Oxytocin Mediates the Estrogen-Dependent Contractile Activity of Endothelin-1 in Human and Rabbit Epididymis
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内分泌学杂志 2005年第8期
Andrology Unit (A.M., L.V., R.M., C.C., G.F., Ma.M.) and Endocrinology Unit (P.F.), Department of Clinical Physiopathology; Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction (S.F.), Departments of Pharmacology and Clinical Physiopathology; Department of Anatomy, Histology and Forensic Medicine (G.B.V., Mi.M.); and Departments of Urology (N.M.) and Pharmacology (F.L.), University of Florence, Florence 50139, Italy
Address all correspondence and requests for reprints to: Mario Maggi, M.D., Andrology Unit, Department of Clinical Physiopathology, University of Florence, Viale G. Pieraccini, 6, 50139 Florence Italy. E-mail: m.maggi@dfc.unifi.it.
Abstract
Epididymis is a sex steroid (androgen + estrogen)-sensitive duct provided with spontaneous motility, allowing sperm transport. We previously reported that the oxytocin (OT) receptor (OTR) mediates an estrogen-dependent increase in epididymal contractility. Because endothelin (ET)-1 also regulates epididymal motility, we tested its sex steroid dependence in a rabbit model. We demonstrated that estrogens up-regulate responsiveness to ET-1, which is reduced by blocking aromatase activity (letrozole, 2.5 mg/kg) or by triptorelin (2.9 mg/kg)-induced hypogonadism, whereas it is fully restored by estradiol valerate (3.3 mg/kg weekly) but not by testosterone enanthate (30 mg/kg weekly). However, changing sex steroid milieu did not affect either ET-1, its receptor gene, or protein expression. Two structurally distinct OTR-antagonists [(d(CH2)5 1, Tyr(Me)2, Orn8)-OT and atosiban] almost completely abolished ET-1 contractility, without competing for [125I]ET-1 binding, suggesting that OT/OTR partially mediates ET-1 action. Immunohistochemical studies in human and rabbit epididymis demonstrated that both OT and its synthesis-associated protein, neurophysin I, are expressed in the epithelial cells facing the muscular layer, suggesting local OT production. Quantitative RT-PCR demonstrated a high abundance of OT transcripts in human epididymis. OT transcript was also originally detected and partially sequenced in rabbit epididymis. To verify whether ET-1 regulates OT release, we used rabbit epididymal epithelial cell cultures. These cells expressed a high density of [125I]ET-1 binding sites and responded to ET-1 with a dose-dependent OT release. Hence, we propose that an ET-1-induced OT/OTR system activation underlies the estrogen-dependent hyperresponsiveness to ET-1. These local sources might promote the spontaneous mo-tility necessary for sperm transport.
Introduction
THE HUMAN EPIDIDYMIS is an elongated and convoluted single duct, continuous with the lumina of vasa efferentes in the most proximal portion to the testis (caput) and with the vas deferens in the most distal one (cauda). In the caput epididymis and in the middle portion (corpus), the contractile cells are sparse, forming a loose layer around the tubule. Conversely, in the cauda there are three distinct muscular layers with increasing thickness in close proximity to the vas deferens. The main functions of the epididymis are: 1) the reabsorption of testicular fluid, 2) the storage of released spermatozoa from the testis, and 3) the facilitation of sperm transport toward the cauda, because it is generally accepted that epididymal spermatozoa are immotile. In addition, the cauda epididymis participates in the first part of the ejaculatory process (emission), enabling stored semen to be propelled toward the farther portions of the genital tract. The caput and corpus epididymis have little innervation (1). In these regions, spontaneous rhythmic peristaltic movements are predominant, allowing a constant basal flow of sperm from the epididymis to the vas deferens, even in the absence of nervous stimulation (2). In contrast, the cauda is provided with a great number of nerve fibers that coordinate the forceful contractile activity necessary for semen emission (1).
