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Am J Physiol Cell Physiol 293: C305-C312, 2007. First published May 2, 2007; doi:10.1152/ajpcell.00608.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Reduced urea flux across the blood-testis barrier and early maturation in the male reproductive system in UT-B-null mice

Lirong Guo,1,* Dan Zhao,1,2,* Yuanlin Song,2 Yan Meng,1 Huashan Zhao,1 Xuejian Zhao,1 and Baoxue Yang2

1Research Center of Prostate Diseases, Department of Reproductive Pathophysiology, School of Basic Medicine, Jilin University, Changchun, Jilin province, China; and 2Department of Medicine, University of California, San Francisco, California

Submitted 6 December 2006 ; accepted in final form 28 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A urea-selective urine-concentrating defect was found in transgenic mice deficient in urea transporter (UT)-B. To determine the role of facilitated urea transport in extrarenal organs expressing UT-B, we studied the kinetics of [14C]urea distribution in UT-B-null mice versus wild-type mice. After renal blood flow was disrupted, [14C]urea distribution was selectively reduced in testis in UT-B-null mice. Under basal conditions, total testis urea content was 335.4 ± 43.8 µg in UT-B-null mice versus 196.3 ± 18.2 µg in wild-type mice (P < 0.01). Testis weight in UT-B-null mice (6.6 ± 0.8 mg/g body wt) was significantly greater than in wild-type mice (4.2 ± 0.8 mg/g body wt). Elongated spermatids were observed earlier in UT-B-null mice compared with wild type mice on day 24 versus day 32, respectively. First breeding ages in UT-B knockout males (48 ± 3 days) were also significantly earlier than that in wild-type males (56 ± 2 days). In competing mating tests with wild-type males and UT-B-null males, all pups carried UT-B-targeted genes, which indicates that all pups were produced from breeding of UT-B-null males. Experiments of the expression of follicle-stimulating hormone receptor (FSHR) and androgen binding protein (ABP) indicated that the development of Sertoli cells was also earlier in UT-B-null mice than that in wild-type mice. These results suggest that UT-B plays an important role in eliminating urea produced by Sertoli cells and that UT-B deletion causes both urea accumulation in the testis and early maturation of the male reproductive system. The UT-B knockout mouse may be a useful experimental model to define the molecular mechanisms of early puberty.

urea transporter; Sertoli cell; testis; male sexual function; spermatogenesis

THE UREA TRANSPORTERS (UTs) are a family of membrane proteins that play a role in vectorial transcellular urea transport in response to a urea gradient (1–4, 22, 23, 29). In mammals, at least seven urea transporters (UT-A1 to UT-A6 and UT-B) have been characterized (1, 24, 27, 29). UT-A1, UT-A2, UT-A3, and UT-A4 are expressed in the kidney (UT-A4 was only found in the rat) (23, 34). UT-A5 is expressed in the testis (10). UT-A6 localizes to the colon (28). UT-B is expressed in the kidney, erythrocyte, brain, heart, liver, colon, bone marrow, spleen, lung, skeletal muscle, bladder, prostate, and testis (7, 15). Phenotype analysis of UT-A1/A3, UT-A2, and UT-B knockout mice has confirmed the role of UTs in transepithelial and endothelial urea transport in the kidney (8, 32, 35). Deletion of UTs results in a urea-selective urine-concentrating defect (11, 34). The functional significance of UTs in other tissues, where the urea concentration is probably not very high, is less clear. In the testis, UT-A5 is expressed in the outer cell layer of seminiferous tubules (10), and UT-B is expressed in Sertoli cells of seminiferous tubules (9, 31).

