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BOR - Papers in Press, published online ahead of print October 25, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.056168
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BIOLOGY OF REPRODUCTION 76, 241–249 (2007)
DOI: 10.1095/biolreprod.106.056168
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Multiple Pathways for Cationic Amino Acid Transport in Rat Seminiferous Tubule Cells1

Virginie Cérec 3 4, Claire Piquet-Pellorce 3 4, Hamdy A.A. Aly 3 5, Anne-Marie Touzalin 3 4, Bernard Jégou 3 4, and Françoise Bauché 2 3 4

Inserm,3 U625, Rennes F-35042, France University Rennes 1,4 GERHM, IFR 140, Campus de Beaulieu, Rennes F-35042, France Department of Pharmacology and Toxicology,5 Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt

ABSTRACT

Arginine and ornithine are known to be important for various biological processes in the testis, but the delivery of extracellular cationic amino acids to the seminiferous tubule cells remains poorly understood. We investigated the activity and expression of cationic amino acid transporters in isolated rat Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids. We assessed the L-arginine uptake kinetics, Na+ dependence of transport, profiles of cis inhibition of uptake by cationic and neutral amino acids, and sensitivity to trans stimulation of cationic amino acid transporters, and studied the expression of the genes encoding them by RT-PCR. Our data suggest that L-arginine is taken up by Sertoli cells and peritubular cells, principally via system y+L (SLC3A2/SLC7A6) and system y+ (SLC7A1 and SLC7A2), with system B0+ making a minor contribution. By contrast, system B0+, associated with system y+L (SLC3A2/SLC7A7 and SLC7A6), made a major contribution to the transport of cationic amino acids in pachytene spermatocytes and early spermatids. Sertoli cells had higher rates of L-arginine transport than the other seminiferous tubule cells. This high efficiency of arginine transport in Sertoli cells and the properties of the y+L system predominating in these cells strongly suggest that Sertoli cells play a key role in supplying germ cells with L-arginine and other cationic amino acids. Furthermore, whereas cytokines induce nitric oxide (NO) production in peritubular and Sertoli cells, little or no upregulation of arginine transport by cytokines was observed in these cells. Thus, NO synthesis does not depend on the stimulation of arginine transport in these somatic tubular cells.

arginine, cytokines, germ cells, nitric oxide, Sertoli cells, spermatogenesis, testis, transport

INTRODUCTION

Certain cationic amino acids, such as L-arginine or L-ornithine, have long been known to be important for testicular function. For example, the polyamines putrescine, spermidine, and spermine, which stimulate growth, are generated from L-ornithine and play key roles in spermatogenic DNA synthesis and the control of spermatogenesis [1, 2]. L-arginine is a precursor of nitric oxide (NO), the synthesis of which is catalyzed by three different NO synthases [3]: inducible, endothelial, and neuronal. All three have been found in the testis [4–6], and NO is thought to be involved in the regulation of spermatogenesis, inflammation-mediated infertility, and the programmed cell death of germ cells [6–8]. L-arginine and L-lysine also are required for the synthesis of the highly basic arginine rich-proteins, transition proteins, and protamines produced during the postmeiotic stages of spermatogenesis [9]. Chromatin remodeling during normal spermiogenesis in mammals involves the replacement of histones by transition proteins and then by protamines [10]. Intracellular availability of cationic amino acids may contribute largely to the regulation of these important testicular processes. As L-lysine, L-arginine, and L-ornithine are essential or semiessential amino acids, their transport across the plasma membrane is a critical step likely to be crucial for normal testicular function. However, little is currently known about cationic amino acid transport in the seminiferous tubules.

Cationic amino acids are transported across mammalian cell membranes by four different carrier systems: y+, y+L, bo+, and B0+ (Table 1), which differ in substrate specificity and affinity, sodium dependence, mechanism, tissue expression, and regulation [11].


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  		TABLE 1. Cationic amino acid transport systems.

System y+ is a high-affinity, Na+-independent transport system with marked selectivity for cationic amino acids. It mediates the transport of these amino acids across the membrane in both directions and displays trans stimulation (i.e., stimulation of transport by the substrate on the trans side of the membrane). It also catalyzes the transfer of some neutral amino acids, but such transport occurs only in the presence of sodium and with a very low affinity [11]. Three genes, Slc7a1, Slc7a2, and Slc7a3 (previously known as Cat1, Cat2, and Cat3, respectively), encode membrane transporters with system y+ activity. Slc7a1 is constitutively expressed in most cell types, except those in the liver [12], which express only Slc7a2. Differential tissue-specific splicing of the Slc7a2 mRNA give rise to two different SLC7A2 proteins: a low-affinity cationic amino acid transporter (isoform 1, previously known as CAT-2A) expressed primarily in the liver [13], and a high-affinity isoform (isoform 2, previously known as CAT-2B) present in several organs, including the testis [11]. The expression of Slc7a3 seems to be restricted to the brain in rodents [14, 15], whereas it also is observed in peripheral tissues in humans [16].

