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Endocrinology, doi:10.1210/en.2003-0962
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Endocrinology Vol. 145, No. 4 1823-1834
Copyright © 2004 by The Endocrine Society

Oxytocin Receptor Is Expressed in the Penis and Mediates an Estrogen-Dependent Smooth Muscle Contractility

Linda Vignozzi, Sandra Filippi, Michaela Luconi, Annamaria Morelli, Rosa Mancina, Mirca Marini, Gabriella Barbara Vannelli, Simone Granchi, Claudio Orlando, Stefania Gelmini, Fabrizio Ledda, Gianni Forti and Mario Maggi

Andrology Unit, Endocrinology Unit (R.M.), and Clinical Biochemistry Unit (C.O., S.G.), Department of Clinical Physiopathology; Department of Anatomy (G.B.V.), Histology, and Forensic Medicine; and Department of Pharmacology (F.L.), University of Florence, 50139 Florence, Italy

Address all correspondence and requests for reprints to: Prof. Mario Maggi, Department of Clinical Physiopathology, University of Florence, V.le G. Pieraccini 6, 50139 Florence, Italy. E-mail: m.maggi{at}dfc.unifi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oxytocin (OT) is released by the posterior pituitary during male orgasm and is supposed to participate in the ejaculatory process. We now report evidence demonstrating the presence of an OT receptor gene (real-time RT-PCR and Northern blot) and protein (immunohistochemistry, Western blot, and binding studies) expression in the rabbit and human corpus cavernosum (CC) and its possible involvement in postorgasmic penile detumescence. OT receptor is expressed in the penis at a concentration similar to that present in other portions of the male genital tract and mediates CC contractility. OT-induced CC contractility is clearly regulated by the changing sex steroid milieu. In fact, we found that in a rabbit model of hypogonadotropic hypogonadism (induced by a single administration of the long-acting GnRH agonist triptorelin pamoate, 2.9 mg/kg), OT responsiveness was strongly reduced and was completely restored by estradiol valerate (3.3 mg/kg weekly), but not by testosterone enanthate (30 mg/kg weekly). As we found that CC expresses both subtypes of estrogen receptors and P450 aromatase, we hypothesized a physiological role for endogenous estrogens in regulating OT responsiveness. We therefore treated adult rabbits with an aromatase inhibitor (letrozole, 2.5 mg/kg) or an antiestrogen (tamoxifen, 0.25 mg/kg) for 3 wk. Both treatments significantly reduced CC responsiveness to OT stimulation. In conclusion, these findings indicate that OT might participate in inducing postorgasmic penile flaccidity and suggest a new role for estrogens in the male: regulation of CC responsiveness to OT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE LAST 20 yr molecular events underlying the initiation of penile erection have been studied extensively. However, only a few studies have investigated postorgasmic penile detumescence (1, 2, 3, 4, 5, 6). Essentially, penile erection follows the relaxation of trabecular smooth muscle cells with a consequent increase in arterial inflow, whereas detumescence is the result of muscle contraction, which, in turn, by decreasing trabecular spaces, reduces the blood supply. The autonomic nervous system coordinates this series of events. The thoracolumbar adrenergic system regulates smooth muscle contraction and flaccidity. Conversely, the lumbosacral cholinergic system is primarily involved in allowing smooth muscle relaxation and penile erection. However, the thoracolumbar adrenergic system also mediates the first phase of the ejaculatory process with the collection of the seminal bolus in the posterior part of the urethra, whereas the propulsion of the semen out of the urethra is mediated again by the lumbosacral cholinergic system. Semen ejaculation is usually accompanied by a prompt detumescence, which characterizes the postejaculatory refractory period. A refractory period is always present after each ejaculation and is characterized by feeling of sexual satiety, decreased sexual drive, and disappearance of all signs of sympathetic activation (hyperventilation, tachycardia, and hypertension). It is generally assumed, without any compelling evidence, that the sympathetic system itself mediates the postejaculatory penile detumescence and sexual refractivity (2, 5). In this study we propose a novel effector of postorgasmic penile flaccidity: the neurohypophyseal hormone oxytocin (OT).

