School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
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ABSTRACT |
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The blood-seminiferous tubule barrier is responsible for maintaining the unique microenvironment conducive to spermatogenesis. A key feature of the blood-testis barrier is selective permeability to solutes and water transport, conferred by the Sertoli cells of the seminiferous tubules (SMTs). Movement of fluid into the lumen of the seminiferous tubule is crucial to spermatogenesis. By Northern analysis, we have shown that 4.0-, 3.3-, 2.8-, and ~1.7-kb UT-A mRNA transcripts and a 3.8-kb UT-B mRNA transcript are detected within rat testis. Western analysis revealed the expression of both characterized and novel UT-A and UT-B proteins within the testis. Immunolocalization studies determined that UT-A and UT-B protein expression are coordinated with the developmental stage of the SMT. UT-A proteins were detected in Sertoli cell nuclei at all stages of tubule development and in residual bodies of stage VIII tubules. UT-B protein was expressed on Sertoli cell membranes of stage II-III tubules. Using in vitro perfusion, we determined that a phloretin-inhibitable urea pathway exists across the SMTs of rat testis and conclude that UT-B is likely to participate in this pathway.
spermatogenesis; urea flux; Sertoli cell; residual bodies
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INTRODUCTION |
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THE MOVEMENT OF UREA across plasma membranes is modulated by specialized, phloretin-inhibitable transporter proteins that are the products of two closely related genes, UT-A (Slc14a2) and UT-B (Slc14a1) (1, 6, 12, 14). The products of these genes show a wide tissue distribution in that UT-A and UT-B are both expressed in several tissues including kidney, heart, liver, colon, and testis (2, 5, 11, 29; reviewed in Ref. 22). UT-B, but not UT-A, is also expressed in erythrocyte membranes (16, 17). In functional terms, members of this family of proteins are characterized as facilitative urea transporters and do not mediate water movement (7, 20).
In rat kidney, seven cDNAs (see Fig. 1) encoding four members of the
UT-A urea transporter family, UT-A1, UT-A2, UT-A3, and UT-A4, have been
characterized (2, 10, 19, 21; reviewed in Ref. 22). These
isoforms result from differential pre-mRNA splicing of the UT-A gene
and the use of two distinct promoters (14). In addition to
the seven cDNAs characterized within rat kidney, we have recently
isolated and characterized a 1.5-kb cDNA from mouse encoding a novel
member of the UT-A family, UT-A5, which is expressed exclusively in the
testis (7). By in situ hybridization, we localized UT-A5
to an outer cell layer of all seminiferous tubules (SMTs) and showed
that the expression of UT-A5 mRNA increases 20 days after birth
(7). This increase coincides with the initiation of seminiferous tubular fluid production and, consequently, the transit
of mature sperm along the SMTs into the male reproductive tract. UT-B
mRNA is also present in Sertoli cells and is upregulated in the early
stages of spermatogenesis (29). Although the precise cellular location of UT-B protein has not been determined, it has been
suggested that UT-B expressed in Sertoli cells may mediate the exit of
urea formed during the synthesis of arginine and ornithine (29).
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The SMT epithelium is made up of a complex association of somatic and germ cells (18). The Sertoli cell layer is generally regarded as the major barrier for solute entry into and out from the SMT lumen. It is a component of the blood-testis barrier and is responsible for generating the ionic gradients that drive fluid secretion. The SMT secretes a fluid that is, in part, responsible for the flow of mature spermatozoa out of the SMT into the rete testis and epididymus (18). Several different factors are known to influence the rate of SMT fluid secretion. At present, however, the complete mechanism of fluid secretion is not fully understood (9).
Because both UT-A and UT-B transcripts are highly expressed in rat testis (7, 10, 29), we focused our research on this tissue to begin to understand the role that urea transporters play in testicular function. Initially, we undertook a detailed analysis of the expression of UT-A and UT-B mRNA transcripts and determined the location of UT-A and UT-B proteins in rat testis using UT-A and UT-B peptide-targeted antisera. We also hypothesized that UT-A and/or UT-B urea transporters expressed in the testis mediate urea movement across the SMT membrane. To test this hypothesis, we measured phloretin-inhibitable urea flux across the rat SMT using in vitro tubular perfusion.
