotech, St Albans, Herts, UK); the yield and purity of RNA was estimated by spectrophotometry. Single strand cDNA was generated from 5 µg poly A+ RNA by reverse transcription using 1.6 ng oligo (deoxythymidine)12–18 primer and Superscript reverse transcriptase, according to the manufacturer's instructions (Gibco BRL, Paisley, UK). cDNA (10 ng) was then diluted 25 times in double-distilled H2O and amplified by PCR using primers corresponding to nucleotide positions 182–201 (5'-CACCTCCTGAAAAACCCAAG-3'; forward primer) and 724–743 (5'-CCATGGTCTGGCTTGCAGCG-3'; reverse primer) of the PRL-R open reading frame. The reaction was carried out in PCR buffer (50 mmol/l KCl, 2 mmol/l MgCl2 and 20 mmol/l Tris–HCl, pH 8.3), 200 µmol/l deoxy-NTPs, 25 pmol forward and reverse primers and 1 IU Taq polymerase (Perkin-Elmer, Warrington, Cheshire, UK) in a total volume of 50 µl. Samples were subjected to 35 cycles of 94°C for 40 s, 52°C for 75 s and 72°C for 150 s. After a 10 min final extension at 72°C, the products were visualized on a 1% agarose gel using ethidium bromide staining.
Histology and immunohistochemistry
Testis, vas deferens and other tissues were placed immediately in Bouin's fixative for 6 h before transfer to 70% ethanol, and subsequent dehydration and embedding in wax blocks. Sections were cut and mounted on slides coated with 2% 3-aminoproyltriethoxysilane (TESPA) in acetone. Slides were then dried overnight at 50°C before dewaxing in histoclear (National Diagnostics, Hull, UK). Tissues were rehydrated in graded ethanol and washed in water followed by Tris-buffered saline (TBS; 0.05 mol/l Tris–HCl, pH 7.4, 0.85% NaCl). Sections were treated with 10% hydrogen peroxide in methanol for 30 min and blocked for 30 min with normal swine serum (NSS) diluted 1:5 in TBS with 5% bovine serum albumin (BSA).
The primary antibody for PRL-R (kindly donated by Dr P.M.Ingleton, School of Medicine, University of Sheffield) was raised against a 16 amino acid synthetic peptide corresponding to residues 53–68 of the external domain of the rat PRL-R (Nevalainen et al., 1996). The polyclonal antibody was validated for use in human tissue based on the manner that pre-absorption of the antibody with the corresponding synthetic peptide totally blocked the immunostaining (Nevalainen et al., 1999). The polyclonal antibody was diluted in NSS/TBS/5% BSA (as above) and incubated overnight at 4°C. Control sections were incubated with non-immune rabbit serum. All sections were washed twice in TBS (5 min each), incubated for 30 min with biotinylated swine anti-rabbit Ig (Dako, Bucks, UK), diluted 1:500 in NSS/TBS. Sections were washed again twice in TBS (5 min each) and incubated with peroxidase conjugated to avidin–biotin complex (Dako) for 30 min at room temperature. Colour reaction was developed by incubation in a mixture of 0.05% 3,3'-diaminobenzidine (DAB; Sigma) in 10 ml 0.05 mol/l Tris–HCl buffer (pH 7.4) and 0.033% hydrogen peroxide. Sections were subsequently counterstained using haematoxylin.
Immunoprecipitation and Western blotting
Tissue was homogenized and lysed in 150 mmol/l NaCl, 10 mmol/l Tris (pH 7.4), 1 mmol/l EDTA, 10% glycerol, 0.6% NP40, 10 µg/ml aprotonin, 1 mmol/l phenylmethylsulphonyl fluoride and 1 mmol/l sodium orthovanadate. Cytoplasmic extracts were prepared by centrifugation for 2 min at 10 000 g. For analysis of phosphorylation of JAK 2 and STAT 5, 50 µg of protein was incubated with 10 µl monoclonal anti-phosphotyrosine antibody (5 mg/ml; Affiniti, Exeter, UK) for 1 h at 4°C. Samples were then incubated overnight at 4°C with M-450 Dynabeads conjugated to rat anti-mouse IgG2b (Dynal, Wirral, UK). The complexes were washed three times in PBS using a Dynal MPC magnet (Dynal), and boiled for 5 min in sample buffer [125 mmol/l Tris–HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 2.5% dithiothreiotol, 20% glycerol, 0.05% Bromophenol Blue].