During the last 2 decades, it has been clarified that epididymal motility is promoted not only by sympathetic and parasympathetic nerves but also by a complex interplay between locally produced peptides, such as endothelin (ET)-1 (3, 4, 5) and steroidal or peptide hormonal regulators (see Refs. 6 and 7 for reviews). In fact, receptors for the neurohypophysial hormone oxytocin (OT) have been demonstrated and localized in both the epithelial and muscular layers of the epididymis of several animal species (5, 8, 9, 10, 11), including the monkey and human (5, 12, 13, 14). An intense muscular immunolabeling for the OT receptor (OTR) has been found mostly in the cauda epididymis (5, 10, 11), although it is also present in the peritubular myoid cells of the caput and corpus (5). In these regions, OTR mediates an OT-induced in vitro (5, 7, 11, 15, 16, 17) and in vivo contractility (2, 18, 19), most probably assisting sperm progression through the duct. Conversely, epithelial OTR is more abundant in the caput (2, 5), where it is involved in functions still only partially clarified, such as ET-1 release (5). Indeed, we recently reported that OT stimulates ET-1 release from rabbit epididymal epithelial (rEE) cells in culture (5). ET-1 was originally characterized as an endothelium-derived potent constrictor peptide, which is cleaved from a bigger precursor (Big-ET) by a specific peptidase family [ET-converting enzyme (ECE)-1] and is involved in regulating vascular tone (20). Later on, it was recognized that ET-1 acts through two distinct receptor subtypes, ETA and ETB, and it is largely present in the reproductive tissues of both sexes (see Refs. 21 and 22 for reviews). Concerning the epididymal duct, ET-1 and ECE-1 are expressed by the basal epithelial cells of the lumen, facing the muscular layers (3, 5, 23, 24) where ETA receptors are present (3, 25). Exposure of human (3) or rabbit (5) epididymal strips to nanomolar concentrations of ET-1 elicited vigorous contractions, mostly through ETA receptor activation. Interestingly, ETA receptors are also present in the epithelial cells of human caput epididymis (24). The stimulation of rat epididymal epithelial cells by ET-1 induced changes in short-circuit current and anion secretion (26, 27). These findings generated the hypothesis that ET-1 plays a role in the control of water and electrolyte transport through the antiport Na+-H+ exchange (27).
Epididymal functions are tightly regulated by testicular androgens: testosterone (T) and dihydrotestosterone. However, the rudimentary epididymal phenotype derived by the LH receptor knockout in mice cannot be completely rescued by T replacement (28), suggesting that other LH-dependent testicular substances are essential for fully restoring epididymal features. Because high concentrations of estrogens are present in the epididymis and ER and ER? (estrogen receptors) are widely expressed, it has been speculated that estrogens play physiological functions (11, 29, 30, 31, 32, 33). Accordingly, mice genetically lacking the subtype of estrogen receptors (ER knockout) are subfertile, showing efferent ductule dilatation and fluid overload, due to an impaired synthesis of several proteins involved in epididymal fluid reabsorption (34), such as sodium/hydrogen exchanger-3 (35). We believe that estrogens control not only epididymal fluid reabsorption but also its contractility. In fact, we recently demonstrated that estrogens, but not androgens, up-regulate OT-induced contractility in the rabbit epididymal duct (11). The aim of the present study was to investigate whether estrogens regulate responsiveness to another potent stimulator of epididymal motility, such as ET-1, and whether the estrogen-sensitive OT-OTR system is involved in mediating ET-1 effects.
Materials and Methods
Chemicals
Noradrenaline (NA), the ETB selective agonist (N-Suc-[Glu9, AL11, 15]-ET-1 [8, 21] (IRL1620) and DMEM were purchased from Sigma (St. Louis, MO). Potassium chloride (KCl) was supplied from Merck & Co., Inc. (Darmstadt, Germany). (d(CH2)5 1, Tyr(Me)2, Orn8)-OT (OTA) was purchased from Bachem AG (Bubendorf, Switzerland). 1-Deamino-2-D-Tyr(OEt)-4-Thr-8-Orn-OT (atosiban) was supplied by Ferring AB (Limhamn, Sweden). ET-1, ET-3, and BQ123 were purchased from Calbiochem (La Jolla, CA). Testosterone enanthate (T) and estradiol valerate (E2v) were supplied by Schering AG (Berlin, Germany); letrozole was a gift from Novartis Pharmaceuticals (Basel, Switzerland). Triptorelin pamoate was supplied by Ipsen (Milan, Italy). The anti-OT polyclonal antibody and the antineurophysin polyclonal antibody were purchased from Chemicon International (Temecula, CA). [125I]-ET-1 (2000 Ci/mmol) was purchased from Amersham Biosciences (Freiburg, Germany).
Epididymal and corpora cavernosal tissue preparations
Human tissues from male genital tract (testis, vas deferens, epididymis, corpora cavernosa, and prostate) and uterus samples were collected during surgery for benign diseases, as already reported (36). All the uterine samples were from normal cycling women, in the secretive phase, according to endometrial histology. Rabbit tissues (epididymis, corpora cavernosa, placenta, and uterus) were obtained from New Zealand White rabbits. The animals were killed by a lethal dose of pentobarbital. Human and rabbit samples were prepared and stored as previously described (36). All human tissue samples were collected after the approval from the Hospital Committee for Investigation in Humans (Azienda Ospedaliera Careggi, Florence, Italy; Protocol no. 6783–04) and after receiving consent from the informed patients. All the animal experiments were performed in accordance to the Italian Ministerial Law no. 116/92 and approved by the Institutional Animal Care and Use Committee of the University of Florence.
