| The urea transporters (UT) are a family of membrane proteins that play a role in vectorial transcellular urea transport in response to a urea gradient. In mammals, at least seven urea transporters, UT-A1 to UT-A6 and UT-B, have been characterized. UT-A1, UT-A2, UT-A3 and UT-A4 are expressed in the kidney. UT-A5 is expressed in testis. UT-A6 localizes to the colon. UT-B is expressed in kidney, erythrocytes, brain, heart, liver, colon, bone marrow, spleen, lung, skeletal muscle, bladder, prostate and testis. 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 kidney. Deletion of UTs results in urea-selective urine concentrating defect. The functional significance of UTs in other tissues, where urea concentration is probably not very high, is less clear. In testis, UT-A5 is expressed in the outer cell layer of seminiferous tubules and UT-B is expressed in Sertoli cells of seminiferous tubules.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 expression of UT-B in Sertoli cells facilitates exit of urea generated during the synthesis of polyamines. The study here utilized transgenic UT-B deficient mice to test the hypothesis that UT-B deletion results in decreased the urea permeability across blood-testis barrier and thus, altered male reproductive function.Objective :The primary objective of this investigation was to study the role of urea transporter UT-B in facilitating urea transport between Sertoli cells and blood. The secondary objective of this investigation was to study the effect of UT-B deletion on the male reproductive function.Methods:UT-B knockout mice. Transgenic knock-out mice deficient in UT-B protein were gifted from University of California, San Francisco, USA. UT-B null mice did not express detectable UT-B protein in any organ. Measurements were done in litter-matched mice produced by intercrossing heterozygous mice.Reverse transcription PCR and fluorescence-based real-time PCR. Total RNA from mouse tissues was reverse transcribed with oligo(dT). PCR was carried out by the Gene Amp PCR system 9700 and with Taq DNA polymerase using primers for hypothalamus GnRH, pituitary gland GnRHR and FSH, testes FSHR. PCR products were electrophoresed on a 1.5 % agarose gel.Fluorescence-based real-time PCR was carried out by the LightCycler with LightCycler FastStart DNA MasterPLUS SYBR Green I kit. Primers for androgen-binding protein (ABP) and follicle stimulating hormone receptor (FSHR) were designed. 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 used as the calibrator. Results are reported as calibrated ratios. All samples are normalized to the reference gene. Concentration ratios for each sample are then calibrated so that results are reported as a normalized ratio:Normalized ratio = ratio of sample (target / reference) / ratio of calibrator (target / reference).Urea permeability measurement. Mice were anesthetized during the experiment by methoxyflurane inhalation 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) was infused by tail vein. Blood and tissues were obtained at 5 min after [14C]urea infusion. [14C]urea radioactivity in blood and tissues homogenization was assayed by scintillation counting. The urea distribution rates were expressed as ratio of [14C]urea radioactivity (cpm) in tissue to that in serum per unit weight.Tissue urea measurement. 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. Urea concentration was measured by colorimetry using a commercial kit.Histological examination. One testis per mouse was subjected to histological examination at selected ages from day 10 to day 84. For light microscopic analysis, testis tissue samples were fixed in Bouin's fixative overnight, embedded in paraffin, cut in 3μm-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. The 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, 20μg/ml PMSF, 1μg/ml pepstatin A, and 1μg/ml leupeptin (pH 7.4), and centrifuged at 4000 g for 15 min to remove whole cells, nuclei, and mitochondria. Total protein was assayed in the supernatant fractions using the BioRad DC protein assay kit (BioRad, Richmond, CA) and loaded on a 12 % SDS-PAGE gel (10μg/lane). Proteins were transferred to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI) and immunoblotted with 1:1000 dilution of rabbit polyclonal serum raised against an C-terminal peptide (NH2-DNRIFYLQNKKRMVESPL-COOH) of mouse UT-B by standard procedures.Immunofluorescence. Testis tissue samples were fixed with 4 % paraformaldehyde in PBS for 4 h, infiltrated with 30 % sucrose in PBS overnight. Tissues were immunostained by standard procedures as described. Mating studies. A 28-day-old UT-B male and a wildtype male from the same litter were mated with a 70-day-old wildtype female in each competing mating group. Two wildtype males were mated with a wildtype female in each control group. The animals were maintained under well-controlled conditions of temperature (22 oC), 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. The 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.Results and discussion:UT-B expression in mouse testis was first determined by RT-PCR. DNA fragments for UT-B were amplified from testis cDNA in wildtype mice (+/+) and UT-B heterozygous mice (+/-). No positive band was found in UT-B null mice (-/-). A 46 kD protein was found in testes and red blood cells in wildtype mice, but not in UT-B knockout mice, by Western blot analysis. Immunofluorescence confirmed UT-B protein expression in Sertoli cells in wildtype mice using antibody against UT-B. Staining of testis from UT-B knockout mice was negative.