Department of Experimental Medicine, Histology and Embriology Section, University of Palermo, Palermo, Italy Immunohistochemical and Biomolecular Identification of Orphanin FQ, eNOS, Atrial natriuretic Factor and Oxytocin in Rat Seminal Vesicles A. Mauro 1 , M. Buscemi 1 , F. Cappello 2 , M. L. Uzzo 1 , E. Farina-Lipari 2 , A. Martorana 3 and A. Gerbino 1 * Addresses of authors: 1 Department of Experimental Medicine, Histology and Embriology Section, 2 Department of Experimental Medicine, Human Anatomy Section, 3 Department of Human Patology, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy; *Corresponding author: Tel.: 0039-091-6553584; fax: 0039-091-6553586; e-mail: aldogerbino@tiscali.it With 3 figures and 2 tables Received January 2009; accepted for publication May 2009 Summary In previous studies performed on rodents, we detected the presence of adreno-cholinergic and peptidergic innervation in seminal vesicles and other organs of the male genital system, such as prostate and deferent duct, in which we also investi- gated the expression of NOS and NADPH-diaphorase. During this project, we focused our attention on the expression of some peptides involved in local control of smooth muscle relaxation, contractility, vasodilatation and control of blood flow in rat seminal vesicles. We investigated, through immuno- histochemistry and RT-PCR, the presence of four peptides: orphanin, eNOS, ANF and oxytocin. Immunohistochemistry was used to detect the presence of the proteins, whereas RT- PCR analysis confirmed gene expression of orphanin, eNOS and ANF, but not oxytocin. In our opinion, orphanin, eNOS and ANF could have paracrine effects regulating the function of seminal vesicles, whereas oxytocin, which may reach this anatomical district through the blood flow, may have a hor- monal action. This is a pilot study that, with further investi- gation, may allow to better clarify the role of these molecules in the control of seminal vesicle tissuesÕ homeostasis. Introduction Rat (Rattus norvegicus) seminal vesicles are two tubular structures located between the urinary bladder and the rectum. Each vesicle is made up of a single convoluted duct that folds on itself and gives rise to well anastomosed ampullary and tubular diverticula; for this reason, seminal vesicles can be considered as branched tubulo-alveolar glands. Histologically, seminal vesicles are notable for their tortuous pathways, diverticula, pseudo-stratified columnar epithelium and cuboi- dal cells along the basal layer. In the lower part of the gland, the duct of the seminal vesicle joins the deferent duct and forms the ejaculatory conduct. Each vesicle is enveloped by a fibromuscular sheath. Three layers can be identified: the external adventitial one; the intermediate muscular one and finally the internal mucosa. The muscular layer is richly innervated by the orthosympathic nervous system and is composed of two smooth muscle layers: the external longitu- dinal one and the internal circular one. The mucosa is structurally organized as a net; such organization significantly increases the surface area. The mucosa is composed of a columnar epithelium with a thin core of connective tissue, where numerous capillaries are present. The secretion of the seminal vesicles represents the major part (85%) of the seminal liquid, whereas the rest is produced by the prostate gland. The secretion is slightly alkaline and contains proteins, enzymes, fructose, mucus, vitamin C, flavins, phosphorylcholine and prostaglandins. The high fruc- tose concentrations provide energy for the spermatozoa as they travel through the female reproductive system. The fluid is expelled under sympathetic contraction of the smooth muscle wall. In rodents, one of the functional properties of seminal vesicle secretion is to contribute to the clot formation of ejaculated semen and to influence the metabolism, mobility and surface properties of spermatozoa, to induce capacitation and suppress the immunocompetence of the female genital tract (Peitz, 1988; Metafora et al., 1989). In this paper, we investigate the expression of orphanin FQ, eNOS (endothelial Nitric Oxide Synthase), ANF (atrial natriuretic factor) and oxytocin in rat seminal vesicles, as the expression of these molecules in this organ has not been studied previously. Orphanin FQ/Nociceptin (OFQ/N) is a peptide structurally similar to the oppioid dinorphin. It is a heptadecapeptide that binds to the NOP1 receptor (nociceptin/orphanin FQ peptide receptor). The NOP1 receptor is closely homologous to the traditional opioid receptors; it is a typical G-protein-coupled receptor with seven transmembrane domains. Its ligand is known as nociceptin or orphanin FQ, having been isolated by two groups independently (Meunier et al.,1995; Reinscheid et al., 1995). Orphanin is mainly involved in the mediation and modulation of pain, but it is also involved in anxiety, stress, memory, learning, locomotor activity, tolerance, dependence and withdrawal phenomena. Supraspinal (intracerebroventric- ular) injection of OFQ/N in mouse has a hyperalgesic action; i.e., a decrease in nociceptive threshold (increase in nociceptive sensitivity) (Meunier et al., 1995; Reinscheid et al., 1995). High dose of spinally administered OFQ/N has inhibitory effects producing behavioural analgesia and/or anti-hyperalgesia/ anti-allodynia (Henderson and McKnight, 1997). The nitric oxide synthase (NOS) is responsible for the synthesis of nitric oxide (NO) from the terminal nitrogen atom of l-arginine in the presence of NADPH and O 2 . Three isoforms are known: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3 or constitutive/cNOS). All three isoforms (presumed to be homodimers) share a carboxyl-terminal reductase domain homologous to the cytochrome P450 reductase, and Ó 2009 Blackwell Verlag GmbH Anat. Histol. Embryol. 38, 443–448 (2009) doi: 10.1111/j.1439-0264.2009.00968.x ISSN 0340–2096