The Sertoli cells in seminiferous tubules have significant arginase activity that hydrolyzes arginine into urea and ornithine. Ornithine is then further metabolized in the polyamine pathway. Thus, Sertoli cells must excrete large amounts of urea arising from the polyamine pathway. Another key function of Sertoli cells is to form a blood-seminiferous tubule barrier that is responsible for maintaining the unique microenvironment conducive to spermatogenesis. The seminiferous tubule epithelium is made of a complex association of somatic and germ cells. The Sertoli cell layer is generally regarded as the major barrier for solute entry into and out from the seminiferous tubule lumen. It has been suggested that UT-A5, UT-B, or both transport urea across the seminiferous tubules (9). Tsukaguchi et al. (31) suggested that expression of UT-B in Sertoli cells facilitates the exit of urea generated during the synthesis of polyamines.

The present study utilized transgenic UT-B-deficient mice to test the hypothesis that UT-B deletion results in decreased urea permeability across the blood-testis barrier and, thus, altered male reproductive function. Therefore, the primary objective of this investigation was to study the role of a known UT in facilitating urea transport between Sertoli cells and blood. Using in vivo models, we found that UT-B deletion reduced urea transport across the blood-testis barrier. The secondary objective of this investigation was to investigate spermatogenesis in UT-B-null mice. The pattern of the first wave of spermatogenesis as a function of sexual maturity was defined in the UT-B-null males versus wild-type males from the same litter. Using the time of first appearance of elongated sperm in the testis and the first breeding episode in a competing mate test, we unexpectedly found that spermatogenesis and fertilizing ability occur 1 wk earlier in UT-B-null male mice compared with wild-type male mice, which indicates that UT-B deletion causes early maturation in the male reproductive system.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
UT-B knockout mice. Transgenic knockout mice deficient in UT-B protein were generated by targeted gene disruption as previously reported (35). UT-B-null mice did not express detectable UT-B protein in any organ. The adult male wild-type and UT-B-null mice used in this study had a CD1 genetic background. Measurements were done in litter-matched mice produced by intercrossing heterozygous mice. All animal procedures were approved by the University of California-San Francisco Committee on Animal Research.

RT-PCR and fluorescence-based real-time PCR. Total RNA from mouse tissues was reverse transcribed with oligo(dT) (SuperScript II preamplification kit, Invitrogen, Carlsbad, CA). PCR was carried out by the Gene Amp PCR system 9700 and with Taq DNA polymerase (Invitrogen) using the following primers: 5'-CCTTCCCCTGCTGGAAATGCC-3' (sense) and 5'-CTATGTGGCTGTCTTCATCTG-3' (antisense) for UT-A1 and UT-A3; 5'-TTTCTCCAGTCCTATCTGAG-3' (sense) and 5'-ACGGTCTCAGAGCTCTCTTC-3' (antisense) for UT-A2; 5'-TGACAGTGAGACGCAGTGAAG-3' (sense) and 5'-GGAAAGTGTGTGCTTGGGTG-3' (antisense) for UT-A3 and UT-A5; 5'-TCTTCTCAAACAAGGGCGAC-3' (sense) and 5'-TTGCTGAGCACGGAGCTCAA-3' (antisense) for UT-B; and 5'-TGTATGCCTCTGGTCGTACC-3' (sense) and 5'-CAGGTCCAGACGCAGGATG-3' (antisense) for beta-actin as a reference. Primer sequences were derived from published sequences with GenBank Accession Nos. AF366052 (UT-A1), AF367359 (UT-A2), AF258602 (UT-A3), AF258601 (UT-A5), AF448798 (UT-B), and NM007393 (beta-actin). Each primer set was checked using a BLAST search to ascertain that the sequences were unique for each mouse UT. PCR products of all primer sets were between 100 and120 bp and crossed an exon-intron boundary to prevent genomic DNA contamination. PCR products were electrophoresed on a 1.5% agarose gel.

Fluorescence-based real-time PCR was carried out by the LightCycler with the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics, Indianapolis, IN). Primers for androgen-binding protein (ABP) and follicle-stimulating hormone (FSH) receptor (FSHR) were designed as follows: 5'-TGCAACCTGGACTGTTCTTCCCTC-3' (sense) and 5'-GGTCCTTGGCTCAAGACCACTTTG-3' (antisense) for ABP; and 5'- AGTCACTGCTGTGCCTTTGCAAAC-3' (sense) and 5'-TGATGGCCAGGATGCTGATAAACC-3' (antisense) for FSHR. Real-time PCR was carried out according to the manufacturer's instructions. beta-Actin was used as the reference gene, and pooled wild-type cDNA was used as the calibrator. Results are reported as calibrated ratios. All samples were normalized to the reference gene. Concentration ratios for each sample were then calibrated so that the results are reported as the normalized ratio as follows: normalized ratio = ratio of sample (target/reference)/ratio of calibrator x (target/reference).