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 that in wildtype mice (7.6±0.1 mM and 46.9±1.5 mmol/kg tissue weight). Total testis urea content was 335.4±43.8 g in UT-B null mice vs. 196.3±18.2 g in wildtype mice (p < 0.01). There was no difference in brain and liver between UT-B null and wildtype mice. Urea distribution measurements were done using knockout mice to determine whether UT-B facilitates urea movement through blood-brain barrier and blood-testis barrier. Renal blood vessels were ligated to prevent confounding due to differential renal excretion in wildtype and UT-B null mice. [14C]urea was injected intravenously into both wildtype and UT-B null mice, and [14C]urea radioactivity in serum, brain, liver, spleen and testis was measured 5 min post-injection. Similar serum [14C]urea radioactivity was found in wildtype and UT-B null mice. Tissue [14C]urea distribution were normalized to serum values and reported as a percentage of serum [14C]urea radioactivity. High urea radioactivity was found in 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 %). Brain had low urea radioactivity in both wildtype (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 wildtype mice (18±3.1 %) (p < 0.01). At 84 days of age, the UT-B mice had lower body weight (33.1±3.0 g) than wildtype mice (36.4±2.1 g) as previously reported. There was no significant difference in the ratio of kidney weight and liver weight to body weight. However, the UT-B null mice had significantly lower spleen weight and higher testis weight. The testes in UT-B null mice (103.7±6.9 mg) were significantly larger than that in wildtype mice (80.3±6.7 mg). The ratio of testis weight to body weight was 0.31±0.02 % in UT-B null mice and 0.22±0.03 % in wildtype mice. Estimate of water content using wet-to-dry weight ratios showed no significant difference in testes from wildtype (82.9±0.4 %) vs. UT-B null (82.6±0.2 %) mice.Histological examination of testis showed no significant difference in features or distribution of stages of the spermatogenesis in all seminiferous tubules of UT-B null mice and wildtype mice at adult age. The cellular integrity of the seminiferous epithelium also appeared to be normal in UT-B null mice. The tubular and luminal diameters of seminiferous tubules were similar in UT-B null and wildtype males. Because of the increased testicular weight in UT-B null mice and the localization of UT-B to Sertoli cells in wildtype mice, sperm number and morphology were examined. There was no significant difference in sperm number in the cauda epididymes from wildtype mice (11.2±2.1 x 105 sperm/mouse) vs. UT-B null mice (12.6±2.4 x 105 sperm/mouse). Analysis of sperm morphology showed < 0.4 % abnormal sperm in both wildtype and UT-B knockout mice.Testicular development was earlier in UT-B null mice. This is reflected by the testicular size in UT-B null and wildtype males. Testis weight of UT-B males was significantly greater from day 17 onward. Interestingly, at 24 days of age, numerous elongating spermatids were observed in the UT-B males, but none were present in wildtype males. By day 28, elongating spermatids were present in all UT-B null mice. On the other hand, before day 28, no wildtype males demonstrated elongating spermatids that were observed at day 32. By day 36 elongating spermatids were present in all wildtype males. Once elongated spermatids were present, there were no differences in the morphology or localtion between UT-B null and wildtype males.In view of the differences observed in testicular developmental patterns in UT-B null males, we examined their fertility by conducting competing mate studies. A 35-day-old UT-B null male and a wildtype male from the same litter were mated with a 70-day-old wildtype female in a competing mate group. 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 targeted UT-B gene and were produced by UT-B null males. The number and gender ratio of pups sired by the UT-B males in the competing mate groups were similar to those produced by the wildtype males in control groups. These data suggest relatively early attainment of sexual maturity in UT-B male mice.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 wildtype mice at 10 days old, and then decreased subsequently. At 24 days old, both FSHR and ABP mRNA expression levels in UT-B null mice were significantly lower than those in wildtype mice. Peak FSHR and ABP mRNA expression occurred earlier in UT-B nulls mice compared to wildtype mice (10 days vs 17 days after birth). Earlier FSHR and ABP mRNA expression peaks in UT-B null mice compared to wildtype mice indicate earlier Sertoli cell development in UT-B null mice. 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. Testis urea concentration in UT-B null mice (34.3±1.6 mM) was significntly higher than wildtype mice (31.4±0.5 mM) at 10 days old. The difference of testis urea concentration between genotypes became more significant with age. Serum urea concentration in UT-B null mice was significantly higher than that in wildtype mice at all ages, though not varied with age. Despite differences in urea concentration, there were no significant differences in testis histology to a blinded researcher between UT-B null mice and wildtype mice at 10 days and 17 days old.More than 90% serum testosterone comes from Leydig cells in the testis. The testosterone is the fundamental substance for spermatogenesis. The results show that the serum testosterone level in 4w and 7w mice was significantly higher in UT-B null males than wildtype males, but there is no difference between the two groups at 9w age old. By semi-quantitative RT-PCR, we found that the mRNA expression levels of P450scc, 3β-HSD and 17β-HSD, rate-limiting enzymes for testosterone synthetic process were significantly higher in UT-B null males than wildtype males.The mRNA level of hypothalamus GnRH, pituitary GnRHR and FSH in 4w and 7w UT-B mull males were determined by semi-quantitative RT-PCR. All of these mRNA levels were significantly higher than those of the wildtype male mice. These data suggest that earlier sexual maturation in the UT-B null males might be relative to the change of the hypothalamic-pituitary-gonadal axis (HPGA).Conclusions:In summary, the results in this study provide evidence that UT-B is expressed in Sertoli cells in testis and contributes to urea permeability across the blood-testis barrier. Deletion of UT-B results in the accumulation of urea in the testis. The development of the male reproductive system of UT-B null mice occurs earlier than in wildtype mice. The data suggest that the accumulation of urea in the testes could cause the premature development of Sertoli cells, which in turn initiate early spermatogenesis. The earlier sexual maturation in the UT-B null males may be relative to the change of the hypothalamic-pituitary-gonadal axis (HPGA). Further studies are needed to explore the molecular mechanisms by which UT-B deletion promotes the observed maturation of male reproductive system.The results in this study has never been reported, they are the original findings.
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