OT neurons are equally present in the paraventricular and supraoptic nuclei of the hypothalamus of both females and males without any evident sexual dimorphism (7). However, a clear role for OT is recognized only in the female. OT is considered to be the most readily available agent that mediates the milk ejection reflex and uterus contractility (8, 9). Debackere and colleagues (10) in the early sixties postulated that OT could be released in the male circulatory system during sexual activity. In fact, using a cross-circulation technique, they found that manual stimulation, per rectum, of seminal vesicles prompted a milk ejection response in the female partner. Further studies demonstrated that a surge of OT occurs during male sexual activity, peaking during (11, 12, 13, 14) or after (15, 16) orgasm and detumescence. This evidence in humans is matched by data from many other species (17, 18, 19). Because pharmacological administration of OT increases the number of ejaculated spermatozoa in different animal species (20), including human (21), a physiological role for OT during ejaculation has been hypothesized. OT receptor (OTR) has been identified in several portions of the male genital tract (MGT) (21, 22, 23, 24, 25), but never in the penis. We now report evidence that OTR is present in both rabbit and human penile tissues and mediates smooth muscle contractility and, therefore, penile detumescence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Noradrenaline, phenylephrine HCl, oxytocin, tamoxifen, [Thr4,Gly7]OT, [deamino-Cys1,D-Arg8]vasopressin (DDAVP), reagents for immunohistochemistry and SDS-PAGE, peroxidase-conjugated antimouse rat secondary antibodies, and polyethylene glycol-8000 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). [d(CH2)51,Tyr(Me)2,Orn8]vasotocin (OTA) and [Phe2,Orn8]vasotocin were purchased from Bachem AG (Bubendorf, Switzerland). BQ-123 sodium was purchased from RBI (Natick, MA). d(CH2)5[Tyr(Me)2,Thr4,Orn8[125I]Tyr9-NH2]vasotocin ([125I]OTA; 2200 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). Endothelin-1 was purchased from Calbiochem (La Jolla, CA). Testosterone enanthate (T) and estradiol valerate (E2v) were supplied by Schering AG (Berlin, Germany); the BM enhanced chemiluminescence system was purchased from Roche (Milan, Italy). Reagents for protein measurement were obtained from Bio-Rad Laboratories (Munich, Germany). The mouse monoclonal antibodies raised against the sequences of human OTR SVWDANAPKEAS (298–309, antibody 3–12) and PPGAEGNRTAGPPRRNEALAR (20–40, antibody 2F8) were gifts from Dr. T. Kimura (Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka, Japan); the mouse monoclonal antibody (CHINA/1F3) raised against the NH2 terminus of OTR was a gift from Dr. S. Deaglio (Laboratory of Cell Biology, Department of Genetics, University of Turin, Turin, Italy); the monoclonal rat H222 antibody against human estrogen receptor {alpha} (ER{alpha}) was a gift from Prof. G. Greene (University of Chicago, Chicago, IL); the polyclonal goat antiserum for detection of human ERß (N-19) was purchased from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA); rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase was a gift from Dr. C. Yarborough (Hauptman-Woodward Medical Research Institute, Buffalo, NY). Letrozole was a gift from Novartis Pharma (Basel, Switzerland). Triptorelin pamoate was supplied by Ipsen (Milan, Italy). Tamoxifen (TAM) was solubilized in sesame oil; the other substances were dissolved daily in double-distilled water, and further dilutions to the final concentrations were made in Krebs’ solution.

Tissue collection
Gene expression analysis was performed on commercially available human RNAs obtained from Stratagene (La Jolla, CA), including liver, stomach, and heart. The remaining RNAs were derived from human tissues collected during surgery for benign diseases after obtaining the approval of the hospital committee for investigations in humans and the patient’s informed consent. Female tissues were all from normally cycling women. Corpora cavernosa (CC) were obtained from patients undergoing penile prosthesis implantation (n = 6; age range, 40–67), correction of Peyronie’s disease (n = 1; 52 yr old), or congenital curvature of the penis (n = 1; 24 yr old). Rabbit corpora cavernosa, epididymis, testis, seminal vesicles, prostate, colon, and uterus were obtained from New Zealand White rabbits, weighing approximately 3 kg. The animals were killed by a lethal dose of pentobarbital. Human and rabbit penis were removed, and the CC were carefully dissected free from the tunica albuginea. CC were immediately fixed in Bouin’s solution and embedded in paraffin for immunohistochemistry. For in vitro contractility studies, CC preparations were immediately placed and maintained in cold Krebs solution until the beginning of the experiments. Tissue specimens were fresh-frozen for RNA preparation, Western blot analysis, and binding studies. The local ethical committee for investigations in animals of the University of Florence approved the study.

RT-PCR
Total RNA from rabbit CC, epididymis, testis, seminal vesicles, colon, and uterus was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentrations were determined by spectrophotometric analysis at 260 nm. RT-PCR experiments were performed as previously reported (21). Briefly, total RNA (500 ng) was retrotranscribed for 30 min at 50 C, denatured for 2 min at 95 C, and amplified for 35 cycles with the following cycles: 45 sec at 95 C, 1 min at 55 C, and 1 min at 70 C. The specific primers amplify a 317-bp region of the rabbit OTR mRNA sequence, as deposited in GenBank at NCBI (accession no. AF023851). The sequence of the sense primer (position 110–129) was 5'-ATG TTT GCC TCC ACC CAC AT-3'; the sequence of the antisense primer (position 398–427) was 5'-CCA GAT CTT GAA GCT GAT GA-3'. The integrity of total RNA was verified by performing RT-PCR for the rabbit housekeeping nonmuscle {gamma}-actin ({gamma}-ACT). The {gamma}-ACT-specific primers covered a 328-bp region of the rabbit {gamma}-ACT sequence, as deposited in GenBank at NCBI (accession no. X60733). The sequence of the sense primer (position 317–336) was 5'-ACA TGG AGA AGA TCT GGC AC-3'; the sequence of the antisense primer (position 626–645) was 5'-CAT GAG GTA GTC GGT CAG GT-3'. The amplified cDNA was run on a 2% agarose gel; the specific band was excised from the gel and purified from agarose using the Concert Gel Extraction Systems Kit (Invitrogen, Milan, Italy). After purification, the amplified cDNA was custom-sequenced (MGW-Biotech, Florence, Italy) to confirm the correspondence of sequence. The contamination of genomic DNA was excluded by performing amplification without retrotranscription.

