Mol. Hum. Reprod. Advance Access originally published online on December 10, 2004
Molecular Human Reproduction 2005 11(2):99-106; doi:10.1093/molehr/gah138
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Identification, characterization and biological activity of oxytocin receptor in the developing human penis
1Department of Clinical Physiopathology, Andrology and Endocrinology Unit, 2Department of Anatomy, Histology and Forensic Medicine, 3Department of Pharmacology and Clinical Physiopathology, Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, University of Florence, 50139 Florence, Italy
4 To whom correspondence should be addressed at: Department of Clinical Physiopathology, University of Florence, V.le G. Pieraccini, 6, 50139 Florence Italy. Email: m.maggi{at}dfc.unifi.it
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Abstract |
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Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT–PCR in human fetal tissues (11th–12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log10 unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.
Key words: development/oxytocin receptor/penis/smooth muscle cell
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Introduction |
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During the initial stages of development, the external genitalia of male and female fetuses are identical and include the genital tubercle, an undifferentiated structure that will form the penis in males and the clitoris in females. In humans, this ambisexual stage lasts until the 8th–10th week of gestation. Immediately, the dimorphic phase takes place under the sex steroids' influence. Most of the penile structure derived from the mesodermal cells, constitute the large corporal bodies, the connective tissue and the dermis. The corporal tissue is first recognized as the dense mesenchymal condensation within the developing penis, which will eventually constitute the so-called corpus cavernosum urethrae (CCU; Gilpin and Gosling, 1983
). This mesenchymal compartment is initially characterized by an avascular dense agglomerate of fibromuscular cells, which is surrounded by a series of convoluted blood vessels (Yamada et al., 2003). During morphogenesis, these surrounding neo-vascular structures must invade the developing CCU in order to form the trabecular structure of the adult penis (Granchi et al., 2002). In the meantime, the urethral folds migrate towards each other and fuse in the midline to sustain the tubular penile urethra. This fusion process begins proximally in the perineal region, but extends distally as the phallus elongates and differentiates, establishing a midline mesenchymal continuity along the penile shaft (Yamada et al., 2003).
Little is known, to date, concerning the biological mechanisms underlying penile mesenchymal differentiation into its various derivates. Based on our recent report on the gene and protein expression of oxytocin receptor (OTR) in human adult penis, we initiated studies on the OTR in the human developing penis. OTR is a G-protein-coupled receptor that binds with high affinity to all the neurohypophysial hormones and, as recently proposed, it is a candidate gene for morphogenesis regulation (Zingg and Laporte, 2003; Cassoni et al., 2004). Here we describe the presence of a functional OTR in the developing fetal penis and in its derived smooth muscle cell cultures, leading us to consider OTR as a new candidate involved in the development of CCU and penile organogenesis.
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Chemicals
Oxytocin (OT), [deamino-Cys1, D-Arg8]-vasopressin (DDAVP), reagents for immunohistochemistry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), peroxidase-conjugated anti-mouse secondary antibody and polyethylene glycol (PEG)-8000 were purchased from Sigma (St Louis, MO). [d(CH2)51, Tyr(Me)2,Orn8]-vasotocin (OTA) and (Phe2,Orn8)-vasotocin [(Phe2,Orn8)VT] were purchased from Bachem AG (Bubendorf, Switzerland). d(CH2)5[Tyr(Me)2, Thr4,Orn8,[125I]-Tyr9-NH2]-vasotocin ([125I]-OTA; 2200 Ci/mmol) was purchased from NEN Life Science (Boston, MA, USA). BM enhanced-chemiluminescence system was purchased from Roche Diagnostics (Milan, Italy). Reagents for protein measurement were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The mouse monoclonal antibody (CHINA/1F3) raised against the NH2 terminus of the OTR was a generous gift from Dr S. Deaglio (Laboratory of Cell Biology, Department of Genetics, University of Turin, Turin, Italy).