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METHODS |
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Northern Analysis
Northern analysis and reverse transcription-polymerase chain reaction (RT-PCR) experiments (see RT-PCR and Southern Blotting) were performed to determine the molecular identity of the urea transporter transcripts present in testis. Total RNA was isolated from the testis and kidney inner medulla (IM; positive control) of Wistar rats as described previously (21), separated (15 µg/lane) in a 1% agarose gel in the presence of 2.2 M formaldehyde, and transferred to Hybond-N filters (Amersham Pharmacia). Several identical filters were prepared in this way. Filters were probed using the following 32P-labeled cDNA probes: probe 1, full-length (3,974 bp) rat UT-A1 (GenBank accession no. U77971); probe 2, a 272-bp AccI fragment corresponding to nucleotides 1-272 at the 5' terminus of rat UT-A1; probe 3, a 145-bp fragment corresponding to nucleotides 1785-1929 at the 3' terminus of rat UT-A3 (GenBank accession no. AF041788); probe 4, a 272-bp EcoR1 fragment corresponding to nucleotides 1-272 at the 5' terminus of rat UT-A2 (GenBank accession no. U09957); and probe 5, an 836-bp Cla1 fragment corresponding to nucleotides 3128-3974 at the 3' terminus of UT-A1. A schematic representation of these probes and the transcripts they are predicted to detect is shown in Fig. 1. Full-length (1,412 bp) rat UT-B (GenBank accession no. NP062219) was also used as a probe. Hybridization was 16 h at 42°C (50% formamide), and washing was at 65°C in 0.1× SSC (sodium chloride-sodium citrate), 0.1% SDS for 40 min.RT-PCR and Southern Blotting
Because of the low abundance of UT-A4 mRNA in the kidney (10), RT-PCR was employed to determine the presence of UT-A4 in kidney and testis. Total RNA (1 µg) from rat kidney IM or testis was reverse transcribed by using oligo (dT), Superscript II reverse transcriptase (Invitrogen), and the manufacturer's recommended protocol. PCR amplification was performed by using 0.1 µl of RT reaction, 20 pmol of forward (5'-CCTTGAGCACCGTCTTCGC) and reverse (5'-CAGGTGATGTTGGGTGTGG) gene-specific primers, 2.5 mM dNTPs, 1× PCR buffer containing 2 mM MgCl2, and 0.2 units of TaKaRa Ex Taq DNA polymerase (TaKaRa Biomedicals) in a total volume of 20 µl. The primers were designed to amplify a 154-bp UT-A4 product and/or a 1,543-bp UT-A1 product. After an initial denaturation step at 96°C for 2 min, PCR cycling conditions were 30 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 60 s, and a final extension at 72°C for 8 min. After electrophoresis through a 1% agarose gel, products were capillary transferred to Hybond-N filters and probed using probe 1 at high stringency (final wash at 65°C in 0.1× SSC-0.1% SDS).Antibodies
To determine the location of UT-A and UT-B proteins in the SMT, the following antisera were used: antiserum Q2, which was raised to the carboxy-terminal 14 amino acids (H2N-TAKRSDEQKPPNGG-COOH) of rat UT-A3 (26). This epitope is present in UT-A3 and putatively in rat UT-A5, although a cDNA encoding the rodent isoform has not been isolated. Antiserum L194, which was raised to the carboxy-terminal 19 amino acids (H2N-QEKNRRASMITKYQAYDVS-COOH) of rat UT-A1. This epitope is present in UT-A1, UT-A2, and UT-A4 (15, 25). L194 and Q2 were kind gifts from Dr. M. A. Knepper (National Heart, Lung, and Blood Institute, Bethesda, MD). Antiserum 3007 was raised to the carboxy-terminal 20 amino acids (H2N-SEENRIFYLQNKKSAVDRPL-COOH) of rat UT-B (28) and was a kind gift from Dr. M.-M. Trinh-Trang-Tan (Institut National de la Santé et de la Recherche Médicale U76, Paris, France).Protein Preparation and Immunoblotting