Northern blot analysis
Total RNA (30 µg) was fractionated on a 1.2% agarose gel containing 8% formaldehyde, transferred onto nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Milan, Italy), and baked at 80 C for 2 h. Membranes were prehybridized for 1 h and hybridized overnight at 65 C with Church and Gilbert buffer solution as previously described (26). The probe for the detection of OTR mRNA was derived from RT-PCR of total CC rabbit RNA, as described in the previous section. The probe was labeled with 5'-[{alpha}-32P]deoxy-CTP by a random priming kit (Roche) and chromatographed (Nu-Clean D25 disposable spun columns, IBI, New Haven, CT) before use. The hybridized nylon membranes were submitted to autoradiography using Hyperfilm-MP (Amersham Pharmacia Biotech) and an X-OMAT regular intensifying screen (Eastman Kodak Co., Rochester, NY) at -80 C for various exposition times.

Real-time quantitative RT-PCR assay
Real-time RT-PCR assay was used to measure specific OTR, ER{alpha}, and ERß mRNA levels in normal human tissues, including CC. The assay was performed according to the fluorescent TaqMan methodology as previously described (27, 28). Total RNA was isolated from human tissue samples using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Before the quantitative assay, cDNA was prepared by RT-PCR according to the TaqMan RT kit (PE Applied Biosystems, Foster City, CA) protocol using 400 ng total RNA for each sample. The PCR primers and fluorogenic probe, reported in Table 1Go, were designed using Primer-Express software (PE Applied Biosystems) according to TaqMan requirements. Designs were based on NCBI GenBank human OTR, ER{alpha}, and ERß sequences. 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 (PE Applied Biosystems). 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 sec and 60 C for 1 min.


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  		TABLE 1. Primers and probes used

 
Absolute mRNA quantitation of human OTR.
External reference calibration curve was obtained by cloning a 390-bp OTR cDNA fragment, produced by RT-PCR (primer sense, 5'-CCTTCATCGTGTGCTGGACG-3'; position 1469–1489; primer antisense, 5'-CTAGGAGCAGAGCACTTATG-3'; position 1839–1859 of the human OTR mRNA sequence, as deposited at the NCBI GenBank, accession no. NM_00916), using RNA from human breast as template. Cloning was performed using the TOPO TA cloning kit (Invitrogen) according to the manufacturer’s instructions. RNA sense transcript was first obtained from OTR cloned fragment using the Ribomax in vitro transcription kit (Promega Corp., Madison, WI), and then retrotranscribed to cDNA according to the PE Applied Biosystem protocol. Known starting concentrations, ranging from 4 x 106 to 4 x 101 molecules, were used to obtain the standard curve. The sequence detection software generated a calibration curve vs. the known quantity of standard cDNA and then determined unknown sample concentrations by interpolation. All measurements were made in duplicate. Results were expressed as OTR mRNA molecules per microgram of total RNA.

Relative mRNA quantitation of human ER{alpha}/ERß.
Quantitative values were obtained from the threshold cycle (Ct) number at which the increase in fluorescent signal, associated with an exponential increase in PCR products, can be detected. The maximum change in Ct ({Delta}Ct) values of the sample was determined by subtracting the average of duplicate Ct values of the reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), quantitated using a predeveloped control assay provided by PE Applied Biosystems] from the average of duplicate Ct values of the target gene. The relative gene expression levels were also normalized to an internal calibrator, consisting of human testis RNA. Results were expressed as 1/N target x 103. N target was calculated as follows (29): N target = 2 ({Delta} Ct sample - {Delta} Ct calibrator).

Immunohistochemistry
Immunohistochemical studies were carried out as previously described (30). For studies in tissues, human and rabbit penile sections (fixed in Bouin’s solution and embedded in paraffin) were incubated first for 1 h in 2% fetal calf serum in PBS to block nonspecific antibody binding. Sections were then incubated overnight at 4 C with two different antihuman OTR antibodies: mouse monoclonal Ab 2F8 (diluted 1:1500) and IgM mouse CHINA/1F3 (diluted 1:200). The sections were then incubated with the correspondent specific Ig peroxidase conjugates for 30 min (dilution, 1:1000). Demonstration of peroxidase activity and controls for the specificity of the antisera were performed as previously described (30). The specificity of the anti-OTR antibodies in this study was controlled by 1) omission of the primary antibodies, and 2) preabsorption of the primary antibodies with myometrial cell microsomes, expressing a high density (15 pmol/mg protein) of OTR (31). OTR2F8 and CHINA/1F3 antibodies (working dilution) were incubated with 1 mg/ml myometrial membranes overnight at 4 C. After an additional 60-min incubation with 4% polyethylene glycol, the unbound antibodies were separated by rapid centrifugation and used for immunohistochemistry and Western studies. The slides were photographed using a Microphot-FX microscope (Nikon, Kogaku, Tokyo, Japan).