Urea permeability measurements. Mice were anesthetized during the experiment by methoxyflurane inhalation (Methofane, Mallinckrodt Veterinary, Mundelein, IL) in 100% O2. Mice were kept supine throughout the study. An incision was made in the abdominal wall. Renal blood vessels were tied with 3-0 silk purse-string sutures; 0.1 ml of [14C]urea (1 mCi/ml, New England Nuclear, Bedford, MA) was infused in the tail vein. Blood and tissues were obtained at 5 min after [14C]urea infusion. [14C]urea radioactivity in the blood and tissue homogenization was assayed by scintillation counting. Urea distribution rates were expressed as the ratio of [14C]urea radioactivity [in counts/min (cpm)] in tissue to that in serum per unit weight.

Tissue urea measurements. Tissue homogenates were obtained by homogenizing tissue in a 15-fold excess of distilled water, and the supernatant after centrifugation was then assayed for urea. The urea concentration was measured by colorimetry using a commercial kit (Roche Diagnostic).

Histological examination. One testis per mouse was subjected to histological examination at selected ages from days 10 to 84. For light microscopic analysis, testis tissue samples were fixed in Bouin's fixative overnight, embedded in paraffin, cut in 3-µm-thick sections, and stained with hematoxylin and eosin. Histological examinations were performed by a reviewer who was blinded to the genotypes. The inner and outer diameters of seminiferous tubules were recorded. Elongated spermatids were observed in serial sections (>5) of whole testis from each animal.

Western blot analysis. Tissues were homogenized with a glass Dounce homogenizer in 250 mM sucrose containing 1 mM EDTA, 20 µg/ml PMSF, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin (pH 7.4) and centrifuged at 4,000 g for 15 min to remove whole cells, nuclei, and mitochondria. Total protein was assayed in supernatant fractions using the Bio-Rad DC protein assay kit (Bio-Rad, Richmond, CA) and loaded on a 12% SDS-PAGE gel (10 µg/lane). Proteins were transferred to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI) and immunoblotted with a 1:1,000 dilution of rabbit polyclonal serum raised against a COOH-terminal peptide (NH2-DNRIFYLQNKKRMVESPL-COOH) of mouse UT-B by standard procedures (16).

Immunofluorescence. Testis tissue samples were fixed with 4% paraformaldehyde in PBS for 4 h, infiltrated with 30% sucrose in PBS overnight, frozen in OCT with liquid nitrogen, and cut in 3-µm-thick sections using a cryostat. Tissues were immunostained by standard procedures as previously described (36).

Sperm count and shape analysis. Cauda epididymes were isolated and minced in 1 ml PBS. Tissue fragments were removed by filtration through mesh. Suspended sperm were counted using a hemacytometer. An aliquot of the suspension was smeared on a glass slide, fixed in methanol for 5 min, and stained in eosin for 1 h. One thousand sperm were examined for each mouse at x400 total magnification. Abnormal sperm were recorded as hookless, banana-like, or amorphous as described by Wyrobek and Bruce (33).

Mating studies. A 35-day-old UT-B-null male mouse and a wild-type male mouse from the same litter were mated with a 70-day-old wild-type female in each competing mating group. Two wild-type males were mated with a wild-type female in each control group. Animals were maintained under well-controlled conditions of temperature (22°C), light, and humidity with food and water provided ad libitum. The age of male mice was recorded when the first litter was delivered in each group. Breeding ages of male mice were deduced by deducting 21 days from the ages at delivery time. The size of the litter and gender of pups were recorded. All pups were genotyped as mentioned above.