Tissue collection
Human fetal tissues were collected after spontaneous or therapeutic abortions. Legal abortions were performed in authorized hospitals and certificates of approval were obtained from each patient. The use of human fetal tissues for research purposes was approved by the Hospital Committee for Investigations in Humans (Azienda Ospedaliera Careggi, Florence, Italy protocol no 6783-04). Human adult myometrial cells were prepared from myometrial samples obtained from three normal cycling women undergoing hysterectomy for benign pathology, after the approval of the Hospital Committee for Investigations in Humans and after receiving the patients' informed consent.
Cell cultures
Human fetal penile smooth muscle cells (hfPSMC) were prepared from four samples of the fetal male external genitalia (MEG) (11th–12th week of gestation) obtained after spontaneous or therapeutic abortion, as previously described (Granchi et al., 2002; Crescioli et al., 2003). Briefly, penile tissues were mechanically dispersed and treated with 1 mg/ml bacterial collagenase for 15 min at 37°C. The fragments were then collected, washed in phospate-buffered saline (PBS) and cultured in a 1:1 (vol/vol) of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin in a fully humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells began to emerge within 24–48 h and were used within the fifth passage.
Human adult myometrial cells were prepared as previously described (Maggi et al., 1994). Briefly, 1 mg/ml collagenase was added to the finely minced, stripped uterine tissue for 12 h at 37°C. Digested fragments were then washed twice in PBS and cultured in a 1:1 (vol/vol) of DMEM and Ham's F-12, supplemented with 10% FBS, 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin in a fully humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells were cultured and used for up to five passages.
Real-time RT–PCR
Real-time RT–PCR assay was used to measure specific OTR mRNA levels in normal human fetal tissues, including penis. The assay was performed according to the fluorescent TaqMan methodology as already published (Vignozzi et al., 2004). Briefly, total RNA was isolated from human tissue samples using TRIZOL (Invitrogen, CA, USA) according to the manufacturer's instructions. Total RNAs from human cell cultures (hfPSMC and myometrial cells) were obtained using the RNeasy Mini Kit according to the manufacturer's instructions (Quiagen, Milan, Italy). An amount of 400 ng of total RNA was used to synthesize cDNA from each sample by RT–PCR, following the TaqMan Reverse Transcription Kit protocol (Applied Biosystems, CA, USA). As previously reported (Vignozzi et al., 2004), PCR primers (primer sense: 5'-CCTTCATCGTGTGCTGGACG-3', position 1469–1489; primer antisense: 5'-CTAGGAGCAGAGCACTTATG-3', position 1839–1859 of human OTR mRNA sequence) and the fluorogenic probe were designed using the Primer-Express software (Applied Biosystems, CA, USA) according to TaqMan's requirements. Designs were based on NCBI Genbank human OTR sequences (accession number NM_00916). The PCR mixture (25 µl final volume) consisted of the respective primers (300 nM each), probe (200 nM) and 12.5 µl Universal Master Mix (Applied Biosystems, CA, USA). Amplification and detection were performed with the ABI Prism 7700 system with one step at 50°C for 2 min, one step at 95°C for 10 min, and 40 cycles at 95°C for 30 s and 60°C for 1 min. The external reference calibration curve was obtained by cloning a 390 bp OTR cDNA fragment, as previously described (Vignozzi et al., 2004). All measurements were carried out in duplicates. Results were expressed as OTR mRNA molecules/µg total RNA.