SDS-PAGE and Western blot analysis
Frozen samples were directly suspended in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 0.25% Nonidet P-40, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride] and homogenized (Teflon-glass) for protein analysis. The homogenates were centrifuged at 2000 rpm for 10 min at 4 C, and the protein content of the supernatants was evaluated according to Bradford’s method using Coomassie reagent (Bio-Rad Laboratories). After protein measurement, aliquots containing 30 µg protein were diluted in reducing 2x SB [Laemmli sample buffer = 62.5 mM Tris (pH 6.8), 10% glycerol, 20% sodium dodecyl sulfate, 2.5% pyronin, and 100 mM dithiothreitol] 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 (Roche)-TTBS (0.1% Tween 20, 20 mM Tris, and 150 mM NaCl), washed in TTBS, and incubated overnight with primary antibodies (5 µg/ml for 3-12 antibody, 1:1000 for 2F8 and CHINA/1F3 antibodies in BM blocking buffer-TTBS) or with myometrial microsome-preabsorbed (see above) CHINA/1F3, followed by peroxidase-conjugated secondary IgG (1:3000). Finally, reacted proteins were revealed using the BM enhanced chemiluminescence system.

Membrane preparation and binding studies
Membranes from CC and colon tissues were prepared as described previously (22, 32, 33). Protein concentration was assayed using a commercial protein assay kit (Bio-Rad Laboratories). For OT binding studies, aliquots of membranes (0.075 mg/ml) were incubated (in a final volume of 0.25 ml) with [125I]OTA (15–50 pM) in a buffer containing 50 mM Tris-maleate, pH 7.6, with 10 mM MgSO4, 1 mM benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor in the presence of 0.1% BSA at 22 C for 60 min with or without increasing concentrations of various unlabeled compounds: OTA (the corresponding unlabeled peptide), OT, [Phe2,Orn8]VT (a selective V1 vasopressin agonist), and DDAVP (the selective V2 vasopressin agonist). All measurements were performed in triplicate. After incubation, membrane suspensions were filtered through Whatman GF/B filters (Clifton, NJ) that had been presoaked in ice-cold 50 mM Tris (pH 7.4) in 0.1% BSA using the M-48R 48 well cell harvester (Brandel, Gaithersburg, MD). Filters were washed twice with 2 ml ice-cold 50 mM Tris, pH 7.4. Radioactivity retained by filters was counted in a {gamma}-counter at 70% efficiency. Binding results were simultaneously fitted using the LIGAND program (34).

Contractility studies
Human and rabbit strips were vertically mounted under 1.8 g resting tension in organ chambers containing 10 ml Krebs solution at 37 C and gassed with 95% O2 and 5% CO2 at pH 7.4. The solution had the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 2.5 mM CaCl2, and 10 mM glucose. The preparations were allowed to equilibrate for at least 90 min; during this period the bath medium was replaced every 15 min. Changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, San Giorgio di Piano, Bologna, Italy). A high potassium salt solution (KCl), made by equimolar substitution of sodium by potassium, increased tonic tension, with a maximum effect obtained at 80 mM. This value was taken as 100%, and the increase recorded in the presence of different concentrations of OT (0.01–10000 nM) and its analogs referred to this value. Cumulative drug concentrations were added, at 7-min intervals, to the bath to obtain a concentration-dependent curve; a 30- to 60-min pretreatment with selected antagonist (OTA) was performed before the concentration-response curve for the agonist.

Experimental hypogonadism and sex steroid replacement.
New Zealand White male rabbits (weighing ~2.5 kg; n = 27) were divided into five groups. One group was kept intact (controls; n = 12). Another group was treated with a single administration of 2.9 mg/kg of the long-acting GnRH analog triptorelin pamoate (n = 9). After 15 d, a subset of GnRH-treated rabbits (n = 6) was supplemented with a pharmacological dose of T (30 mg/kg weekly; n = 3) or with E2v (3.3 mg/kg weekly; n = 3). Two months after triptorelin pamoate administration and 1 wk after the last supplementation of T/E2v, rabbits were killed, and blood was drawn from the heart for sex steroid measurement. Another group of sexually mature, intact animals (weighing ~3 kg) was treated for 3 wk with letrozole, an aromatase inhibitor (35) (2.5 mg/kg daily; n = 3) dissolved in the drinking water or with the estrogen antagonist TAM (0.250 mg/kg daily; n = 3).

T and E2 measurement.
T and E2 plasma levels were measured with an Automated Chemiluminescence System (Bayer Diagnostics, East Walpole, MA) as previously described (36, 37). Briefly, extraction was performed by mixing samples with 4 vol diethyl ester for 15 min, centrifuging for 5 min at 2000 rpm, and freezing the aqueous phase in dry ice. The organic phase was recovered, evaporated to dryness under a nitrogen stream, and reconstituted in the assay buffer.