Male Wistar rats were killed by halothane anesthesia followed by cervical dislocation. The kidney IM and testis were then removed and homogenized in 5 ml of ice-cold isolation solution in a handheld Dounce homogenizer. This solution contained 300 mM mannitol and 12 mM HEPES, pH 7.6. The protease inhibitors pepstatin (final concentration 1 µg/ml), leupeptin (final concentration 2 µg/ml), and phenylmethylsulfonyl fluoride (PMSF; final concentration 1 µg/ml) were added to the isolation solution before homogenization. Homogenates were centrifuged at 2,000 g for 15 min at 4°C. Supernatants were saved and centrifuged at 200,000 g for 30 min at 4°C. Pellets were resuspended in isolation solution. Total protein concentrations were determined by using a protein assay kit (Bio-Rad), and sample concentrations were adjusted to 10 µg/µl in isolation solution. Laemmli buffer (5×) was added, and samples were heated to 60°C for 15 min before being loaded onto 6-8% polyacrylamide gels. SDS-PAGE was performed by using a Full-Range Rainbow Molecular Weight Marker (Amersham Pharmacia) for size determination, and the proteins were transferred electrophoretically to nitrocellulose membranes (Gelman Sciences). After being blocked for 1 h in 5% nonfat dry milk prepared in wash buffer [15 mM Tris (pH 8.0), 150 mM NaCl, and 0.1% Tween 20], membranes were probed with affinity-purified antibodies for 18 h at 4°C. Three 10-min washes in an excess of wash buffer were performed before addition of goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (HRP; Dako) at a final concentration of 0.075 µg/ml after dilution in 5% nonfat dry milk/wash buffer. Antibody-antigen reactions were visualized using luminol-based chemiluminescence (Amersham Pharmacia).Immunohistochemistry
Testis from Wister rats were removed and emersion-fixed in Bouin's solution for 2 h. Testis were cut transversely into three equally sized segments and fixed in Bouin's solution for a further 2 h. The tissue was dehydrated in an ascending series of ethanol concentrations (70-100%) and embedded in paraffin wax. Sections (5 µm) were mounted on Superfrost Plus slides (VwR International) and allowed to dry overnight at 37°C. After xylene treatment and rehydration of sections in a descending series of ethanol concentrations, endogenous peroxidase was blocked by 30 min of incubation in 3% hydrogen peroxide in methanol. Antigen retrieval was performed by boiling sections for 10 min in a solution containing 25 mM Tris · HCl (pH 8.0), 10 mM EDTA, and 50 mM glucose before sections were incubated overnight at 4°C with affinity-purified primary antibodies. Labeling was visualized with either an HRP-conjugated secondary antibody (P448; goat anti-rabbit 1:200; Dako), followed by incubation in diaminobenzidine, or, for fluorescent microscopy, with an affinity-purified indocarbocyanine (Cy3)-conjugated goat anti-rabbit secondary antibody (1:300, Jackson ImmunoResearch Laboratories).In Vitro Perfusion
We used in vitro perfusion to determine whether a phloretin-inhibitable urea pathway exists across the SMT. Male Wistar rats were killed as described in Protein Preparation and Immunoblotting. The testis was removed and separated from the testicular capsule. A randomly selected segment of SMT was manually dissected, mounted on glass pipettes, and secured in place with silk thread. The length of a perfused tubule varied from 0.8 to 7.1 mm with a mean of 3.5 ± 0.2 mm (n = 71). The tubule lumen and the peritubular surface were perfused with a physiological solution (in mM: 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 mannitol) containing [14C]urea (final concentration 4 µCi/ml; Amersham Pharmacia) and [3H]inulin (final concentration 0.4 µCi/ml; Amersham Pharmacia) as a volume marker. The final osmolarity of this solution was 310 ± 5 mosmol/kg. To prevent transtubular osmotic gradients, luminal and peritubular solutions were matched to within ±1 mosmol/kg. SMTs were perfused at 1.5 µl/min with a syringe pump (Infors). In some perfusion experiments, phloretin (0.5 mM final concentration, stock dissolved in ethanol), which is a known inhibitor of urea transporters, was included. An equivalent concentration of ethanol (0.1% vol/vol) was added to controls. Timed collections were made, and the isotope activity in the collectate was measured by liquid scintillation counting (Canberra Packard model 1900 CA) using a full-spectrum dpm program. Tubule images were captured with a digital imaging system (Electrim), and the length of the perfused tubule was measured using ScionImage (ScionCorp). The collection efficiency of the [3H]inulin marker was calculated from the equation