Data analysis. Statistical comparisons between UT-B-null and wild-type mice were made using Student's t-test.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
UT-B expression in the mouse testis was first determined by RT-PCR. Figure 1A shows that DNA fragments for UT-B were amplified from testis cDNA in wild-type mice (+/+) and UT-B heterozygous mice (+/–). No positive band was found in UT-B null mice (–/–). A 45-kDa protein was found in testes and red blood cells in wild-type mice but not in UT-B knockout mice by Western blot analysis (Fig. 1B). Immunofluorescence confirmed UT-B protein expression in Sertoli cells in wild-type mice using an antibody against UT-B (Fig. 1C, left), consistent with previous findings in the rat (9). Staining of the testis from UT-B knockout mice was negative (Fig. 1C, right).


Figure 1
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  		Fig. 1. Urea transporter (UT)-B expression in the testis. A: UT-B mRNA expression in testes from the indicated genotypes by RT-PCR analysis (top). beta-Actin was used as the reference gene (bottom). B: Western blot of mouse red blood cells (RBCs) and testes using polyclonal UT-B antiserum. C: immmunofluorescence of UT-B antibody staining of testes of wild-type mice (left; +/+) and UT-B-null mice (right; –/–). +/–, Heterozygous mice. L and B indicate the lumen and basal layer of the tubule, respectively. Arrowheads indicate positive staining. Scale bar = 50 µm.

 
RT-PCR was done to determine whether other UT transcripts are detectable in the mouse testis. Reverse-transcribed cDNA was the template for UT-specific primers that amplify the coding sequences of UT-A isoforms. Figure 2A, top, depicts the DNA fragments amplified from testis cDNA using common primers for UT-A3 and UT-A5. There were no detectable PCR products from common primers for UT-A1 and UT-A3, nor from primers for UT-A2. The positive control, shown in Fig. 2A, bottom, used kidney cDNA, which is known to express UT-A1, UT-A2, and UT-A3 with the same primers used for the testis. These results indicate that UT-A1, UT-A2, and UT-A3 are not expressed in the mouse testis, but UT-A5 is expressed in the mouse testis. Relative quantification of UT-A5 mRNA in wild-type and UT-B-null testes was determined by fluorescence-based real-time RT-PCR. The normalized, calibrated ratios of UT-A5 transcript levels are shown in Fig. 2B. There was no difference in UT-A5 mRNA expression in UT-B-null mice compared with wild-type mice.


Figure 2
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  		Fig. 2. Transcript expression of UT-A isoforms in the mouse testis. A: regular RT-PCR analysis. PCR was performed using testis (top) and kidney (bottom) cDNA as the template and with UT-A1 and UT-A3 common primers, UT-A2 primers, UT-A3 and UT-A5 common primers, and beta-actin primers. B: fluorescence-based real-time RT-PCR. Real-time PCR was carried out by a LightCycler and with the LightCycler FastStart DNA MasterPLUS SYBR Green I kit. mRNA expression levels for each sample are expressed relative to the wild-type testis level, which was arbitrarly considered equal to 1.0 in each individual comparison. Data are means ± SE; n = 6 mice of each genotype.