Statistical analysis
Results are expressed as the mean ± SEM for n number of experiments. Statistical analysis was performed by t test for paired or unpaired data, with ANOVA followed by Fisher’s test to evaluate the differences between groups, and P < 0.05 was taken as significant. The half-maximal effective concentration (EC50) and the half-maximal inhibitory concentration (IC50) values were calculated using the computer program ALLFIT (38).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Northern analysis (Fig. 1AGo) and RT-PCR (Fig. 1BGo) revealed specific transcripts for OTR in all parts of the rabbit MGT investigated, including the CC. To evaluate and quantitate OTR gene expression in human MGT and CC, we set up a new TaqMan real-time quantitative RT-PCR method. The results are shown in Fig. 1CGo. OTR mRNA was widely expressed in the MGT at virtually similar concentrations as in other OT-responsive tissues, such as placenta (39, 40), kidney, and heart (41). However, OTR mRNA abundance in MGT was 2–3 orders of magnitude lower than that in the classical OT targets, such as uterus and breast. The expression of OTR mRNA was very low in the human colon (Fig. 1CGo) and was almost undetectable in rabbit one (Fig. 1BGo). To investigate OTR localization in rabbit and human penis, we performed immunohistochemical studies using two distinct specific antisera: 2F8 and CHINA/1F3. The left panels of Fig. 2Go show results in humans, and the right panels show results in rabbit penile tissue. Note that similar results were obtained with the two antisera (Fig. 2Go, A and E: 2F8; Fig. 2Go, B and F: CHINA/1F3). OTR-positive staining was observed in the endothelial cells and smooth muscle cells of the arteriolar wall and trabecular spaces. The specificity of staining was evaluated through the complete absence of labeling obtained by omitting the primary antibodies in CC sections (not shown) or using the preabsorbed anti-OTR antisera (Fig. 2Go, D and H). Figure 3Go shows OTR protein expression in different human and rabbit tissues, as detected by different antisera (antibody 3-12: A, B, and D; antibody 2F8: D; CHINA/1F3: C and D). All three different antibodies stained a protein with the expected molecular mass (21, 24, 42) that completely disappeared after preabsorption of OTR antisera with an OTR-enriched tissue (see Fig. 3DGo for CHINA/1F3). OTR protein expression approximately reflects its mRNA distribution (see Fig. 1Go). To further characterize OTR found in human CC, we performed competition experiments using [125I]OTA as labeled ligand. Figure 4AGo shows results from homologous competition curves for [125I]OTA in CC (n = 5) and colon (n = 1) membranes. Results from a typical self- and cross-displacement study obtained in human CC homogenates are shown in Fig. 4BGo. Quantitative analysis (LIGAND program) of 10 competition curves performed in four separate experiments indicated the presence of a homogeneous class of binding sites, corresponding to the classic OTR (binding capacity, 9.65 ± 1.15 fmol/mg protein). This site has high specificity for OT (Kd, 280 ± 190 pM) and for the OT antagonist, OTA (Kd, 20 ± 9 pM). Conversely, it binds with lower specificity [Phe2,Orn8]VT (a selective V1 vasopressin agonist; Kd, 1.3 ± 0.884 nM) and with an extremely lower affinity DDAVP (the selective V2 vasopressin agonist; Kd, 85 ± 55.25 nM). Similar results were obtained from the analysis of homologous competition curves for [125I]OTA performed in pooled CC from 12 adult rabbits (Kd for OTA, 8.6 ± 5.6 pM; binding capacity, 6.1 ± 1.6 fmol/mg protein). Binding sites for OTA were undetectable in colon membranes. To evaluate the biological activity of OTR in human and rabbit CC, we performed contractility studies. Figure 5AGo shows the effects of OT, [Thr4,Gly7]OT (a selective OTR agonist), [Phe2,Orn8]VT, and DDAVP on isolated preparations of human CC. Increasing concentrations of OT induced a consistent enhancement of tone with a maximal effective concentration (Emax) of 38.3 ± 6.7% (n = 5). The rank order of potency of several neurohypophyseal hormone agonists in inducing human CC contractions indicates that this effect is mediated by OTR. In fact, the selective OTR agonist, [Thr4,Gly7]OT, induced a similar increase in CC contractility as OT (OT EC50, 11 ± 2.7 nM; [Thr4,Gly7]OT EC50, 24 ± 7 nM). Conversely, both [Phe2,Orn8]VT and DDAVP (selective V1 and V2 agonists, respectively) were less potent than OT in stimulating contractility. In human CC, the selective OT antagonist OTA almost completely counteracted (maximum inhibitory concentration, 69.8 ± 8.4%) the stimulatory effect of OT (1 µM), with an IC50 of 74 ± 5 nM (Fig. 5BGo). Virtually identical results were derived from contractility studies performed in rabbit preparations (not shown). In rabbit preparations, OT induced a dose-dependent increase in tone, with Emax comparable to that observed in human CC (Emax, 48.9 ± 6.7%; n = 6; see also Fig. 6AGo). Maximal responsiveness to OT was not affected by removing the endothelial layer [Emax control with endothelium, 42 ± 11.2% (n = 3); Emax without endothelium, 51.9 ± 13.4% (n = 3)] or blocking the endothelin A receptor for endothelin-1 (ET-1) with BQ123 [Emax control, 47 ± 9% (n = 3); Emax BQ123 (100 nM), 61 ± 5% (n = 3)], indicating that in rabbit CC preparations, only OTR present in the muscular compartment is involved in mediating the contractile activity of OT and that the latter does not involve ET-1. We previously observed that OT responsiveness in rabbit epididymis was greatly regulated by changing the sex steroid milieu (36). Therefore, to verify whether sex steroids could also influence OT responsiveness in the rabbit CC, we performed contractility experiments in the previously described pharmacological model of hypogonatropic hypogonadism (36, 37). Briefly, adult rabbits were treated once with the long-acting GnRH agonist triptorelin (2.9 mg/kg) and after 2 wk were supplemented weekly with vehicle, T (30 mg/kg), or E2v (3.3 mg/kg). Hormonal values at the time of contractility experiments (2 months after GnRH analog administration and 1 wk after the last administration of T or E2v) are reported in Table 2Go. Figure 6AGo shows the effect of OT on CC contractility in the different groups of rabbits. Figure 6BGo shows results obtained in the same rabbits with ET-1, a potent stimulator of CC contractility (21, 36, 37). Chronic treatment with the GnRH agonist induced a dramatic decrease in OT and ET-1 sensitivity (P < 0.01 vs. control). T supplementation completely restored ET-1 responsiveness, but did not rescue sensitivity to OT. Conversely, estrogen treatment of hypogonadal rabbits induced the opposite effect; it completely restored sensitivity to OT, but did not affect the reduced responsiveness to ET-1. This indicates a peculiar sex steroid regulation of CC sensitivity to contractile hormones.