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Statistics
All values are quoted as means ± SE. Analysis was performed by t-tests or one-way ANOVA as appropriate by using SigmaStat for Windows, with significance assumed at the 5% level. If ANOVA indicated a significant difference, comparison between groups was performed with the Student-Newman-Keuls method. ![]() |
RESULTS |
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Northern Analysis, RT-PCR, and Southern Blotting
To determine the identity of urea transporter transcripts present in testis, we performed Northern analysis with probes designed to selectively recognize UT-A isoforms (Fig. 1). Kidney IM mRNA was used as a positive control for transcript identification. Probe 1 detected 4.0-, 3.6-, 3.0-, and 2.1-kb mRNA transcripts in kidney IM (Fig. 1A). In terms of molecular weight, these corresponded to the transcripts encoding UT-A1, UT-A3b, UT-A2, and UT-A3, respectively. In testis, four mRNA transcripts of ~4.0, 3.3, 2.8, and 1.7 kb were detected. Probe 2 detected mRNA transcripts of 4.0, 3.6, and 2.1 kb in kidney IM (Fig. 1B). On the basis of molecular weight, these relate to UT-A1, UT-A3b, and UT-A3 transcripts, respectively (2, 19). Transcripts of the size predicted for UT-A1b (3.5 kb) or UT-A4 (~2.5 kb) were not detected, confirming their low abundance, as previously reported (2, 10). Even after prolonged autoradiography, no mRNA transcripts were detected in testis using probe 2. Probe 3 detected transcripts of 3.6 and 2.1 kb in kidney IM (Fig. 1C). These represent UT-A3b and UT-A3 transcripts, respectively. Probe 3 detected transcripts of 3.3 and 1.7 kb in testis. The identity of the 3.3-kb transcript remains unknown. However, the 1.7-kb transcript is probably the rat homolog of UT-A5 because in the mouse, UT-A3 and UT-A5 share identical 3 untranslated regions (UTRs) (equivalent to probe 3) (7). Probe 4 detected a 3.0-kb band in kidney IM (Fig. 1D), the size expected for the UT-A2 transcript (21). Even after prolonged exposure, no mRNA transcripts were detected in testis using probe 4. Probe 5 detected transcripts of 4.0 and 3.0 kb in kidney IM (Fig. 1E). These represent the UT-A1 and UT-A2 transcripts, respectively. Even after prolonged autoradiography, no mRNA transcripts were detected in testis using probe 5. Table 1 summarizes the results of Northern analysis with UT-A-specific cDNA probes. The full-length UT-B probe detected a 3.8-kb mRNA transcript (Fig. 1F) in kidney IM and testis, as previously reported (29).
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RT-PCR, with a primer set optimized to differentiate between UT-A1 and
UT-A4, followed by high-stringency Southern blot analysis detected
UT-A1 and UT-A4 transcripts in kidney IM but only UT-A1 in testis (Fig.
2).
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Identification of Immunoreactive UT Proteins in Testis
Antiserum Q2.
Western analysis with affinity-purified antiserum Q2, raised to the 14 COOH-terminal amino acids of rat UT-A3, identified several bands in
kidney IM and testis samples enriched for plasma and vesicular
membranes. In kidney IM (Fig.
3A), the strongest protein
band was ~44 kDa in size. A broad diffuse band was also observed at
55-65 kDa. In testis, two protein bands at 46 and 70 kDa were
evident, along with a less intense band at 60 kDa (Fig. 3C).
These signals were ablated after preincubation of Q2 antiserum with
immunizing peptide (Fig. 3, B and D). Also, no signals were observed when antiserum Q2 was excluded and only the
secondary antiserum was applied (not shown).