For Western blotting, samples were subjected to SDS–PAGE and then transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). For analysis of ERK phosphorylation, 50 µg of protein from each sample was analysed. Membranes were incubated with antibodies against ERK 
, JAK 2, STAT 5 (Santa Cruz Biotechnology, Santa Cruz CA, USA) or phosphorylated ERK (T202/Y204, Cell Signaling, New England Biolabs, Beverly, MA, USA), each diluted 1000-fold in 2% dried skimmed milk/TBST (20 mmol/l Tris–HCl pH 7.4, 500 mmol/l NaCl, 0.1% Tween 20). Membranes were washed briefly in TBST and incubated with secondary antibodies conjugated to horse radish peroxidase (Amersham, Buckinghamshire, UK), in 2% milk/TBST. Membranes were again washed in TBST and proteins detected using the ECL+ detection kit (Amersham).
Statistical analysis
The degree of phosphorylation of proteins in response to PRL was measured at four time points by densitometry, and the significance of the changes was analysed by analysis of variance (ANOVA) with repeated measures.
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RT–PCR of PRL-R mRNA
The expression of PRL-R was analysed initially by RT–PCR, using RNA extracted from testis and vas deferens. A single 312 bp transcript, corresponding to a region in the extracellular domain of the human PRL-R mRNA, was amplified in all samples of both testis and vas deferens tissue, indicative of the presence of PRL-R mRNA in these tissues (Figure 1
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Immunohistochemistry of PRL-R
To determine the localization of PRL-R, immunohistochemistry was performed using sections prepared from human testis, vas deferens, epididymis, prostate, seminal vesicle and bladder. In the testis biopsies of all six subjects, weak but consistent immunostaining was localized to the Leydig cells in the interstitial tissue and stronger staining localized to the germ cells at different stages of spermatogenesis in the seminiferous tubules. Representative micrographs are shown in Figure 2a,c
. The most marked staining was evident in the cytoplasm of the germ cells in the developmental stages extending from pachytene spermatocytes to elongating spermatids. The spermatogonia, early dividing spermatocytes and final differentiating sperm were devoid of staining, as were the Sertoli cells (Figure 2c). Control sections of testis were totally devoid of staining (Figure 2b). In vas deferens collected from 10 different subjects, and in epididymis and seminal vesicle samples collected from three other subjects, immunostaining for PRL-R was consistently evident in the epithelium, with minimal staining in the underlying stroma and muscle tissue (Figure 2d,f,g); control sections of vas deferens were again unstained (Figure 2e). Immunostaining for PRL-R was also consistently present in the epithelium of the prostate in three subjects (data not shown; demonstrated previously) (Nevalainen et al., 1997).
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Intracellular signalling in the vas deferens
Intracellular signalling pathways were examined by investigating tyrosine phosphorylation of JAK 2 and STAT 5 and tyrosine and threonine phosphorylation of ERK 1 and 2, following stimulation of fresh vas deferens tissue with human PRL (Figure 3
). This was repeated using fresh tissue from three different subjects attending for vasectomy. Tyrosine phosphorylation of JAK 2 and STAT 5 was measured by an immunoprecipitation procedure using an antibody against phosphorylated tyrosine, followed by Western blotting using antibodies against JAK 2 and STAT 5. Phosphorylation of ERK 1 and 2 were measured by Western blotting using an antibody raised against ERK, phosphorylated on specific tyrosine and threonine residues, and the density of the signal was measured by densitometry. Rapid phosphorylation, with a significant increase in signal intensity (P < 0.05, ANOVA), was observed following treatment with PRL for ERK 1 and 2 (evident from 5 min), JAK 2 (10 min) and STAT 5 (20 min) (Figure 3). No increase in phosphorylated proteins was observed in the absence of PRL.