Western blot
Frozen samples of human fetal tissues were directly suspended in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.25% NP-40, 1 mM Na3VO4, 1 mM PMSF) and homogenized (Teflon–glass) for protein analysis. The homogenates were centrifuged at 500 g for 10 min at 4°C and the protein content of supernatants was evaluated according to Bradford's method using Coomassie reagent (BIO-RAD Labs, Hercules, CA). After protein measurement, aliquots containing 30 µg of proteins were diluted in reducing 2 x SB (Laemmli's sample buffer = 62.5 mM Tris pH 6.8, 10% glycerol, 20% SDS, 2.5% pyronin and 100 mM dithioteithrol) and loaded onto 10% SDS-PAGE. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes. Membranes were blocked for 2 h at room temperature in 10% BM blocking buffer (BM, Roche Diagnostics, Milan, Italy)-TTBS (0.1% Tween-20, 20 mM Tris, 150 mM NaCl), washed in TTBS and incubated overnight with primary antibody (1:1000 CHINA/1F3 antibody in BM blocking buffer-TTBS) followed by peroxidase-conjugated secondary immunoglobulin G (1:3000). Finally, reacted proteins were revealed by a BM enhanced-chemiluminescence system.
Immunohistochemistry
Immunohistochemical studies were carried out as previously described (Vignozzi et al., 2004). Briefly, human fetal penile sections (fixed in Bouin's solution and embedded in paraffin) were incubated for 1 h in 2% FBS in PBS to block non-specific antibody binding. Sections were then incubated overnight at 4°C with a previously validated (Vignozzi et al., 2004) anti-human OTR IgM mouse antibody, CHINA/1F3 (dilution 1:200) and then with the corresponding specific immunoglobulin peroxidase-conjugated for 30 min (dilution 1:1000). Demonstration of peroxidase activity and controls for specificity of the antiserum were performed as previously described (Vignozzi et al., 2004). The slides were photographed using a Nikon Microphot-FXA microscope (Nikon, Kogaku, Tokyo, Japan).
Binding assay
HfPSMC were grown in 24-well dishes in DMEM/Ham F12 (vol/vol, 1:1) supplemented with 10% FBS, until sub-confluence. At the time of the experiment cells were starved for 24 h and then washed twice with DMEM containing 20 mM HEPES, 10 mM MgSO4 and 0.5% bovine serum albumin (BSA), pH 7.4 and were incubated in 200 µl of the same medium at room temperature for 60 min, with fixed concentrations of [125I] OTA in the presence or absence of increasing concentrations of the following unlabelled ligands: OTA (the corresponding unlabelled peptide; 0.01–100 nM), OT (0.01–100 nM), (Phe2,Orn8)VT (a selective V1 vasopressin agonist; 0.01 nM–1 µM) and DDAVP (the selective V2 vasopressin agonist; 1 nM–100 µM). After incubation, cells were extensively washed with ice-cold PBS, 0.1% BSA, solubilized in 0.1 N NaOH, and the cell-bound radioactivity was determined in a gamma counter. Measurements were performed in triplicate. Cell counts between wells routinely varied by <10%.
Intracellular free Ca2+ measurements
To test OT actions on intracellular free Ca2+ ([Ca2+]i) concentrations, hfPSMC cells were plated and grown in DMEM/Ham F12 (vol/vol, 1:1) added with 10% serum FBS. At subconfluence they were starved for 24 h and then trypsinized with 0.1% trypsin for 3 min. Cells were resuspended and loaded with 2 µM fura-2/AM (Sigma) at 37°C for 40 min in a serum-free culture medium. The cells were further incubated for 40 min with a serum-free medium to wash the unincorporated fura-2/AM. The cells were then centrifuged and resuspended in 2 ml Krebs–Henseleit HEPES buffer containing (135 mM NaCl, 53 mM KCl, 3 mM MgSO4, 11.7 mM HEPES, 8.6 mM NaHEPES, 1.25 mM CaCl2, and 55 mM glucose, pH 7.0) on ice until [Ca2+]i measurements were performed using the Hitachi F-2000 double wavelength fluorometer (Hitachi, San Jose, CA, USA). Fluorescence measurements were converted to values of [Ca2+]i by determining maximal fluorescence (Fmax) with 0.2% (wt/vol) digitonin (Sigma) followed by minimal fluorescence (Fmin) with 3 µM EGTA. [Ca2+]i was calculated according to the method proposed by Grynkiewicz et al., (1985) using the 340:380 nm. Increasing concentrations of OT (0.1 nM–1 µM) were used for the [Ca2+]i determination. The specificity of the response was evaluated by pre-incubation, 2 min before OT stimulus (100 nM), with increasing concentrations of OTA (0.1 nM–1 µM). Results were obtained from three separate experiments performed at least in triplicate. Data were expressed as the mean±SEM of the increase percentage, %=([Ca2+]peak–[Ca2+]basal)/[Ca2+]basal.
