Identification, localization and functional in vitro and in vivo activity of oxytocin receptor in the rat penis

in Journal of Endocrinology

We recently found that the oxytocin receptor (OTR) is expressed in the human and rabbit corpus cavernosum and mediates contractility in vitro. The present study extended our investigations to the rat, and explored whether OTR regulates penile detumescence in vivo. Real-time RT-PCR quantitatively characterized the distribution of OTR mRNA in the male genital tract. Specific transcripts for OTR were expressed in all the tissues investigated. Penile expression of OTR was comparable to that observed in testis and prostate. Western blot analysis detected a single band of the expected molecular mass for OTR in all tissues examined, including rat penis. Expression of OTR protein in rat penile extracts was further confirmed by binding studies, using the OTR selective radiolabeled ligand 125I-OTA (Kd=17 ± 6.5 pM, Bmax=15.7 ± 5 fmoles/mg protein). OTR was immunolocalized to the endothelial and smooth muscle compartments of cavernous spaces and blood vessels. In rat corpus cavernosum strips, oxytocin (OT) and an OTR selective agonist ([Thr4,Gly7]OT) induced identical increases in tension, while different vasopressin agonists were less active. In vivo, OT intra-cavernous injection (ICI) dose-dependently inhibited intracavernous pressure (ICP) increase elicited by either electrical stimulation of the cavernous nerve or ICI of papaverine with similar IC50s (117.7 ± 37 mU). The OTR antagonist, atosiban, counteracted the contractile effect of OT both in vitro and in vivo. Atosiban alone significantly increased ICP at lower stimulation frequencies (2 Hz=P<0.001 and 4 Hz=P<0.05 vs control), but not at the maximal frequency (16 Hz). Our data showed that OTR is present in the rat penis and mediates contractility both in vitro and in vivo, therefore suggesting a role for OT in maintaining penile detumescence.

Abstract

We recently found that the oxytocin receptor (OTR) is expressed in the human and rabbit corpus cavernosum and mediates contractility in vitro. The present study extended our investigations to the rat, and explored whether OTR regulates penile detumescence in vivo. Real-time RT-PCR quantitatively characterized the distribution of OTR mRNA in the male genital tract. Specific transcripts for OTR were expressed in all the tissues investigated. Penile expression of OTR was comparable to that observed in testis and prostate. Western blot analysis detected a single band of the expected molecular mass for OTR in all tissues examined, including rat penis. Expression of OTR protein in rat penile extracts was further confirmed by binding studies, using the OTR selective radiolabeled ligand 125I-OTA (Kd=17 ± 6.5 pM, Bmax=15.7 ± 5 fmoles/mg protein). OTR was immunolocalized to the endothelial and smooth muscle compartments of cavernous spaces and blood vessels. In rat corpus cavernosum strips, oxytocin (OT) and an OTR selective agonist ([Thr4,Gly7]OT) induced identical increases in tension, while different vasopressin agonists were less active. In vivo, OT intra-cavernous injection (ICI) dose-dependently inhibited intracavernous pressure (ICP) increase elicited by either electrical stimulation of the cavernous nerve or ICI of papaverine with similar IC50s (117.7 ± 37 mU). The OTR antagonist, atosiban, counteracted the contractile effect of OT both in vitro and in vivo. Atosiban alone significantly increased ICP at lower stimulation frequencies (2 Hz=P<0.001 and 4 Hz=P<0.05 vs control), but not at the maximal frequency (16 Hz). Our data showed that OTR is present in the rat penis and mediates contractility both in vitro and in vivo, therefore suggesting a role for OT in maintaining penile detumescence.

Keywords:

Introduction

The penis resides in the flaccid, non-erect, state for the majority of the time, due to an active contraction of smooth muscle cells (SMC) in penile vessels and lacunar spaces. Penile SMC contraction is favored by a rapid breakdown of the relaxing, nitric oxide-related, cGMP, through an increased penile expression of phosphodiesterase type 5 (Morelli et al. 2004), and by the activity of a complex array of contractile neurotransmitters and neuro-modulators, which has only partly been characterized. It is generally assumed that the release of noradrenaline from sympathetic adrenergic terminals plays a primary role in determining vasoconstriction in penile vascular beds (Maggi et al. 2000, Andersson 2003). However, modulatory peptides such as endothelin-1 might also participate in this process, in both physiological (Granchi et al. 2002) and pathological (Filippi et al. 2003a) conditions. Interestingly, we recently described the presence of the oxytocin receptor (OTR) gene and protein in rabbit and human cavernous tissue in a similar concentration to that found in other portions of the male genital tract (Vignozzi et al. 2004), classically considered the main male target of oxytocin (OT), such as the epididymis (Maggi et al. 1987, Einspanier & Ivell 1997, Ivell et al. 1997, Frayne & Nicholson 1998, Filippi et al. 2002a,b). In the epididymis, OTR mediates an increase in both in vitro (Hib 1974, Filippi et al. 2002a,b, 2003b) and in vivo (Cross 1955, Hib 1977, Nicholson et al. 1999, Assinder et al. 2002, Studdard et al. 2002) contractility and sperm output. Also in the penis OTR mediates in vitro contractility and therefore we hypothesized that OT participates in maintaining penile SMC contraction and flaccidity (Vignozzi et al. 2004).

OT was originally characterized as a hormone with a permissive role in female reproduction: facilitating uterine contraction and milk ejection (Cross 1955, Soloff & Swartz 1974, Roberts et al. 1976, Markle et al. 1978, Soloff et al. 1979, Filippi et al. 2003b). In males, OT was found to be one of the most potent agents to induce penile erection by electrical or pharmacological stimulation of paraventricular nuclei in rats, rabbits and monkeys (see Argiolas 1992 and Gimpl & Fahrenholz 2001 for reviews). Nevertheless, an additional and peripheral role for OT has been proposed. In fact, in all the studies performed so far (Ogawa et al. 1980, Carmichael et al. 1987, Murphy et al. 1987, Uckert et al. 2003), a clear-cut surge of OT plasma levels has been described during male sexual activity, peaking during orgasm and detumescence, with still unknown physiological significance. Since OT cannot easily cross the blood–brain barrier, a central effect of this peptide released by the posterior pituitary during orgasm is rather unlikely. Conversely, a peripheral action of OT should be considered, in particular at the level of the male genital tract.

The primary goal of the present study was to extend our previous observations obtained in other species (Vignozzi et al. 2004) to the rat and to verify whether or not OT has any effect in an in vivo model of penile erection. Hence, we investigated the pharmacological stimulation with OT or inhibition with atosiban (an OTR antagonist) of OTR in rat corpus cavernosum (CC) on intracavernous pressure (ICP) elicited by electrostimulation (ES) of the cavernous nerve and intracavernous injection (ICI) of papaverine. Essentially, we found that local administration of OT decreased and atosiban increased rat penile erectile response.

