1 School of Biological Science, University of Manchester, Manchester M13 9PT, United Kingdom; and 2 Seconda Universita' degli Studi di Napoli, Facolta' di Medicina e Chirurgia, 1680138 Napoli, Italy
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ABSTRACT |
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Urea movement across plasma membranes is modulated by specialized transporter proteins that are products of two genes, termed UT-A and UT-B. These proteins play key roles in the urinary concentrating mechanism and fluid homeostasis. We have isolated and characterized a 1.4-kb cDNA from testes encoding a new isoform (UT-A5) belonging to the UT-A transporter family. For comparison, we also isolated a 2.0-kb cDNA from mouse kidney inner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNA has a putative open reading frame encoding a 323-amino acid protein, making UT-A5 the smallest UT-A family member in terms of molecular size. Its putative topology is of particular interest, because it calls into question earlier models of UT-A transporter structure. Expression of UT-A5 cRNA in Xenopus oocytes mediates phloretin-inhibitable urea uptake and does not translocate water. The distribution of UT-A5 mRNA is restricted to the peritubular myoid cells forming the outermost layer of the seminiferous tubules within the testes and is not detected in kidney. UT-A5 mRNA levels are coordinated with the stage of testes development and increase 15 days postpartum, commensurate with the start of seminiferous tubule fluid movement.
urea transporter; membrane protein; testes; mouse
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INTRODUCTION |
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UREA MOVEMENT ACROSS PLASMA membranes is modulated by specialized transporter proteins that play key roles in fluid homeostasis (1, 6). These proteins are important for modulating plasma and cellular levels of urea and play a central role in the urinary concentrating mechanism. In mammals they are derived from two genes, UT-A (Slc14a1) and UT-B (Slc14a2), that occur in tandem on chromosome 18 (5, 12, 13). These genes give rise to proteins that share a high degree of homology and are functionally similar.
To date, four splice variants of the UT-A gene have been characterized in rat. These proteins, UT-A1 (19), UT-A2 (21), UT-A3, and UT-A4 (8), are predominantly expressed in the kidney but are suggested to be also present in heart, liver, colon, and testes (6, 8, 9). The UT-B gene gives rise to two transcripts by differentially utilizing alternate polyadenylation signals (10). In contrast to UT-A, both transcripts encode the same protein and have a much wider tissue distribution (3).
The role urea transporters play in the renal concentrating mechanism is well established. However, the part played by urea transporters in other tissues is much less clear. Tsukaguchi and colleagues (22) recently localized UT-B (termed UT3 by the authors) to the Sertoli cells of the testis. This raises the question whether the transporter is implicated in urea movement across the blood testis barrier or, as Tsukaguchi et al. suggested, to allow exit of urea formed during the synthesis of protamine and polyamines for spermatogenesis. Fluid movement into the seminiferous tubules (SMTs) is a central component of the spermatogenic process (18), and the presence of high concentrations of urea in the seminiferous tubular fluid would favor osmotic water flow. Isotopic studies using [14C]urea have shown that urea moves into the SMT across the blood-testis barrier and reaches concentrations greater than those found in plasma (23). It is unlikely that urea generated in Sertoli cells during the synthesis of protamine and polyamines is responsible for this build up of urea; hence, a mechanism must exist for the movement of urea against its concentration gradient into the SMT.
Recent studies in our group and by others have identified unique mRNA species homologous to UT-A in mouse and rat testis (8). Because the proteins these transcripts encode may play a key role in urea movement across the blood-testis barrier, our aim was to determine the molecular structure of the transcript and determine the cells in which it is located and whether it is implicated in testicular fluid movement.
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METHODS |
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Clone Isolation
Clones were isolated from either an unamplified, size selected (>1 kb) MF1 mouse testesBoth strands of the cDNA clones were sequenced using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems), and data were analyzed using the Lasergene program (DNAstar).
