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Testis |
Division of Reproductive Biology,3 Department of Biochemistry and Molecular Biology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
Department of Anatomy and Cell Biology,4 McGill University, Montreal, Quebec, Canada H3A 2B2
Joint Diseases Laboratory,5 Shriners Hospital for Children, Montreal, Quebec, Canada H3G 1A6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, Sertoli cells, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The most studied member of the cathepsin family in Sertoli cells is cathepsin L. Cathepsin L is detected in Sertoli cells but not in germ cells, but unlike cathepsins A and D, it is expressed in a stage-specific manner [5, 8], with maximal expression at stages VI–VII. At these stages, most if not all cathepsin L is secreted as a proenzyme [9, 10]. Of particular interest is the apparent accumulation of the protein around the heads of step 19 elongating spermatids at the time of spermiation, suggesting a role for this protein in the release of sperm from Sertoli cells [10].
Although gene knockouts exist for several of the cathepsins, a reproductive phenotype has been reported only for the furless mouse, which contains a point mutation rendering cathepsin L enzymatically inactive [11]. These mice have a 12-fold increase in seminiferous tubule atrophy and, additionally, a 32% reduction in the number of step 7 spermatids per Sertoli cell [12] in what otherwise appears to be normal seminiferous tubules. However, because these mice are fertile, we hypothesized that other members of the cathepsin family may accumulate in a similar location in Sertoli cells as cathepsin L, and complement its function. To test this hypothesis, we first utilized cDNA microarray technology to identify other members of the cathepsin gene family expressed by Sertoli cells isolated from the adult rat testis. We found that one other member of the cathepsin family, cathepsin K, was highly expressed in these cells, and other cathepsins, namely S, H, D, B, E, and C/J, were absent or expressed at very low levels.
Cathepsin K is a secreted cysteine protease that has been shown to be highly expressed by cells involved in bone resorption and remodeling [13–17], as well as by skin and ovary. In the present study, we demonstrate that cathepsin K is expressed by Sertoli cells at the same intracellular sites and at the same stages of the cycle of the seminiferous epithelium as cathepsin L. On the basis of its expression pattern and localization, we suggest that cathepsin K may play a role in the testis that is similar or complementary to that of cathepsin L.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague-Dawley rats were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN), or Charles River Laboratories (St. Constant, QC, Canada). Rats were housed in a vivarium under a 14L:10D cycle and provided water and rat chow at libitum. All procedures were performed under protocols approved by the Johns Hopkins University School of Public Health Animal Care and Use Committee, or in accordance with guidelines established by the McGill University Animal Care Committee.
Sertoli Cell Isolation
Sertoli cells were isolated following methods described previously [18] omitting the 10-min trypsin digestion. Briefly, two decapsulated testes were incubated in 0.5 mg/ml of collagenase in 1x Hanks solution (pH 7.4) at 34°C, with shaking for 15 min to eliminate the interstitial cells, and then washed three times. To separate the Sertoli and germ cells, the tubules were incubated in a mixture of enzymes (0.1% collagenase, 0.2% hyaluronidase, 0.04% DNase I, and 0.03% trypsin inhibitor in 1x Hanks pH 7.4) at 34°C, with shaking for 40 min. The Sertoli cells were pelleted by centrifugation, washed in 1x Hanks, and repelleted a total of three times. The Sertoli cells were then resuspended in 1x Hanks, subjected to hypotonic shock in a dilute Hanks solution (1:3.5; Hanks:water final dilution), and collected by centrifugation. The cells were resuspended in 1x Hanks and filtered through 53 µm nylon mesh, then washed and resuspended in F12/Dulbecco modified Eagle medium (1:1) tissue culture media. Sertoli cell number and purity were estimated by hemocytometer and light microscopy analyses as described previously [18]. In each Sertoli cell preparation, an average of 7–8 million Sertoli cells per testis was obtained, at approximately 80% purity. Germ and myoid cells comprised the contaminants.