Cell chemotaxis
HfPSMC migration in response to OT was investigated as chemotaxis in Boyden's chamber (Nuclepore Inc., Pleasanton, CA, USA). Chemotaxis was evaluated using polyvinylpyrrolidone-free polycarbonate filters with an 8-µm pore size coated with 20 µg/ml type I collagen (BD Biosciences, Bedford, MA, USA). OT (100 nM, for 5 h) was added to the lower wells, and the cells (40 x 103 cells in 50 µl) were seeded in the presence or absence of OTA (100 nM) into the upper wells of the chamber, which was incubated at 37°C for 5 h. Platelet-derived growth factor-bb (PDGF-bb, 5 ng/ml for 5 h) was chosen as a positive control. A serum-free culture medium in the absence of any stimulus was taken as a basal migration. Methanol-fixed cells were stained with Diff-Quick (DADE Behring AG, Switzerland), and cell migration was measured by microscopic evaluation of the number of cells that moved across the filter into 10 random fields. Each experimental point was performed at least five times. Results were obtained from three independent experiments. Data were expressed as the mean±SEM of the increase percentage, considering the respective controls as 100%.
Proliferation assay
To take the growth measurement, 2 x 104 hfPSMC were seeded into 12-well plates in the growth medium. After 24 h, the medium was removed, the cells were washed twice in PBS and incubated in phenol red- and serum-free medium. After 24 h of starvation, the cells were incubated for 48 h in phenol red- and serum-free medium with 0.1% BSA containing increasing concentrations of OT (0.1–100 nM). OT's specific effect on cell growth was evaluated through the simultaneous incubation of equimolar concentration of OT and OTA (100 nM) and OTA alone (100 nM). The cells in phenol red- and serum-free medium containing 0.1% BSA and the vehicle were used as controls. After 48 h of stimulation, the cells were trypsinized and each experimental point was derived from the haemocytometer counting and then averaging from six to nine different fields for each well. In the same experiment, each experimental point was repeated in triplicate and the experiments were performed three times. Results were expressed as percentage variation (mean±SEM) over the control.
Statistical analysis
Results are expressed as the mean±SEM in the indicated number of experiments. Statistical analysis was performed using the one-way analysis of variance and paired or unpaired Student's t-test when appropriate. P<0.05 was taken as significant. Half-maximal response effective concentration (EC50) and half-maximal response inhibitory concentration (IC50) values were calculated using the computer program ALLFIT (De Lean et al., 1978). The binding data were evaluated quantitatively with non-linear least-squares curve fitting using the LIGAND computer program (Munson and Rodbard, 1980).
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Results |
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The absolute quantitation of OTR mRNA expression in different human fetal tissues, including penis, is reported in Figure 1. We found a relatively high expression of the OTR gene in all the fetal samples investigated. In particular, human fetal penis expressed only a half log10 unit lower level of OTR mRNA (penis; 4.1±0.9 x 104 molecules/µg of total mRNA; n=7) than the classical OT target tissues, such as the central nervous system (10±2.2 x 104 molecules/µg of total mRNA; n=3) and uterus (8.1 x 104 molecules/µg of total mRNA; n=1), considered positive controls.
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OTR protein expression in human fetal tissues, as detected by western blot analysis using a specific antiserum (CHINA/1F3), is reported in Figure 2. In the fetal corpora cavernosa, as well as in other human tissues investigated, we found a single specific band of the expected molecular weight (about 55 kDa; Vignozzi et al., 2004
).
