1Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso; 2Instituto de Bioquímica, Universidad Austral de Chile, Valdivia, Chile; and 3Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 16 October 2003 ; accepted in final form 5 November 2004
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ABSTRACT |
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cell differentiation; seminiferous tubules; spermatogenesis; testicle; meiosis
In this study, we have explored the hypothesis that both high- and low-affinity isoforms of L-lactate transporters are expressed and functional in spermatogenic cells and that MCT isoform expression changes during spermatogenesis.
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MATERIALS AND METHODS |
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Rat spermatogenic cell populations were prepared from the testicles of adult Sprague-Dawley rats (5070 days old) as described by Romrell et al. (36). Rats were housed with free access to food and water using a 12:12-h light-dark cycle. The animals were lightly narcotized by exposure to CO2 for 45 s and then killed by cervical dislocation. A 95% O2-5% CO2 atmosphere was maintained throughout the tissue enzymatic digestion procedure. The pachytene spermatocyte (85 ± 5% purity) and round spermatid fractions (92 ± 4% purity) were identified by their size as well as by the typical aspect of the nucleus stained with Hoechst H33342 [GenBank] (Molecular Probes, Eugene, OR) (31). The round spermatid fraction contained cells between stages 1 and 7. Our method of vital cell identification did not allow a further classification of rat spermatids at these stages of development. The isolated cells were used within 4 h after the purification procedures. All animal experimentation was conducted in accordance with the American Physiological Societys "Guiding Principles for Research Involving Animals and Humans," and all animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of each university.
RNA Purification
Total RNA was obtained from 1 million purified spermatogenic cells using a Stratagene Absolutely RNA Miniprep RT-PCR kit (Stratagene, La Jolla, CA). After extraction, the RNA was quantified using a UV spectrometer to monitor the absorbance ratio at 260/280 nm. The integrity of the RNA was checked by running the sample in 1% agarose-formaldehyde gels. Samples were maintained at 20°C or 80°C for long-term storage.
One-Step RT-PCR
The RT-PCR reaction was performed using a Stratagene one-step RT-PCR kit. A standard protocol with a 0.6 µM concentration of each primer, 2 µg of template RNA, and the reagent master mix was used to amplify the fragments during the temperature cycles. Control reactions without the RT cycle were performed. To detect the rat MCT mRNA, we used the following set of primers: 1) MCT1 sense (5'-GTCTACGACCTATGTTGGG-3') and antisense (5'-CCTCCGCTTTCTGTTCT-3') to obtain a 320-bp product, 2) MCT2 sense (5'-GGGGCTGGGTTGTAGT-3') and antisense (5'-GACGGTGAGGTAAAGTTCTA-3') to obtain a 296-bp product, 3) MCT3 sense (5'-CGCTGCTCTAAGAACATCTCATC-3') and antisense (5'-TCTGGCCTCGTGCCTCAT-3') to obtain a 238-bp product, and 4) MCT4 sense (5'-GGCAGTCCCGTGTTCCTTT-3') and antisense (5'-GCACCTTCTTGAGCCCTGTTAT-3') to obtain a 421-bp product. Primers were obtained from Life Technologies (custom primers; GIBCO-BRL, Grand Island, NY). cDNA products were separated on a 2% agarose gel, and images were acquired using a Stratagene Eagle Eye II imaging system.
MCT Sequences
All of the MCT sequences were obtained from GenBank. Accession numbers were D63834 (MCT1), X97445 (MCT2), AF059258 (MCT3), and U87627 (MCT4) (12).
cDNA Extraction and Sequencing
PCR products were extracted from the agarose gel using a QIAquick gel extraction kit (Qiagen, Santa Clarita, CA). After extraction, the PCR products were cloned into a TOPO-TA cloning vector (Invitrogen, Carlsbad, CA) according to the manufacturers instructions. Transformed cells were grown overnight at 37°C, and positive colonies were subcultured in 3 ml of Luria-Bertani medium containing ampicillin. Plasmids were purified using a standard alkaline lysis/polyethylene glycol precipitating protocol and analyzed in a 2% agarose gel after EcoRI digestion. Plasmids containing the MCT sequences were sent to the Iowa State University DNA sequencing facility for sequencing.
Preparation of RNA Probes
Probes for in situ hybridization were made with the same sense primers used for RT-PCR. Digoxigenin-UTP-linked probes anti-MCT1 and anti-MCT2 mRNA were prepared using a master mix containing sense primer (35 pmol/µl), digoxigenin-11-2'-deoxyuridine-5'-triphosphate, dATP (9 mM), CoCl2 (5 mM), and terminal deoxynucleotidyl transferase (25 U) for 1 h at 37°C. The probes were stored at 20°C.