 
Urea concentrations in serum and testes in UT-B-null mice (9.3 ± 0.6 mM and 57.5 ± 2.6 mmol/kg tissue weight, respectively) were significantly higher than those in wild-type mice (7.6 ± 0.1 mM and 46.9 ± 1.5 mmol/kg tissue weight). Total testis urea content was 335.4 ± 43.8 µg in UT-B-null mice versus 196.3 ± 18.2 µg in wild-type mice (P < 0.01). There were no differences in the brain and liver between UT-B-null and wild-type mice (Fig. 3A). Urea distribution measurements were done using knockout mice to determine whether UT-B facilitates urea movement through the blood-brain barrier and blood-testis barrier. Renal blood vessels were ligated to prevent confounding due to differential renal excretion in wild-type and UT-B-null mice. [14C]urea was injected intravenously into both wild-type and UT-B-null mice, and [14C]urea radioactivity in the serum, brain, liver, spleen, and testis was measured 5 min postinjection. Similar serum [14C]urea radioactivity was found in wild-type (14,999 ± 1,980 cpm/10 µl) and UT-B-null mice (15,933 ± 1,618 cpm/10 µl). Tissue [14C]urea distributions were normalized to serum values and reported as a percentage of serum [14C]urea radioactivity. High urea radioactivity was found in the liver and spleen from both wild-type (34 ± 2.7% and 25 ± 2.8%) and UT-B-null mice (31 ± 3.5% and 27 ± 2.0%). The brain had low urea radioactivity in both wild-type (4.6 ± 0.8%) and UT-B-null mice (4.3 ± 0.1%). However, [14C]urea in testes from UT-B-null mice (7.5 ± 2.3%) was significantly lower than that from wild-type mice (18 ± 3.1%, P < 0.01; Fig. 3B).


Figure 3
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  		Fig. 3. Urea concentrations and transport in tissues. A: urea concentration in plasma and homogenized organs. Organs from 84-day-old mice were homogenized in water. Urea concentrations in supernatants of centrifuged homogenates were measured using a kit purchased from Roche Diagnostic. B: kinetics of [14C]urea uptake and organ morphology in wild-type versus UT-B-null mice. After renal blood flow was blocked, a bolus of [14C]urea was injected intravenously, and the blood, brain, liver, spleen, and testis were sampled at 5 min. [14C]urea accumulation in different organs was normalized to serum. cpm, Counts per minute.Values are means ± SE of 4 mice. *P < 0.01 vs. wild-type mice.

 
At 84 days of age, the UT-B-null mice had a lower body weight (33.1 ± 3.0 g) than wild-type mice (36.4 ± 2.1 g), as previously reported (16). There were no significant differences in the ratio of kidney weight and liver weight to body weight. However, UT-B-null mice had significantly lower spleen weight and higher testis weight. Testes in UT-B-null mice (103.7 ± 6.9 mg) were significantly larger than those in wild-type mice (80.3 ± 6.7 mg; Fig. 4A, inset). The ratio of testis weight to body weight was 0.31 ± 0.02% in UT-B-null mice and 0.22 ± 0.03% in wild-type mice (Fig. 4A). Estimate of water content using wet-to-dry weight ratios showed no significant differences in testes from wild-type (82.9 ± 0.4%) versus UT-B-null (82.6 ± 0.2%) mice (Fig. 4B).


Figure 4
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  		Fig. 4. Comparison of testis weight of UT-B-null mice and wild-type mice. Organs were isolated and weighed at day 84 postpartum. A: organ weights are shown as a percentage of body weight (means ± SE, n = 6). *Level of significance at P < 0.05. Inset, testis morphology in wild-type versus UT-B-null mice at day 84 postpartum. B: water content of testes. The percentage of water content was calculated by the difference between wet and dry weight, divided by wet weight.

 
Histological examination of the testis showed no significant differences in the features or distribution of stages of spermatogenesis in all seminiferous tubules of UT-B-null mice and wild-type mice at adult age. Representative tubules showing the most advanced stage of spermatogenesis in wild-type and UT-B-null mice at an age of 84 days are shown in Fig. 5A. The cellular integrity of the seminiferous epithelium also appeared to be normal in UT-B-null mice. Tubular and luminal diameters of seminiferous tubules were similar in UT-B-null and wild-type males (Fig. 5B). Because of the increased testicular weight in UT-B-null mice and the localization of UT-B to Sertoli cells in wild-type mice, sperm numbers and morphology were examined. There were no significant differences in sperm numbers in cauda epididymes from wild-type mice (11.2 ± 2.1 x 105 sperm/mouse) versus UT-B-null mice (12.6 ± 2.4 x 105 sperm/mouse). Analysis of sperm morphology showed <0.4% abnormal sperm in both wild-type and UT-B knockout mice.