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To investigate OTR localization in the developing human penis we performed immunohistochemical studies. Figure 3 shows different magnifications of human MEG transversal sections. In these sections, dense mesenchymal bodies forming the CCU can be clearly distinguished. Between the ventral portion of the remodelling penile urethra and the developing CCU, a crown of convoluted blood vessels was present. These blood vessels were to invade the CCU, to give rise to the forthcoming lacunar spaces. An intense scattered immunopositivity was revealed in the whole dense mesenchymal body (Figure 3 panels A and B, magnification x40 and x50, respectively). Intense positive staining was also detected in the penile blood vessels (Figure 3 panels A and B). As shown at a higher magnification (x250; Figure 3 panel C), all mesenchymal cells of the developing penis demonstrated positive OTR immunoreactivity. Figure 3 panel D shows the negative control obtained by pre-absorbing the primary antibody CHINA/1F3 with OTR-rich membranes, as previously described (Vignozzi et al., 2004
), and counterstaining with haematoxylin.
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To further characterize the expression of OTR in the mesenchymal compartment we cultured hfPSMC from penile explants of fetuses at the 11th–12th week of gestation, as previously described (Granchi et al., 2002
; Crescioli et al., 2003). Figure 4 (panel A, inset) shows that hfPSMC cultures express a very high OTR mRNA level (1.1±0.2 x 107molecules/µg of total mRNA, n=7) comparable to or even higher than adult myometrial cell levels (0.1±0.02 x 107 molecules/µg of total mRNA, n=3). Simultaneous analysis of 21 homologous competition curves for [125I]OTA performed in hfPSMC cultures indicates the presence of a high density (163 400±4800 sites/cell), high affinity (Kd=0.22±0.02 nM), homogeneous class of binding sites having pharmacological properties similar to the classic OTR (see Figure 4, panels A and B). In fact, in hfPSMC OTR binds with high affinity OT (Kd=0.07±0.001 nM), while it shows a relatively lower selectivity for the V1 vasopressin receptor agonist, Phe2,Orn8-VT (Kd=3.2±0.7 nM) and for the V2 vasopressin receptor agonist, DDAVP (Kd=153±33.6 nM). Binding parameters (including Bmax) were similar to those previously reported by our group in human myometrial cells (Maggi et al., 1994).
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In order to evaluate whether OTR mediated biological activity in hfPSMC, we performed [Ca2+]i measurement. Figure 5 panel A shows the typical calcium waveforms in response to increasing concentrations of OT (0.1 nM–1 µM). The basal [Ca2+]i in fura-2-loaded hfPSMC was 123.6±10.6 nM (n=21). The addition of OT induced an immediate initial transient increase in [Ca2+]i reaching its peak within 40 s. Figure 5 panel B shows the results of a typical dose–response experiment. The effect of OT was dose-dependent with EC50=4.2±2.2 nM (n=3). To test the effect of the OT antagonist, OTA, on OT-induced [Ca2+]i mobilization (Figure 5 panel C), we stimulated hfPSMC with 100 nM OT. This concentration of OT stimulated an increase in [Ca2+]i =205.07±46.1 nM (n=3). Pre-treatment with increasing concentrations of OTA progressively blunted the stimulatory effect of OT. Mathematical analysis (program ALLFIT, De Lean et al., 1978
) of the experimental results (n=3) indicates IC50=3.6±1.2 nM for OTA.
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To further evaluate the biological activity of OTR in hfPSMC, we performed migration assays using Boyden's chamber technique (Figure 6). In fact, as hfPSMC was cultured from the early gestation fetal explants of penile tissue, they preserved their motion properties. As expected, PDGF-bb (5 ng/ml, for 5 h) induced a sustained migration across filters coated with type I collagen (368±37.5%, data not shown). OT (100 nM for 5 h) also induced a substantial increase in the migratory effect (187.3±9.3%, P<0.001). OT-mediated hfPSMC chemotaxis was abrogated by pre-treatment with OTA (100 nM), bringing the number of migrated cells (111.2±10.2%) to a level not statistically different from the control. Enhanced cell migration was not seen when hfPSMC were incubated with OTA alone (111.8±6.2%).