System y+L is a high-affinity transport system for both neutral and cationic amino acids, with a unique profile of differential Na+ dependence: the transport of neutral amino acids is coupled to Na+, whereas the transport of basic amino acids is Na+ independent [11, 17]. It mediates an obligatory exchange of neutral and cationic amino acids, transport being most efficient when extracellular neutral amino acids coupled to Na+ are exchanged for intracellular cationic amino acids [17–19]. System y+L is a heterodimeric amino acid transporter comprising a common heavy chain, SLC3A2 (previously known as 4F2hc), associated with one of two light chains, SLC7A7 or SLC7A6 (previously known as y+LAT-1 and y+LAT-2, respectively) [17–19]. Slc7a7 transcripts are detected in only a few organs, including small amounts in the testis [18], whereas Slc7a6 and Slc3a2 mRNA have a wider tissue distribution and are produced in larger amounts in the testis [19, 20].

The b0+ amino acid transport system is also a heterodimeric complex, comprising the b0+ amino acid transporter (SLC7A9, previously known as b0+AT) and the protein related to the b0+ amino acid transporter (SLC3A1, previously known as rBAT) [17]. The Na+-independent transport activity mediated by b0+ displays high affinity for cationic or neutral amino acids and involves an obligatory exchange mechanism favoring the uptake of cationic amino acids [21]. Slc3a1 and Slc7a9 transcripts are produced mostly in intestinal and renal tubular cells [22, 23].

Unlike systems y+, y+L, and bo+, all of which are Na+-independent cationic amino acid transporters, B0+ is an Na+-dependent, high-affinity transport system for both neutral and cationic amino acids [11]. This carrier system is not widely expressed; it has been identified only in the lung and colon at high levels and in the testis at low levels [24, 25].

In this study, we aimed to define the properties of L-arginine transport in rat somatic seminiferous tubule cells (Sertoli cells, peritubular cells) and in meiotic and postmeiotic germ cells (pachytene spermatocytes, early spermatids). We investigated the roles of the various cationic amino acid carriers involved in supplying the testis with arginine by determining the kinetic parameters of L-arginine uptake by these cultured rat testicular cells. We also evaluated the Na+ dependence, cis inhibition and trans stimulation of such transport. In addition, expression of the genes encoding the various components of the cationic amino acid transport systems was investigated by RT-PCR. We have shown previously that NO production increases in Sertoli cells and peritubular cells exposed to a combination of cytokines, including interleukin 1{alpha} (IL1{alpha}), tumor necrosis factor {alpha} (TNF{alpha}), and interferon {gamma} (IFN{gamma}) [4]. We therefore investigated the effects of such cytokine treatment on L-arginine uptake by Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids.

MATERIALS AND METHODS

Materials

Dulbecco minimum essential medium (DMEM), Ham F12 medium, L-arginine, L-lysine, L-leucine, and all other reagents used for cell culture were obtained from Gibco (Cergy-Pontoise, France). L-[2,3,4,5-3H]arginine monohydrochloride was purchased from Amersham Biosciences (Orsay, France). Recombinant rat IL1{alpha}, TNF{alpha}, and IFN{gamma} were obtained from R&D Systems (Lille, France). All other reagents were supplied by Sigma Chemical Co. (St. Louis, MO).

Enriched Cell Preparations and Cultures

Male Sprague-Dawley rats were purchased from Elevage Janvier (Le Genest, France). Procedures relating to the care and use of animals were approved by the French Ministry of Agriculture according to the French regulations for animal experimentation. Sertoli cells and peritubular cells were prepared from 20-day-old Sprague-Dawley rats as described by Toebosch et al. [26]. Sertoli cells (106 cells/ml) were cultured in Ham F12/DMEM medium (1/1 [v/v]) without phenol red that was supplemented with gentamicin (50 µg/ml), insulin (5 µg/ml), and transferrin (2.5 µg/ml) in six-well plates (2 ml/well) at 32°C in a humidified incubator (5% CO2/95% air). After 4 days in culture with the medium changed daily, cells were incubated for 24 h in a transferrin-free medium containing 0.1% bovine serum albumin (BSA) with or without a combination of IL1{alpha} (10 U/ml), TNF{alpha} (100 U/ml), and IFN{gamma} (100 U/ml). The medium was recovered, centrifuged, and used for nitrite content determination, and the cells were used for L-arginine transport assays. At the end of the incubation period, cell viability was assessed by evaluating mitochondrion-dependent MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, as previously described [27]. The purity of preparations was estimated by microscopy (>98% under these conditions).

Peritubular cells were added to 75-cm2 culture flasks containing the same medium as used for Sertoli cells, but supplemented with 10% fetal calf serum (FCS) and cultured at 32°C in a humidified incubator (5% CO2/95% air) for 24 h. Cells then were washed with phosphate-buffered saline (PBS) to remove contaminating nonadherent germ cells and were incubated in the conditions described above. The medium was renewed every 48 h, and cells were treated with trypsin-EDTA (0.5 mg/ml, 0.25 M) and subcultured at half density when confluent, usually at 7 and 14 days. Peritubular cells were released by trypsin treatment and cultured to confluence in six-well plates, this process generally taking about a week. Twenty-four hours before L-arginine transport assays, cells were transferred to transferrin- and FCS-free culture medium containing 0.1% BSA with or without IL1{alpha} (10 U/ml), TNF{alpha} (100 U/ml), and IFN{gamma} (100 U/ml). At the end of the incubation period, the medium was recovered and centrifuged for nitrite content determination, and the peritubular cells (pellet) were immediately used for L-arginine transport assays or MTT viability tests. The peritubular cell preparations used were about 98% pure, as assessed by alkaline phosphatase staining [28].

