Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Functional α1‐ and β2‐adrenergic receptors in human osteoblasts

Central (hypothalamic) control of bone mass is proposed to be mediated through β2‐adrenergic receptors (β2‐ARs). While investigations in mouse bone cells suggest that epinephrine enhances both RANKL and OPG mRNA via both β‐ARs and α‐ARs, whether α‐ARs are expressed in human bone cells is controversial. The current study investigated the expression of α1‐AR and β2‐AR mRNA and protein and the functional role of adrenergic stimulation in human osteoblasts (HOBs). Expression of α1B‐ and β2‐ARs was examined by RT‐PCR, immunofluorescence microscopy and Western blot (for α1B‐ARs). Proliferation in HOBs was assessed by ³H‐thymidine incorporation and expression of RANKL and OPG was determined by quantitative RT‐PCR. RNA message for α1B‐ and β2‐ARs was expressed in HOBs and MG63 human osteosarcoma cells. α1B‐ and β2‐AR immunofluorescent localization in HOBs was shown for the first time by deconvolution microscopy. α1B‐AR protein was identified in HOBs by Western blot. Both α1‐agonists and propranolol (β‐blocker) increased HOB replication but fenoterol, a β2‐agonist, inhibited it. Fenoterol nearly doubled RANKL mRNA and this was inhibited by propranolol. The α1‐agonist cirazoline increased OPG mRNA and this increase was abolished by siRNA knockdown of α1B‐ARs in HOBs. These data indicate that both α1‐ARs and β2‐ARs are present and functional in HOBs. In addition to β2‐ARs, α1‐ARs in human bone cells may play a role in modulation of bone turnover by the sympathetic nervous system. J. Cell. Physiol. 220: 267–275, 2009. © 2009 Wiley‐Liss, Inc.     

Testicular morphology and expression of aromatase in testes of mice with the mosaic mutation (Atp7a mo-ms)

The aim of this study was to determine whether testicular cells of mice with the mosaic mutation, associated with abnormal copper metabolism, are able to aromatize androgens to estrogens, and what is the putative role of estrogens in the gonad of the mutant male. Mosaic is a lethal mutation; affected males usually die on about day 16. Those, which survive to reach sexual maturity, are valuable research subjects. In testes of young and adult mutants, histological analysis revealed the presence of many degenerating seminiferous tubules besides normal-looking ones. Additionally, high numbers of apoptotic germ cells were observed, especially in young mutants when compared with the controls. Positive immunostaining for aromatase was found in cultured Leydig cells and testicular sections of both control and mutant males. The intensity of immunostaining was always stronger in the mosaic mice. In both groups, Western-blot analysis revealed the presence of aromatase protein as a single band of approximately 55 kDa. In the mosaic males, levels of testosterone in cultured Leydig cells, whole testes, and in blood plasma were lower than in those of the respective controls. On the contrary, estradiol concentrations were always higher in the mutants. Both in vivo and in vitro studies indicate that morphological and functional changes in the testes of the mosaic mice mainly result from defective copper metabolism. The higher level of endogenous estrogens can additionally enhance morphological alterations within the testes. It seems also likely that excess estrogens may affect the survival rate of the mosaic males.     

Evidence for the presence of androgen receptors in human Leydig cells

Localization of androgen receptors (ARs) in the human testis Leydig cells was examined with an AR assay and Northern blot analysis. Leydig cells, highly purified on a Percoll gradient, were used for the experiments. AR concentration in the total cell extract containing both the cytosol and nuclear fractions in Leydig cells was measured using [3H]methyltrienolone. ARs in Leydig cells showed a high affinity for [3H]methyltrienolone and the Kd and Bmax of the receptors were 1.24 nM and 11.7 fmol/mg protein, respectively. Northern blot analysis, using a 32P-labeled full-length human AR complementary DNA (cDNA) detected a 9.5-kb hybridizing band in the total RNA extracted from Leydig cells. These data can be interpreted as evidence of the existence of ARs in human Leydig cells.     