In Situ Hybridization
Cells or tissue sections (5 µm) were placed on silane-treated slides and fixed for 10 min with HistoChoice (Amresco, Solon, OH). The slides were then washed three times and left to dry at room temperature. Subsequently, acetone (100%) fixation was performed for 20 min. The slides were stored at 70°C for up to 1 mo. For hybridization, the cell or tissue slides were washed with PBS, treated with -mercaptoethanol (15 mM, 8 min), and washed again with 2x SCC buffer (300 mM NaCl and 30 mM sodium citrate, pH 7.2). The samples were incubated with the hybridization mixture [0.5 ml of formamide, 100% (GIBCO-BRL); 0.2 ml of 20x SSC; 0.2 ml of dextran sulfate (50%; Sigma); 29 µl of salmon DNA (8.7 mg/ml); 25 µl of yeast transfer RNA (10 mg/ml); 10 µl of 100x Denhardts solution; 4 µl of EDTA (500 mM); and 32 µl of H2O] for 30 min at 37°C. The hybridization mixture was eliminated without washing, and the samples were incubated overnight with the complete hybridization solution (hybridization mix + probe) at 37°C. The probe dilution was 1:200 in hybridization mixture. For detection purposes, antidigoxigenin Fab coupled to alkaline phosphatase was used (1:500 dilution). The immune reactions were performed using a standard protocol. Substrate solution was composed of 1 µl of levamisole (240 mg/ml), 4.5 µl of nitro blue tetrazolium dye (75 mg/ml), and 3.5 µl of 5-bromo-4-chloro-3-indolyl phosphate (50 mg/ml) in 1 ml of Tris·HCl buffer (in mM: 100 Tris·HCl, 100 NaCl, and 50 MgCl2, pH 9.5). Control experiments were performed using 200x cold probe (sense primer) together with 1x digoxigenin-labeled sense primer. The samples were histologically mounted on slides, and images were acquired using a Zeiss Axioscope microscope equipped with a Nikon DXM1200 digital camera.
Immunohistochemistry and Immunocytochemistry of MCT1, MCT2, and MCT4
Affinity-purified antibodies against MCT1, MCT2, and MCT4 isoforms and the corresponding antigenic peptides were obtained from Alpha Diagnostic International (San Antonio, TX). The expression of MCT1, MCT2, and MCT4 isoforms in rat testes was determined by performing histochemical analysis of thin sections prepared from archived paraffin-embedded tissue blocks according to the method described by Zambrano et al. (41). Paraffin was removed by incubating the sections in xylene followed by absolute alcohol, and it was rehydrated by immersion in graded alcohol solutions. Immunocytochemistry of isolated pachytene spermatocytes and round spermatids was performed on cell smears, similarly to the tissue sections. Endogenous peroxidase was inactivated by treating the tissue sections and cells with 3% H2O2 for 15 min at room temperature. Sections and cell smears were incubated in PBS containing 5% skim milk, followed by incubation overnight at room temperature with affinity-purified anti-MCT1, anti-MCT2, and anti-MCT4 isoform antibodies (in 1% BSA-PBS), pH 7.4, and 0.3% Triton X-100. Tissue sections and cell smears were washed and incubated with anti-rabbit IgG-horseradish peroxidase (1:100 dilution; Amersham, Arlington Heights, IL) for 2.5 h at room temperature. Immunostaining was performed using 0.05% 3,3'-diaminobenzidine and 0.03% H2O2. Cells and tissue sections were counterstained with hematoxylin. The antibodies preadsorbed with the respective peptides were used as controls.
Determination of [14C]L-Lactate Flux
The radioactive flux measurements were performed at 18°C. Approximately 7 million cells/ml were suspended at the different L-lactate concentrations described in RESULTS. [2-3H]-D-Mannitol was added to the cell suspension at a final concentration of 10 µCi/ml. Subsequently, L-[U-14C]lactic acid sodium salt was added to the cell suspension at time 0 at a concentration of 3 µCi/ml. After the desired times had elapsed, 0.5 ml of the cell suspension was layered on top of a dibutylphthalate-dodecane mixture (1 ml; 1.025 g/ml density) in an Eppendorf microcentrifuge tube and centrifuged for 1 min at 15,000 g. The supernatant on top of the dibutylphthalate-dodecane layer was removed, and the upper part of the tube was rinsed four times with distilled water. The dibutylphthalate-dodecane mixture was then removed, and the cell pellet was suspended in 250 µl of 0.1 M NaOH and 0.1% Triton X-100. Aliquots of the supernatant and cell pellet suspension were mixed with scintillation liquid and counted in a Beckman 1800 scintillation counter with 3H and 14C counting windows. Intracellular [14C]-L-lactate concentration was obtained by correcting for extracellular space trapping in the cell pellet as described previously (30). The concentration of L-lactate entering the cells was calculated using the external specific radioactivity, which was obtained from the supernatant radioactive counts and the external L-lactate concentration.