Materials and Methods

Chemicals and tissues

Phenylephrine HCl, papaverine HCl, oxytocin, [Thr4,Gly7]oxytocin, [deamino-Cys1, d -Arg8]vasopressin (DDAVP), reagents for immunocytochemistry, for SDS-PAGE and peroxidase-conjugated anti-mouse secondary antibody were purchased from Sigma (St Louis, MO, USA). (d(CH2)51,Tyr(Me)2,Orn8)vasotocin (OTA) and (Phe2,Orn8)vasotocin were purchased from Bachem AG (Bubendorf, Switzerland). d(CH2)5[Tyr(Me) 2,Thr4,Orn8, 125I-Tyr9-NH2]-vasotocin (125I-OTA) was purchased from NEN Life Science (Boston, MA, USA). Atosiban was from Ferring AB (Limhamn, Sweden). Male rat genital tissues including CC, brain, liver, cornea and uterus were obtained from adult Sprague-Dawley (SD) rats (Harlan, S. Pietro al Natisone, Italy). The Local Ethical Committee for Investigations in Animals of the University of Florence approved the study. Human CC samples were from patients undergoing penile prosthesis implantation (n=4) with the approval of the Hospital Committee for Investigation in Humans and with informed consent.

Isolation of RNA and cDNA synthesis

Total RNA was extracted from frozen tissues using TRIZOL (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. RNA concentration and quality were measured by spectrophotometric analysis at 260 and 280 nm. RNA integrity was assessed by electrophoresis in agarose gel. For each sample 400 ng total RNA were reverse-transcribed to cDNA in a final volume of 80 μl using a TaqMan Reverse Transcription kit (Applied Biosystems, Forster City, CA, USA) under the following conditions: 10 min at 25 °C, 30 min at 48 °C, 5 min at 95 °C.

Real-time quantitative RT-PCR

The mRNA quantitative analysis was performed according to the fluorescent TaqMan methodology as already published (Vignozzi et al. 2004). PCR primers and probe for OTR mRNA quantitation were purchased as an Assay-On-Demand (AoD) gene expression product from Applied Biosystems. The beta-2 microglobulin gene was chosen as the reference gene and the corresponding AoD product was provided by Applied Biosystems. The PCR mixture (25 μl final volume) consisted of 1× final concentration of AoD mix, 1× final concentration of Universal PCR Master Mix (Applied Biosystems), and 25 ng cDNA. Amplification and detection were performed with the ABI Prism 7700 Sequence Detection System with the following thermal cycler conditions: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles at 95 °C for 30 s and 60 °C for 1 min. Each measurement was carried out in duplicate. The analysis of the results was based on the comparative Ct method according to the manufacturer’s instructions (Applied Biosystems), where Ct represents the cycle number at which the fluorescent signal, associated with an exponential increase in PCR products, crossed a given threshold. The maximum change in Ct values of the sample (Ctsample) was determined by subtracting the average of duplicate Ct values of the reference gene from the average of duplicate Ct values of the target gene.

Immunohistochemistry

Immunohistochemical studies were carried out as previously described (Maggi et al. 1991, Vignozzi et al. 2004) in both rat CC and mammary gland. Briefly, rat penile and breast sections (fixed in Bouin’s solution and embedded in paraffin) were subsequently incubated, first for 1 h in 2% FCS in PBS to block non-specific antibody binding, overnight at 4 °C with the IgM mouse CHINA/1F3 (diluted 1:200), then with the corresponding specific immunoglobulin peroxidase conjugates for 30 min (dilution 1:1000). Demonstration of peroxidase activity and controls for specificity of the antisera were performed as previously described (Maggi et al. 1991, Vignozzi et al. 2004). The specificity of the anti-OTR antibodies in this study was controlled by (1) omission of the primary antibodies and (2) pre-absorption of the primary antibodies with myometrial cell microsomes, expressing a high density (15 pmoles/mg protein) of OTR (Maggi et al. 1994a). 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. The slides were photographed using a Nikon Microphot-FX microscope (Nikon, Kogaku, Tokyo, Japan).

SDS-PAGE and Western blot analysis

Protein from male rat reproductive tissues, liver, uterus and human CC were prepared as previously described (Vignozzi et al. 2004). Briefly, frozen samples were homogenized in lysis buffer. Aliquots containing 30 μg proteins were diluted in reducing 2 × SB (Laemmli’s sample buffer) and loaded onto 10% SDS-PAGE. After separation, 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 anti-OTR CHINA/1F3 antibody (1:1000 in BM blocking buffer-TTBS) followed by peroxidase-conjugated secondary IgG (1:3000). Finally, reacted proteins were revealed by the BM enhanced-chemiluminescence system (Roche Diagnostics).

Membrane preparation and binding studies

Membranes from rat CC were prepared as previously described (Maggi et al. 1987, 1994b, Peri et al. 1997, 1998, Vignozzi et al. 2004). Briefly, aliquots of membranes (0.075 mg/ml) were incubated (in a final volume, 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. Then, membrane suspensions were filtered through Whatman GF/B filters by using the Brandel M-48R. Radioactivity was counted in a γ-counter. Results were simultaneously fitted using the LIGAND program (Munson & Rodbard 1980).

Contractility studies

As previously reported for rabbit (Filippi et al. 2002b, Vignozzi et al. 2004), rat CC strips were attached to an isometric muscle bath system and equilibrated for at least 60 min at 37 °C in Krebs-bicarbonate solution containing (in mM) NaCl 118.1, KCl 4.7, KH2PO4 1.0, MgSO4 1.0, NaHCO3 25.0, CaCl2 2.5 and glucose 11.1 in a bath chamber, gassed with 95% O2 and 5% CO2. Tissue resting force was set at 500 mg and changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, San Giorgio di Piano, Bologna, Italy). High potassium salt solution (KCl) increased the 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 (1–10 000 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-min pre-treatment with a selective antagonist (atosiban) was performed before the concentration–response curve for the agonist.

Physiological erection studies

SD rats (280–360 g) were anesthetized with pentobarbital (Abbot, Campoverde d’Aprilie, LT, Italy) 45 mg/kg intraperitoneal injection. Mean arterial pressure (MAP) was continuously monitored via the femoral artery. Both crura were exposed and each was perforated with a 26-gauge needle connected to PE-50 tubing for ICP recording and drug delivery. MAP and ICP were recorded via a pressure transducer (World Precision Instruments Inc, Sarasota, FL, USA) connected to a recorder TA240 (Gold Inc, Cleveland, OH, USA). For ES, a bipolar platinum electrode attached to an ST6 stimulator (Bio-medica Mangoni Inc, Pisa, Italy) was mounted on the cavernous nerve. ES (width 5 ms, duration 30 s, 2.5 V) at different frequencies (1, 2, 4, 8, 16, 32 Hz) was performed, with the maximal ICP obtained at 16Hz. OT (Fort Dodge Veterinaria, S.A., Girona, Spain) ICI was tested at this frequency at the following doses (in mU) 600, 200, 60, 20, 6 and 2, 15 min after the first ES and 5 min before the second stimulation. Atosiban (45 μg) plus OT (60 mU) was administered according to the previously described protocol, and atosiban was delivered 5 min before OT injection. Papaverine (0.35 mg) was found to elicit the optimal response as reported previously (Chen et al. 1992, Martinez-Pineiro et al. 1994). The effect of OT and OT plus antagonist was also investigated on the increase in ICP induced by this pharmacostimulation with the only difference from the ES protocol being that there was a 30-min wait between the two responses. No more than two doses were tested for each animal. The effect of the OT antagonist atosiban (45 μg), alone, on erectile response was tested by ES of the cavernous nerve at varying stimulation frequencies (2, 4, 16 Hz). Atosiban was intracavernously injected 10 min after the first ES and 5 min before the second ES. The erectile response elicited by electro- or pharmaco-stimulation were quantified by calculating the ratio of max ICP/MAP × 100. The max ICP is the maximal ICP rise, with MAP being the mean artery pressure during the plateau phase.