Pachytene spermatocytes and early spermatids were isolated from 90-day-old Sprague-Dawley rat testes by trypsin treatment and centrifugal elutriation, as described by Meistrich et al. [29]. Cell fractions enriched in pachytene spermatocytes and early spermatids (>95% and 90% purity, respectively, as evaluated by flow cytometry) were cultured in 75-cm2 flasks at densities of 2.5 x 106 and 8 x 106 cells/ml, respectively, in PBS supplemented with CaCl2 (0.88 mM), MgCl2 (0.5 mM), lactate (6 mM), glucose (5.6 mM), BSA (0.4%), L-arginine (0.7 mM), and gentamycin (50 µg/ml), in the presence or absence of IL1{alpha} (10 U/ml), TNF{alpha} (100 U/ml), and IFN{gamma} (100 U/ml), at 32°C in a humidified incubator (5% CO2/95% air). After culture for 24 h, the cell suspensions were centrifuged (100 x g for 5 min), the medium was recovered for nitrite content determination, and the cells were resuspended in PBS supplemented with either 140 mM NaCl or choline chloride (PBS-choline) for L-arginine transport assays. Germ cell viability, evaluated by trypan blue exclusion, was about 95%.

Measurement of L-arginine Transport

L-arginine transport was determined by measuring the rate of influx of L-[3H]arginine using various methods according to the cell type tested, because Sertoli and peritubular cells adhere to plates, whereas pachytene spermatocytes and early spermatids do not. Sertoli or peritubular cells in six-well plates were washed (3 ml/well) with Hepes buffer (5 mM KCl, 0.9 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 25 mM Hepes, pH 7.4) supplemented with 140 mM NaCl or choline chloride, and were incubated for 3 min at 32°C in 3 ml of the same buffer. The medium was removed, and the cells were further incubated at 32°C with 1 ml Hepes buffer supplemented with 10 to 150 µM L-arginine and L-[3H]arginine (2 µCi/ml and 4 µCi/ml for Sertoli and peritubular cell assays, respectively) for the times indicated (30 sec to 10 min). Arginine uptake was stopped by rapidly removing the radioactive supernatants, immediately transferring the cells onto ice, washing them three times with 3 ml ice-cold PBS in each case, and lysing them with 1 ml of 0.2% sodium dodecyl sulfate in 0.2 M NaOH. The protein content of cell lysates was determined by the Lowry method [30], and radioactivity corresponding to total (specific and nonspecific) L-[3H]arginine uptake was evaluated by liquid scintillation counting. The specific transport of L-[3H]arginine was determined by correcting for nonspecific uptake, as evaluated in cells incubated in Hepes buffer containing L-[3H]arginine in the presence of 10 mM unlabeled L-arginine. In cis inhibition assays the specific uptake of 50 µM L-[3H]arginine was assessed as described above, in Hepes buffer supplemented with 10 mM L-lysine or L-leucine. In trans stimulation experiments, cells were first incubated for 2 h in Hepes buffer supplemented with 10 mM L-arginine or L-leucine and washed, and the specific transport of 50 µM L-[3H]arginine was then determined as previously described.

Pachytene spermatocytes and early spermatids suspended in PBS or PBS-choline (2.5 x 106 and 8 x 106 cells/ml, respectively) were incubated at 32°C in the presence of L-[3H]arginine (2 µCi/ml) with either 10 to 150 µM L-arginine (total uptake) or 10 mM L-arginine (nonspecific uptake) for 30 sec to 10 min. At the end of the incubation period, 5 ml ice-cold PBS was added to the cell suspension, and the mixture was centrifuged immediately at 4°C (100 x g for 5 min). Cells were resuspended in 5 ml ice-cold PBS, centrifuged as above, and lysed with 1 ml of 0.2% sodium dodecyl sulfate in 0.2 M NaOH. Aliquots of lysates were used for protein determination by the Lowry method and for the liquid scintillation counting of radioactivity. In all experiments, nonspecific uptake was subtracted from total uptake to determine the specific transport of L-[3H]arginine. In cis inhibition assays, the specific uptake of 50 µM L-[3H]arginine was measured as described above, in PBS supplemented with 10 mM L-lysine or L-leucine. In trans stimulation experiments, cells were first incubated at 32°C for 2 h in PBS supplemented with 10 mM L-arginine or L-leucine, then centrifuged (100 x g for 5 min) and resuspended in PBS for determination of the specific transport of 50 µM L-[3H]arginine, as described above. Km and Vmax for arginine uptake were determined with GraphPad Inplot software (San Diego, CA.)