Figure 5
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  		Fig. 5. Tubular diameters of seminiferous tubules. A: testis tissue morphology in wild-type versus UT-B-null mice at day 84 postpartum. Testis tissue sections were stained with hematoxylin and eosin. Scale bar = 100 µm. B: seminiferous tubular diameter (outer) and luminal diameter (inner) of the seminiferous epithelium were measured and recorded by the double-blind method.

 
Testicular development was earlier in UT-B-null mice. This is reflected by the testicular size in UT-B-null and wild-type males. The testis weight of UT-B-null males was significantly greater from day 17 onward (Fig. 6A). Interestingly, at 24 days of age, numerous elongating spermatids were observed in UT-B-null males, but none were present in wild-type males (Fig. 6B). By day 28, elongating spermatids were present in all UT-B-null mice. On the other hand, before day 28, no wild-type males demonstrated elongating spermatids, which were observed at day 32. By day 36, elongating spermatids were present in all wild-type males. Once elongated spermatids were present, there were no differences in the morphology or location between UT-B-null and wild-type males.


Figure 6
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  		Fig. 6. Testis development in UT-B-null mice. A: age-related changes in kidney and testis weights. The left kidney and left testis were isolated and weighed. The x-axis shows the age of the mice, and the y-axis shows the organ percentage of body weight (n = 6). *Level of significance at P < 0.05. B: histological comparison of the testis at different ages. The cross section shows the most advanced stage of representative seminiferous tubules at each age group for wild-type and UT-B knockout male mice. Arrows indicate elongating spermatids. I, L, and B indicate the interstitium, lumen, and basal layer of tubule, respectively. Scale bar = 50 µm.

 
In view of the differences observed in testicular developmental patterns in UT-B-null males, we examined their fertility by conducting competing mate experiments. A 35-day-old UT-B-null male and a wild-type male from the same litter were mated with a 70-day-old wild-type female in a competing mate group. As shown in Fig. 7, the time to the first litter in the competing mate groups (69 ± 3 days, n = 7) was significantly earlier than that (77 ± 2 days, n = 7) in the control groups, which indicates that the breeding ages were 48 ± 3 days in the competing mating groups and 56 ± 2 days in the control groups. Interestingly, the genotypes of all pups in the competing mate groups were UT-B heterozygotes, which suggests that all pups in competing mate groups carried the targeted UT-B gene and were produced by UT-B-null males. The numbers and gender ratios of pups sired by UT-B-null males in the competing mate groups were similar to those produced by wild-type males in the control groups. These data suggest a relatively early attainment of sexual maturity in UT-B-null male mice.


Figure 7
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  		Fig. 7. Breeding performance of maturing male mice. Male (M) mice at 35 days of age were housed with 10-wk-old wild-type female (F) mice. Data are shown for 7 pairs of competing mates (left) and 7 pairs of wild-type controls (right) and are means ± SE.

 
FSHR and ABP mRNA expression levels in testes were measured at certain ages by real-time RT-PCR to determine Sertoli cell development. Both FSHR and ABP mRNA expression levels were significantly higher in UT-B-null mice than those in wild-type mice at 10 days of age and then decreased subsequently (Fig. 8, A and B). At 24 days old, both FSHR and ABP mRNA expression levels in UT-B-null mice were significantly lower than those in wild-type mice. Peak FSHR and ABP mRNA expression occurred earlier in UT-B-null mice compared with wild-type mice (10 vs. 17 days after birth). The earlier FSHR and ABP mRNA expression peaks in UT-B-null mice compared with wild-type mice indicate earlier Sertoli cell development in UT-B-null mice. The urea concentration in the testis and serum in young mice was measured to help elucidate the relationship between urea accumulation and early puberty in UT-B-null mice. The testis urea concentration in UT-B-null mice (34.3 ± 1.6 mM) was significntly higher than that in wildtype mice (31.4 ± 0.5 mM) at 10 days old. The difference of testis urea concentration between genotypes became more significant with age (Fig. 8C). The serum urea concentration in UT-B-null mice was significantly higher than that in wild-type mice at all ages, although it did not vary with age (Fig. 8D). Despite differences in urea concentration, there were no significant differences in testis histology to a blinded researcher between UT-B-null mice and wild-type mice at 10 and 17 days old (data not shown).