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Discussion |
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The data presented here demonstrate that PRL-R is expressed in the human testis and in various male accessory glands. This is based on RT–PCR amplification of the receptor mRNA extracted from testis and vas deferens, and the localization of the receptor protein by immunocytochemistry in a range of reproductive tissues. Furthermore, incubation studies with fresh tissues of human vas deferens show that the addition of PRL activates phosphorylation of several proteins known to mediate the intracellular effects of PRL in target cells. These data provide clear evidence for functional activation of PRL-R in a clinically accessible human male reproductive tissue.
Various isoforms of PRL-R, differing in the intracellular domain, have been characterized in man, and these are differentially expressed in the many PRL target organs (Kline et al., 1999; Hu et al., 2001). Long forms are expressed in the testis, and long and intermediate forms in the prostate. In the human testis, the abundance of PRL-R mRNA is low compared with many tissues (Kline et al., 1999), and there is evidence that a consistent portion of PRL-R present in the testis is in a truncated form which may block, or alter, the function of the long form of the receptor (Hu et al., 2001). The methodology used in the current study was directed at the extracellular domain of PRL-R (cDNA primers for RT–PCR, and antibody for immunocytochemistry). Thus, it was not possible to confirm the expression of the different isoforms of PRL-R in the human testis and other tissues. In the rat, different isoforms of PRL-R have been identified, and both are expressed in the testis and accessory glands (Ouhtit et al., 1993). Hence, it is likely that a similar diversity of PRL-R expression exists in human testis and accessory glands; this may permit tissue-specific responses to PRL within the male reproductive tract.
The current immunohistochemical studies localized for the first time the PRL-R protein to the Leydig cells and germ cells of the human testis. This is contrary to the results of an earlier study which failed to detect PRL-R in human testicular tissue using a radioreceptor method, although the receptor was readily demonstrated in the rat (Wahlstrom et al., 1983). This was despite the use of fresh human testicular tissue, and a comprehensive study of the binding characteristics of human PRL in testis homogenates. The most likely explanation for the discordance is that PRL-R is expressed at low abundance in the human testis, and it requires a sensitive immunohistochemical or molecular amplification method to resolve the localization or gene expression in the tissue. The current study also confirmed PRL-R expression in the secretory, adsorptive or lining epithelium in the various accessory male structures. The pattern of immunostaining in the testis is similar to that described in the red deer and ram (Jabbour et al., 1998a; Jabbour and Lincoln, 1999). Expression of the receptor in Leydig cells is consistent with a role of PRL in the control of testicular steroidogenesis. Evidence in rodent species suggests that PRL influences testosterone secretion through the stimulation and maintenance of expression of LH receptors in Leydig cells (Klemcke et al., 1984; Takase et al., 1990) or by regulating specific enzymatic steps in androgen biosynthesis (Chandrashekar and Bartke, 1988). The effect of PRL on steroidogenesis in the adrenal gland is also well documented in animals and man (Glasow et al., 1996).
The presence of PRL-R in the differentiating germ cells in the testis is consistent with the view that PRL acts directly within the seminiferous tubules to affect spermatogenesis, as well as acting indirectly through gonadal steroid secretion. In the immature hypophysectomized rat, treatment with PRL stimulated an increase in the number of primary spermatocytes (Dombrowicz et al., 1992), while in hypoprolactinaemic subfertile men, exogenous PRL, or treatment with metoclopramide to promote PRL secretion, increased sperm density, reduced sperm abnormalities and acted to restore fertility (Uferio et al., 1995). Other studies in the ram indicate that the effects of PRL in the testis are largely dependent on the concurrent secretion of LH and FSH (Lincoln et al., 2001). Taken together, the data indicate that PRL acts within the testis to facilitate and augment the actions of LH and FSH in promoting full testicular activity.