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  		FIG. 1. OTR mRNA expression in rabbit and human MGT. A and B, OTR gene expression in rabbit MGT. A, Northern analysis for OTR in CC and other portions of the male MGT. A major band at 2.7 kbp was expressed by MGT, and rabbit uterus was used as a positive control. Each lane was loaded with 20 µg total RNA. Corresponding ethidium bromide staining of the gel is shown below the blot. B, RT-PCR products from rabbit MGT total RNA using OTR-specific (upper panel) and {gamma}-ACT-specific (lower panel) primers. Rabbit uterus total RNA was used as a positive control. {gamma}-ACT mRNA amplification was used as housekeeping gene. MWM, Molecular weight marker. C, Expression of OTR mRNA in different human tissues, including CC, as quantitated by real-time RT-PCR. The products are derived from total RNA using specific primers and a specific fluorogenic probe for OTR. Data are reported as OTR mRNA molecules per micrograms of total RNA ± SEM on a logarithmic scale. Human uterus and breast total RNA were used as positive controls. n, Sample number.

 


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  		FIG. 2. Immunolocalization of OTR in transversal sections of human (left panels) and rabbit (right panels) CC. White arrows indicate the lacunar spaces; black arrowheads indicate the blood vessels. The left panels (A–C) show an intense immunopositivity for OTR (brown staining) in the endothelial (hatched arrows) and smooth muscle cells (black arrows) of the arteriolar wall and lacunar spaces. Similar results were obtained in rabbit CC (right panels, D–F). Immunostaining was obtained using two different antibodies against human OTR: 2F8 (dilution, 1:1500; magnification, x50; A and E) and CHINA/1F3 (dilution, 1:200; magnification, x50; B and F). A higher magnification (x100) is also shown for human (C) and rabbit (G) corpora cavernosa. Control sections were obtained incubating the primary antibodies for OTR with OTR-enriched microsomes (see D and H for CHINA/1F3; magnification, x50) or omitting the primary antibodies (not shown).

 


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  		FIG. 3. Western blot detection of OTR in human and rabbit tissues. Thirty micrograms of proteins obtained from different regions of MGT were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and probed for OTR expression with monoclonal 3-12 antibody (5 µg/ml; A, B, and D), 2F8 antibody (1:1000; D), or CHINA/1F3 antibody (1:1000; C and D). A single band of about 55 kDa molecular mass (arrowhead) is present in both human (A, C, and D) and rabbit (B and C) CC as well as in all samples of MGT or in uterus and breast, which were used as positive controls. D, The positivity detected by CHINA/1F3 completely disappeared after preabsorption of the antibody with myometrial microsomes expressing a high density of [125I]OTA-binding sites (15 pmol/mg protein). Breast, uterus, and placenta were used as positive controls, whereas spermatozoa were used as a negative control for OTR. Molecular mass markers (kilodaltons) are indicated to the left of the blots.

 


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  		FIG. 4. Characterization of OTR in human CC (binding studies). A, Homologous competition curve for [125I]OTA in CC ({square}) and colon ({bullet}), used as a negative control. Ordinate: B/T, Bound to total ratio for [125I]OTA; abscissa: total concentration (molar) of labeled and unlabeled OTA. B, Competition curves between [125I]OTA and unlabeled OT ({square}), OTA (X), [Phe2, Orn8]VT (+), and DDAVP ({diamond}). Ordinate: B/T, Bound to total ratio for [125I]OTA; abscissa: total concentration (molar) of unlabeled competitors. Binding results from four separate experiments (two different samples) were simultaneously fitted using the program LIGAND.

 


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  		FIG. 5. Characterization of OTR in human CC (contractility studies). A, Effect of increasing concentrations of OT ({bullet}), [Thr4,Gly7]OT ({blacktriangleup}), [Phe2,Orn8]VT ({blacksquare}), and DDAVP ({diamondsuit}) on the basal contractility of human CC preparations. Ordinate, Increase in basal tone, expressed as percentage of the maximal response obtained with KCl (80 mM); abscissa, molar concentration of the agonists. Data are expressed as the mean ± SEM for least four separate experiments. B, Effect of increasing concentrations of OTA, a specific OT antagonist, on the tone induced by a fixed concentration of OT (1 µM) in the human CC (n = 4). Relative IC50 and EC50 are reported in the text.

 


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  		FIG. 6. Effect of triptorelin-induced hypogonadism and sex steroid replacement on responsiveness to OT and ET-1 in rabbit CC. Effects of increasing concentrations of OT (A) and ET-1 (B) on the basal tone of CC preparations in untreated ({bullet}; n = 12 in nine separate animals) and in hypogonadal rabbits without ({blacksquare}; n = 6 in three separate animals) or with ({blacktriangleup}; n = 6 in three separate animals) weekly administration of T (30 mg/kg) or E2v (3.3 mg/kg; {blacktriangledown}; n = 6 in three separate animals). Ordinate, Contractile activity, expressed as percentage of the maximal response obtained with KCl (80 mM); abscissa, concentration of the agonist. Data were expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01 (vs. control).