Xenopus Oocyte Expression Experiments
These were performed as previously described (21). cDNA encoding hAQP2 (4) was a generous gift of Dr. P. Deen (Univ. of Nijmegen). The plasmids containing rUT-A2 (21), mUT-A3, mUT-A5, and hAQP2 were linearized using Not I, EcoR I, Hind III, or Hinc II, respectively, and cRNA was prepared using the mCAP RNA capping kit (Stratagene). The cRNAs for rUT-A2 and hAQP2 were prepared using T7 polymerase, and cRNA for mUT-A3 and mUT-A5 was prepared using T3 polymerase. After injection oocytes were incubated for 3 days, and then [14C]urea uptake was measured as previously described (21).Quantifying Oocyte Osmotic Water Permeability
Values for the osmotic water permeability (Pf) were obtained by measuring the initial rate of oocyte swelling on exposure to a hypotonic solution containing (in mM) 27 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES. A silhouette image of the oocyte was captured every 10 s using a digital imaging system (Electrim). The sampled images were analyzed using Scion Image (Scion), a personal computer conversion of NIH Image for Macintosh. In all calculations, the oocyte is assumed to be a sphere of average initial surface area of 0.045 cm2 and volume 0.0009 cm3 (15). Pf was computed from the equation: Pf = [V0×d(V/V0)/dt]/[S×Vw×(osmiNorthern Analysis
To study the distribution of urea transporter mRNA in mouse tissue, poly(A+) RNA was isolated from MF1 mouse forebrain, cerebellum, heart, lung, liver, spleen, kidney cortex, kidney IM, testes, epididymis (head, middle, and tail), small intestine, colon, and skeletal muscle by the guanidinium isothyocyanate method followed by an oligo(dT)-cellulose (Amersham Pharmacia Biotech) batch method. Poly(A+) RNA (3 µg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred to Hybond-N filters (Amersham Pharmacia Biotech). Filters were probed using either a 32P-labeled 206-bp fragment corresponding to nucleotides 1,744-1,949 at the 3'-terminus of mUT-A3 (probe 1) or a 32P-labeled Hinc II-Hinf I fragment corresponding to nucleotides 1-168 at the 5'-terminus of UT-A5 (probe 2). Probe 1 would be expected to recognize isoforms UT-A3 and UT-A5; probe 2 would recognize the UT-A5 isoform only. Hybridization was for 16 h at 48°C (50% formamide) and washing at 55°C (medium stringency) or 65°C (high) in 0.1× SSC-0.1% SDS.In Situ Hybridization
Digoxigenin-11-UTP (DIG)-labeled probes were synthesized by in vitro transcription (DIG RNA Labeling Kit, Boehringer Mannheim). Sense and antisense probes corresponding to nucleotides 1-168 of UT-A5 (probe 2, in TOPOII, Invitrogen) were generated after linearization of the plasmid DNA with Hind III or Not I, using T7 (sense) or SP6 (antisense) RNA polymerase, respectively.Mouse testes were removed and fixed in 4% paraformaldehyde (PFA) in
PBS for 1 h. Testes were then sliced transversely in half and
fixed in 4% PFA for a further 1 h. Testes were rinsed in 30% sucrose-PBS and left overnight at 4°C in fresh 30% sucrose. Samples were then frozen in liquid nitrogen and stored at 80°C.
Cryosections (10 µm) were mounted on Superfrost Plus slides (Merck)
and postfixed in 4% PFA-PBS for 20 min at room temperature. Mounted
sections were rinsed in PBS, incubated for 10 min in a buffer
containing 2 µg/ml proteinase K (Sigma), 40 µg/ml RNAse A (Sigma),
20 mM Tris · HCl, and 2 mM CaCl2, pH 7.4, acetylated for 20 min in 0.1 M triethanolamine-0.25% acetic anhydride,
and rinsed in PBS. Next, sections were dehydrated through 70/80/100%
ethanol and left to air dry for 30 min. Slides were prehybridized at
42°C for 2 h in a solution containing 50% formamide, 50 mM
Tris · HCl (pH 7.5), 25 mM EDTA, 22.5 mM NaCl, 0.1% SDS, 0.25 mg/ml yeast tRNA, and 2.5× Denhardt's. Hybridization was performed at
42°C for 18 h in a solution containing 10 ng/µl of probe, 50%
formamide, 25 mM Tris · HCl (pH 7.5), 9.7 mM DTT, 42 mM NaCl,
75 ng/ml yeast tRNA, 100 µg/ml salmon sperm DNA, 1.25× Denhardt's,
and 12.5% dextran sulfate. Sections were rinsed twice in 2× SSC,
followed by consecutive 1-h washes at 49°C in 1× SSC-50% formamide,
0.5× SSC-50% formamide, and 0.25× SSC-50% formamide. After a 5-min rinse in 0.25× SSC, detection was performed using a 1:500 dilution of
anti-digoxigenin Fab fragments (Boehringer-Mannheim) and
5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium
substrate. Sections were developed in substrate solution for 4-6
h, rinsed in distilled water, and placed under a coverslip with
Glycergel (Dako).
