Identification, localization and functional activity of oxytocin receptors in epididymis

Abstract

Oxytocin (OT) is a neurohypophysial hormone with unclear physiological functions in the male. Several previous studies indicated that OT might have a role in the ejaculatory process, stimulating sperm release from the epididymal storage. In this study we investigated on the presence and function of OT receptor (OTR) in rabbit and human epididymis. By using RT-PCR, Western and binding studies, we found that OTR gene and protein is expressed in the human epididymis and stimulates in vitro contractility. The immunolocalization of OTR suggests that the receptor is not only present in the smooth muscle cells of the human epididymis but also in the epithelial compartment. Experiments performed in rabbit epididymal epithelial (rEE) cells in culture indicate that OT induces the release of an other potent stimulator of epididymal contractility, endothelin-1 (ET-1), Blocking the ET_A subtype of the ET-1 receptors, by using a specific antagonist (BQ-123), partially counteracts the contractile effect of OT, suggesting positive interactions between the two peptides in regulating epididymal contractility. Finally, to investigate whether an acute OT administration increases sperm release also in humans, we treated oligozoospermic patients with an intravenous bolus of OT (2.5 IU), just before sperm collection. In a small, single blind study, we found that OT almost doubled sperm retrieval when compared with vehicle administration. Our results indicate that OT might have physiological functions also in the male, controlling epididymal motility and sperm progression through the male genital tract.

Introduction

The epididymis is the portion of the genital tract more proximal to the male gonad. It plays a crucial role in the storage and maturation of spermatozoa produced by the testis. Sperm release from the epididymis is the first step during the emission and the ejaculatory processes and is essentially promoted by the contractile activity of its smooth muscle cells. The epididymis is formed by three distinct regions: the caput, the corpus and the cauda. Each of these regions is provided of contractile cells, although with a different extent. Indeed, while in the caput contractile cells form a loose layer around tubules, in the cauda these cells are replaced by thick smooth muscle cells organized in three distinct layers. The contractile activity of the epididymis is essentially regulated by the noradrenergic output from the sympathetic intermediate and inferior spermatic nerves (Mithcell, 1935). Sympathetic fibers are more represented in the distal portion of the epididymis than in the proximal one, according to the distribution of the smooth muscle cells (Baumgarten et al., 1971). Beside sympathetic innervation, also hormonal substances seem to be involved in the regulation of epididymal motility and semen emission, as adrenaline from the adrenal medulla (Cross and Glover, 1958, Kihara et al., 1998) and oxytocin (OT) from posterior pituitary.

OT was originally characterized as a hormone with a permissive role for female reproduction: facilitating uterine contraction during parturition and milk ejection during lactation (Dale, 1906, Ott and Scott, 1910). In the female, expression and activity of the OT receptor (OTR) is clearly up-regulated by estrogens and down-regulated by progesterone (Maggi et al., 1991, Ivell and Walther, 1999). Although magnocellular neurons of the hypotalamo-posterior pituitary system contain similar amounts of OT in both sexes (Robinson and Jones, 1982), the exact role of OT in the male remained undefined for a long time. However, up to now, there is substantial evidence that OT might be physiologically involved in facilitating semen emission. Indeed plasma OT concentrations are increased at ejaculation in the ram (Sharma et al., 1972), rabbit (Stoneham et al., 1985), bulls (Peeters et al., 1983) and even man (Ogawa et al., 1980, Murphy et al., 1987). OT, in turn, facilitates semen emission in virtually all the animal species tested by increasing the number of sperm per ejaculate (Kihlstrom and Melin, 1963, Knight and Lindsay, 1970, Knight, 1974, Voglmayr, 1975, Sharma and Hays, 1976, Agmo et al., 1978, Berndtson and Igboeli, 1988, Nicholson et al., 1999). Quite recently these findings were also confirmed, in a preliminary report, in humans. Indeed, in azoospermic subjects, OT treatment significantly increased the recovery of a sufficient amount of sperm to circumvent testicular sperm extraction prior than ICSI program (Rolf et al., 2000; oxytocin treatment prior to ICSI may prevent the necessity for testicular biopsy and testicular spermatozoa extraction (TESE.) in azoospermic or severe oligozoospermic patients. Abstract of the 16th Annual meeting of the ESHRE, Bologna, Italy). The effect of OT on sperm ejection seems to be partially mediated by an increased release of sperms from epididymis. Indeed, OT increases epididymal contractility ‘in vivo’ (Melin, 1970, Hib, 1977) and ‘in vitro’ (Hib, 1974). This effect is mediated by specific receptors, described, for the first time, in the pig epididymis by using mathematical analysis of families of competition curves between labeled OT, labeled arginine vasopressin (AVP), its cognate peptide, and selective analogs (Maggi et al., 1987). Later on, our findings were confirmed by other group in several animal species including monkey (Einspanier and Ivell, 1997, Frayne and Nicholson, 1998) and human (Frayne and Nicholson, 1998) by using specific anti-OTR antibodies. Positive immunostaining was mainly present in the smooth muscle cells of the cauda epididymis; however, epithelial cells were also labeled, throughout the entire epididymis. While it is quite possible that OTR positivity in the epididymal smooth muscle cells is somehow related to the control of their contractile activity, the significance of OTR positivity in the epithelial cells of the epididymis it less clear, at the present time. It has been hypothesized that epithelial OTR might be involved in the regulation of testosterone signaling via stimulation of 5 α reductase activity (Nicholson, 1996, Frayne and Nicholson, 1998, Jenkin and Nicholson, 1999). Another possible function of OTR in epithelial cells is the regulation of the release of other contractile agents as endothelin-1 (ET-1), as it has been demonstrated in the uterus (Orlando et al., 1990, Maggi et al., 1993). Because we previously found that epithelial cells of the epididymis express ET-1 gene and protein, while the smooth muscle cells express the receptors and were responsive to their stimulation (Peri et al., 1997, Peri et al., 1998), we raised the question whether the OTR might be involved in the regulation of the release of ET-1 also in epididymis. In this study we provide evidence that OTR (gene and protein) is expressed in human epididymis and stimulates contractility. We also demonstrated that in rabbit epididymis OT partially mediates its contractile activity by allowing ET-1 release from epithelial cells. In addition, we provided preliminary evidence that in oligospermic human subjects, OT administration significantly increases the numbers of ejaculated motile sperms.