Seminiferous Tubule Microdissection
Seminiferous tubule segments were isolated by transillumination-assisted microdissection [1]. Tubules (120 cm total) at stages I, II–III, IV–V, VI, VIIab, VIIcd, VIII, IX–XI, XII, and XIII–XIV were dissected from each of four testes (30 cm each testis), and staged tubules were pooled.
Germ Cell Isolation
Pachytene spermatocytes, round spermatids, and elongating spermatids were isolated by unit gravity sedimentation (Staput Glass Shop ProScience, Scarborough, ON, Canada), accordingly to methods described previously [19]. The purities of the isolated pachytene spermatocyte, round spermatid, and elongating spermatid preparations were approximately 90%, 90%, and 85%, respectively.
RNA Isolation and Complementary DNA Microarray Analysis
Total RNA was purified from freshly isolated pachytene spermatocytes, round and elongating spermatids, Sertoli cells, and seminiferous tubular segments, using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Total RNA (3 µg) from freshly isolated Sertoli cells was denatured at 75°C for 2 min, and then incubated at 46°C for 60 min in a 25 µl (final volume) solution containing 0.2 units of Superscript II (Invitrogen), 2 µl of CDS primer mix (Clontech, Palo Alto, CA), 10 mM dithiothreitol, 20 µM dNTPs mix (-dATP), 50 µCi of 
-32P[dATP] and single-strength, first-strand synthesis buffer (Invitrogen). Free radiolabeled nucleotides were removed from the cDNA using Sephadex G-50 spin columns (Amersham Pharmacia, Piscataway, NJ). Rat Atlas 1.2 microarrays (Clontech) were hybridized overnight at 68°C in ExpressHyb hybridization solution (Clontech). Arrays were then washed in 2x saline-sodium citrate (SSC)/1.0% sodium dodecyl sulfate (SDS) for 30 min at 65°C; 1x SSC/0.5% SDS for 30 min at 65°C; and 0.1% SSC/0.1% SDS for 30 min at 65°C. Arrays were then placed in phosphor screen cassettes (Amersham Pharmacia) for 14–18 h. Phosphorescence was detected using a Typhoon 9200 PhosphorImager (Amersham Pharmacia), and quantified with Atlas Image version 2.0 software (Clontech). A gene was considered expressed if the intensity was 150% above background intensity. RNA was analyzed from five independent Sertoli cell preparations.
Generation of a Complementary DNA for Cathepsin K
The cDNA clone for cathepsin K (781 to 1186 of the reported cathepsin K cDNA sequence, accession number NM_031560) was generated for Northern blot analyses by reverse transcriptase-polymerase chain reaction (RT-PCR). Testis total RNA (3 µg) was reverse transcribed in a 20-µl reaction at 46°C for 60 min, using 0.2 units of Superscript II (Invitrogen) and 50 ng of oligo-dT primer in single-strength first-strand synthesis buffer. PCR was performed in a reaction volume of 50 µl containing 0.5 µl of the RT reaction, single-strength buffer, 20 µM dNTPs, 1.5 mM MgCl2, 400 nM antisense primer, 400 nM sense primer, and 0.5 units AmpliTaqR DNA Polymerase (Perkin Elmer, Boston, MA). The PCR conditions were 35 cycles at 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min, and a final extension of 72°C for 3 min. The antisense primer for the PCR reaction was 5'-CAGAGGCCACAACTCTCAGAA-3', and the sense primer was 5'-GTGTCCATCGATGCAAGCTT-3'. The PCR product was cloned into p-GemT Easy Vector (Promega, Madison, WI) and sequenced for verification of the insert.
Northern Blot Analysis
Total RNA (10 µg) from each sample was fractionated on a 1% agarose/formaldehyde gel, transferred overnight to a nylon membrane (Hybond-N, Amersham Pharmacia) and UV cross-linked (UV Stratagene 1800; Stratagene, La Jolla, CA). Complementary DNA fragments (see below) were radiolabeled with (-32P)dATP using the Rad Prime DNA Labeling Kit (Invitrogen). Northern blots were hybridized overnight at 65°C with labeled cDNA probes in ExpressHyb hybridization solution (Clontech) with 10 µg/ml of sheared salmon sperm DNA. Following hybridization, blots were washed in 2x SSC/1.0% SDS for 30 min at 65°C, 1x SSC/0.5% SDS for 30 min at 65°C, and 0.1% SSC/0.1% SDS for 30 min at 65°C. Blots were then placed in phosphor screen cassettes (Amersham Pharmacia) for 12–14 h. Phosphorescence was detected using a Typhoon 9200 and ImageQuant software (Amersham Pharmacia).