Intracellular pH Measurements
The intracellular pH (pHi) of rat spermatogenic cells was estimated on the basis of fluorescence measurements of intracellular 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cells were incubated in Krebs-Henseleit (KH)-L-lactate-Ca2+ medium with 5 µM BCECF-AM (Molecular Probes, Eugene, OR) for 30 min at room temperature in a 95% O2-5% CO2 atmosphere. After loading the probe, the cells were washed three times and suspended in KH-Ca2+ medium. To inhibit the main H+ transport system in spermatids and pachytene spermatocytes (25), dicyclohexyl carboxiimide, a nonspecific H+-ATPase inhibitor, was added to the cells at 18 or 30°C 20 min before the pHi measurements. The excitation fluorescence ratio of 505/445 nm was determined using an emission wavelength of 535 nm in a Spex FluoroMax-2 fluorometer (HORIBA Jobin Yvon, Edison, NJ). pHi measurements were performed at a cell density of 2 million cells/ml. After a basal fluorescence ratio was obtained, different L-lactate concentrations were added to the cell suspension and the fluorescence ratio was recorded until it reached a new steady-state level. Calibration of intracellular BCECF was performed as described by Rink et al. (34). The buffering capacity of spermatogenic cells was determined by measuring pHi and using NH4Cl pulses as described by Boron (5), and we found values of 21 ± 7 mM/pH unit (N = 3) and 25 ± 5 mM/pH unit (N = 3) for pachytene spermatocytes and round spermatids, respectively, at pH 7.2.
pHi Data Analysis
Initial velocities of pHi changes were determined by performing linear regression analysis of the pHi vs. time records 1 min before and 45 s after L-lactate addition. L-Lactate-dependent pHi changes were calculated by subtraction of the rates of pHi changes in the presence and absence of L-lactate, respectively. The rates of L-lactate-dependent pHi changes at different L-lactate concentrations were corrected by the buffering capacity of the cells on the basis of estimated buffering capacity (see above; see also Ref. 5) and the volume per million cells from the average diameter of round spermatids (11 µm) and pachytene spermatocytes (16 µm). L-Lactate-dependent H+ uptake was expressed as picomolar H+ per minute per million cells.
Software
For primer design, we used Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). Alignments were performed using ALN3 software, version 1.1. Images were processed with Adobe Photoshop version 5.5 (Adobe Systems, San Jose, CA) and with TotalLab 1.11 software (Nonlinear Dynamics, Newcastle upon Tyne, UK). pHi measurement and kinetic data analysis were performed with Microcal Origin software version 5.0 (OriginLab, Northampton, MA). Statistical analysis was performed with GraphPad InStat software, version 3.05 (GraphPad Software, San Diego, CA).
Chemicals and Isotopes
L-[U-14C]lactic acid sodium salt was obtained from Amersham Pharmacia (Little Chalfont, UK); its specific activity was 152 mCi/mmol. [2-3H]-D-Mannitol was obtained from ICN Biomedicals (Irvine, CA); its specific activity was 20 Ci/mmol. Unless stated otherwise, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
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RESULTS |
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Figure 1A shows the time course of [14C]-L-lactate uptake by rat round spermatids at 18°C. More than 95% of the L-lactate uptake was inhibited by 2 mM -cyano-4-OH cinnamate (CHC), an inhibitor of MCT-mediated transport (39). In subsequent experiments, 40-s sampling was used to obtain initial velocities of L-lactate uptake in round spermatids. Figure 1B shows the relationship between the initial rate of L-lactate uptake by rat round spermatids and the concentrations of L-lactate in the medium in the absence and presence of CHC. These data indicate that L-lactate uptake in these cell was >95% mediated by MCT-mediated transport. At <4 mM external L-lactate concentration, the uptake had a tendency to saturate (see Fig. 1B, inset). Raising the L-lactate concentration to >4 mM led to an increment in the uptake of this compound by mechanisms that were also inhibited by CHC (Fig. 1B). These results strongly suggest that the MCT-mediated L-lactate transport was occurring in both high- and low-affinity transport systems. Because of isotope dilution, >10 mM L-lactate concentrations were not tested in these types of measurements, and hence the possible saturation properties of the low-affinity, MCT-mediated transport system were not evident using this experimental strategy. On the basis of the analysis of the L-lactate-dependent H+ transport (see below), we simulated the kinetic behavior of [14C]-L-lactate transport using a high-affinity hyperbola, together with a low-affinity sigmoid curve (see solid and dashed lines in Fig. 1B). The kinetic parameters in these curves were as follows: hyperbola, Km 1.0 mM and Vmax 150 pmol/min/million cells; sigmoid, L-lactate concentration giving half maximal uptake (K0.5) 27 mM, Vmax 3,100, and Hill coefficient (nH) 2 (fixed).