Statistical analysis

Results are expressed as means ± s.e.m. for n experiments. Statistical analysis was performed with Student’s t-test followed by Fisher’s test when appropriate. P<0.05 was taken as significant. Values of half-maximal response inhibiting concentrations (IC50) were calculated by using the ALLFIT program (De Lean et al. 1978).

Results

The quantitative distribution of OTR mRNA in the rat male genital tract is shown in Fig. 1A. Specific transcripts for OTR are expressed in all the tissues investigated, including penis. Penile expression of OTR was comparable to that observed in testis and prostate, although one log unit lower than in the epididymis, while the rat cornea and liver, considered as negative controls (Assinder et al. 2004), reveal the lowest level of OTR mRNA. To further characterize OTR in rat CC, we performed binding studies using the OTR selective radiolabeled ligand 125I-OTA, according to the previously described protocol (Maggi et al. 1987, Peri et al. 1997, 1998, Vignozzi et al. 2004). Figure 1B shows the simultaneous fitting, using the LIGAND program (Munson & Rodbard 1980) of homologous competition curves for 125I-OTA, derived from three separate experiments. 125I-OTA binds with high affinity to a homogeneous class of binding sites. These sites are expressed in rat CC at a density similar to that which we have already described for the OTR in human and rabbit penis (Vignozzi et al. 2004). Binding parameters are reported in Table 1. We also performed Western blot analysis of OTR in male rat genital tissues, using rat uterus and human CC as positive controls and rat liver as a negative control (Fig. 1 inset). In agreement with previous studies in humans and rabbits (Vignozzi et al. 2004), a single band of the expected molecular mass was detected in all tissues examined, including rat CC.

To localize OTR in rat cavernous tissue we performed immunohistochemical studies (Fig. 2A,B,C), using the same antibody as employed for Western blot studies (CHINA/1F3) and compared them with the results using mammary gland (Fig. 2D,E,F), considered as a positive control. An OTR positive staining was observed in the endothelial cells and in the smooth muscle cells of the arteriolar wall and trabecular spaces (Fig. 2A,B).

A similar pattern of OTR expression was demonstrated in the mammary gland, where both the epithelial and the surrounding smooth muscle compartment were positively stained (Fig. 2D,E). The specificity of staining was evaluated through the complete absence of labeling obtained by omitting the primary antibody or pre-absorbing it with human myometrial membranes enriched with OTR (Fig. 2C,F), as previously described (Vignozzi et al. 2004).

To pharmacologically characterize OTR expression in the rat penis, we performed in vitro contractility studies in CC strips using several neurohypophysial hormone analogs showing selectivity for the different receptor subtypes. The OT and the selective OTR agonist [Thr4, Gly7]OT induced a sustained and virtually identical increase in tension (Emax=77.2 ± 2.8%), while the selective V1 and V2 agonists were definitively less active (Fig. 3A). In addition, increasing the concentration of the OT antagonist, atosiban, dose-dependently counteracted the contractile effect of 1 μM OT (Fig. 3B).

The in vivo effect of locally administered OT on physiological erections was also studied. The baseline ICP and MAP, recorded at the beginning of the experiment, were 10.8 ± 0.35 mmHg and 125.1 ± 1.6 mmHg respectively (n=60). ES of the cavernous nerve induced a frequency-dependent increase in ICP (data not shown), reaching a maximum ICP (68.1 ± 1.64 mmHg) at 16 Hz. Papaverine (0.35 mg) ICI also induced a sustained increase in ICP (45.6 ± 1.39 mmHg). Figure 4 (left side) shows the results from a typical experiment of ES (upper tracing) and papaverine (lower tracing). As shown on the right side of Fig. 4, OT (200 mU) blunted the effect of both stimulators. Figure 5A shows that this inhibitory effect of OT is dose-dependent, with similar IC50s for both ES- and papaverine-induced responses (shared IC50=117.7 ± 37 mU). The OT antagonist atosiban (45 μg intra-cavernously) completely abrogated the inhibitory effect of OT (60 mU, a dose close to the IC50) on both the ES and the papaverine-induced ICP increase (Fig. 5B).

Finally, to evaluate whether blocking endogenous of OT action would increase ES-induced erection, we administered atosiban (45 μg) alone by ICI during ES with increasing frequencies (2–16 Hz). The results are shown in Fig. 6. Atosiban significantly increased the mean ICP/MAP ratio at lower stimulation frequencies (2 Hz= P<0.001 and 4 Hz=P<0.05 vs control, n=5 for each frequency), but not at the maximal frequency (16 Hz). Interestingly, after atosiban ICI, the maximal ICP/MAP ratio induced by 4 Hz stimulation (56.3 ± 1.9) was not different from the value obtained with the maximal (16 Hz) stimulation (59.02 ± 2.1). In unstimulated preparations, atosiban slightly, but not significantly, increased the basal ICP/MAP ratio when compared with control rats (control=9.67 ± 0.96, atosiban=10.8 ± 1.65, n=15, P=0.11).

Discussion

The present study extended a previous observation on the presence of biologically active OTR in the penis of rabbits and humans (Vignozzi et al. 2004) to rats. Also it shows, for the first time, that exogenous injection of OTR ligands into rat CC affects penile erection in vivo.

To identify, characterize and localize OTR in rat CC, we performed combined real-time RT-PCR, radioligand binding, Western blot, immunohistochemical and in vitro contractility studies. Results obtained from all these different methods support the view that OTR is expressed and biologically active in the rat penis as previously reported by us in the rabbit and human penis (Vignozzi et al. 2004). Interestingly, the OTR gene is expressed in the penis in similar concentrations as those found in the prostate and testis, classically considered male targets for OT (Bathgate & Sernia 1994, Einspanier & Ivell 1997, Ivell et al. 1997, Frayne & Nicholson 1995, 1998, Assinder et al. 2000, 2004, Whittington et al. 2001). In the latter tissues, OTR regulates steroidogenesis (Nicholson et al. 1991, Nicholson & Jenkin 1995, Frayne & Nicholson 1995) and contractility (Bodanszky et al. 1992, Frayne et al. 1996, Harris & Nicholson 1998). In the penis, OTR is mainly localized in smooth muscle and regulates its in vitro contractile tone.

The main finding of this study is the original demonstration that intracavernous delivering of OT led to a dose-dependent inhibition of the ICP increase elicited by either ES or papaverine. Concomitant administration of the OTR antagonist, atosiban, completely abolished the OT-induced detumescence. Another important finding of this study is that ICI of atosiban facilitated a sub-maximal erectile response induced by ES of the cavernous nerve, at lower stimulation frequencies. This supports the view that OTR mediates penile detumescence in vivo and suggests that endogenous OT may exert a tonic role in maintaining penile flaccidity, which can be unmasked by the pharmacological block of its receptor. If these in vivo findings in the rat model are confirmed also in humans, we hypothesize that intracavernous injection of OT could be clinically helpful in treating low-flow priapism, as an alternative therapy to adrenergic agonists which are not completely devoid of dangerous systemic side-effects. In addition, the use of atosiban to increase the erectile response to other vasorelaxant stimuli might be envisaged.