Figure 8
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  		Fig. 8. Changes of follicle-stimulating hormone receptor (FSHR) and androgen binding protein (ABP) expression and urea concentration with age. A: transcript expression of FSHR evaluated by real-time PCR in testes of wild-type and UT-B-null mice. The mRNA expression level for each sample is expressed relative to expression in wild-type testes at 10 days old, which was arbitrarily considered equal to 1.0 in each individual comparison. B: transcript expression of ABP. C: testis urea concentration. D: serum urea concentration. Values are means ± SE of 4 mice. *P < 0.05 and **P < 0.01 vs. wild-type mice.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purpose of this study was to analyze the extrarenal phenotype of UT-B-null mice, with a focus on the male reproductive system, because UT-B is expressed in the testis and phenotypic differences can be measured. The testis is a major site of UT-B expression in humans and rats (7, 9, 24, 30, 31). In our study, we studied UT-B expression in the mouse testis. Previously, we (35) had been unable to detect UT-B in the wild-type testis by Northern blot, although we were able to detect UT-B in other tissues. Using RT-PCR, we were able to detect weak expression of UT-B in this study. We confirmed UT-B protein expression in the mouse testis by immunoblot analysis. UT-B expression localization within the testis has been previously investigated. Immunocytochemistry in the rat (9) showed UT-B expression in the plasma membrane of Sertoli cells found in stage II–III tubules. Doran et al. (7) reported UT-B in cells lining the periphery of seminiferous tubules, with expression diminished toward the center of the lumen, suggesting that these cells cease expressing UT-B as they mature. UT-B protein expression in mouse Sertoli cells, measured by immunofluorescence, demonstrated the same pattern as that found in the rat. These results suggest that UT-B plays a role in urea transport in Sertoli cells. However, the physiological function of UT-B in the male reproductive system was unclear.

A recent study (7) has demonstrated that UT-A is expressed in multiple layers of the rat seminiferous tubule periphery. To determine whether UT-A isoforms are expressed in the mouse testis and function as a testicular UT, we looked for UT-A expression by RT-PCR using cDNA primers derived from 1) a sequence common to UT-A1 and UT-A3, 2) a UT-A2 sequence, and 3) a sequence common to UT-A3 and UT-A5. Using these methods, UT-A5 alone was found in the mouse testis. Because a UT-A5 antibody is unavailable, we were unable to confirm the presence of UT-A5 by immunoblot detection.

To explore the regulation of UT-A5 in UT-B-null testes, we performed RT-PCR to evaluate UT-A5 transcript levels in UT-B-null mice compared with wild-type mice. UT-A5 expression at the mRNA level was not different in UT-B-null mice compared with wild-type mice, which indicates that UT-A5 is not involved in UT-B-mediated testicular urea transport.

Urea is produced during the synthesis of arginine and ornithine in Sertoli cells (31). Urea in Sertoli cells might be expected to accumulate without a functional UT present in the cell membrane. To test the hypothesis that UT-B expressed in Sertoli cells facilitates the exit of urea and provides a pathway for urea movement across the blood-testis barrier, we studied urea transport by infusing a single bolus of radioactive urea in both wild-type and UT-B-null mice with ligated renal vessels. At 5 min postinfusion, there was a significant reduction in radioactive urea in UT-B-null testes compared with wild-type testes. Additionally, higher testis urea concentrations were noted in UT-B-null mice compared with wild-type mice at baseline. These two lines of evidence suggest that deletion of UT-B decreases urea flux across the blood-testis barrier and blocks the exit of urea from Sertoli cells. However, the serum urea concentration was significantly higher in UT-B-null mice than in wild-type mice, despite reduced urea exit from the testes of UT-B-null mice. This serum concentration difference is likely attributable to UT-B-mediated renal urea concentrating ability (35).