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Round spermatids. Consistent with the [14C]-L-lactate transport measurements and with the fact that MCT-mediated transport is an L-lactate-H+ cotransporter, the decrease in pHi induced by external L-lactate added to a suspension of round spermatids was inhibited by CHC (Fig. 2A). To express the data in the same units as those used to express L-lactate uptake, we corrected the L-lactate-dependent H+ uptake data using the average buffering capacity of the cells (see MATERIALS AND METHODS) and the average diameter of round spermatids (11 µm; range, 912.5 µm).
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It is known that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) seems to inhibit MCT1 and MCT2, but not MCT4 (29). Consistent with this property of DIDS, this compound produced a partial inhibition of L-lactate-dependent H+ uptake in round spermatids at 30°C (see Fig. 4). Eadie-Hofstee analysis of the DIDS-insensitive component of this uptake (Fig. 4, inset) showed that it was well described by a single component that presented the kinetic parameters Km 20 ± 9 mM and Vmax 366 ± 127 pmol/min/million cells. Thus DIDS was able to differentiate, and at the same time corroborated, that round spermatids have at least two components of L-lactate transport kinetically corresponding to what has been described as L-lactate transport mediated by high-affinity MCT (MCT1 and/or MCT2) and low-affinity MCT4.
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Similarly to the findings for round spermatids, DIDS was able to inhibit partially the L-lactate-dependent H+ uptake in pachytene spermatocytes (Fig. 6). The Eadie-Hofstee analysis of the DIDS-insensitive component of this uptake showed that it was well described by a single component that presented the kinetic parameters Km 17.4 ± 2.7 mM and Vmax 4,835 ± 586 pmol/min/million cells.
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The L-lactate transport measurements indicated that L-lactate entered both pachytene spermatocytes and round spermatids using MCT transporters. Furthermore, our kinetic data strongly suggest that at least two different transport systems for L-lactate were present in both pachytene spermatocytes and round spermatids. Thus, to test the hypothesis of a differentiation of MCT expression during the meiotic-postmeiotic transition in spermatogenesis, we studied the expression of the MCT1, MCT2, MCT3, and MCT4 transcripts in pachytene spermatocytes and round spermatids using RT-PCR. Figure 7, A and B, shows the pattern of agarose gel electrophoresis of the RT-PCR products for MCT1, MCT2, and MCT4. We did not find any detectable expression of MCT3 in pachytene spermatocytes or round spermatids. The RT-PCR products presented the expected number of bases for the MCT1, MCT2, and MCT4 amplification products. Furthermore, sequencing of the RT-PCR products showed that their aligned sequences were 100% identical to the sequences flanked by the primers designed. Simultaneous RT-PCR for MCT1 consistently showed a less strong signal in round spermatids than in pachytene spermatocytes (Fig. 7A). MCT2 and MCT4 expression did not show obvious differences between cell types (Fig. 7, A and B).
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Our RT-PCR data of MCT isoforms in pachytene spermatocytes and round spermatids suggest that MCT1 was decreased in round spermatids. Thus, to test directly for the presence of the MCT1 and MCT2 transcripts in spermatogenic cells, we performed in situ hybridization to MCT1 and MCT2 mRNA in pachytene spermatocytes and round spermatids. Figure 8, A and C, shows the control in situ hybridization performed in the presence of excess unlabeled probes for MCT1 and MCT2, respectively, in round spermatids. Figure 8, B and D, shows the hybridization with digoxigenin-labeled probes for MCT1 and MCT2, respectively, in round spermatids. These images show that MCT1 transcripts were not detected using this technique in round spermatids. In contrast, pachytene spermatocytes showed positive staining for both MCT1 and MCT2 (Fig. 9, A and C, respectively; controls, Fig. 9, B and D).