Membranes were probed with labeled cDNAs for cathepsin K, clusterin [20], and cathepsin L [5], and with ribosomal protein S2 (ChoB) as a control for RNA loading [21]. Northern blot analysis of RNA from isolated Sertoli cells was repeated five times and from tubule segments or isolated germ cells, twice.
Tissue Preparation and Immunocytochemistry for Cathepsin K
Four rats were anesthetized with sodium pentobarbital (Somnitol; MTC Pharmaceuticals, Hamilton, ON, Canada). The testes were fixed with Bouins fixative by retrograde perfusion through the abdominal aorta for 10 min. After perfusion, the testes were removed, cut in half, and placed in Bouins for 72 h. Thereafter, the tissue was washed for several days in 70% alcohol, dehydrated, and embedded in paraffin using standard protocols.
A polyclonal rabbit anti-rat cathepsin K antibody was used at a dilution of 1:200 in PBS pH 7.4. The antibody was generated against the peptide CGGITNLASFPKM, representing the eleven C-terminal residues of rat cathepsin K, plus an N-terminal cysteine for coupling and a glycine spacer residue, and was prepared by solid phase synthesis using Fmoc chemistry. An ovalbumin-peptide conjugate was prepared with the bifunctional reagent, N-hydroxysuccinimidylbromoacetate, and used for rabbit immunization. Conjugation, immunization, and characterization of the resulting antiserum were carried out as described previously [22, 23]. Affinity-purified immunoglobulin was prepared using a Sulfolink column (Pierce, Rockford, IL) substituted with the above peptide. The antibody specifically stains osteoclasts in decalcified bone sections [24]. Five-micrometer-thick paraffin sections of the testis were deparaffined in Histoclear and hydrated in a series of graded ethanol solutions. During hydration, residual picric acid was neutralized in 70% ethanol containing 1% lithium carbonate, and endogenous peroxidase activity was quenched in 70% ethanol containing 1% H2O2. The tissue was then washed in distilled H2O containing 300 mM glycine to block free aldehyde groups. Immunoreactions were enhanced with the avidin-biotin complex method, applying the Elite Vectastain ABC alkaline phosphatase kit (Vector Laboratories). Following hydration of the tissue, the slides were incubated for 20 min with a blocking agent at room temperature in order to prevent nonspecific binding of primary antibody. This was followed by incubation with the primary antibody for 30 min, diluted secondary antibody for 30 min, Elite Vectastain ABC reagent for 30 min, and peroxidase substrate: 0.05% 3,3-diaminobenzidine tetrahydrochloride with 0.03% hydrogen peroxide in tris-buffered saline (TBS). After thorough washing with distilled H2O, the sections were counterstained with 0.1% methylene blue, dehydrated in ethanol and Histoclear, and mounted with coverslips using Permount. Negative controls consisted of incubating slides in PBS instead of primary antibody and using preimmune serum at a dilution of 1:200 in PBS.
Cathepsin L Localization
Three rats were anesthetized and their testes were fixed by perfusion with Bouins fixative through the testicular artery. Slices of testes were embedded in polyester wax [25], cut into 12 µm sections, and placed on glass slides treated with Vectabond reagent (Vector Laboratories). Following dewaxing, the sections were incubated sequentially in 1% lithium carbonate in 70% ethanol and 300 mM glycine in water at room temperature. After water and TBS washes, sections were incubated for 20 min in 1.5% goat serum in TBS, and then overnight in 60 µg/ml of either anti-cathepsin L immunoglobulin G (IgG) or preimmune IgG. To reduce background staining, preparations were absorbed against Escherichia coli lysates. Following incubation with primary IgG, tissue sections were washed in TBS and incubated for 1 h with 15 µg biotinylated anti-rabbit IgG in 2 ml TBS plus 30 µl normal goat serum. Sections were washed again with TBS, incubated for 1 h with 2 µg Fluorescein Avidin DCS diluted to 100 µl with TBS, washed three times with TBS, and then incubated for 15 min with 1 µg propidium iodide and 25 µg DNase-free RNase in 100 µl of 20 mM Tris-HCl pH 7.4. Slides were then washed sequentially with water and TBS, and mounted in Vectashield (Vector Laboratories).
