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Andrology Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy
1 Department of Anatomy, Histology and Forensic Medicine, University of Florence, 50139 Florence, Italy
(Requests for offprints should be addressed to M Maggi; Email: m.maggi{at}dfc.unifi.it)
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Abstract |
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Introduction |
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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.
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Materials and Methods |
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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 1x final concentration of AoD mix, 1x 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 x 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 
-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 x 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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.
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), 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).
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). 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).
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(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).
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. 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgements |
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Funding |
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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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Received in final form 17 November 2004
Accepted 7 December 2004
Made available online as an Accepted Preprint 8 December 2004
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