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Antiserum L194.
was raised to the 19 COOH-terminal amino acids of rat UT-A1 and was
predicted to recognize UT-A1, UT-A2, and UT-A4. In accordance, Western
analysis with antiserum L194 identified several bands in kidney IM and
testis samples enriched for plasma and vesicular membrane proteins. In
kidney IM, we observed a broad protein band centered between 55 and 60 kDa, and less intense bands were evident at 98 and 120 kDa (Fig.
5A). In contrast, the testis
showed a different banding pattern, consisting of intense protein bands at 103 and 140 kDa, alongside weaker bands at 115 and 160 kDa (Fig.
5C). All bands were absent when antiserum was preincubated with immunizing peptide (Fig. 5, B and D) or when
primary antiserum was excluded (not shown).
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Antiserum 3007.
Western analysis with antiserum 3007, raised to the 20 COOH-terminal
amino acids of rat UT-B, detected an intense protein band of 98 kDa in
kidney IM and a number of less intense protein bands between 38 and 55 kDa (Fig. 7A). In testis, the
predominant protein bands were at 48 and 98 kDa, with a weaker protein
band at 70 kDa (Fig. 7B).
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In vitro perfusion of SMTs.
Addition of 5 mM urea to the bath resulted in the rapid appearance of
urea in the SMT lumen, with the rate of urea influx reaching a steady
state after about 20 min (Fig.
8A). Likewise, applying an
outward urea gradient caused urea to appear in the bath (Fig.
8B). Thus the imposed urea gradient determined the direction
of urea movement. Addition of 0.5 mM phloretin to the bath or lumen,
irrespective of the direction of the urea gradient, significantly
reduced the rate of urea influx (Fig. 8, A and
B). This finding strongly implicated UT-A, UT-B, or both in
transport of urea across the SMT.
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DISCUSSION |
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Urea transporters are highly expressed in the kidney where they play a central role in the urinary concentrating mechanism. These proteins are also expressed in several extrarenal tissues including liver, brain, colon, and testis (22). Urea is synthesized in the liver, and the role of urea transporters in this tissue, although yet to be proven, is probably associated with egress of urea from liver cells (11). Far less is known about the role these proteins play in other tissues not primarily associated with urea metabolism. We focused our research on the testis after the isolation of a novel cDNA from mouse testis encoding a phloretin-sensitive UT-A urea transporter (7). We observed that UT-A5 mRNA was located in an outer cell layer of all SMTs and that detectable levels of UT-A5 mRNA appeared around 20 days of age. This appearance coincided with the formation of the blood-testis barrier and the commencement of solute and water movement across the SMT (18). To clarify the role of urea transporters in the rat testis, we first undertook a detailed analysis of the expression of UT-A and UT-B mRNA transcripts. Second, we determined the location of UT-A and UT-B proteins in rat testis using peptide-targeted antisera. Finally, using in vitro perfusion of SMTs, we measured phloretin-inhibitable urea flux across SMT epithelia.
The results of Northern analysis clearly show the presence of UT-A transcripts in testis that differ in size from those detected in kidney IM. Using the 3' UTR of UT-A3, we detected 1.7- and 3.3-kb transcripts in testis that were not present in kidney IM. This result is in accord with that reported previously by Karakashian et al. (10), who, using an almost identical probe to our probe 3, also detected the 1.7- and 3.3-kb transcripts in testis but not in kidney.