Confocal microscopy was performed with an OZ confocal laser scanning microscope (Thermo NORAN, Middleton, WI) using a green krypton-argon laser (488–568 nm) and red helium-neon laser (630 nm). Images were collected in 0.5-µm increments in a 10 µm thick z-series and saved as an RBG movie file. Collected images were then examined using a Silicon Graphic Indy computer (Silicon Graphics, Mountain View, CA) with Intervision (Thermo Noran) software.
Microscopic Analysis of Acidic Compartments
Seminiferous tubules at stages VI, VII, VIII, IX–XI, and XII were isolated by transillumination-assisted microdisection [26]. The identity of the stages was verified by examination of spermatogenic cells expressed from small segments cut from the ends of the tubules by phase-contrast microscopy. Tubules were incubated for 50 min in Lysosensor blue DND-167 (Molecular Probes, Eugene, OR) diluted 1:250 in Hams F12/DMEM. Lysosensor blue DND-167 fluoresces at pH of 4.5 to 6.0 with a pKa of 5.1. Incubations were conducted at 34°C in 5% CO2/95% air. Tubules were then washed once with 0.14 M NaCl, 10 mM HEPES pH 7.4, and individual tubules were placed between a microscope slide and glass coverslip. Sufficient medium was removed by capillary action to slightly flatten the tubule. The tubule was then immediately examined with a Nikon Eclipse E800 microscope, 20x Plan Fluor lens, DAPI excitation filter, 505 nm dichroic mirror, and a barrier filter with a band width of 515–555 nm (Nikon, Inc, Melville, NY). Images were captured for 200–300 msec at 200x magnification with a Princeton 5 MHz cooled interlined CCD camera (Princeton Instruments, Trenton, NJ). This experiment was repeated three times, each time with sections from a different rat.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Clontech Atlas 1.2 rat cDNA microarray contains cDNAs for eight cathepsin family members: cathepsins L, D, E, K, H, S, B, and C/J. Five replicates were run, probing the membranes with cDNA prepared from freshly isolated Sertoli cells [18] from five separate sets of animals. Overall, of the 1186 genes represented on the array membrane, approximately 330 of the genes were detected, including known Sertoli cell genes such as the highly expressed clusterin (Fig. 1). Genes encoding two members of the cathepsin family of proteases, cathepsins K and L, were highly expressed (Fig. 1). In contrast, the transcriptional expression of each of cathepsins D, H, S, B, E, and C/J was below the sensitivity of the microarray assay. The genes for tissue inhibitor of metaloproteases-2 (TIMP2) and -3 (TIMP3) as well as proteasome iota subunit, amino peptidase, and urokinase-type plasminogen activator (uPA) also were highly expressed.
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To corroborate the results of the microarray analyses, a cDNA clone of cathepsin K was generated by RT-PCR and used for Northern blot analyses of RNA from freshly isolated Sertoli cells, and from Staput-purified pachytene spermatocytes, round spermatids, and elongating spermatids. The Sertoli cells contained high levels of cathepsin K and clusterin mRNAs, whereas the pachytene spermatocytes, round spermatids, and elongating spermatids did not (Fig. 2). (Note: the clusterin mRNA that was detected in the pachytene spermatocyte preparations almost certainly is due to contaminating Sertoli cells.) All the samples contained the ribosomal S2 (ChoB) mRNA, which was used to control for RNA loading.