The universal distribution of PRL-R in the epithelium of the epididymis, vas deferens, seminal vesicles and prostate confirms that PRL also plays a role in the regulation of accessory gland function in man. Animal studies clearly demonstrate that PRL acts in association with androgens to stimulate the activity of the accessory glands. For example, the production of secreted proteins from cultured rat prostate cells is markedly enhanced by addition of PRL (Nevalainen et al., 1996). In transgenic rodent models, over-expression of PRL, or its receptor, is associated with enlargement of the accessory glands, while under-expression is associated with reduced functional activity (Wennbo et al., 1997). The clinical observation that suppression of PRL enhances the effectiveness of steroid withdrawal for the treatment of malignant disease of the prostate (Rana et al., 1995 ) also supports an action for PRL in the human accessory glands. The presence of PRL-R in the epithelial cells in the tissues of the male reproductive tract suggests that PRL is potentially involved in the regulation of secretion, absorption, and/or the control of transport of fluids across the cell membrane. This is consistent with a conserved function of PRL in epithelial tissues across species (Nicoll, 1974).
As well as demonstrating functional PRL-R within the vas deferens, the current results support the view that PRL signals through more than one intracellular pathway to regulate the tissue responses. In the vas deferens, PRL activated rapid tyrosine phosphorylation of JAK 2 and STAT 5 protein. The temporal sequence is similar to that observed in other PRL target-tissues including rat mammary gland (Jahn et al., 1997) and ovary (Ruff et al., 1996), ram testis (Jabbour and Lincoln, 1999) and human endometrium (Jabbour et al., 1998b). Upon activation, STAT proteins dimerize, translocate to the nucleus and bind to STAT-regulatory elements in the promoters of target genes to influence transcription (Schindler and Darnell, 1995). In the mammary gland, activation of STAT 1 and STAT 5 proteins up-regulate transcription of genes for ß-casein (Gouilleux et al., 1994), ß-lactoglobulin (Burdon et al., 1994) and whey acidic protein (Li and Rosen, 1995) to mediate the effects of PRL on milk synthesis and secretion. The target genes for PRL in the vas deferens epithelium are still unknown, but are likely to include genes encoding secretory proteins.
The demonstration of rapid phosphorylation of ERK proteins in response to PRL in human vas deferens indicates that PRL also acts via the ERK pathway to affect the function of the secretory epithelium. The involvement of both the JAK/STAT and ERK pathways acting together to influence differentiation has been documented in other cell types (Lewis et al., 1998). In the human endometrium, PRL induces the JAK/STAT and ERK pathways in the epithelial cells during the secretory phase of the menstrual cycle (Jabbour et al., 1998b; Gubbay et al., 2001). The activation of ERK by PRL in terminally differentiated secretory epithelium, as in the human vas deferens, indicates that PRL signals through ERK, as well as through the JAK/STAT pathway, to provide divergent control of more than one cellular response. In the current study, only fresh tissue from human vas deferens was available for tissue culture, thus it was not possible to establish the pattern of phosphorylation of the second messenger proteins in the different male reproductive tissues.
In conclusion, this study provides clear support for the view that PRL is a regulator of reproductive function in the human male. PRL appears to act in the Leydig cells of the testis to promote steroidogenesis, and in the germ cells of the seminiferous tubules of the testis to promote the efficiency of spermatogenesis, actions that augment the more dominant progonadal effects of LH and FSH. PRL also acts in the epithelia of the efferent ducts and the male accessory sex glands, in conjunction with the gonadal steroid hormones, to regulate the secretory/adsorptive functions of these male tissues. Our results are consistent with the pleiotrophic character of PRL, as a hormone with multiple target tissues signalling through multiple pathways. A role for PRL within the male reproductive tract, suggests that abnormalities in circulating levels of PRL within the body, or abnormalities in PRL-R function, may have a significant impact on male fertility.
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We wish to thank Sheila Boddy for expert technical assistance with the molecular studies, Dr Alastair Campbell for assistance with the collection of human testicular tissue, Mr Ian Wallace for resectioned vas deferens specimens, Dr Pamela Ingleton for providing R120 anti-prolactin receptor antibody, Dr Stewart Irvine for assistance in obtaining clinical materials and ethical approval, and Mike Miller and Sheila McPherson for expert assistance with the immunohistochemical studies. Purified preparations of human prolactin were generously provided by joint arrangement of NHPP, NIDDK, NICHHD and the US Department of Agriculture.
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1 To whom correspondence should be addressed. E-mail: g.lincoln{at}hrsu.mrc.ac.uk
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Submitted on December 5, 2001; accepted on April 11, 2002.
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