In rat testis, a 4.0-kb transcript was detected by probe 1, the full-length UT-A1 probe, and RT-PCR analysis also showed that nucleotides 1068-2610 of UT-A1 were present in testis. Taken together, these results suggest the presence of UT-A1 mRNA. However, probe 2 bound to UT-A1 in kidney IM, whereas the 4.0-kb transcript in testis was not recognized by probe 2. Furthermore, probe 5 detected UT-A1 mRNA in kidney, but the 4.0-kb transcript in testis was not recognized. These results suggest that the 4.0-kb transcript in testis is not UT-A1 mRNA but possibly a similar molecule with alternate 5' and 3' sequences. We also detected a 2.8-kb transcript in testis using our full-length UT-A1 probe. This transcript was not recognized by any other UT-A probe. The finding that probe 2, corresponding to the 5' UTR of UT-A1, did not detect the 1.7-, 2.8-, 3.3-, or 4.0-kb transcripts indicates that they may have an alternative 5' sequence to UT-A1. It is conceivable that the 1.7-kb transcript is the rat homolog of UT-A5. This transcript lacks the 5' sequence of UT-A1 and, hence, would not be detected by probe 2. Instead, it has a unique 5'UTR and an alternative initiation codon at M139 of mouse UT-A1 (M138 of rat UT-A1). With regard to the 2.8-, 3.3-, and 4.0-kb transcripts, their pattern of hybridization to our set of cDNA probes does not equate to any known UT-A transcripts, and we therefore conclude that they represent novel, possibly tissue-specific, transcripts that encode novel UT-A isoforms. Clearly, experiments aimed at resolving the structure of these novel transcripts, such as 5' rapid amplification of cDNA ends, are required.
Immunohistochemical studies with three peptide-targeted antisera were used to determine the expression profiles of UT-A and UT-B proteins in testis. In rat, UT-A3 is a 460-residue protein with 100% identity to the first 399 amino acids of UT-A1 (10). Antiserum Q2 was raised to the COOH-terminal 14 amino acids of rat UT-A3, 13 of which are present in UT-A1. Terris et al. (26) have shown that antiserum Q2 binds to 44- and 67-kDa proteins in kidney IM. They showed that these bands represented UT-A3 in different glycosylation states. However, no signals corresponding to UT-A1 protein were observed at 98 or 117 kDa, prompting them to conclude that the Q2 antiserum preferentially bound to UT-A3 (26). In our analysis, the Q2 antibody identified a broad band at ~44 kDa and a protein smear between 55 and 65 kDa in kidney IM. Therefore, our results are in accord with those of Terris et al. and suggest that Q2 identified UT-A3 in different states of glycosylation. In contrast to the protein bands observed in the kidney, the strongest signals in testis emanated from 46- and 70-kDa proteins, whereas there was a weaker signal of 60 kDa. These results indicate that the UT-A isoforms detected by Q2 in the testis are different from those detected in kidney IM. These proteins are likely to represent some of the products of the 1.7-, 2.8-, 3.3-, or 4.0-kb transcripts.
Antiserum L194 has been shown to recognize UT-A1, UT-A2, and UT-A4 isoforms (15, 30). In our hands, L194 detected a broad protein band between 55 and 60 kDa in kidney IM corresponding to UT-A2 and possibly UT-A4, as well as distinct proteins of ~98 and 120 kDa corresponding to UT-A1. The sizes of these proteins are very similar to those reported previously (15, 30). In testis, intense protein bands of 103 and 140 kb were evident alongside faint bands of 115 and 160 kDa. Because Northern analysis could not detect any mRNA transcripts in testis for the characterized rat UT-A isoforms, these proteins are likely to represent a novel UT-A protein(s) that possesses the COOH-terminal UT-A1 epitope.
Western analysis with an anti-UT-B serum (antiserum 3007) identified a prominent immunoreactive protein of 98 kDa in kidney IM and weaker protein bands between ~38 and 55 kDa. In testis, we observed strong bands at 48 and 98 kDa and a weaker band at 70 kDa. These results are in accord with a recent study using an antiserum raised to the 19 COOH-terminal amino acids of human UT-B, which reported the expression of 41- to 54-kDa and 98-kDa protein bands in human kidney IM and 48- and 98-kDa protein bands in testis (27). The presence of a single 3.8-kb UT-B mRNA transcript in rat testis suggests that only one major UT-B isoform is expressed. Therefore, the multiple bands present on Western blots may represent UT-B in different states of glycosylation and/or multimers of UT-B.