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Finally, to determine whether OTR also regulates penile cell proliferation, as previously described for the sex steroid receptors (Crescioli et al., 2003
), we conducted growth studies in hfPSMC. As shown in Figure 7, 48 h exposure to increasing concentrations of OT significantly increased hfPSMC proliferation, with EC50=4±1.0 nM. Incubation of hfPSMC with OTA (100 nM) completely blocked the mitogenic effect of OT. Interestingly, we found that OTA alone retains an anti-proliferative effect, decreasing cell proliferation below the level of untreated control cells (66.3±3.5%; P<0.01 versus control).
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Discussion |
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This study represents the first demonstration that human fetal penis expresses OTR, which regulates migration and proliferation of penile mesenchymal cells. We therefore hypothesized that OTR might be a new factor involved in supporting correct development of the MEG.
In recent years the classical well-known role of OTR, promoting labour and lactation, has been greatly extended. New insights on its biological activities have been demonstrated, including morphogenic actions in the commitment towards distinct phenotypes and the proliferation of both normal and malignant cells (Zingg and Laporte, 2003; Cassoni et al., 2004). In the heart, OT, the natural ligand for OTR, stimulated differentiation of P19 embryonic (Paquin et al., 2002) and adult cardiac (Matsuura et al., 2004) stem cells in beating cardiomyocytes, characterized by specific contractile proteins and sarcomeric structures. OTR also mediated myoepithelial cell differentiation and proliferation in mouse organotypic cultures (Sapino et al., 1993) and pushed fetal mouse fibroblasts towards a mammary epithelial phenotype (Wang et al., 2003). In humans, OTR has been proposed as a growth and differentiating modulator for bone cells (Copland et al., 1999; Colucci et al., 2002) and myoblasts, where it induced fusion and myotubule formation (Breton et al., 2002).
The effects of OT on the development of external genitalia have not yet been studied, although, it was recently reported that soon after birth, weekly intraperitoneal injections of OT to the female rat disrupted the timing of vaginal opening and delayed sexual maturation (Withuhn et al., 2003). We now report, for the first time that human penile cells express extremely high concentrations of OTR. The fetal penile expression of OTR is more than 10-fold higher than in corresponding adult tissue (Vignozzi et al., 2004) and rather comparable to other classic target tissues, such as uterus and kidney, or brain and heart, whose development have been postulated to be under OTR control (Paquin et al., 2002; Carter, 2003). The OTR gene and protein expression in isolated smooth muscle cells from developing penis was even higher, reaching levels (gene: 1.1 x 107 molecules/µg total RNA; protein: 1.7 x 105 sites/cell) comparable to those observed in human myometrial cells in culture (the present report and Maggi et al., 1996) or uterine tissue (Vignozzi et al., 2004). In addition, pharmacological characterization of the OTA binding site expressed by hfPSMC indicates that this site binds neurohypophysial hormone analogues with affinity constants similar to those previously reported in the human myometrial cells (Maggi et al., 1994, 1996). In human myometrial cells, it has been shown that OTR mobilized intracellular calcium stores (Maggi et al., 1994, 1996; Tahara et al., 2000) and induced proliferation (Tahara et al., 2000). Similar results were reported in other human cell types, such as vascular endothelial cells (Thibonnier et al., 1999), trophoblasts (Cassoni et al., 2001a,b), osteoclasts (Colucci et al., 2002) and now originally shown in the fetal penile cells. Conversely, in several neoplastic lineages OTR does not couple to an intracellular calcium increase and does not stimulate growth but, paradoxically, mediates anti-proliferative effects (Cassoni et al., 1997, 1998, 2000), most probably through the activation of cAMP–PKA pathways (Cassoni et al., 2001a). Hence, it has been proposed that cell-specific OTR coupling to distinct G-proteins mediates these opposite proliferative effects (Bussolati and Cassoni, 2001). However, more recently, this concept has been partially revised (Zingg and Laporte, 2003; Cassoni et al., 2004): the divergent effects of OTR on cell growth have been related more to a different receptor sub-cellular trafficking than to an activation of distinct signalling pathways. In the fetal penile cells, intracellular trafficking of OTR and calcium downstream signal transduction has not been studied, and therefore needs further investigation. Interestingly, in human ovarian carcinoma cells, OT not only inhibits cell proliferation but also decreases cell migration (Morita et al., 2004). As timely and adequate cell migration is a key event in organogenesis and penile tissue remodelling, we next investigated the effect of OTR activation on this process in hfPSMC. We found opposite results with respect to those reported in the ovarian carcinoma cells: OT stimulated a sustained increase in migration that was completely reverted by the simultaneous addition of the OTR antagonist, OTA. Thus, OTR mediates two critical steps in the penile morphogenesis: cell proliferation and migration.