Nitrite Assay

Nitrite accumulation in culture supernatants was used as an indicator of cellular NO synthesis and was determined with the Griess reagent, as previously described [31]. Nitrite concentrations were determined using a standard curve obtained with NaNO2 solutions prepared with culture medium.

RNA Extraction and RT-PCR Analysis

Total RNA was extracted from the whole testis, and preparations enriched in various cell types were produced with the SV Total Isolation System (Promega, Charbonnières, France). Complementary DNAs were prepared from 10 µg RNA in the presence of 200 ng random hexadeoxynucleotides and 200 U Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. The same reaction mixture without reverse transcriptase was used as a negative control to check for possible genomic DNA contamination. Actin was amplified as a control for RNA quality, efficient reverse transcription, and quantification purposes. PCR was carried out as recommended by Perkin-Elmer, starting with 100 ng cDNA as a template. The sequences of the oligonucleotide primers used to amplify the genes encoding components of systems y+ (Slc7a1, Slc7a2, Slc7a3), y+L (Slc3a2, Slc7a7, Slc7a6), b0+ (Slc7a9), and B0+ (Slc6a14) were designed on the bases of sequences in the GenBank database using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), except for the Slc7a2-derived primers, which were as previously reported by Hattori et al. [32] (Table 2). All PCR amplifications were carried out over 35 cycles, with the exception of actin amplification, which was carried out over 25 cycles. For each gene amplification, the PCR products obtained were directly sequenced and shown to be 100% identical to the published mRNA sequence (GenBank references in Table 2). RNA samples from two independent cell preparations were analyzed for each cell type.


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  		TABLE 2. RT-PCR primer sequences and conditions.

Statistical Analysis

All values are means ± SEM of measurements in at least three independent cell preparations with three pooled replicates per experiment. We used paired Student t-tests or ANOVA, followed by Tukey test for multiple comparisons and using SPSS software (SPSS Inc., Chicago, IL) to analyze the specific arginine transport data. Values of P < 0.05 were considered significant.

RESULTS

Time Course of Arginine Uptake

The time course of the specific transport of 50 µM L-[3H]arginine into Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids was determined (Fig. 1). The rate of uptake differed considerably according to the cell type considered. Sertoli cells had the highest rate of transport (1128 ± 30 pmol 30 sec–1 mg–1 protein) and pachytene spermatocytes, the lowest (51 ± 12 pmol 30 sec–1 mg–1 protein), whereas peritubular cells and early spermatids had intermediate rates (401 ± 24 and 156 ± 50 pmol 30 sec–1 mg–1 protein, respectively).


Figure 76020901
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  		FIG. 1. Time courses of L-arginine uptake by rat seminiferous tubular cells. Specific L-arginine transport in isolated Sertoli cells (open squares), peritubular cells (closed squares), early spermatids (open circles), and pachytene spermatocytes (closed circles) was measured in sodium-containing buffers. Results are means ± SEM of three separate experiments, each performed in triplicate.

Arginine uptake by germ cells (pachytene spermatocytes and early spermatids) was approximately linear for the first 10 min, whereas it decreased after 30 sec in somatic tubular cells (Sertoli or peritubular cells). We therefore subsequently determined the initial rates of L-[3H]arginine transport over 4 min for germ cells, but only 30 sec for somatic tubular cells. Under these conditions, diffusion processes were not involved, with levels of nonspecific arginine uptake remaining low in all cell types, consistently accounting for 4% to 13% of the total arginine uptake (data not shown).

Kinetic Parameters of Arginine Influx

We characterized the kinetic parameters of L-arginine transport by measuring the specific uptake of radiolabeled L-arginine using 10 to 150 µM arginine, as physiological arginine concentrations of about 50 µM have been reported for plasma [33], with even lower levels reported for seminiferous tubule fluid [34]. Plots of specific L-[3H]arginine uptake as a function of extracellular L-arginine concentration and Eadie-Hofstee transformations of the data are shown in Figure 2. As suggested by the linearity of the Eadie-Hofstee plots (Fig. 2B), L-arginine uptake by Sertoli cells or peritubular cells was mediated either by a single carrier or by several transporters with similar affinities for L-arginine. The Km values obtained for Sertoli and peritubular cells were 56 ± 4 µM (n = 5) and 55 ± 6 µM (n = 3), respectively. These two types of somatic cells, therefore, had transporters with similarly high affinities for arginine. However, the maximum transport velocity was three times higher in Sertoli cells (Vmax = 2055 ± 172 pmol 30 sec–1 mg–1 protein) than in peritubular cells (Vmax = 626 ± 67 pmol 30 sec–1 mg–1 protein). By contrast, L-arginine uptake by germ cells was biphasic (Fig. 2C), suggesting the involvement of at least two types of transporters with different affinities for L-arginine. In early spermatids and pachytene spermatocytes, the components with the highest affinity had Km of 105 ± 8 µM (n = 3) and 85 ± 21 µM (n = 3), respectively, whereas the lower-affinity components had Km of 195 ± 17 µM (n = 3) and 191 ± 23 µM (n = 3), respectively.