To begin to understand the role of urea transporter proteins within the testis, we localized candidate UT-A and UT-B proteins using immunohistological techniques. Immunofluorescence studies using antiserum Q2 showed labeling of Sertoli cell nuclei in all SMTs, indicating that expression of the Q2 immunoreactive protein was not dependent on the stage of spermatogenesis. The role UT-A proteins play in Sertoli cell nuclei is very much open to speculation, because there is no evidence to suggest the formation of urea in Sertoli cell nuclei (4). It is difficult to explain these results in terms of urea transport, suggesting that urea transporters may actually fulfill a function different from and perhaps even independent of urea transport.
Antiserum L194 showed strong immunoreactivity in residual bodies of stage VIII SMTs. What possible physiological role does a urea transporter protein expressed in residual bodies fulfill? Residual bodies arise during the transformation of spermatids to spermatozoa. Cytoplasmic loss is essential for the formation of the compact spermatozoa head, and the residual bodies serve to compartmentalize the cytoplasm and organelles before phagocytosis by the Sertoli cell (23). Densification of the spermatids is associated with volume reduction due to substantial fluid loss, possibly mediated by aquaporins 7 and 8 (3, 24). Urea transporters expressed in residual bodies would allow urea to quickly exit the residual bodies and thus facilitate volume reduction.
In contrast to the results for antisera Q2 and L194, antiserum 3007 strongly labeled the Sertoli cells of stage II-III tubules. This localization is in strong accord with the report by Tsukaguchi et al. (29), who, using in situ hybridization, reported that UT-B mRNA is expressed within Sertoli cells in a subpopulation of SMTs. Tsukaguchi et al. did not precisely identify the stage of spermatogenesis expressing UT-B mRNA, but, in agreement with our study, they were able to exclude stages VII and VIII. Specifically, we found UT-B protein to be expressed primarily in the apical plasma membranes of Sertoli cells, but often in basolateral plasma membranes, as well. Because the Sertoli cell layer is generally regarded as the major barrier for solute entry into and out from the SMT lumen (18), UT-B is ideally situated to mediate urea flux out of Sertoli cells or across the SMT epithelia.
To test the hypothesis that urea transporters expressed in the testis provide a pathway for movement of urea across the SMT epithelium, we devised a method to perfuse isolated rat SMTs in vitro. By imposing a 5 mM urea gradient across the SMT membrane, we observed urea movement into or out of the SMT in the direction of the urea gradient. This movement suggests that urea was diffusing across the SMT cell membranes and Sertoli cells and that the blood-testis barrier formed by the tight junction between cells was no hindrance to diffusion. Introduction of 0.5 mM phloretin, to either the bath or the luminal perfusate, significantly reduced urea movement. Because urea transport by UT-B proteins is inhibited by phloretin (22), this finding strongly suggests that UT-B transporters mediate urea movement across SMT epithelia.
Tsukaguchi et al. (29) suggested that expression of UT-B in Sertoli cells may facilitate exit of urea generated during the synthesis of polyamines. Because UT-B is expressed on Sertoli cell membranes, this finding may indeed be true, although our data suggest that urea would pass into both the SMT lumen and blood rather than in one direction, as might be expected if urea was destined for the blood to be excreted.
In conclusion, we have identified four UT-A transcripts in testis that are not present in kidney IM and have shown that urea transporter proteins are expressed in a stage-dependent manner within the SMTs. We have also established that a phloretin-inhibitable urea pathway exists across the rat SMT epithelium and that UT-B is likely to participate in this pathway.
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ACKNOWLEDGEMENTS |
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We thank Dr. P. T. Hurley for help with the light microscopy and Drs. G. S. Stewart and D. T. Ward for helpful comments.
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FOOTNOTES |
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This work was supported by the Biotechnology and Biological Sciences Research Council (34/D10935), Wellcome Trust (043322/Z/94), and the Royal Society (C. P. Smith).
Address for reprint requests and other correspondence: C. P. Smith, School of Biological Sciences, G38, Stopford Bldg., Univ. of Manchester, Oxford Rd., Manchester M13 9PT, UK (E-mail: cpsmith{at}man.ac.uk).
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.
First published February 13, 2002;10.1152/ajpcell.00567.2001
Received 26 November 2001; accepted in final form 7 February 2002.
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