Little is known about factors that coordinate human external genitalia growth and differentiation at present. Despite substantial species-specific anatomical and endocrine differences, the animal model has been habitually used to study the most frequent ailments in newborn males: cryptorchidism and hypospadias (Yamada et al., 2003). In this report, we studied human fetal MEG at the 11th–12th week of gestation, just when penile differentiation takes place and the genital tubercle develops to form the phallus. We observed that OTR was specifically localized both in the mesenchymal CCU and in the convoluted blood vessels surrounding it, suggesting a putative role for OTR in these compartments.
At the 11th–12th week of gestation, the CCU smooth muscle cells migrate and proliferate in order to support the midline urethral fusion. If it does not occur properly, hypospadias can result (Gilpin and Gosling, 1983). Since we found that OTR is abundantly expressed by the penile endothelial and mesenchymal cells, it is possible that OTR might play some role in the pathogenesis of hypospadias.
It is intriguing that OTR expression is essentially under estrogen control (Maggi et al., 1992; Gimpl and Fahrenholz, 2001) and that both subtypes of estrogen receptors have been recently described in the developing human penis (Crescioli et al., 2003). The role of OTR in the puzzling ‘estrogen hypothesis’ for testicular dysgenesis syndrome and MEG abnormalities (Sharpe, 2003) needs to be studied further.
At present, the physiological source of the putative OTR ligand(s) is unclear. In the fetal pituitary gland, OT, vasopressin and vasotocin are detectable from the 10th week of fetal life (Smith and McIntosh, 1983; Khan-Dawood and Dawood, 1984). Interestingly, all these neurohypophysial peptides bind with high affinity to the human OTR (Maggi et al., 1990). Human fetal membranes might be an additional source of OT (Chibbar et al., 1993), although their contribution in the earliest stage of pregnancy is unknown. An alternative and relatively attractive hypothesis is that the penile tissues not only express OTR, but also synthesize OTR's own ligand. Accordingly, we found that OTA alone significantly reduced hfPSMC proliferation, suggesting an autocrine production of OT, which in turn, could locally regulate penile growth.
In conclusion, we have demonstrated that OTR is highly expressed in the penis during fetal life, and that it modulates migration and proliferation of CCU smooth muscle cells at the 11th–12th week of gestation, when the fetal urethra must develop in parallel with the elongation of the phallus.
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Acknowledgements |
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The authors wish to thank Dr S. Deaglio (Department of Genetics, University of Turin, Turin, Italy), for kindly providing the anti-OTR monoclonal mouse antibody (CHINA/1F3). This study was supported by grants from COFIN2002-MIUR (Progetti di Ricerca di Rilevanza Nazionale) and from ‘Centro di Ricerca, Trasferimento e Alta Formazione MCIDNENT’ of the University of Florence, Florence, Italy. This study was supported by a grant from the University of Florence, Florence, Italy.
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Submitted on September 27, 2004; resubmitted on November 12, 2004; accepted on November 17, 2004.
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