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Expression of the MCT1, MCT2, and MCT4 isoforms at the protein level in rat round spermatids and pachytene spermatocytes is shown in Figures 10, AF, and 11, AF, respectively. It can be shown that pachytene spermatocytes expressed the MCT1 and MCT4 isoforms, but the expression of MCT2 was less evident. In fact, MCT2 immunoreactivity in pachytene spermatocytes demonstrated faint localization to what is likely the Golgi apparatus in the cell. Round spermatids showed strong MCT1 immunoreactivity but weaker immunoreactivity for MCT2 and MCT4. These data observed with isolated cells were corroborated with seminiferous tubule sections, which are shown in Fig. 12. MCT1 immunoreactivity was observed in the interstitial cells and also in surrounding spermatogenic cells in the seminiferous tubules. A MCT2-related histochemical reaction was strongly observed in interstitial cells as well as on the tails of spermatozoa in the lumina of the tubules (see also Ref. 6), with a weaker reaction observed in seminiferous tubule cells. A strong MCT4-related histochemical reaction was observed in interstitial cells, with a weaker reaction observed in seminiferous tubule cells.
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DISCUSSION |
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As noted in the introductory text, the metabolic properties of spermatogenic cells suggest that they can either export glycolytic L-lactate or take up L-lactate from the medium, depending on the availability of glucose and L-lactate in the external environment. The environmental availability of L-lactate and glucose is controlled by the hormonally regulated glycolytic activity of Sertoli cells, which are the sustentacular cells in the seminiferous epithelium. Hence, transport and metabolism of these metabolic substrates would be part of the signals connecting the physiological activities of these different cell types.
In the present study, we have found that L-lactate-H+ transport properties of spermatogenic cells, which show at least two components of L-lactate transport, are in agreement with the expression of MCT4 (a low-affinity/high-capacity transport system), together with MCT1 (intermediate affinity) and/or MCT2 (high affinity) in pachytene spermatocytes and round spermatids. The expression of these isoforms and the differentiation of the expression of MCT1 and MCT2 at the spermatid stages of spermatogenic cell development were confirmed by our RT-PCR results and in situ hybridization. The cytochemistry of MCT1, MCT2, and MCT4 showed that MCT1 was present in pachytene spermatocytes and in round spermatids. MCT2 was weakly evident at the protein level in pachytene spermatocytes and apparently was intracellularly localized, even though the RNA transcript was clearly detected. MCT4 was present in both cell types at both the mRNA and protein levels. These data are in agreement with our kinetic data suggesting the existence of at least two components of L-lactate transport in these cells, consisting of both high-affinity/low-capacity and low-affinity/high-capacity L-lactate uptake of this monocarboxylate transporter. The tendency to saturate at 5 mM, followed by a further increase in uptake at higher L-lactate concentrations, was reproducible in our experiments with pachytene spermatocytes and round spermatids. In oocytes and when expressed alone, MCT4-mediated transport does not show sigmoid kinetics (20). However, in differentiated muscle cells that contained both high- and low-affinity MCT transport systems, Beaudry et al. (2) showed that, similarly to spermatogenic cells, a sigmoid relationship between L-lactate uptake and L-lactate concentration was obtained. The mechanism of this sigmoid relationship between L-lactate uptake and L-lactate concentration is not well understood. However, it has been shown that MCT1 and MCT4 can be regulated by expression of and interaction with CD70 and CD147, a family of plasma membrane glycoproteins (8). Furthermore, it was recently demonstrated that coexpression of MCT1 and the Na+-HCO3 transporter in oocytes can induce activation of MCT1 through a pHi-related mechanism, strongly suggesting that complex kinetic behavior can be expected in cells expressing several isoforms of MCT and other acid-base transporters (3).
The evidence that the high-affinity isoform L-lactate transporter MCT2 shows greater expression at the protein level in round spermatids strongly suggests that this isoform has a role in or is a consequence of cell differentiation in the spermatogenic process. The fact that L-lactate can maintain low [Ca2+]i with K0.5 of 0.7 mM (32) suggests, first, that MCT1 and MCT2 transport capacity is adequate to supply reducing equivalents to the mitochondria to maintain physiological levels of intracellular ATP (and activity of transport ATPases) in these cells and, second, that MCT4 appears to play a role in these cells that is not related to the entry of L-lactate into oxidative metabolism.
Our results are consistent with the idea that spermatogenic cells differentially express L-lactate transporter isoforms that could allow coordination of metabolism and [Ca2+]i (32) by the hormonally regulated metabolic and transport activity of Sertoli cells. This metabolic signaling from Sertoli cells could be coupled to spermatogenic cell metabolism, channeling glycolytic L-lactate toward MCT4 and external L-lactate toward MCT1 or MCT2 and cell mitochondrial oxidative metabolism. This working hypothesis describes an integrated physiological role for the experimentally observed spermatogenic, cell metabolic, and membrane transport differentiation, which has remained puzzling thus far (see, e.g., Ref. 10).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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