In vitro contractility
The experiments were carried out as previously described (5, 11). Briefly, rabbit epididymal strips were vertically mounted under 700 mg resting tension in organ chambers containing 10 ml Krebs solution (5, 11). Changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, Casalecchio Reno, Bologne, Italy). NA (0.1–10 μM) increased the tonic tension in a concentration-dependent manner, with a maximum effect obtained at 10 μM (taken as 100%). ET-1 cumulative concentrations (0.01–1000 nM) were added to the bath at 7-min intervals. Preexposure to NA (10 μM) did not significantly affect ET-1 responsiveness in terms of milligrams of tension generated (not shown). Selective antagonists were added 30–60 min before the concentration-response curve for the agonist.
Experimental hypogonadism and sex steroid replacement
New Zealand White male rabbits (weighing 3 kg; n = 24) were divided into the following four groups and treated as previously described (11, 33, 36, 37): 1) controls (n = 6), 2) treated with the long-acting GnRH analog triptorelin pamoate (2.9 mg/kg; n = 18), 3) treated with the GnRH analog plus T (30 mg/kg weekly; n = 6), and 4) treated with the GnRH analog plus E2v (3.3 mg/kg; n = 6). Another group of sexually mature, intact animals (weighing 3 kg) was treated for 3 wk with letrozole (2.5 mg/kg daily; n = 4), an aromatase inhibitor (38), dissolved in drinking water, or with vehicle (n = 4) as already described (11).
Isolation of RNA
Total RNA was extracted from frozen tissues using TRIZOL (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and checked for quantity and quality as previously described (33, 36). Total RNA from human brain was purchased from Stratagene (La Jolla, CA).
Real-time RT-PCR for OT mRNA expression in human tissues
The quantitative assay was performed according to the fluorescent TaqMan method, as already reported (33). The primers and probe for OT mRNAs were gene expression products purchased from Applied Biosystems (Foster City, CA). The glyceraldehyde-3-phosphate dehydrogenase gene was chosen as the reference gene. Amplification and detection were performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Each measurement was carried out in duplicate. The mRNA quantitation was based on the comparative cycle threshold method according to the manufacturer’s instructions (Applied Biosystems) and as previously described (33).
RT-PCR for OT mRNA expression in rabbit epididymis
Total RNA (250 ng) from rabbit samples (epididymis and uterus) and human brain was reverse-transcribed for 30 min at 50 C, denatured for 2 min at 95 C, and amplified for 35 cycles with the following steps: 45 sec at 95 C, 1 min at 58 C, and 1 min at 70 C. Because no rabbit OT sequence is deposited in the GenBank at the National Center for Biotechnology Information, the primer design was based on homology to the human, bovine, rat, and mouse sequences. The sense primer was 5'-CGC CTG CTA CAT CCA GAA CT-3' (corresponding to nucleotides 71–80 of the human OT cDNA sequence); the antisense primer was 5'-CGG CAG GTA GTT CTC CTC CT-3' (corresponding to nucleotides 244–263 of the human OT cDNA sequence). The amplified cDNA was run on a 2% ethidium bromide-stained agarose gel and visualized under UV light. To confirm the amplification specificity, the product of expected 193 bp was sequenced using the ABI-Prism 310 automatic sequencer (Applied Biosystems). Sequencing was performed according to the Applied Biosystems protocol and showed a homology (rabbit vs. human) of 95%.
RT-PCR for ET-1, ET-1 receptors, and ECE-1 mRNA expression in rabbit epididymis
By using the Superscript One Step RT-PCR kit (Life Technologies, Inc., Milan, Italy), total RNA from rabbit epididymis (500 ng) was reverse-transcribed and then amplified for 28 cycles (ET-1, ECE-1, ETA, and ETB) or 23 cycles (-actin) with the following steps: 45 sec at 95 C; 1 min at 55 C for ETA, ETB, and -actin or 58 C for ET-1 and ECE-1; 1 min at 70 C. RT-PCR for -actin was performed to verify the integrity of the total RNA.
