567 Identification, localization and functional in vitro and in vivo activity of oxytocin receptor in the rat penis X H Zhang, S Filippi, L Vignozzi, A Morelli, R Mancina, M Luconi, S Donati, M Marini, G B Vannelli1, G Forti and M Maggi Andrology Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy 1Department of Anatomy, Histology and Forensic Medicine, University of Florence, 50139 Florence, Italy (Requests for offprints should be addressed to M Maggi; Email: [email protected]fi.it) Abstract We recently found that the oxytocin receptor (OTR) is blood vessels. In rat corpus cavernosum strips, oxytocin expressed in the human and rabbit corpus cavernosum and (OT) and an OTR selective agonist ([Thr4,Gly7]OT) mediates contractility in vitro. The present study extended induced identical increases in tension, while different our investigations to the rat, and explored whether OTR vasopressin agonists were less active. In vivo, OT intra- regulates penile detumescence in vivo. Real-time RT- cavernous injection (ICI) dose-dependently inhibited PCR quantitatively characterized the distribution of OTR intracavernous pressure (ICP) increase elicited by either mRNA in the male genital tract. Specific transcripts for electrical stimulation of the cavernous nerve or ICI of OTR were expressed in all the tissues investigated. Penile papaverine with similar IC50s (117·7 37 mU). The expression of OTR was comparable to that observed in OTR antagonist, atosiban, counteracted the contractile testis and prostate. Western blot analysis detected a single effect of OT both in vitro and in vivo. Atosiban alone band of the expected molecular mass for OTR in all tissues significantly increased ICP at lower stimulation fre- examined, including rat penis. Expression of OTR protein quencies (2 Hz=P<0·001 and 4 Hz=P<0·05 vs control), in rat penile extracts was further confirmed by binding but not at the maximal frequency (16 Hz). Our data studies, using the OTR selective radiolabeled ligand showed that OTR is present in the rat penis and mediates 125 I-OTA (Kd=17 6·5 pM, Bmax=15·7 5 fmoles/mg contractility both in vitro and in vivo, therefore suggesting a protein). OTR was immunolocalized to the endothelial role for OT in maintaining penile detumescence. and smooth muscle compartments of cavernous spaces and Journal of Endocrinology (2005) 184, 567–576 Introduction receptor (OTR) gene and protein in rabbit and human cavernous tissue in a similar concentration to that found in The penis resides in the flaccid, non-erect, state for the other portions of the male genital tract (Vignozzi et al. majority of the time, due to an active contraction of 2004), classically considered the main male target of oxy- smooth muscle cells (SMC) in penile vessels and lacunar tocin (OT), such as the epididymis (Maggi et al. 1987, spaces. Penile SMC contraction is favored by a rapid Einspanier & Ivell 1997, Ivell et al. 1997, Frayne & breakdown of the relaxing, nitric oxide-related, cGMP, Nicholson 1998, Filippi et al. 2002a,b). In the epididymis, through an increased penile expression of phosphodieste- OTR mediates an increase in both in vitro (Hib 1974, rase type 5 (Morelli et al. 2004), and by the activity of a Filippi et al. 2002a,b, 2003b) and in vivo (Cross 1955, Hib complex array of contractile neurotransmitters and neuro- 1977, Nicholson et al. 1999, Assinder et al. 2002, Studdard modulators, which has only partly been characterized. It is et al. 2002) contractility and sperm output. Also in the generally assumed that the release of noradrenaline from penis OTR mediates in vitro contractility and therefore we sympathetic adrenergic terminals plays a primary role in hypothesized that OT participates in maintaining penile determining vasoconstriction in penile vascular beds SMC contraction and flaccidity (Vignozzi et al. 2004). (Maggi et al. 2000, Andersson 2003). However, modula- OT was originally characterized as a hormone with a tory peptides such as endothelin-1 might also participate in permissive role in female reproduction: facilitating uterine this process, in both physiological (Granchi et al. 2002) and contraction and milk ejection (Cross 1955, Soloff & pathological (Filippi et al. 2003a) conditions. Interestingly, Swartz 1974, Roberts et al. 1976, Markle et al. 1978, Soloff we recently described the presence of the oxytocin et al. 1979, Filippi et al. 2003b). In males, OT was found Journal of Endocrinology (2005) 184, 567–576 DOI: 10.1677/joe.1.05885 0022–0795/05/0184–567 2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/25/2021 06:53:46AM via free access 568 X H ZHANG and others · OTR in the rat penis to be one of the most potent agents to induce penile the manufacturer’s instructions. RNA