The seminiferous tubule epithelium is made up of a complex association of Sertoli cells and germ cells. Spermatogenesis is absolutely dependent on the function of Sertoli cells. Without the physical and metabolic support of Sertoli cells, germ cell differentiation into mature spermatozoa could not occur (5, 6, 25). The Sertoli cell layer is also generally regarded as the major barrier for solute entry into and out from the seminiferous tubule lumen. UT-B is ideally situated to mediate urea flux out of Sertoli cells or across seminiferous tubule epithelia.

In this study, UT-B deletion caused significant increase in testis size starting at 17 days old. Since water content was not different, the abnormally large testicular size in UT-B-null mice could suggest the possibility of a concurrent abnormality in spermatogenesis. However, we did not find impaired fertility or abnormalities in sperm morphology or sperm numbers in UT-B-null mice. Interestingly, comparison of testicular histology of UT-B-null versus wild-type mice showed that elongating spermatids appeared in UT-B-null testes ~1 wk earlier than in wild-type mice. Also, UT-B-null males started breeding earlier than wild-type mice by ~1 wk. Taken together, these results suggest that sexual maturation occurs earlier in the UT-B-null male reproductive system. If we were to extrapolate this finding to a similar situation in humans, there would be an estimated sexual maturation ahead of 1–2 yr.

Sertoli cell development was also found to be significantly earlier in UT-B-null mice than in wild-type mice. We used FSHR and ABP, which are good markers for determining Sertoli cell development and function (12), to investigate Sertoli cells of UT-B-null mice. Briefly, the rationale for using these markers is as follows: FSHR is specifically expressed in testicular Sertoli cells and ovarian granulose cells (13, 21, 26). FSH binds to FSHR located on Sertoli cells and stimulates Sertoli cells to produce ABP. FSHR signaling pathways are required for Sertoli cell function and testicular size (17, 18). Decreases in testicular weight have been found in FSH-beta-deficient mice and FSHR knockout mice (18, 19). ABP produced by Sertoli cells is considered absolutely essential for maintaining qualitative and quantitative spermatogenesis. Linder et al. (14, 20) reported that maximal mRNA for FSHR and ABP occur in stages VII–IX and stages XIII–III of the seminiferous epithelium, respectively. In this study, we found an earlier elevation of FSHR and ABP mRNA expression levels in UT-B-null males compared with wild-type males. Urea accumulation in UT-B-null testes was also found in mice at 10 days old and beyond, which may account for the early reproductive maturation seen in our study. However, our work does not rule out that a secondary effect due to increased serum urea concentration (increased intracellular urea) may be the cause of early puberty.

In summary, the results in this study provide evidence that UT-B contributes to urea permeability across the blood-testis barrier. Deletion of UT-B results in the accumulation of urea in the testis. In this study, development of the male reproductive system of UT-B-null mice occurs earlier than in wild-type mice. This unanticipated finding suggests that the accumulation of urea in the testes could cause the premature development of Sertoli cells, which in turn initiate early spermatogenesis. Further studies are needed to explore the molecular mechanisms by which UT-B deletion promotes the observed maturation of the male reproductive system.


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 ABSTRACT
 METHODS
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 DISCUSSION
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-35124 and DK-66194, American Heart Association Grant 0365027Y (to B. Yang), and National Natural Science Foundation of China Grant 30370572 (to X. Zhao).


   ACKNOWLEDGMENTS
 
The authors thank Dr. A. S. Verkman and Dr. Shannon S. Sullivan for a critical reading of the manuscript, Dr. Jeff M. Sands for UT-B antibody, and Liman Qian for mouse breeding and genotyping.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Yang, 1246 Health Sciences East Tower, Univ. of California, San Francisco, CA 94143-0521 (e-mail: Baoxue.Yang{at}ucsf.edu) or X. Zhao, Dept. of Reproductive Pathophysiology, School of Basic Medicine, Jilin Univ., Changchun, 130021, Jilin province, China (e-mail: pro_2{at}jlu.edu.cn)

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.

* L. Guo and D. Zhao contributed equally to this work. Back


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