Primers used are described (primer, sequence, length, source) as follows: 1) ETA, 5'-GGC TCT TCG GCT TCT ATT TC-3' (sense) and 5'-CAT CGG TTC TTG TCC ATC TC-3' (antisense), 250 bp, AF311974; 2) ETB, 5'-TAC CCA AAG AAG GGA GGA CA-3' (sense) and 5'-CAG GAA GCA ACA GCT CGA TA-3' (antisense), 400 bp, AB043704; 3) ET-1, 5'-CAA GCG GTG CTC CTG CTC CT-3' (sense) and 5'-GCT CGT GCA CTG GCA CCT GTT-3' (antisense), 193 bp, X59931; 4) ECE-1 (39), 5'-GCA CCC TCA AGT GGA TGG AC-3' (sense) and 5'-CCG GAA ACA CGA TCT CGT TC-3' (antisense), 309 bp, Z35307; and 5) -ACT, 5'-ACA TGG AGA AGA TCT GGC AC-3 (sense) and 5'-CAT GAG GTA GTC GGT CAG GT-3' (antisense), 328 bp, X60733.
Binding studies on epididymal membranes
Epididymal membrane preparations from rabbits and binding studies with [125I]ET-1 were carried out as previously described (40). Briefly, aliquots of membranes (0.075 mg/ml) were incubated at 37 C for 60 min in the presence of [125I]ET-1 with or without increasing concentrations of the following unlabeled peptides: ET-1 (0.01–100 nM), OTA (0.01 nM-100 μM), and atosiban (0.01 nM-100 μM). All measurements were performed in triplicate. Afterward, membrane suspensions were filtered through Whatman GF/B filters (Whatman International Ltd., Maidstone, UK) and the retained radioactivity was counted in a -counter. The results were simultaneously fitted using the LIGAND program (41).
Measurements of ET-like immunoreactivity in epididymal tissues
ET-like immunoreactivity was evaluated in rabbit epididymal tissues as already published (39). Briefly, samples were individually pulverized under liquid nitrogen, and 150 mg of each sample was incubated with 1 ml chloroform/methanol [2:1 (vol/vol)] 0.1% trifluoroacetic acid (TFA) for 18 h at 0 C to 4 C. After the addition of 1 ml double-distilled water containing 0.1% TFA, vigorous mixing, and brief centrifugation, the aqueous protein-containing phase was applied to a preactivated 100-mg Sep-Pak Vac C18 cartridge (Waters, Milford, MA). After washing with 10 ml H2O/0.1% TFA (vol/vol), elution from the cartridge was performed with 2 ml of an eluant mix (60% H2O, 39.9% acetonitrile, 0.1% TFA). After removal of the solvent in an Univapo 150H Speed-Vac (UniEquip, Martinsreid, Munich, Germany), the nearly dried residues were dissolved in 250 μl assay buffer. The concentration of ET-like immunoreactivity in these tissue extracts was determined by using a commercially available ELISA kit (Cayman Company, Ann Arbor) according to the manufacturer’s instructions. This assay did not discriminate between the different ET isoforms (i.e. ET-1 and ET-3) and did not recognize the biologically inactive precursor, Big-ET-1.
Immunocytochemistry
Immunohistochemical studies were carried out as previously described (3, 42). Sections were incubated in 2% fetal calf serum in PBS (1 h) and with primary antibody (anti-OT, 10 μg/ml; antineurophysin-1, 1:2500 vol/vol) overnight at 4 C. Control sections were incubated with pure mouse IgM in place of primary antibody.
OT measurement in conditioned medium from rEE
rEE cells were prepared as previously described (5). Cells were growth in DMEM. After 24 h of starvation, cells were incubated with increasing concentrations (0.1–1000 nM) of ET-1. After 24 h, the conditioned media were collected and kept frozen at –80 C until the assay was conducted. Experiments were performed in triplicate for each cell preparation. Cells in DMEM and vehicle were used as the control. OT secretion was measured using the Correlate-EIA Oxytocin Kit (Assay Designs, Inc., Ann Arbor, MI), following the manufacturer’s instruction. All the samples analyzed were above the detection limit of this method. OT release was expressed as picograms per million cells.
Binding assays of ET receptors on rEE
Cells were incubated in 200 μl of the same growth medium at room temperature, with fixed concentrations (70 pmol/liter) of [125I]ET-1 (2200 Ci/mmol) in the presence or absence of increasing concentrations of the following unlabeled ligands: ET-1 (0.1–100 nM), ET-3 (1 nM-10 μM), the ETB agonist IRL1620 (1 nM-10 μM), the ETA antagonist BQ123 (0.01–100 nM). After 60 min, cells were washed twice with ice-cold PBS, 0.1% BSA and solubilized in 0.1 N NaOH. The cell-bound radioactivity was determined in a -counter. Measurements were performed in triplicate. Cell counts between wells routinely varied by less than 10%.