Although it is unclear which is the physiological source of penile OT, it is interesting to note that the affinity constants of the OTR which we identified in the penis in this (Kd=17 pM) as well as in a previous study (Vignozzi et al. 2004) are compatible with the circulating concentration of OT. Interestingly, circulating OT substantially increases during male orgasm (Ogawa et al. 1980, Carmichael et al. 1987, Murphy et al. 1987, Uckert et al. 2003). Orgasm and semen ejaculation are usually accompanied by a prompt detumescence, which characterizes the post-ejaculatory refractory period. Hence, it is possible that OT plays a physiological role in mediating post-orgasmic penile detumescence. However, it is also possible that OT is generated locally in the penile tissue, as has been described for other male genital tissues such as testis (Nicholson et al. 1984, 1986, Foo et al. 1991, Nicholson & Hardy 1992, Einspanier & Ivell 1997), epididymis (Harris et al. 1996, Assinder et al. 2000) and prostate (Gemmell & Sernia 1989, Nicholson 1996, Jenkin & Nicholson 1999, Assinder et al. 2004, Whittington et al. 2004), exerting an autocrine/paracrine role in regulating cavernous smooth muscle tone.

In conclusion, we have demonstrated for the first time that OTR exists in rat CC and mediates contractility in vitro and in vivo, suggesting a possible role for this receptor in regulating penile erection.

Table 1

Binding parameters derived from the simultaneous fitting (using the LIGAND program) of 3 homologous competition curves for 125I-OTA performed in corpora cavernosa from 3 independent membrane preparations

Affinity constant, Kd (pM)17.0 ± 6.50
Binding capacity, Bmax (fmoles/mg protein)15.7 ± 5.00
Non specific binding (% of total)2.0 ± 0.04
Figure 1
Figure 1

Characterization of OTR in rat CC. (A) Quantitative detection of mRNA for OTR in rat tissues by real-time RT-PCR. Data are expressed as means ± s.e.m. of arbitrary units (a.U.) calculated according to the comparative Ct method. (B) Binding studies. Homologous competition curve for 125I-OTA in CC. Ordinate: B/T, bound to total ratio for 125I-OTA. Abscissa: total concentration (molar) of labeled+unlabeled OTA. Insert: Western blot detection of OTR in rat reproductive tissues. A single band at the expected molecular mass of 55 kDa is present at different levels in all rat genital tissues analyzed. The human CC and rat uterus were run as positive controls and the rat liver as a negative control. Molecular mass markers (kDa) are indicated to the left of the blot.

Citation: Journal of Endocrinology 184, 3; 10.1677/joe.1.05885

Figure 2
Figure 2

Immunolocalization of OTR in transverse sections of rat CC (A,B,C) and in rat breast (D,E,F). Panels A (magnification ×40) and B (magnification ×200) show an intense immunopositivity for OTR both in the endothelial (arrows heads) and smooth muscle cells (arrows) of the arteriolar wall and lacunar spaces. Panels D (magnification ×40) and E (magnification ×200) show the presence of positive labeling in myoepithelial cells of ducts and alveoli (black arrows). Immunostaining was obtained with CHINA/1F3 antibody (dilution 1:200). Panels C and F show the control sections obtained by pre-incubating the primary antibodies for OTR with OTR-enriched microsomes (magnification ×40) and are counterstained with hematoxylin.

Citation: Journal of Endocrinology 184, 3; 10.1677/joe.1.05885

Figure 3
Figure 3

Characterization of OTR in rat CC (contractility studies). (A) Effect of increasing concentrations of OT (solid squares), [Thr4,Gly7]OT (open diamonds), [Phe2,Orn8]VT (solid circles) and DDAVP (solid triangles) on the basal contractility of rat CC preparations. Ordinate: increase in basal tone, expressed as a percentage of the maximal response obtained with KCl (80 mM). Abscissa: molar concentration of the agonists. Data are expressed as means ± s.e.m. of at least 4 separate experiments. (B) Effect of increasing concentrations of atosiban, an OT antagonist, on the tone induced by a fixed concentration of OT (1 μM) in rat CC (n=4).

Citation: Journal of Endocrinology 184, 3; 10.1677/joe.1.05885

Figure 4
Figure 4

Representative ICP tracings. Erectile response elicited by electrical stimulation (ES; 16 Hz, 2.5 V, 5 ms, 30 s) of the rat cavernous nerve (upper tracing) and by papaverine (PAP; 0.35 mg) intracavernous injection (lower tracing) is shown. Tracings on the right side demonstrate ICP changes induced by ES (upper tracing) and papaverine (lower tracing) before and after intracavernous administration of 200 mU OT.

Citation: Journal of Endocrinology 184, 3; 10.1677/joe.1.05885

Figure 5
Figure 5

Effects of OT on ICP. (A) Increasing doses of OT (2–600 mU) on intracavernous pressure induced by electrical stimulation (ES; 16 Hz, 2.5 V, 5 ms, 30 s, open squares) and papaverine (0.35 mg, solid circles), normalized as a percentage of mean arterial pressure (max ICP/MAP ×100). The ICP rise in the absence of OT injection is taken as control. The baseline ICP/MAP (%) value (grey column) is also provided. Values are expressed as means ± s.e.m. of at least 4 separate rats for each dose. (B) Effect of OT (60 mU) with (grey columns) or without (solid columns) the OT antagonist atosiban (45 μg) on ICP increase (max ICP/MAP×100) induced by ES (16 Hz, 2.5 V. 5 ms, 30 s) or papaverine (0.35 mg) ICI. The percentage of max ICP/MAP increases induced by ES (horizontally hatched columns) and by papaverine (PAP, vertically hatched columns) before OT injection are taken as control. The baseline ICP/MAP (%) value (open column) is also represented. Values are shown as means ± s.e.m. of at least 4 separate experiments. ***P<0.01 vs baseline; **P<0.05 vs either PAP or OT+atosiban+PAP; °°P<0.05 vs either ES or OT+atosiban+ES.

Citation: Journal of Endocrinology 184, 3; 10.1677/joe.1.05885

Figure 6
Figure 6

Effects of the OT antagonist atosiban, alone, on erectile response. The ICP increase was induced by electrical stimulation (ES; 2.5 V, 5 ms, 30 s) at varying stimulation frequencies (2, 4, 16 Hz). Atosiban (45 μg) was intracavernously injected 10 min after the first ES and 5 min before the second ES. The first response (before atosiban injection) is taken as control (solid column). Values were normalized as a percentage of the mean arterial pressure (max ICP/MAP ×100) and are shown as means ± s.e.m. (*P<0.001 and °P<0.05 vs control, n=5 for each frequency).

Citation: Journal of Endocrinology 184, 3; 10.1677/joe.1.05885

We thank Dr S Deaglio (Laboratory of Cell Biology, Department of Genetics, University of Turin, Turin, Italy) for kindly providing the anti-OTR monoclonal mouse antibody (CHINA/1F3).

Funding

This study was supported by a grant from Cofin 2002-MIUR (Progetti di Ricerca di Rilevanza Nazionale). The Andrology Unit belongs to the Centro di Ricerca, Trasferimento e Alta Formazione MCIDNENT of the University of Florence, Florence, Italy. The authors declare that there is no conflict of interest that would prejudice the impartiality of this work.