concentration and erection by electrical or pharmacological stimulation of quality were measured by spectrophotometric analysis at paraventricular nuclei in rats, rabbits and monkeys (see 260 and 280 nm. RNA integrity was assessed by electro- Argiolas 1992 and Gimpl & Fahrenholz 2001 for reviews). phoresis in agarose gel. For each sample 400 ng total RNA Nevertheless, an additional and peripheral role for OT has were reverse-transcribed to cDNA in a final volume of been proposed. In fact, in all the studies performed so far 80 µl using a TaqMan Reverse Transcription kit (Applied (Ogawa et al. 1980, Carmichael et al. 1987, Murphy et al. Biosystems, Forster City, CA, USA) under the following 1987, Uckert et al. 2003), a clear-cut surge of OT plasma conditions: 10 min at 25 C, 30 min at 48 C, 5 min at levels has been described during male sexual activity, 95 C. peaking during orgasm and detumescence, with still unknown physiological significance. Since OT cannot Real-time quantitative RT-PCR easily cross the blood–brain barrier, a central effect of this peptide released by the posterior pituitary during orgasm is The mRNA quantitative analysis was performed accord- rather unlikely. Conversely, a peripheral action of OT ing to the fluorescent TaqMan methodology as already should be considered, in particular at the level of the male published (Vignozzi et al. 2004). PCR primers and probe genital tract. for OTR mRNA quantitation were purchased as an The primary goal of the present study was to extend our Assay-On-Demand (AoD) gene expression product from previous observations obtained in other species (Vignozzi Applied Biosystems. The beta-2 microglobulin gene was et al. 2004) to the rat and to verify whether or not OT has chosen as the reference gene and the corresponding any effect in an in vivo model of penile erection. Hence, we AoD product was provided by Applied Biosystems. The investigated the pharmacological stimulation with OT or PCR mixture (25 µl final volume) consisted of 1 final inhibition with atosiban (an OTR antagonist) of OTR in concentration of AoD mix, 1 final concentration of rat corpus cavernosum (CC) on intracavernous pressure Universal PCR Master Mix (Applied Biosystems), and (ICP) elicited by electrostimulation (ES) of the cavernous 25 ng cDNA. Amplification and detection were per- nerve and intracavernous injection (ICI) of papaverine. formed with the ABI Prism 7700 Sequence Detection Essentially, we found that local administration of OT System with the following thermal cycler conditions: decreased and atosiban increased rat penile erectile response. 2 min at 50 C, 10 min at 95 C, and 40 cycles at 95 Cfor 30 s and 60 C for 1 min. Each measurement was carried Materials and Methods out in duplicate. The analysis of the results was based on the comparative Ct method according to the manufac- Chemicals and tissues turer’s instructions (Applied Biosystems), where Ct rep- resents the cycle number at which the fluorescent signal, Phenylephrine HCl, papaverine HCl, oxytocin, associated with an exponential increase in PCR products, 4 7 1 8 [Thr ,Gly ]oxytocin, [deamino-Cys , -Arg ]vasopressin crossed a given threshold. The maximum change in Ct (DDAVP), reagents for immunocytochemistry, for SDS- values of the sample (Ctsample) was determined by subtract- PAGE and peroxidase-conjugated anti-mouse secondary ing the average of duplicate Ct values of the reference gene antibody were purchased from Sigma (St Louis, MO, 1 2 8 from the average of duplicate Ct values of the target gene. USA). (d(CH2)5 ,Tyr(Me) ,Orn )vasotocin (OTA) and (Phe2,Orn8)vasotocin were purchased from Bachem AG 2 4 8 (Bubendorf, Switzerland). d(CH2)5[Tyr(Me) ,Thr ,Orn , Immunohistochemistry 125 9 125 I-Tyr -NH2]-vasotocin ( I-OTA) was purchased Immunohistochemical studies were carried out as pre- from NEN Life Science (Boston, MA, USA). Atosiban viously described (Maggi et al. 1991, Vignozzi et al. 2004) was from Ferring AB (Limhamn, Sweden). Male rat in both rat CC and mammary gland. Briefly, rat penile and genital tissues including CC, brain, liver, cornea and breast sections (fixed in Bouin’s solution and embedded in uterus were obtained from adult Sprague-Dawley (SD) paraffin) were subsequently incubated, first for 1 h in 2% rats (Harlan, S. Pietro al Natisone, Italy). The Local FCS in PBS to block non-specific antibody binding, Ethical Committee for Investigations in Animals of the overnight at 4 C with the IgM mouse CHINA/1F3 University of Florence approved the study. Human CC (diluted 1:200), then with the corresponding specific samples were from patients undergoing penile prosthesis immunoglobulin peroxidase conjugates for 30 min implantation (n=4) with the approval