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Stage-specific expression of cathepsin K mRNA was investigated by Northern blot analyses of RNA from seminiferous tubule segments representing stages I, II–III, IV–V, VI, VIIab, VIIcd, VIII, IX–XI, XII, and XIII–XIV. Figure 3 shows that cathepsin K mRNA was maximally expressed at stages VI–VIIab, and expressed at considerably lower levels at stages I–V and VIIcd–XIV. The expression of cathepsin L mRNA was found to be similar, though not identical, to that of cathepsin K in these preparations; expression was maximal at stages VI–VII and at lower levels at other stages. In contrast, the expressions of clusterin and ChoB were high at all stages of the cycle.
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Immunolocalization of Cathepsins K and L
Testicular sections that were treated either without primary antibody or with preimmune serum revealed an absence of reaction product throughout the entire testis, including the seminiferous epithelium and interstitial space (Fig. 4a). Leydig cells stained for cathepsin K at all stages of the cycle (Fig. 4a insert). Seminiferous tubules staining was variably depending on the stage of the cycle (Fig. 4, b–d). Staining was detectable in the cytoplasm of Sertoli cells at stages VI–VIII of the cycle (see Fig. 4, b and c, which show stages VI and VII, respectively), extending from the base of the epithelium to the lumen; but at other stages of the cycle (Stage IX, Fig. 4d for example), Sertoli cell cytoplasm was far less reactive. These results were consistent with the expression of cathepsin K mRNA at particular stages of the cycle.
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When cathepsin K protein staining was visualized at higher magnifications, it became evident that at least some reaction product was present in Sertoli cells at all stages of the cycle, appearing punctuate or granular, which is suggestive of lysosomal compartmentalization (Fig. 5, a–d, stages VI, IX, XII, and XIV, respectively). Close association of cathepsin K-reactive lysosomes with the heads of elongating spermatids embedded in the seminiferous epithelium was noted (Fig. 5, c and d). Cathepsin K protein staining also was associated with residual bodies within Sertoli cells (Figs. 4d and 5b).
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To compare the distribution cathepsin L protein with that of cathepsin K, cathepsin L was localized in rat testis sections by confocal microscopy. No immunostaining was observed when anti-cathepsin L IgG was replaced with preimmune IgG (not shown). Similar to cathepsin K, cathepsin L staining was present throughout Sertoli cells in tubules at stages VI and VII (Fig. 6). Cathepsin L staining was also present around the heads of a subset of elongating spermatids (Fig. 6, stage VI), and was associated with residual bodies once they were released from spermatids (Fig. 6, late stage VIII). Cathepsin L staining remained associated with late residual bodies until their dissolution within the Sertoli cell cytoplasm (Fig 6, stage X). These results indicate that the distribution of cathepsins L and K in the seminiferous epithelium show a great deal of overlap.
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Localization of Acidic Microenvironments Within Seminiferous Tubules
Both cathepsins K and L are secreted proteases synthesized as proenzymes, and both require a pH below 6.0 for autoprocessing and induction of enzymatic activity [5, 13, 14, 27]. Therefore, if cathepsin K and L participate in the degradation of residual bodies, their pH should be lower than 6.0. To determine whether the pH of residual bodies was below 6.0, seminiferous tubules at specific stages of the cycle were incubated with the pH sensitive fluorescent dye, Lysosensor-Blue (pKi 5.5), to localize regions of acidified microenvironments (Fig. 7). In stage VI, but more so in stages VII–VIII tubules, this dye detected small acidic spheres, which were assumed to be lysosomes and shown by electron microscopy analysis to be prominent in the Sertoli cell cytoplasm at stages VII–VIII [28]. However, at stages IX–XII, the pH-sensitive dye detected large, spherical structures that were the appropriate size for late residual bodies in the basal compartment of Sertoli cells. These results are consistent with the theory that cathepsins K and L undergo autoprocessing within residual bodies and subsequently participate in the dissolution of these structures.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition, early residual bodies also were reactive for cathepsin K, suggesting that cathepsin K might become associated with residual bodies during their phagocytosis by Sertoli cells. Cathepsin K also was present in granules that are associated with the heads of late spermatids embedded in the Sertoli cells at stages I–VI, as well as with granules observed at the base of Sertoli cells at stages VII–VIII and XIII–XIV. Although electron microscopic analyses were not performed, it is likely that the cathepsin K-containing granules are lysosomes; lysosomes have been shown to be present at the base of Sertoli cells and next to elongating spermatids by routine electron microscopy and by electron microscope immunocytochemistry, using various lysosomal markers [4, 6, 28, 30, 31]. Thus, Sertoli cells, in addition to secreting cathepsin K at stages VI–VIII, appear to target cathepsin K from the Golgi apparatus to lysosomes, as has been seen for other Sertoli cell lysosomal enzymes, including SGP-1 and beta-hexosaminidase [29, 30].