 

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  		TABLE 2. Effect of a GnRH analog (triptorelin) with or without T/E2v replacement on sex steroid plasma levels

 
Although we confirmed that androgens are essential for ET-1 responsiveness (36), we now show that ER activation is required for restoring complete OT sensitivity. However, to date no information has been available on either local formation of estrogens or the presence of their receptors in the penis, although we have recently described them in the human fetal external genitalia (43). We therefore studied the expression of ER and P450 aromatase in CC and set up a series of experiments to investigate whether endogenous estrogens regulate OT sensitivity in the penis. Using TaqMan real-time quantitative RT-PCR, we first measured ER{alpha} and ERß gene expression in human CC and compared their relative mRNA levels to those found in epididymis and breast, typical estrogen target tissues in males and females, respectively. As shown in Fig. 7Go, A and B, we detected similar concentration of ER{alpha} mRNA in CC and epididymis. However, ER{alpha} in male tissues was at least 10-fold less represented than that in the breast. Concerning ERß mRNA, the lowest density was observed in CC (6- to 7-fold lower than epididymis and almost 50-fold lower than breast). Hence, CC expresses both ER{alpha} and ERß genes, the first being predominant (almost a 60-fold difference). In addition, as shown in Fig. 7CGo, CC also expresses the relative proteins and even P450 aromatase (Fig. 7DGo), although to a lower extent than in epididymis or other portions of the MGT. Similar results were obtained in the rabbit (not shown). To evaluate the role of endogenous estrogens in OT-induced CC contractility, we treated sexually mature, intact rabbits with letrozole (2.5 mg/kg) or TAM (0.25 mg/kg) for 3 wk to block estrogen formation or action, respectively. Letrozole significantly reduced circulating E2 compared with control levels (control, 194.2 ± 1.5; letrozole, 144.1 ± 22 pM, P < 0.05). As shown in Fig. 8Go, both treatments induced a 50% decrease in CC responsiveness to OT.



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  		FIG. 7. Expression of ER and P450 aromatase in human CC. A, Quantitative analysis using real-time RT-PCR of ER{alpha} (right bars) and ERß (left bars) gene expression in CC ({square}) and other female (breast; ) and male (epididymis; {blacksquare}) estrogen target tissues. Results are expressed as the reciprocal (x103) of N target on a log scale. N target = 2 ({Delta}Ct sample - {Delta}Ct calibrator), the internal calibrator employed was human testis. B, Comparative Ct results. The table reports the Ct data (mean ± SEM) used to determine the amount (N target) of ER{alpha}, ERß, and GAPDH (housekeeping gene) in human CC, epididymis, breast, and testis (used as internal calibrator). C and D, Western blot detection of ER{alpha}, ERß, and P450 aromatase in human CC. Thirty micrograms of proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. ER{alpha} expression (C, upper blot) was probed with rat monoclonal H222 antibody, and a band of about 65 kDa molecular mass (arrowhead) was detected in both human CC samples tested as well as in epididymis (positive control). A different nitrocellulose membrane was probed for ERß (C, lower blot) using goat polyclonal antiserum N-19. A single band of the expected molecular mass for ERß (55 kDa) was present in human CC and in human epididymis and placenta, which were used as positive controls. D, Nitrocellulose membranes loaded with proteins derived from human CC, different regions of MGT, and placenta were probed for aromatase using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. A single band at the expected molecular mass was present in all tissues investigated. Molecular mass markers (kilodaltons) are indicated.

 


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  		FIG. 8. Effect of estrogen ablation in sexually mature, intact rabbits. Responsiveness of CC to OT was tested in untreated rabbits (A and B; {bullet}; n = 6 in three separate animals) or in animals treated daily for 3 wk with letrozole (2.5 mg/kg; A; {blacksquare}; n = 6 in three separate animals) or TAM (0.250 mg/kg; B; {blacksquare}; n = 6 in three separate animals). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with KCl (80 mM); abscissa, concentrations of oxytocin (nanomolar). Data were expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01 (vs. control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
OTR expression has been reported in several portions of the MGT, including testis (23, 24, 25, 44), epididymis (21, 22, 23, 24, 25), vas deferens (22, 23, 25), and prostate (24, 25), but it has never been reported in penile tissue. In this study we have for the first time identified and characterized the OTR gene (RT-PCR and Northern analysis) and protein (Western and binding studies) in human and rabbit CC. In addition, by using real-time RT-PCR, we originally quantitated OTR gene expression in several human tissues, including the penis. We found very high OTR mRNA expression in uterus and mammary gland (1 x 106 molecules/µg total RNA) and 2- to 3-log unit less in the other studied tissues, including the MGT. However, in the MGT, CC showed virtually the same OTR gene expression as in the epididymis, which is generally considered the main physiological target for OT in the male (20). In CC, as in the epididymis and other classic OT target tissues, OTR mediated a dose-dependent increase in contractility. Accordingly, OTR was localized in the smooth muscle compartment in both rabbit and human CC. However, the endothelial layer also expressed it. Hence, endothelial cells of penile microcirculation express OTR, as previously described for other endothelial compartments of small (45, 46) and large vessels (47, 48, 49). However, to date the exact biological significance of endothelial OTR has not been identified. In a previous study of epithelial endometrial cells in culture, we found that the OTR expressed by these cells was involved in mediating an OT-dependent release of ET-1 (50). This finding was later confirmed in epididymal epithelial cells (21). In epididymis, OT-mediated ET-1 release significantly contributed to the final contractile effect of ET-1, because BQ-123 (an ET-1 antagonist) counteracted responsiveness to OT (21). As endothelial cells of CC express ET-1 (51), we tested the effect of BQ-123 or endothelium removal on OT responsiveness in rabbit CC. Our results indicated that neither ET-1 nor the endothelium integrity substantially contribute to the net contractile effect of OT in CC strips. Hence, endothelial OTR does not regulate smooth muscle cell tone, and its function deserves further study.