Figure 76020902
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  		FIG. 2. Kinetic analysis of L-arginine uptake by rat seminiferous tubular cells. Specific transport of L-[3H]arginine was measured in isolated Sertoli cells (Sertoli; open squares), peritubular cells (Peritub; closed squares), early spermatids (ES; open circles) and pachytene spermatocytes (PS; closed circles) in sodium-containing buffers (A). Eadie-Hofstee transformation of the data for Sertoli cells (open squares) and peritubular cells (closed squares; B), and for early spermatids (open circles) and pachytene spermatocytes (closed circles; C). Results are shown as means ± SEM of three to five separate experiments.

Na+ Dependence of Transport

We evaluated the Na+ dependence of L-arginine transport by replacing the sodium chloride in the uptake buffer with choline chloride and determining L-[3H]arginine influx as previously described. L-arginine uptake by Sertoli cells or peritubular cells was found to be largely Na+ independent, as 87.5% ± 3.5% (n = 8) and 81.3% ± 3.4% (n = 4), respectively, of the control transport determined in the presence of Na+ occurred in the absence of Na+ (Fig. 3). By contrast, levels of L-arginine influx in early spermatids or pachytene spermatocytes were much lower in the absence of Na+ than in its presence, reaching only 46.0% ± 2.1% (n = 4) and 66.5% ± 3.9% (n = 4), respectively, of the control transport rate. This indicates the major involvement of an Na+-dependent carrier in L-arginine uptake by these germ cells. Cell viability was not affected by the replacement of sodium chloride by choline chloride (data not shown).

Cis Inhibition of L-arginine Uptake

We carried out 50-µM L-[3H]arginine influx assays in the presence of a cationic (10 mM L-lysine) or neutral (10 mM L-leucine) amino acid to differentiate between the transport systems responsible for L-arginine uptake into testicular cells. Indeed, whereas L-lysine competes with L-arginine for transport via systems y+, y+L, b0+, and B0+, L-leucine competition is mostly restricted to systems y+L, b0+, and B0+ [11]. In all cell types, L-[3H]arginine uptake was affected strongly by the presence of L-lysine or L-leucine, and differed significantly from the respective control influx (Fig. 4). Thus, L-lysine strongly inhibited L-arginine transport in Sertoli cells (–98.8% ± 0.7%), peritubular cells (–98.4% ± 0.6%), early spermatids (–92.6% ± 2.6%), and pachytene spermatocytes (–76.5% ± 2.4%). The neutral amino acid L-leucine also efficiently inhibited L-arginine influx into Sertoli cells (–77.3% ± 4.7%), peritubular cells (–71.7 ± 1.0%), early spermatids (–83.5% ± 0.2%), and pachytene spermatocytes (–53.7% ± 0.8%). This strong inhibitory effect of L-leucine suggests a major contribution of systems y+L, b0+, and/or B0+ to L-arginine uptake by these testicular cells.


Figure 76020904
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  		FIG. 4. Inhibition of L-arginine influx by L-lysine and L-leucine in isolated rat Sertoli cells (Sertoli), peritubular cells (Peritub.), early spermatids (ES), and pachytene spermatocytes (PS). Specific uptake of 50 µM L-[3H]arginine was measured in Na+-containing buffers supplemented with 10 mM L-lysine (open bars) or 10 mM L-leucine (closed bars). Control-specific L-arginine uptakes determined in buffer lacking a competing amino acid were: 1011 ± 79 (3), 321 ± 34 (3), 137 ± 29 (3), and 38 ± 3 (3) pmol 30 sec–1 mg protein–1 for Sertoli, Peritub., ES, and PS, respectively. Multiple comparisons of the transport data were evaluated by an analysis of variance followed by Tukey test (*P < 0.05 vs. the corresponding control. {dagger}P < 0.05 lysine vs. leucine inhibition; NS: not significant). The results presented are expressed as percent inhibition of control uptake in the form of means ± SEM for three independent experiments.

Trans Stimulation of L-arginine Transport

One of the characteristics attributed to system y+ is the stimulation of cationic amino acid transport activity by the presence of a cationic amino acid on the opposite site of the plasma membrane [11]. We investigated whether such trans stimulation occurred in testicular cells by measuring L-[3H]arginine influx after incubation of the cells for 2 h in Na+-containing buffer in the absence (control) or presence of 10 mM L-arginine. L-arginine preloading significantly enhanced L-[3H]arginine uptake by Sertoli cells and peritubular cells (187% ± 15% and 135% ± 4% of control L-arginine influx, respectively), whereas no significant effect was observed in germ cells (Fig. 5), distinguishing clearly between somatic and germ cells. These results are consistent with system y+ involvement in L-arginine uptake by Sertoli and peritubular cells.