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  • ChenKK Chan JY Chang LS Chen MT & Chan SH 1992 Intracavernous pressure as an experimental index in a rat model for the evaluation of penile erection. Journal of Urology1471124–1128.

    • Search Google Scholar
    • Export Citation
  • CrossBA1955 The posterior pituitary gland in relation to reproduction and lactation. British Medical Bulletin11151–155.

  • De LeanA Munson PJ & Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: applications to bioassay radioligand assay and physiological dose–response curves. American Journal of Physiology – Endocrinology and Metabolism235E97–E102.

    • Search Google Scholar
    • Export Citation
  • EinspanierA & Ivell R 1997 Oxytocin and oxytocin receptor expression in reproductive tissues of the male marmoset monkey. Biology of Reproduction56416–422.

    • Search Google Scholar
    • Export Citation
  • FilippiS Vannelli GB Granchi S Luconi M Crescioli C Mancina R Natali A Brocchi S Vignozzi L Bencini E Noci I Ledda F Forti G & Maggi M 2002a Identification localization and functional activity of oxytocin receptors in epididymis. Molecular Cell Endocrinology19389–100.

    • Search Google Scholar
    • Export Citation
  • FilippiS Luconi M Granchi S Vignozzi L Bettuzzi S Tozzi P Forti G & Maggi M 2002b Estrogens but not androgens regulate expression and functional activity of oxytocin receptor in rabbit epididymis. Endocrinology1434271–4280.

    • Search Google Scholar
    • Export Citation
  • FilippiS Marini M Vannelli GB Crescioli C Granchi S Vignozzi L Luconi M Ferruzzi P Morelli A Forti G Ledda F & Maggi M 2003a Effects of hypoxia on endothelin-1 sensitivity in the corpus cavernosum. Molecular Human Reproduction9765–774.

    • Search Google Scholar
    • Export Citation
  • FilippiS Vignozzi L Vannelli GB Ledda F Forti G & Maggi M 2003b Role of oxytocin in the ejaculatory process. Journal of Endocrinological Investigation2682–86.

    • Search Google Scholar
    • Export Citation
  • FooNC Carter D Murphy D & Ivell R 1991 Vasopressin and oxytocin gene expression in rat testis. Endocrinology1282118–2128.

  • FrayneJ & Nicholson HD 1995 Effect of oxytocin on testosterone production by isolated rat Leydig cells is mediated via a specific oxytocin receptor. Biology of Reproduction521268–1273.

    • Search Google Scholar
    • Export Citation
  • FrayneJ & Nicholson HD 1998 Localization of oxytocin receptors in the human and macaque monkey male reproductive tracts: evidence for a physiological role of oxytocin in the male. Molecular Human Reproduction4527–532.

    • Search Google Scholar
    • Export Citation
  • FrayneJ Townsend D & Nicholson HD 1996 Effects of oxytocin on sperm transport in the pubertal rat. Journal of Reproduction and Fertility107299–306.

    • Search Google Scholar
    • Export Citation
  • GemmellRT & Sernia C 1989 The localization of oxytocin and mesotocin in the reproductive tract of the male marsupial bandicoot Isoodon macrourus. General and Comparative Endocrinology75103–109.

    • Search Google Scholar
    • Export Citation
  • GimplG & Fahrenholz F 2001 The oxytocin receptor system: structure function and regulation. Physiological Reviews81629–683.

  • GranchiS Vannelli GB Vignozzi L Crescioli C Ferruzzi P Mancina R Vinci MC Forti G Filippi S Luconi M Ledda F & Maggi M 2002 Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells. Molecular Human Reproduction81053–1064.

    • Search Google Scholar
    • Export Citation
  • HarrisGC & Nicholson HD 1998 Characterisation of the biological effects of neurohypophysial peptides on seminiferous tubules. Journal of Endocrinology15635–42.

    • Search Google Scholar
    • Export Citation
  • HarrisGC Frayne J & Nicholson HD 1996 Epididymal oxytocin in the rat: its origin and regulation. International Journal of Andrology19278–286.

    • Search Google Scholar
    • Export Citation
  • HibJ1974 The contractility of the cauda epididymidis of the mouse its spontaneous activity in vitro and the effects of oxytocin. Journal of Reproduction and Fertility36191–193.

    • Search Google Scholar
    • Export Citation
  • HibJ1977 The ‘in vivo’ effects of oxytocin and vasopressin on spontaneous contractility of the rat epididymis. International Journal of Fertility2263–64.

    • Search Google Scholar
    • Export Citation
  • IvellR Balvers M Rust W Bathgate R & Einspanier A 1997 Oxytocin and male reproductive function. Advances in Experimental Medicine and Biology424253–264.

    • Search Google Scholar
    • Export Citation
  • JenkinL & Nicholson HD 1999 Evidence for the regulation of prostatic oxytocin by gonadal steroids in the rat. Journal of Andrology2080–87.

    • Search Google Scholar
    • Export Citation
  • MaggiM Malozowski S Kassis S Guardabasso V & Rodbard D 1987 Identification and characterization of two classes of receptors for oxytocin and vasopressin in porcine tunica albuginea epididymis and vas deferens. Endocrinology120986–994.

    • Search Google Scholar
    • Export Citation
  • MaggiM Vannelli GB Peri A Brandi ML Fantoni G Giannini S Torrisi C Guardabasso V Barni T Toscano V Massi G & Serio M 1991 Immunolocalization binding and biological activity of endothelin in rabbit uterus: effects of ovarian steroids. American Journal of Physiology – Endocrinology and Metabolism260E292–E305.

    • Search Google Scholar
    • Export Citation
  • MaggiM Fantoni G Baldi E Cioni A Rossi S Vannelli GB Melin P Akerlund M & Serio M 1994a Antagonists for the human oxytocin receptor: an in vitro study. Journal of Reproduction and Fertility101345–352.

    • Search Google Scholar
    • Export Citation
  • MaggiM Vannelli GB Fantoni G Baldi E Magini A Peri A Giannini S Gloria L Del Carlo P Casparis D Tomei T & Serio M 1994b Endothelin in the human uterus during pregnancy. Journal of Endocrinology142385–396.

    • Search Google Scholar
    • Export Citation
  • MaggiM Filippi S Ledda F Magini A & Forti G 2000 Erectile dysfunction: from biochemical pharmacology to advances in medical therapy. European Journal of Endocrinology143143–154.

    • Search Google Scholar
    • Export Citation
  • MarkleHV Warr JL & Branda LA 1978 Oxytocin binding sites in lactating rabbit mammary gland. Canadian Journal of Biochemistry56968–976.

    • Search Google Scholar
    • Export Citation
  • Martinez-PineiroL Brock G Trigo-Rocha F Hsu G Lue TF & Tanagho EA 1994 Rat model for the study of penile erection: pharmacologic and electrical-stimulation parameters. European Urology2562–70.

    • Search Google Scholar
    • Export Citation
  • MorelliA Filippi S Mancina R Luconi M Vignozzi L Marini M Orlando C Vannelli GB Aversa A Natali A Forti G Giorgi M Jannini EA Ledda F & Maggi M 2004 Androgens regulate phosphodiesterase type 5 expression and functional activity in corpora cavernosa. Endocrinology1452253–2263..

    • Search Google Scholar
    • Export Citation
  • MunsonPJ & Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Analytical Biochemistry107220–239.