Global Representative cDNA Amplification
The method as described by Brady and Iscove (2) was employed for global representative testes cDNA amplification from mice at different stages of development. Male L97 BL/6 mice were killed 0, 5, 10, 15, 20, 25, 30, and 35 days postpartum. Testes were removed and quickly frozen in N2 and stored atStatistics
The rates of urea uptake and oocyte osmotic water permeability were compared using one-way ANOVA or unpaired Students t-tests as appropriate. If the ANOVA indicated a difference, treatment comparison between groups was performed using the Student-Newman-Keuls method. Values are quoted as means ± SE, and statistical significance is assumed at the 5% level. ![]() |
RESULTS |
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Homology Screening Experiments.
Kidney medulla.
High-stringency screening of a mouse kidney IM cDNA library with probe
pmUT-A1 yielded a 1,971-bp cDNA (GenBank accession no. AF258602).
Analysis of the nucleotide sequence revealed a putative open reading
frame (ORF) from nucleotides 358 to 1,740, a polyadenylation signal
(ATTAAA) at position 1,920, and a poly(A) tail. The ORF predicts a
461-residue protein, the mouse homologue of rat UT-A3 [Fig.
1A (8)]. Mouse
UT-A3 has 93% amino acid identity with rat UT-A3 (8),
68% identity with rat UT-A2 (21), and 63% identity with
rat UT-B [Fig. 2 (22)].
There are two putative N-glycosylation sites (N-I/N-T-W/G)
at N-280 and N-424, four potential protein kinase C (PKC) sites at
Ser-24, Ser-88, Ser-206, and Ser-448, and two potential protein kinase
A (PKA) sites at Ser-85 and Ser-92.
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Testis. High-stringency screening of a mouse testes cDNA library with probe pmUT-A1 yielded a 1,440-bp cDNA (GenBank accession no. AF258601). Analysis of the nucleotide sequence revealed a putative ORF from nucleotides 234 to 1,202 that predicts a 323-residue protein (Fig. 1B), a polyadenylation sequence (ATTAAA) at position 1,382, and a poly(A) tail. In accordance with the nomenclature suggested by Sands et al. (17), we designated the protein encoded by this cDNA UT-A5. The ORF is flanked by 233 bp at the 5'-end, of which the first 176-bp has no homology to other known UT-A transcripts. The 3'-untranslated region (UTR) is 235-bp and has 100% homology to the 3'-UTR of mouse UT-A3. From its predicted start, methionine (M139 of UT-A3) UT-A5 has 100% amino acid identity with mouse UT-A3. There are two putative N-glycosylation sites (N-I/N-T-W/G) at N-142 and N-286 and two PKC sites at Ser-68 and Tyr-310. Deduced amino acid sequences of mouse UT-A3 and UT-A5 and alignments with rat UT-A2, rat UT-A3, and rat UT-B are shown in Fig. 2.
Effect of mUT-A3 or mUT-A5 Expression on Oocyte Urea Permeability
As demonstrated previously (21), expression of the cRNA encoding rUT-A2 elicited a phloretin-sensitive increase in oocyte urea permeability. Urea uptake was increased approximately sevenfold compared with H2O-injected control oocytes (Fig. 3). Expression of cRNA encoding mouse UT-A3 or mouse UT-A5 increased urea uptake approximately five- and threefold, respectively, above water-injected levels. Urea influx into oocytes expressing rat UT-A2, mouse UT-A3, or mouse UT-A5 was substantially reduced by 0.5 mM phloretin (Fig. 3). Phloretin reduced rat UT-A2-mediated influx by 67%, mouse UT-A3-mediated influx by 75%, and mouse UT-A5-mediated influx by 53% compared with respective controls in the absence of inhibitor. Phloretin had no effect on urea uptake in H2O-injected control oocytes. These properties are consistent with those of previously characterized mammalian, phloretin-sensitive, facilitative urea transporters (8, 17, 22).