Figure 76020905
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  		FIG. 5. Trans stimulation of L-arginine uptake by isolated rat Sertoli cells (Sertoli), peritubular cells (Peritub.), early spermatids (ES), and pachytene spermatocytes (PS). The specific uptake of 50 µM L-[3H]arginine was measured in sodium-containing buffers after preincubation of the cells for 2 h in the absence (control; open bars) or presence (closed bars) of 10 mM arginine. Statistical analysis of the transport data was performed using paired Student t-tests (*P < 0.05 vs. the corresponding control). The results presented are expressed as a percent of control and are means ± SEM of three independent experiments.

Expression of Genes Involved in Cationic Amino Acid Transport

We first investigated the expression of genes involved in cationic amino acid transport by RT-PCR on RNA from the whole testis, comparing it with expression in other appropriate organs used as controls. In these experiments (Fig. 6) we identified cDNAs—Slc7a1 and Slc7a2_v2—encoding some of the members of system y+, but we identified no cDNA encoding the low-affinity SLC7A2 isoform or SLC7A3. By contrast, Slc7a2_v1and Slc7a3 transcripts were detected in the liver and brain, respectively. Furthermore, mRNAs for system y+L proteins (Slc3a2, Slc7a7, Slc7a6) and the system B0+ protein (Slc6a14) also were identified in whole testis. By contrast, the cDNA encoding SLC7A9, a component of system b0+, was not identified in testis samples, whereas it was present in gut samples, as expected [23].


Figure 76020906
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  		FIG. 6. Expression of genes involved in cationic amino acid transport in rat adult testis. RT-PCR analyses of total RNA from adult rat testis were performed with primers for the indicated genes, as described in Table 2. Parallel analyses of total RNA from rat liver, spleen, brain, or gut were carried out as a positive control as follows: liver for Slc7a2, Slc7a2_v1, Slc3a2, and Slc7a7; spleen for Slc7a1, Slc7a2_v2, and Slc7a6; brain for Slc7a3; gut for Slc7a9 and Slc6a14. For each RNA analysis, reverse transcription was performed in the presence (+) and absence (-) of reverse transcriptase, and actin amplification was used as an internal control. A control amplification with no DNA (0) is shown alongside the molecular weight markers (M). Identical findings were obtained with two independent RNA preparations.

We assessed the distribution of Slc7a1, Slc7a2_v2, Slc3a2, Slc7a7, Slc7a6, and Slc6a14 within testicular tubules by carrying out RT-PCR on RNA extracted from cultured purified tubular cells. Slc7a1 and Slc7a2_v2 transcripts were restricted to Sertoli cells and peritubular cells, whereas the Slc7a7 transcript was detected only in pachytene spermatocytes and early spermatids (Fig. 7). With concern to Slc3a2, Slc7a6 and Slc6a14 expression, both somatic and germ cells were shown to be positive.


Figure 76020907
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  		FIG. 7. Expression of genes encoding proteins involved in cationic amino acid transport in isolated rat testicular cells. RT-PCR analyses of total RNA from isolated rat Sertoli cells (S), peritubular cells (P), pachytene spermatocytes (PS), and early spermatids (ES) were performed with primers for the indicated genes, as described in Table 2. For each RNA analysis, reverse transcription was performed in the presence (+) and absence (-) of reverse transcriptase, and actin amplification was used as an internal control. A control amplification with no DNA (0) is shown alongside the molecular weight markers (M). Similar findings were obtained with two different RNA preparations.

Effect of Cytokines on L-arginine Transport

Based on our previous demonstration that NO production increases when somatic tubule cells are cultured in the presence of a combination of proinflammatory cytokines—IL1{alpha}, TNF{alpha}, and IFN{gamma} [4]—we assessed the effect of this combination of cytokines on the rate of L-arginine uptake by the various isolated tubular cells. In parallel, we also determined nitrite production to ask whether such production is correlated with the cationic amino acid carrier activity.

In Sertoli cells, L-[3H]arginine transport and nitrite production increased significantly after incubation for 24 h in the presence of the cytokine combination (Fig. 8, A and B). By contrast, this treatment had no significant effect on L-arginine influx (93% ± 4% of control transport; Fig. 8A) when applied to peritubular cells, despite the induction of maximal rates of nitrite production (Fig. 8B) which is consistent with our previous results [4]. Early spermatids and pachytene spermatocytes displayed similar responses to the combination of cytokines: a small but significant decrease in L-[3H]arginine uptake (–16.0% ± 0.3% and –24% ± 5%, respectively, with respect to the corresponding control) and no change in nitrite generation (Fig. 8, A and B).


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  		FIG. 8. Effects of a combination of proinflammatory cytokines (IL1{alpha}, TNF{alpha}, and IFN{gamma}) on L-arginine uptake and nitrite production by isolated rat testicular cells. Sertoli cells (Sertoli), peritubular cells (Peritub.), early spermatids (ES), and pachytene spermatocytes (PS) were cultured for 24 h in medium lacking (control) or containing a mixture of IL1{alpha} (10 U/ml), TNF{alpha} (100 U/ml), and IFN{gamma} (100 U/ml) (treated) before measurement of L-arginine transport and nitrite production. A) Specific uptake of 50 µM L-[3H]arginine by control cells (open bars) and treated cells (closed bars). The data shown are means ± SEM of at least three independent experiments. *P < 0.05 vs. the corresponding control by paired Student t-test. B) NO2– concentration in conditioned medium from control cells (open bars) and treated cells (closed bars). Results are means ± SEM of three independent experiments. *P < 0.05 vs. the corresponding control by paired Student t-test.