    • Search Google Scholar
    • Export Citation
  • MurphyMR Seckl JR Burton S Checkley SA & Lightman SL 1987 Changes in oxytocin and vasopressin secretion during sexual activity in men. Journal of Clinical Endocrinology and Metabolism65738–774.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD1996 Oxytocin: a paracrine regulator of prostatic function. Reviews of Reproduction169–72.

  • NicholsonHD & Hardy MP 1992 Luteinizing hormone differentially regulates the secretion of testicular oxytocin and testosterone by purified adult rat Leydig cells in vitro. Endocrinology130671–677.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD & Jenkin L 1995 Oxytocin and prostatic function. Advances in Experimental Medicine and Biology395529–538.

  • NicholsonHD Swann RW Burford GD Wathes DC Porter DG & Pickering BT 1984 Identification of oxytocin and vasopressin in the testis and in adrenal tissue. Regulatory Peptides8141–146.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD Worley RT Charlton HM & Pickering BT 1986 LH and testosterone cause the development of seminiferous tubule contractile activity and the appearance of testicular oxytocin in hypogonadal mice. Journal of Endocrinology110159–167.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD Guldenaar SE Boer GJ & Pickering BT 1991 Testicular oxytocin: effects of intratesticular oxytocin in the rat. Journal of Endocrinology130231–238.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD Parkinson TJ & Lapwood KR 1999 Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep. Journal of Reproduction and Fertility117299–305.

    • Search Google Scholar
    • Export Citation
  • OgawaS Kudo S Kitsunai Y & Fukuchi S 1980 Increase in oxytocin secretion at ejaculation in male. Clinical Endocrinology1395–97.

  • PeriA Fantoni G Granchi S Vannelli GB Barni T Amerini S Pupilli C Barbagli G Forti G Serio M & Maggi M 1997 Gene expression of endothelin-1 endothelin-converting enzyme-1 and endothelin receptors in human epididymis. Journal of Clinical Endocrinology and Metabolism823797–3806.

    • Search Google Scholar
    • Export Citation
  • PeriA Fantoni G Granchi S Vannelli GB Barni T Amerini S Pupilli C Barbagli G Serio M Maggi M & Forti G 1998 Endothelin-1 is synthesized and biologically active in human epididymis via a paracrine mode of action. Steroids63294–298.

    • Search Google Scholar
    • Export Citation
  • RobertsJS McCracken JA Gavagan JE & Soloff MS 1976 Oxytocin-stimulated release of prostaglandin F2 alpha from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endocrinology991107–1114.

    • Search Google Scholar
    • Export Citation
  • SoloffMS & Swartz TL 1974 Characterization of a proposed oxytocin receptor in the uterus of the rat and sow. Journal of Biological Chemistry2491376–1381.

    • Search Google Scholar
    • Export Citation
  • SoloffMS Alexandrova M & Fernstrom MJ 1979 Oxytocin receptors: triggers for parturition and lactation? Science2041313–1315.

  • StuddardPW Stein JL & Cosentino MJ 2002 The effects of oxytocin and arginine vasopressin in vitro on epididymal contractility in the rat. International Journal of Andrology2565–71.

    • Search Google Scholar
    • Export Citation
  • UckertS Becker AJ Ness BJ Stief CJ Scheller F Knapp WJ & Jonas U 2003 Oxytocin plasma levels in the systemic and cavernous blood of healthy males during different penile conditions. World Journal of Urology20323–326.

    • Search Google Scholar
    • Export Citation
  • VignozziL Filippi S Luconi M Morelli A Mancina R Marini M Vannelli GB Granchi S Orlando C Gelmini S Ledda F Forti G & Maggi M. 2004 Oxytocin receptor is expressed in the penis and mediates an estrogen-dependent smooth muscle contractility. Endocrinology1451823–1834.

    • Search Google Scholar
    • Export Citation
  • WhittingtonK Assinder SJ Parkinson T Lapwood KR & Nicholson HD 2001 Function and localization of oxytocin receptors in the reproductive tissue of rams. Reproduction122317–325.

    • Search Google Scholar
    • Export Citation
  • WhittingtonK Assinder S Gould M & Nicholson H 2004 Oxytocin oxytocin-associated neurophysin and the oxytocin receptor in the human prostate. Cell and Tissue Research 2004 318375–382.

    • Search Google Scholar
    • Export Citation

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      Society for Endocrinology

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    Characterization of OTR in rat CC. (A) Quantitative detection of mRNA for OTR in rat tissues by real-time RT-PCR. Data are expressed as means ± s.e.m. of arbitrary units (a.U.) calculated according to the comparative Ct method. (B) Binding studies. Homologous competition curve for 125I-OTA in CC. Ordinate: B/T, bound to total ratio for 125I-OTA. Abscissa: total concentration (molar) of labeled+unlabeled OTA. Insert: Western blot detection of OTR in rat reproductive tissues. A single band at the expected molecular mass of 55 kDa is present at different levels in all rat genital tissues analyzed. The human CC and rat uterus were run as positive controls and the rat liver as a negative control. Molecular mass markers (kDa) are indicated to the left of the blot.

  • View in gallery

    Immunolocalization of OTR in transverse sections of rat CC (A,B,C) and in rat breast (D,E,F). Panels A (magnification ×40) and B (magnification ×200) show an intense immunopositivity for OTR both in the endothelial (arrows heads) and smooth muscle cells (arrows) of the arteriolar wall and lacunar spaces. Panels D (magnification ×40) and E (magnification ×200) show the presence of positive labeling in myoepithelial cells of ducts and alveoli (black arrows). Immunostaining was obtained with CHINA/1F3 antibody (dilution 1:200). Panels C and F show the control sections obtained by pre-incubating the primary antibodies for OTR with OTR-enriched microsomes (magnification ×40) and are counterstained with hematoxylin.

  • View in gallery

    Characterization of OTR in rat CC (contractility studies). (A) Effect of increasing concentrations of OT (solid squares), [Thr4,Gly7]OT (open diamonds), [Phe2,Orn8]VT (solid circles) and DDAVP (solid triangles) on the basal contractility of rat CC preparations. Ordinate: increase in basal tone, expressed as a percentage of the maximal response obtained with KCl (80 mM). Abscissa: molar concentration of the agonists. Data are expressed as means ± s.e.m. of at least 4 separate experiments. (B) Effect of increasing concentrations of atosiban, an OT antagonist, on the tone induced by a fixed concentration of OT (1 μM) in rat CC (n=4).

  • View in gallery

    Representative ICP tracings. Erectile response elicited by electrical stimulation (ES; 16 Hz, 2.5 V, 5 ms, 30 s) of the rat cavernous nerve (upper tracing) and by papaverine (PAP; 0.35 mg) intracavernous injection (lower tracing) is shown. Tracings on the right side demonstrate ICP changes induced by ES (upper tracing) and papaverine (lower tracing) before and after intracavernous administration of 200 mU OT.