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Effect of Expressing UT-A3 or UT-A5 on Pf
As expected, expression of hAQP2 increased oocyte water permeability. In our hands, hAQP2 increased Pf ~20-fold, in line with previous reports (11). We tested the effect of expressing mouse UT-A3 or UT-A5 on Pf and found no effect on oocyte osmotic water permeability (data not shown).Northern Analysis and Tissue Distribution
Medium stringency Northern analysis using probe 1 resulted in strong signals in testes (1.4 kb) and kidney IM (2.1 kb) (Fig. 4A). These signals corresponded to UT-A5 and UT-A3, respectively. Weaker signals of 3, 3.6, and 4 kb were also detected in IM. The molecular identity of these transcripts remains unresolved. No other transcripts were detected in any other tissues tested. High-stringency Northern analysis using probe 2 gave a strong signal at 1.4 kb in testes. This transcript was not detected in any other tissue, including epididymis (Fig. 4B).
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Localization of UT-A5 mRNA in Testis
In situ hybridization with a UT-A5 antisense probe (probe 2) revealed strong signals in the SMTs (Fig. 5a). Visualization using a higher magnification (Fig. 5b) showed the signal resided in the peritubular myoid cells that form the outermost layer of the SMTs. All tubules were heavily stained. Control experiments with a sense strand probe gave no specific staining (Fig. 5c).
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Global Representative cDNA Amplification
UT-A5 cDNA was not detected in samples amplified from testes of mice aged 0, 5, and 10 days postpartum. Levels increased between 15, 20, and 30 days postpartum (Fig. 6) and remained elevated at 35 days postpartum.
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DISCUSSION |
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Urea transport in mammals is mediated by a family of proteins that are the products of two closely related genes, UT-A (6, 8, 17) and UT-B (14, 17, 22). These genes have been most extensively studied in rat, and, to date, four UT-A isoforms and one UT-B isoform have been characterized. Recent evidence supports the existence of novel UT-A homologues in rat and mouse kidney and testis (8). We set out to discover the molecular identity of a 1.4-kb transcript we had detected in mouse testis as a basis for studying the role of urea transporters in testicular function. We isolated a cDNA with a putative ORF that predicts a 323-amino acid protein, UT-A5. For comparison, we also isolated a cDNA encoding the mouse homologue of UT-A3. The fact that UT-A5 has a unique 176 nucleotide 5'-UTR and thereafter the nucleotide sequence is identical to UT-A3 indicates that UT-A5 is the result of alternative splicing of the UT-A gene. The identity between UT-A3 and UT-A5 begins at bp 715 in mouse UT-A3 (bp 731 in rat UT-A3). The putative initiation codon of mouse UT-A3 occurs at bp 358 (bp 377 in rat); in UT-A5 this is absent and the first possible ORF begins at bp 32 of UT-A5 and terminates at bp 181. The next ORF begins at bp 234 and ends at bp 1202, and we suggest that this is the coding region of UT-A5. Comparison of the amino acid sequences of UT-A3 and UT-A5 reveals that the latter protein does not have the NH2-terminal 139 amino acids. This means that UT-A5 does not have the consensuses for the putative PKC and PKA regulatory sites that are present in the NH2-terminal hydrophilic region of UT-A3. However, all the putative glycosylation and regulatory consensuses COOH-terminal to amino acid 139 of UT-A3 are present in UT-A5. It will be interesting to determine whether these structural differences confer any functional and regulatory differences.
Injection of mouse UT-A3 or UT-A5 cRNA into Xenopus oocytes induced an increase in urea permeability. Although the absolute amount of urea influx was less than that in oocytes expressing rat UT-A2, no direct functional comparison can be made because the levels of UT-A protein expressed were not measured. Characteristic of urea transport by UT-A proteins, phloretin was found to inhibit the urea flux. Expression of UT-A3 or UT-A5 in oocytes did not increase osmotic water permeability. In agreement, UT-B does not translocate water (20) when expressed at levels akin to those found physiologically. Together our findings reinforce the current dogma that UT-A and UT-B proteins do not translocate water.
As a general point, it is interesting to note that UT-A5 has the basic functional properties of other UT-A family members, yet it is by far the smallest UT protein characterized to date. UT-A5 may, therefore, represent the minimal functional unit for the family as a whole.
From a structural viewpoint, UT-A5 raises some interesting questions.