Immunocytochemical evidence for the specific localization of aldose reductase in rat Sertoli cells

Evidence that the enzyme aldose reductase (AR) is specifically located in Sertoli cells is presented by means of an established immunocytochemical technique and with a variety of approaches. By staining tissue sections, the enzyme was shown to be present in Sertoli cells at birth and the intensity of the immunocytochemical stain increased by 5 days of age to that found in the testes of older rats. By means of enzyme dispersion of mature testes; the culture of enriched Sertoli cell preparations from the testes of immature rats; and the collection of newly released testicular spermatozoa in rete testis fluid, it was shown that immunoreactive AR was not present in any testicular cell type except the Sertoli cell. The significance of the specific localization in Sertoli cells of a principal enzyme concerned in the sorbitol or polyol pathway for the conversion of aldose sugars to their corresponding ketoses is discussed.     

Immunoexpression of androgen receptors and aromatase in testes of patient with Klinefelter's syndrome

Klinefelter's syndrome (47, XXY) is the most common chromosome aneuploidy in men and is usually characterized by underdeveloped testes and sterility. The aim of the present study was to detect cellular distribution of androgen receptors (AR) and aromatase in testes of patient with KS. The tissue sections were processed for morphological and immunohistochemical staining. Additionally, levels of FSH, LH, PRL, estradiol, and testosterone were measured in the plasma. Morphological analysis revealed a complete absence of spermatogenesis. No germ cells were present in seminiferous tubules. In some tubules, nests of apparently degenerating Sertoli cells were found. In the interstitium, Leydig cell hyperplasia was observed. Using immunohistochemistry, nuclear AR staining was detected in Sertoli cells and peritubular cells, whereas in Leydig cells the staining was exclusively cytoplasmic. The immunostaining of aromatase was detected in the cytoplasm of Sertoli cells and Leydig cells. Increased levels of gonadotropins and decreased level of testosterone concomitantly with the cytoplasmic localization of AR in Leydig cells might contribute to the impaired testicular function in patient with KS.     

The role of androgen receptors in the masculinization of brain and behavior: what we've learned from the testicular feminization mutation

Many studies demonstrate that exposure to testicular steroids such as testosterone early in life masculinizes the developing brain, leading to permanent changes in behavior. Traditionally, masculinization of the rodent brain is believed to depend on estrogen receptors (ERs) and not androgen receptors (ARs). According to the aromatization hypothesis, circulating testosterone from the testes is converted locally in the brain by aromatase to estrogens, which then activate ERs to masculinize the brain. However, an emerging body of evidence indicates that the aromatization hypothesis cannot fully account for sex differences in brain morphology and behavior, and that androgens acting on ARs also play a role. The testicular feminization mutation (Tfm) in rodents, which produces a nonfunctional AR protein, provides an excellent model to probe the role of ARs in the development of brain and behavior. Tfm rodent models indicate that ARs are normally involved in the masculinization of many sexually dimorphic brain regions and a variety of behaviors, including sexual behaviors, stress response and cognitive processing. We review the role of ARs in the development of the brain and behavior, with an emphasis on what has been learned from Tfm rodents as well as from related mutations in humans causing complete androgen insensitivity.     

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.     

Immunohistochemical localisation of androgen receptor during sex-specific morphogenesis in the fetal mouse

The distribution of androgen receptors (ARs) in paraffin serial sections of day 17 and day 18 male and female mouse embryos was investigated. In the cranial section of the genital tract AR expression was restricted to Wolffian structures while Müllerian ducts and surrounding mesenchyme were AR negative. In the fusion zone with the urogenital sinus the epithelial components of the vaginal bud were clearly distinguished by differential AR expression, which was faint in the Wolffian ducts, totally missing in the Müllerian ducts, and intense in the sinus ridges with the most intense expression in the morphogenetically active mesenchyme, indicating a new mechanism of negative control of vagina formation via androgens. Expression of ARs outside the genital tract was observed: (1) in loose interstitial mesenchyme extending into the retroperitoneal space up to the coeliac artery, indicating androgen effects during ascent of the kidneys and descent of intraperitoneal organs, (2) in the trigone of the bladder indicating androgen involvement in the development of the vesico-ureteral junction, and (3) in loose mesenchyme between striated muscle fibres and around pelvic skeletal elements, indicating mediation of androgen effects on the musculoskeletal system via loose mesenchyme.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains

Agonist stimulation of the β2-adrenergic receptors (β2ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length β2ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized β2ARs in nonlysosomal compartments. Additionally, a lysine-less β2AR (0K-β2AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT β2AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated β2ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the β2AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited β-arrestin2, and internalized just as the WT β2AR. However, only loop 3 and carboxyl tail β2ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the β2AR. We overexpressed and purified the β2AR from HEK-293 cells with or without prior agonist exposure and subjected trypsin-cleaved β2AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the β2AR. These findings introduce a new concept that two distinct domains in the β2AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors.     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

Prostate androgen receptor: immunohistological localization and mRNA characterization

Four androgen receptor (AR) specific monoclonal antibodies were used for the immunohistochemical localization of AR in the human prostate tissue. The prostate tissue consisted of alveoli embedded in fibromuscular stroma and lined with a single layer of columnar secretory epithelial cells. The immunoreactive ARs were found predominantly in the nuclei of epithelial cell, suggesting ARs, like estrogen receptors and progesterone receptors, are mainly nuclear proteins. Northern blot hybridization showed that AR mRNA is about 9 kilobases (kb) and relative abundant in the androgen-sensitive organs, such as ventral prostate, dorsolateral prostate and seminal vesicle.     

Rapid recycling of beta-adrenergic receptors is dependent on the actin cytoskeleton and myosin Vb

For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.     

Targeted deletion of A(3) adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A1433 (50 microM) decreased functional recovery following ischemia in A(3)AR(-/-) but not in wild-type hearts. We also examined myocardial infarct size using an intact model with 30-min left anterior descending coronary artery occlusion and 24-h reperfusion. Infarct size was reduced by over 60% in A(3)AR(-/-) hearts. In summary, targeted deletion of the A(3)AR improved functional recovery and tissue viability during reperfusion following ischemia. These data suggest that activation of A(3)ARs contributes to myocardial injury in this setting in the rodent. Since A(3)ARs are thought to be present on resident mast cells in the rodent myocardium, we speculate that A(3)ARs may have proinflammatory actions that mediate the deleterious effects of A(3)AR activation during ischemia-reperfusion injury.     

The anti-tumor effect of A3 adenosine receptors is potentiated by pulsed electromagnetic fields in cultured neural cancer cells

A(3) adenosine receptors (ARs) play a pivotal role in the development of cancer and their activation is involved in the inhibition of tumor growth. The effects of pulsed electromagnetic fields (PEMFs) on cancer have been controversially discussed and the detailed mechanisms are not yet fully understood. In the past we have demonstrated that PEMFs increased A(2A) and A(3)AR density and functionality in human neutrophils, human and bovine synoviocytes, and bovine chondrocytes. In the same cells, PEMF exposure increased the anti-inflammatory effect mediated by A(2A) and/or A(3)ARs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-tumor effect of A(3)ARs in PC12 rat adrenal pheochromocytoma and U87MG human glioblastoma cell lines in comparison with rat cortical neurons. Saturation binding assays and mRNA analysis revealed that PEMF exposure up-regulated A(2A) and A(3)ARs that are well coupled to adenylate cyclase activity and cAMP production. The activation of A(2A) and A(3)ARs resulted in the decrease of nuclear factor-kappa B (NF-kB) levels in tumor cells, whilst only A(3)ARs are involved in the increase of p53 expression. A(3)AR stimulation mediated an inhibition of tumor cell proliferation evaluated by thymidine incorporation. An increase of cytotoxicity by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation in PC12 and U87MG cells, but not in cortical neurons, was observed following A(3)AR activation. The effect of the A(3)AR agonist in tumor cells was enhanced in the presence of PEMFs and blocked by using a well-known selective antagonist. Together these results demonstrated that PEMF exposure significantly increases the anti-tumor effect modulated by A(3)ARs.