  • View in gallery

    Effects of OT on ICP. (A) Increasing doses of OT (2–600 mU) on intracavernous pressure induced by electrical stimulation (ES; 16 Hz, 2.5 V, 5 ms, 30 s, open squares) and papaverine (0.35 mg, solid circles), normalized as a percentage of mean arterial pressure (max ICP/MAP ×100). The ICP rise in the absence of OT injection is taken as control. The baseline ICP/MAP (%) value (grey column) is also provided. Values are expressed as means ± s.e.m. of at least 4 separate rats for each dose. (B) Effect of OT (60 mU) with (grey columns) or without (solid columns) the OT antagonist atosiban (45 μg) on ICP increase (max ICP/MAP×100) induced by ES (16 Hz, 2.5 V. 5 ms, 30 s) or papaverine (0.35 mg) ICI. The percentage of max ICP/MAP increases induced by ES (horizontally hatched columns) and by papaverine (PAP, vertically hatched columns) before OT injection are taken as control. The baseline ICP/MAP (%) value (open column) is also represented. Values are shown as means ± s.e.m. of at least 4 separate experiments. ***P<0.01 vs baseline; **P<0.05 vs either PAP or OT+atosiban+PAP; °°P<0.05 vs either ES or OT+atosiban+ES.

  • View in gallery

    Effects of the OT antagonist atosiban, alone, on erectile response. The ICP increase was induced by electrical stimulation (ES; 2.5 V, 5 ms, 30 s) at varying stimulation frequencies (2, 4, 16 Hz). Atosiban (45 μg) was intracavernously injected 10 min after the first ES and 5 min before the second ES. The first response (before atosiban injection) is taken as control (solid column). Values were normalized as a percentage of the mean arterial pressure (max ICP/MAP ×100) and are shown as means ± s.e.m. (*P<0.001 and °P<0.05 vs control, n=5 for each frequency).

  • AnderssonKE2003 Erectile physiological and pathophysiological pathways involved in erectile dysfunction. Journal of Urology170S6–S13.

  • ArgiolasA1992 Oxytocin stimulation of penile erection. Pharmacology site and mechanism of action. Annals of the New York Academy of Sciences652194–203.

    • Search Google Scholar
    • Export Citation
  • AssinderSJ Carey M Parkinson T & Nicholson HD 2000 Oxytocin and vasopressin expression in the ovine testis and epididymis: changes with the onset of spermatogenesis. Biology of Reproduction63448–456.

    • Search Google Scholar
    • Export Citation
  • AssinderSJ Rezvani A & Nicholson HD 2002 Oxytocin promotes spermiation and sperm transfer in the mouse. International Journal of Andrology2519–27.

    • Search Google Scholar
    • Export Citation
  • AssinderSJ Johnson C King K & Nicholson HD 2004 Regulation of 5α-reductase isoforms by oxytocin in the rat ventral prostate. Endocrinology1455767–5773.

    • Search Google Scholar
    • Export Citation
  • BathgateRA & Sernia C 1994 Characterization and localization of oxytocin receptors in the rat testis. Journal of Endocrinology141343–352.

    • Search Google Scholar
    • Export Citation
  • BodanszkyM Sharaf H Roy JB & Said SI 1992 Contractile activity of vasotocin oxytocin and vasopressin on mammalian prostate. European Journal of Pharmacology216311–313.

    • Search Google Scholar
    • Export Citation
  • CarmichaelMS Humbert R & Dixen J 1987 Plasma oxytocin increases in the human sexual response. Journal of Clinical Endocrinology and Metabolism6427–31.

    • Search Google Scholar
    • Export Citation
  • ChenKK Chan JY Chang LS Chen MT & Chan SH 1992 Intracavernous pressure as an experimental index in a rat model for the evaluation of penile erection. Journal of Urology1471124–1128.

    • Search Google Scholar
    • Export Citation
  • CrossBA1955 The posterior pituitary gland in relation to reproduction and lactation. British Medical Bulletin11151–155.

  • De LeanA Munson PJ & Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: applications to bioassay radioligand assay and physiological dose–response curves. American Journal of Physiology – Endocrinology and Metabolism235E97–E102.

    • Search Google Scholar
    • Export Citation
  • EinspanierA & Ivell R 1997 Oxytocin and oxytocin receptor expression in reproductive tissues of the male marmoset monkey. Biology of Reproduction56416–422.

    • Search Google Scholar
    • Export Citation
  • FilippiS Vannelli GB Granchi S Luconi M Crescioli C Mancina R Natali A Brocchi S Vignozzi L Bencini E Noci I Ledda F Forti G & Maggi M 2002a Identification localization and functional activity of oxytocin receptors in epididymis. Molecular Cell Endocrinology19389–100.

    • Search Google Scholar
    • Export Citation
  • FilippiS Luconi M Granchi S Vignozzi L Bettuzzi S Tozzi P Forti G & Maggi M 2002b Estrogens but not androgens regulate expression and functional activity of oxytocin receptor in rabbit epididymis. Endocrinology1434271–4280.

    • Search Google Scholar
    • Export Citation
  • FilippiS Marini M Vannelli GB Crescioli C Granchi S Vignozzi L Luconi M Ferruzzi P Morelli A Forti G Ledda F & Maggi M 2003a Effects of hypoxia on endothelin-1 sensitivity in the corpus cavernosum. Molecular Human Reproduction9765–774.

    • Search Google Scholar
    • Export Citation
  • FilippiS Vignozzi L Vannelli GB Ledda F Forti G & Maggi M 2003b Role of oxytocin in the ejaculatory process. Journal of Endocrinological Investigation2682–86.

    • Search Google Scholar
    • Export Citation
  • FooNC Carter D Murphy D & Ivell R 1991 Vasopressin and oxytocin gene expression in rat testis. Endocrinology1282118–2128.

  • FrayneJ & Nicholson HD 1995 Effect of oxytocin on testosterone production by isolated rat Leydig cells is mediated via a specific oxytocin receptor. Biology of Reproduction521268–1273.

    • Search Google Scholar
    • Export Citation
  • FrayneJ & Nicholson HD 1998 Localization of oxytocin receptors in the human and macaque monkey male reproductive tracts: evidence for a physiological role of oxytocin in the male. Molecular Human Reproduction4527–532.

    • Search Google Scholar
    • Export Citation
  • FrayneJ Townsend D & Nicholson HD 1996 Effects of oxytocin on sperm transport in the pubertal rat. Journal of Reproduction and Fertility107299–306.

    • Search Google Scholar
    • Export Citation
  • GemmellRT & Sernia C 1989 The localization of oxytocin and mesotocin in the reproductive tract of the male marsupial bandicoot Isoodon macrourus. General and Comparative Endocrinology75103–109.

    • Search Google Scholar
    • Export Citation
  • GimplG & Fahrenholz F 2001 The oxytocin receptor system: structure function and regulation. Physiological Reviews81629–683.

  • GranchiS Vannelli GB Vignozzi L Crescioli C Ferruzzi P Mancina R Vinci MC Forti G Filippi S Luconi M Ledda F & Maggi M 2002 Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells. Molecular Human Reproduction81053–1064.

    • Search Google Scholar
    • Export Citation
  • HarrisGC & Nicholson HD 1998 Characterisation of the biological effects of neurohypophysial peptides on seminiferous tubules. Journal of Endocrinology15635–42.

    • Search Google Scholar
    • Export Citation
  • HarrisGC Frayne J & Nicholson HD 1996 Epididymal oxytocin in the rat: its origin and regulation. International Journal of Andrology19278–286.

    • Search Google Scholar
    • Export Citation
  • HibJ1974 The contractility of the cauda epididymidis of the mouse its spontaneous activity in vitro and the effects of oxytocin. Journal of Reproduction and Fertility36191–193.