Altough estrogens in the male have been considered as only androgen by-products for a long time, recent observations in animal models and human diseases have substantially changed our view (52, 53). It is generally recognized that normal estrogen formation and activity are essential not only for bone turnover and cardiovascular health, but also for reproductive function. P450 aromatase and ERs are widely expressed in the MGT and are of crucial relevance for male fertility (54). For example, we recently observed that endogenous estrogens, but not androgens, positively regulate OTR gene and protein expression and OT responsiveness in rabbit epididymis (36), and, most probably, epididymal sperm transport (21). Estrogen might also be important for penile development and physiology. Indeed, we previously found that human fetal penis expresses P450 aromatase and both isoforms of ERs, i.e. ER{alpha} and ERß, and that estrogens regulate penile cell growth (43). We now report for the first time that human adult CC also expresses ER{alpha}, ERß, and aromatase. Interestingly, the relative abundance of ER{alpha} in the CC is almost identical to that found in the epididymis, which is generally considered one of the main estrogen targets in the male. To verify whether estrogen formation and activity are essential for OT responsiveness in CC, as observed in epididymis, we employed several experimental models. In a rabbit model of hypogonadotropic hypogonadism (36, 37), we found that estrogens, but not androgens, completely rescued CC hyposensitivity to OT. This positive estrogen regulation of penile contractility seems to be peculiar to OT, because in the same animals, penile responsiveness to ET-1 was regulated in the opposite way, i.e. positively by androgens, but not by estrogens. Moreover, endogenous estrogen deprivation induced by a 3-wk treatment with a P450 aromatase inhibitor, letrozole, or with an ER antagonist, tamoxifen, induced a similar CC hyposensitivity to OT. Therefore, our data indicate that estrogens positively regulate OT responsiveness, as previously observed in other OT target tissues such as the uterus (55) and epididymis (36).

In conclusion, we demonstrate for the first time that OTR gene and protein are expressed by rabbit and human CC and mediate contractility. OTR mRNA abundance in human CC is comparable to the expression of OTR in other well established targets for OT in the male, such as the epididymis. Because in humans, orgasm induces an OT surge, and OT, in turn, mediates CC smooth muscle contraction, we hypothesized that OT may represent a hormonal signal for the universally present, but seldom studied, male postorgasmic penile detumescence and sexual inertia. We also originally demonstrated that both subtypes of ER are present in the CC and are involved in the regulation of OT-induced CC contractility. This suggests an additional new function for estrogens in the male: the regulation of OT responsiveness in the penis. Interestingly, a recent report indicates that estrogen administration to male rats increased postejaculatory latencies and decreased erectile responsiveness to nerve stimulation (56). We speculate that OT might mediate these estrogen effects by activating a contractile program in the penile musculature.


   Acknowledgments
 
We thank Dr. T. Kimura (Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka, Japan), Dr. S. Deaglio (Department of Genetics, University of Turino, Turin, Italy), Prof. G. Greene (University of Chicago, Chicago, IL), and Dr. C. Yarborough (Hauptman-Woodward Medical Research Institute, Buffalo, NY) for kindly providing the anti-OTR monoclonal antibodies 3-12 and 2F8, anti-OTR monoclonal mouse antibody (CHINA/1F3), monoclonal rat H222 antibody, and anti-aromatase antibody, respectively. We also thank Mrs. Mary Forrest for manuscript revision, and Mr. Paolo Ceccatelli and Mr. Mauro Beni (CESAL, University of Florence, Florence, Italy) for technical assistance with animal treatment.


   Footnotes
 
This work was supported by a grant from the University of Florence (Florence, Italy).

Abbreviations: {gamma}-ACT, {gamma}-Actin; CC, corpus cavernosum; Ct, threshold cycle; DDAVP, [deamino-Cys1,D-Arg8]vasopressin; E2, estradiol; EC50, half-maximal effective concentration; Emax, maximal effective concentration; ER, estrogen receptor; ET-1, endothelin-1; E2v, estradiol valerate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IC50, half-maximal inhibitory concentration; MGT, male genital tract; OT, oxytocin; OTA, [d(CH2)51,Tyr(Me)2,Orn8]vasotocin; OTR, oxytocin receptor; T, testosterone enanthate; TAM, tamoxifen.

Received July 29, 2003.

Accepted for publication December 18, 2003.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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