Compared with the primary structure of the other UT-A proteins, the
NH2-terminus of the molecule begins in a region that is
predicted to be membrane spanning. This in itself calls into question
previous putative topological models that incorporate -helical
membrane-spanning domains symmetrically arranged around a putative
extracellular loop (6, 8). Applying the criteria used to
construct these models to UT-A5 reveals that one or more of the
membrane-spanning domains are absent. This implies that either the
previous models are correct and UT-A5 indeed lacks the first
membrane-spanning domains or, and this is what we feel to be more
likely, the models are incorrect and a reevaluation is required based
on empirical findings rather than purely computer-based predictions.
Our Northern analysis showed that, of the tissues tested, UT-A5 is only found in testis. Our analysis is of course constrained by the detection limits of Northern blotting; however, our findings do agree with previously published findings in rat. Karakashian et al. (8) detected 1.7- and 3.3-kb transcripts in rat testis that were not detected in any other tissue analyzed. The 1.7-kb transcript in rat is probably the rat UT-A5 homologue. The larger transcript was not detected in mouse testis, and in rat the molecular identity remains uncharacterized. It is worth noting that UT-A5 mRNA was not detected in kidney cortex or kidney IM. In this study we did not directly address the question whether UT-A5 is present in outer medulla because of the difficulty associated with dissecting outer medullary tissue in mouse. However, we performed Northern analysis on poly(A+) mRNA enriched for outer medullary transcripts and on whole kidney (data not shown) and found no indication of expression of UT-A5. We therefore conclude that UT-A5 is the first nonrenal UT-A isoform.
In situ hybridization showed UT-A5 mRNA to be present in the peritubular myoid cells surrounding the SMTs. The signal was present in all tubules, indicating that expression of UT-A5 is not linked to the maturation stage of spermatocytes. This contrasts in two ways to UT-B mRNA in that UT-B mRNA localizes to Sertoli cells and appears to be upregulated in the early stages of spermatocyte development (22). Because the peritubular cells form a cellular barrier between the testis interstitium and the Sertoli cells, we suggest that UT-A5 plays a role in translocating urea into the SMT and in so doing is implicated in fluid movement into the SMT. This conclusion is supported by the finding that UT-A5 mRNA expression begins around 15 days postpartum, the point at which the barrier to the entry of macromolecules into the SMT and the intratubular secretion of fluid is first detected (16). Previous studies in rats have shown that intravenous injections of [14C]urea result in accumulation of radiolabeled urea in the SMT (23). This implies that urea moves into the SMTs and that movement is against a urea concentration gradient. This suggests an "active" mechanism for movement of urea is present in SMT epithelia. UT-A5 is unlikely to be responsible for "uphill" translocation of urea because UT-A family members are characteristically facilitative transporters. However, we suggest that UT-A5 is involved in urea movement into the SMT and, in conjunction with an as yet uncharacterized active urea transporter protein, provides a means of translocating urea across the peritubular epithelium.
UT-B isoforms have a much broader tissue distribution than UT-A isoforms and have been detected in kidney (12), brain (3), liver (12) and testes (22). With in situ hybridization, UT-B mRNA has been detected in Sertoli cells within rat testis (22). The role of a urea transporter within these cells is unknown. However, it has been suggested that the UT-B expressed in Sertoli cells allows the exit of large volumes of urea formed during the synthesis of polyamines, such as spermidine and spermine, in early spermatocyte development. However, Sertoli cells form part of the blood-testes barrier and must be traversed by solutes entering the SMTs. Therefore, it is possible that as well as serving as an exit pathway for urea formed in Sertoli cells, UT-B may also play a role in the blood testes movement of urea. Consequently, knowing the exact cellular location of both UT-A and UT-B proteins in testis would be of great benefit.
In summary, we isolated and characterized a novel form of the UT-A family of urea transporters named UT-A5. We found its functional characteristics to be similar to those of other known isoforms of the UT-A family. UT-A5 mRNA localized to the peritubular myoid cells of the SMT and was not detected in any other tissue tested. UT-A5 protein is implicated in urea movement across the blood-testis barrier.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. G. Brady, University of Manchester, for expert advice regarding global cDNA amplification.
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FOOTNOTES |
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This work was supported by Biotechnology and Biological Sciences Research Council Grant No. 34/D10935 (C. P. Smith) and the Royal Society (C. P. Smith).
Address for reprint requests and other correspondence: C. P. Smith, School of Biological Sciences, Univ. of Manchester, G38, Stopford Bldg., 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.
Received 9 June 2000; accepted in final form 15 June 2000.
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