Cathepsin L also was found to accumulate around the heads of spermatids and to be associated with residual bodies prior to their engulfment by the Sertoli cells [32], and thus its distribution in Sertoli cells closely paralleled that of cathepsin K. The association of both cathepsins K and L with the early residual bodies prior to their acidification suggest that the two proteases become associated with early residual bodies prior to their phagocytosis by Sertoli cells. Whether or not cathepsins K and L are associated together in the same residual bodies is not known.
Sertoli cells have been shown to be active in endocytosis and phagocytosis [33]. Endocytosis involves the uptake of substances from the lumen by coated pits, and the eventual degradation of these substances by lysosomes. In Sertoli cells, as in epithelial cells of the epididymis, it has been suggested that endocytosis leads to the formation of lysosomes, and that this occurs at most stages of the cycle [34]. However, at stages IX–XII, Sertoli cell lysosomes fuse with the phagocytosed residual bodies for purposes of degrading them, and at these stages a reduction in the number of lysosomes has been documented [34]. Our data suggest that cathepsins K and L are located in lysosmes in the base of Sertoli cells. The delivery of these proteins to late residual bodies would promote their degradation, as is the case for other lysosomal enzymes [4, 30].
The lack of lysosomes in the apical regions of Sertoli cells argues that these structures may not deliver cathepsins K and L to the early residual bodies. However, Sertoli cells also are involved in the secretion of products destined for the lumen [35], and this typically is visualized by light microscope immunocytochemistry as intense staining of the entire cytoplasm [29]. Although particular secretory vesicles of Sertoli cells have yet to be identified [28, 36], it was noted in the present study that at stages VI–VIII, anti-cathepsin K antibody intensely stained the entire Sertoli cell cytoplasm, which is typical of a secretory staining pattern. Previous studies have shown that cathepsin L is secreted by Sertoli cells at these same stages [9]. Thus, cathepsin K, while being targeted from the Sertoli cell Golgi apparatus to lysosomes, may also be secreted into the lumen of the seminiferous tubule. The rationale for this hypothesis is that cathepsin K has been demonstrated to be a secretory product of bone osteoclasts [13, 14, 16]. Such a dual function has been already documented for prosaposin (SGP-1), which exists in both lysosomal and secreted forms in Sertoli cells [37]. Thus, the cathepsin L and cathepsin K localized to early residual bodies may have been secreted by Sertoli cells. Cathepsin K and cathepsin L may functionally complement each other in the seminiferous epithelium.
The similar stage-specific expression and localization of cathepsins K and L in the seminiferous epithelium suggest that these two proteins may have overlapping functions. This may explain the testicular phenotype of furless mice, which express an enzymatically inactive cathepsin L. Although these mice exhibit an increased incidence of seminiferous tubule atrophy and reduced germ cell numbers in what are otherwise morphologically normal tubules, they are nonetheless fertile [12]. Mice that lack cathepsin K are also fertile, though their testicular phenotype has yet to be evaluated [38]. However, our data raise the possibility that mice lacking both cathepsin K and cathepsin L would degrade residual bodies less efficiently than wild-type mice or mice lacking either, but not both, of the two enzymes. We also suggest that the lack of both of these enzymes seems likely to produce a more severe testicular phenotype than has been reported for mice that lack functional cathepsin L only. This has yet to be tested.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Matthew Anway, Washington State University, School of Molecular Biosciences, Pullman WA 99164-4660. FAX: 509 335 2176; manway{at}wsu.edu
Received: 12 April 2003.
First decision: 9 May 2003.
Accepted: 8 October 2003.
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