In subsequent experiments aiming to determine the kinetic parameters of L-arginine uptake by Sertoli cells after exposure to the cytokine combination for 24 h, the Km was found to be similar to (59 ± 6 µM) and the Vmax slightly higher than (119% ± 2%) that of unstimulated cells (Fig. 9).


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  		FIG. 9. Effects of a combination of proinflammatory cytokines (IL1{alpha}, TNF{alpha}, and IFN{gamma}) on the kinetics of L-arginine uptake by Sertoli cells. The specific transport of L-[3H]arginine (10–150 µM) was measured in control (open squares) and cytokine-treated (closed squares) Sertoli cells. The data presented in the Eadie-Hofstee plots are the means ± SEM of three independent experiments.

DISCUSSION

Basic amino acids enter mammalian cells via several different transport systems: the y+, y+L, b0+, and B0+ systems. A single system or various combinations of transport systems may be involved in the transfer of cationic amino acids across the cell membrane, depending on the cell type considered. In this in vitro study, which was designed to characterize L-arginine transport in various rat seminiferous tubules cell types, we have shown that different patterns of transporter expression are associated with somatic and germ cells.

In both Sertoli cells and peritubular cells, L-arginine uptake was observed to be slightly Na+ dependent, fully inhibited by L-lysine but not by L-leucine, and trans stimulated after the loading of cells with L-arginine. These characteristics indicate a minor contribution of system B0+ to L-arginine uptake by these cells and are consistent with the involvement of system y+. However, the strong inhibitory effect of L-leucine on L-arginine influx suggests that system y+L and/or b0+ may also be involved. Our RT-PCR data demonstrated expression of Slc6a14, Slc7a1, Slc7a2_v2, Slc7a6, and Slc3a2 in Sertoli and peritubular cells, fully supporting our hypothesis that systems B0+, y+, and y+L mediate cationic amino acid transport in these cells. The involvement of system b0+ can be ruled out, because we detected no Slc7a9 mRNA in the testis, consistent with previous results [23]. The relative contributions of systems B0+, y+, and y+L to L-arginine transport can be estimated on the basis of Na+ dependence and substrate specificity: Na+-dependent arginine uptake reflects B0+ activity, whereas leucine-insensitive L-arginine influx reflects y+ activity [11]. Our data indicate that systems B0+ and y+ mediate about 15% and 25%, respectively, of L-arginine uptake by Sertoli cells and peritubular cells. Thus, system y+L, which accounts for about 60% of the L-arginine influx, appears to be the predominant cationic amino acid transport system in tubular somatic cells. The various L-arginine transporters in rats seem to have similar affinities, as suggested by the linearity of the Eadie-Hofstee plots observed in our kinetic experiments with Sertoli cells and peritubular cells. The Km (~55 µM) obtained in this study is consistent with previously reported values for human fibroblasts or monocytes [35, 36] and for several types of somatic cells in rats [37–39].

Interestingly, the characteristics of L-arginine influx in early spermatids clearly differ from those in somatic seminiferous tubule cells. A significant fraction of L-arginine uptake by spermatids was Na+ dependent (around 54% of the influx of 50 µM L-arginine), suggesting a major contribution of system B0+. System y+ does not seem to be involved in early spermatids, as 1) L-leucine inhibited L-arginine transport as efficiently as L-lysine; 2) no trans stimulation effect was observed following the prior loading of cells with L-arginine; 3) RT-PCR detected neither Slc7a1 nor Slc7a2_v2 transcripts in these cells (neither Slc7a2_v1 nor Slc7a3 transcripts were detected in the whole testis). Finally, as system b0+ was not detected in the testis, the Na+-independent fraction of L-arginine transport into spermatids may be attributed to system y+L. The RT-PCR identification of transcripts encoding proteins of systems B0+ (Slc6a14) and y+L (Slc7a7, Slc7a6, and Slc3a2) in early spermatids provides further evidence that systems B0+ and y+L mediate arginine uptake in this cell type. These two transporter systems exhibit high but different affinities for L-arginine (Km1: ~100 µM, and Km2: ~200 µM), as illustrated by the biphasic Eadie-Hofstee plots. Such affinities are consistent with those measured in various mammalian cells, ranging from 45 to 341 µM for system y+L [18, 19, 40, 41], and from 100 to 160 µM for system B0+ [24, 42, 43].

Biochemical and RT-PCR analyses, as described above, also implicated systems B0+ and y+L in L-arginine uptake in pachytene spermatocytes. However, L-arginine transport by pachytene spermatocytes was not entirely inhibited by L-lysine or L-leucine. This suggests that, in addition to systems B0+ and y+L, another as yet unknown L-arginine transporter is involved in L-arginine uptake in meiotic germ cells. We have tested all known rat transporters, and no other convincing candidates can be identified from previous studies.