    • Search Google Scholar
    • Export Citation
  • HibJ1977 The ‘in vivo’ effects of oxytocin and vasopressin on spontaneous contractility of the rat epididymis. International Journal of Fertility2263–64.

    • Search Google Scholar
    • Export Citation
  • IvellR Balvers M Rust W Bathgate R & Einspanier A 1997 Oxytocin and male reproductive function. Advances in Experimental Medicine and Biology424253–264.

    • Search Google Scholar
    • Export Citation
  • JenkinL & Nicholson HD 1999 Evidence for the regulation of prostatic oxytocin by gonadal steroids in the rat. Journal of Andrology2080–87.

    • Search Google Scholar
    • Export Citation
  • MaggiM Malozowski S Kassis S Guardabasso V & Rodbard D 1987 Identification and characterization of two classes of receptors for oxytocin and vasopressin in porcine tunica albuginea epididymis and vas deferens. Endocrinology120986–994.

    • Search Google Scholar
    • Export Citation
  • MaggiM Vannelli GB Peri A Brandi ML Fantoni G Giannini S Torrisi C Guardabasso V Barni T Toscano V Massi G & Serio M 1991 Immunolocalization binding and biological activity of endothelin in rabbit uterus: effects of ovarian steroids. American Journal of Physiology – Endocrinology and Metabolism260E292–E305.

    • Search Google Scholar
    • Export Citation
  • MaggiM Fantoni G Baldi E Cioni A Rossi S Vannelli GB Melin P Akerlund M & Serio M 1994a Antagonists for the human oxytocin receptor: an in vitro study. Journal of Reproduction and Fertility101345–352.

    • Search Google Scholar
    • Export Citation
  • MaggiM Vannelli GB Fantoni G Baldi E Magini A Peri A Giannini S Gloria L Del Carlo P Casparis D Tomei T & Serio M 1994b Endothelin in the human uterus during pregnancy. Journal of Endocrinology142385–396.

    • Search Google Scholar
    • Export Citation
  • MaggiM Filippi S Ledda F Magini A & Forti G 2000 Erectile dysfunction: from biochemical pharmacology to advances in medical therapy. European Journal of Endocrinology143143–154.

    • Search Google Scholar
    • Export Citation
  • MarkleHV Warr JL & Branda LA 1978 Oxytocin binding sites in lactating rabbit mammary gland. Canadian Journal of Biochemistry56968–976.

    • Search Google Scholar
    • Export Citation
  • Martinez-PineiroL Brock G Trigo-Rocha F Hsu G Lue TF & Tanagho EA 1994 Rat model for the study of penile erection: pharmacologic and electrical-stimulation parameters. European Urology2562–70.

    • Search Google Scholar
    • Export Citation
  • MorelliA Filippi S Mancina R Luconi M Vignozzi L Marini M Orlando C Vannelli GB Aversa A Natali A Forti G Giorgi M Jannini EA Ledda F & Maggi M 2004 Androgens regulate phosphodiesterase type 5 expression and functional activity in corpora cavernosa. Endocrinology1452253–2263..

    • Search Google Scholar
    • Export Citation
  • MunsonPJ & Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Analytical Biochemistry107220–239.

    • Search Google Scholar
    • Export Citation
  • MurphyMR Seckl JR Burton S Checkley SA & Lightman SL 1987 Changes in oxytocin and vasopressin secretion during sexual activity in men. Journal of Clinical Endocrinology and Metabolism65738–774.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD1996 Oxytocin: a paracrine regulator of prostatic function. Reviews of Reproduction169–72.

  • NicholsonHD & Hardy MP 1992 Luteinizing hormone differentially regulates the secretion of testicular oxytocin and testosterone by purified adult rat Leydig cells in vitro. Endocrinology130671–677.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD & Jenkin L 1995 Oxytocin and prostatic function. Advances in Experimental Medicine and Biology395529–538.

  • NicholsonHD Swann RW Burford GD Wathes DC Porter DG & Pickering BT 1984 Identification of oxytocin and vasopressin in the testis and in adrenal tissue. Regulatory Peptides8141–146.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD Worley RT Charlton HM & Pickering BT 1986 LH and testosterone cause the development of seminiferous tubule contractile activity and the appearance of testicular oxytocin in hypogonadal mice. Journal of Endocrinology110159–167.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD Guldenaar SE Boer GJ & Pickering BT 1991 Testicular oxytocin: effects of intratesticular oxytocin in the rat. Journal of Endocrinology130231–238.

    • Search Google Scholar
    • Export Citation
  • NicholsonHD Parkinson TJ & Lapwood KR 1999 Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep. Journal of Reproduction and Fertility117299–305.

    • Search Google Scholar
    • Export Citation
  • OgawaS Kudo S Kitsunai Y & Fukuchi S 1980 Increase in oxytocin secretion at ejaculation in male. Clinical Endocrinology1395–97.

  • PeriA Fantoni G Granchi S Vannelli GB Barni T Amerini S Pupilli C Barbagli G Forti G Serio M & Maggi M 1997 Gene expression of endothelin-1 endothelin-converting enzyme-1 and endothelin receptors in human epididymis. Journal of Clinical Endocrinology and Metabolism823797–3806.

    • Search Google Scholar
    • Export Citation
  • PeriA Fantoni G Granchi S Vannelli GB Barni T Amerini S Pupilli C Barbagli G Serio M Maggi M & Forti G 1998 Endothelin-1 is synthesized and biologically active in human epididymis via a paracrine mode of action. Steroids63294–298.

    • Search Google Scholar
    • Export Citation
  • RobertsJS McCracken JA Gavagan JE & Soloff MS 1976 Oxytocin-stimulated release of prostaglandin F2 alpha from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endocrinology991107–1114.

    • Search Google Scholar
    • Export Citation
  • SoloffMS & Swartz TL 1974 Characterization of a proposed oxytocin receptor in the uterus of the rat and sow. Journal of Biological Chemistry2491376–1381.

    • Search Google Scholar
    • Export Citation
  • SoloffMS Alexandrova M & Fernstrom MJ 1979 Oxytocin receptors: triggers for parturition and lactation? Science2041313–1315.

  • StuddardPW Stein JL & Cosentino MJ 2002 The effects of oxytocin and arginine vasopressin in vitro on epididymal contractility in the rat. International Journal of Andrology2565–71.

    • Search Google Scholar
    • Export Citation
  • UckertS Becker AJ Ness BJ Stief CJ Scheller F Knapp WJ & Jonas U 2003 Oxytocin plasma levels in the systemic and cavernous blood of healthy males during different penile conditions. World Journal of Urology20323–326.

    • Search Google Scholar
    • Export Citation
  • VignozziL Filippi S Luconi M Morelli A Mancina R Marini M Vannelli GB Granchi S Orlando C Gelmini S Ledda F Forti G & Maggi M. 2004 Oxytocin receptor is expressed in the penis and mediates an estrogen-dependent smooth muscle contractility. Endocrinology1451823–1834.

    • Search Google Scholar
    • Export Citation
  • WhittingtonK Assinder SJ Parkinson T Lapwood KR & Nicholson HD 2001 Function and localization of oxytocin receptors in the reproductive tissue of rams. Reproduction122317–325.

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