Under our experimental conditions, the rate of arginine transport in Sertoli cells is much higher than that in the other seminiferous tubule cells tested. Indeed, as illustrated in Table 3, the rate of L-arginine transport in Sertoli cells was approximately 3, 7, and 22 times higher than in peritubular cells, early spermatids, and pachytene spermatocytes, respectively. Even though this feature was observed in vitro with cultured cells originating from different age animals, it may be of physiological relevance, because Sertoli cells extend from the basement membrane toward the lumen of the seminiferous tubules and are essential constituents of the blood testis barrier [44]. All exogenous nutrients required by the germ cells sequestered in the adluminal compartment, such as spermatocytes and spermatids, must therefore be supplied by Sertoli cells. System y+L has been described as a transporter that preferentially exchanges intracellular cationic against extracellular neutral amino acids in the presence of Na+ [17–19]. System y+L (SLC3A2/SLC7A6) seems to be the major cationic amino acid carrier identified in Sertoli cells, suggesting that these cells can export cationic amino acids efficiently and may therefore play a central role in providing germ cells with L-arginine. Thus, Sertoli cells, which also have high-affinity uptake systems for cationic amino acids (systems y+ and B0+), seem to be particularly well equipped for mediating both the influx of L-arginine from the vascularized interstitium and its efflux toward germ cells within the adluminal compartment of the seminiferous tubules. If appropriate antibodies were available, it would be interesting to further investigate the distribution of these systems in vivo, at either the basolateral or apical pole, to develop a model for the transport of cationic amino acids in the seminiferous tubules. Furthermore, such immunohistological approaches in the whole testis from different age animals would challenge the cellular localization profile of the different arginine transporters that we identified in vitro.


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  		TABLE 3. L-Arginine transport in rat seminiferous tubule cells.

The physiological relevance of system B0+, which mediates a large fraction of L-arginine transport in pachytene spermatocytes and spermatids, remains unclear. System B0+ displays high levels of uptake activity, because it is driven by both membrane potential and Na+ and Cl– gradients. It is therefore tempting to speculate that this transporter allows germ cells to concentrate cationic amino acids. This feature would be of particular importance in this context, especially for spermatids, which must replace their histones by arginine-rich proteins—transition proteins—and then by protamines during spermiogenesis [9].

We also evaluated the role of arginine transport in the NO synthesis induced by the combination of IL1{alpha}, TNF{alpha}, and IFN{gamma}. In Sertoli cells, L-arginine transport was only modestly upregulated by cytokine exposure (about 20%), whereas NO synthesis was strongly induced (by a factor of 10), indicating that increases in L-arginine uptake may not be essential for NO synthase (NOS) activity. This is particularly clear for peritubular cells, in which the response to cytokines involved exclusively the induction of Nos2 expression [4] and high levels of NO production, with no change in the low levels of L-arginine transport observed before stimulation. The lack of need for an increase in L-arginine uptake for sustained NO generation suggests that L-arginine is efficiently supplied by endogenous production in Sertoli cells and peritubular cells. Endogenous arginine is obtained usually by the intracellular degradation of proteins or by regeneration from citrulline, a coproduct of NOS activity through argininosuccinate synthetase and argininosuccinate lyase activities, via the citrulline-NO cycle. These two enzymes have been reported to be present in large amounts throughout the testis [45], but no data are currently available concerning their distribution in the various types of testicular cell. Therefore, further studies are required to determine whether Sertoli cells and peritubular cells can efficiently convert citrulline to arginine under normal conditions and after cytokine treatment.

In conclusion, our in vitro data provide evidence for contrasting patterns of L-arginine uptake ability and cell-specific expression patterns of the various cationic amino acid transporters. Indeed, L-arginine transport in cultured somatic seminiferous tubule cells mostly involves systems y+ and y+L, whereas L-arginine uptake by cultured germ cells is mediated primarily by systems B0+ and y+L (Table 3). This study strongly suggests that Sertoli cells play a key role in supplying arginine or other cationic amino acids to germ cells. Given the importance of arginine in testicular function, this property of a testicular cell type is probably physiologically relevant and provides an interesting basis for further investigations on arginine-dependent biological functions.


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  		FIG. 3. Sodium dependence of L-arginine uptake by isolated rat Sertoli cells (Sertoli), peritubular cells (Peritub.), early spermatids (ES), and pachytene spermatocytes (PS). Specific transport of 50 µM L-[3H]arginine was measured in buffers containing either sodium (control; open bars) or choline (closed bars). Statistical analysis of the transport data was performed using paired Student t-tests (*P < 0.05 vs. the corresponding control). The results presented are expressed as a percent of control and are means ± SEM of four to eight separate experiments.

FOOTNOTES

1Supported by INSERM, Ministère de l'Education Nationale, de la Recherche et de la Technologie. Back

Correspondence: 2FAX: 33 02 23 23 50 55; e-mail: francoise.bauche{at}univ-rennes1.fr

Received: 3 August 2006.

First decision: 1 September 2006.

Accepted: 12 October 2006.

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