Statistical analysis
The results are expressed as the mean ± SEM for n experiments. Statistical analysis was performed with the Student’s t test for paired or unpaired data, with ANOVA followed by Fisher’s test to evaluate the differences between groups, and P < 0.05 was taken as significant. Half-maximal response effective concentration (EC50) and half-maximal response inhibiting concentration (IC50) values were calculated by the computer program ALLFIT (43). The computer program ALLFIT was also used for the analysis of the curves. ALLFIT uses the constrained four-parameter logistic model to analyze families of sigmoid curves, to obtain estimates of EC50 values, and to compare them using an F test and tests of randomness of the residuals around the fitted curves. In particular, the statistical hypothesis that two or more curves share EC50s was tested by first forcing the curves to share this parameter (shared EC50) and then verifying that such constraints have only minimal (not significant) effect on several indicators of "goodness of fit". The binding data were analyzed using the computer program LIGAND (41). ALLFIT and Ligand computer programs were supplied by P. J. Munson (National Institutes of Health, Bethesda, MD).
Results
Estrogens increase responsiveness to ET-1
To verify whether the responsiveness to ET-1 was somehow affected by changing endocrine milieu, we treated adult rabbits with the long-acting GnRH agonist triptorelin (2.9 mg/kg), using a previously described protocol (11, 33, 36, 37). Subsets of rabbits were supplemented, after 2 wk, with vehicle (control), T (30 mg/kg), or E2v (3.3 mg/kg). Table 1 shows the levels of T and 17? estradiol (E2) in the peripheral blood at the time of the experiment. Both T and 17?E2 were significantly decreased by triptorelin (P < 0.01 and P < 0.05, respectively) and completely restored by administration of the corresponding sex steroid. Figure 1A shows the effects of the different pharmacological treatments on epididymal contractility. In control rabbits, increasing concentrations of ET-1 induced a dose-dependent increase in contractility (EC50 = 33 ± 5 nM). The contractile effect of ET-1 was as potent [maximal effect (Emax) = 105.7 ± 3.8%] as 10 μM NA, taken as 100%. Changing sex steroid milieu did not significantly affect the EC50 values for ET-1 (P = 0.184) but significantly changed Emax values. In fact, in hypogonadal rabbits, the maximal contractility elicited by ET-1 was dramatically reduced (Emax = 20.8 ± 3.1%; P < 0.0001) and only partially restored by T administration (Emax = 42.8 ± 3.4%; P < 0.005 vs. hypogonadism; P < 0.0001 vs. control). Conversely, estrogen administration fully restored the ET-1-induced contractility at a level not statistically different from the control (P = 0.236). Because rabbit epididymis expresses P450 aromatase (11), we tested the effect of a subacute endogenous estrogen deprivation, treating adult rabbits with the aromatase inhibitor letrozole (2.5 mg/kg daily for 3 wk), according to previously described experimental design (11, 33). Letrozole administration significantly decreased 17?E2 levels (P < 0.001; Table 2) and Emax for ET-1 (P < 0.0001; control Emax = 118 ± 6.3%; letrozole Emax = 55.8 ± 2.6%; Fig. 1B), without affecting the EC50 values (P = 0.275; shared EC50 = 39.6 ± 7.6 nM). Taken together, these results, strongly suggest that estrogens, but not androgens, regulate ET-1 sensitivity in rabbit epididymis.
TABLE 1. Effect of a GnRH analog (triptorelin), with or without T/E2v replacement, on sex steroid plasma levels
FIG. 1. Effect of changing sex steroid milieu on epididymal responsiveness to ET-1. A, Effect of increasing concentrations of ET-1 on the basal tone of preparations from sexually mature rabbits: untreated (n = 8 in six separate experiments); hypogonadal (GnRH analog) without (n = 7 in six separate experiments) or with hormonal replacement by weekly administration of T (n = 8 in six separate experiments) or E2v (n = 6 in six separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of ET-1. Data were expressed as the means ± SEM. B, Effect of increasing concentrations of ET-1 on the basal tone of preparations from sexually mature intact rabbits, untreated (n = 6, in four separate experiments) or chronically treated with the aromatase inhibitor letrozole (n = 6 in four separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of ET-1. Data are expressed as means ± SEM. The relative EC50 and Emax values are reported in the text.
TABLE 2. Effect of an aromatase inhibitor (letrozole) on sex steroid plasma levels
Sex steroids do not affect ET-1 and its receptor gene and protein expression
We next investigated whether the estrogen-induced change in responsiveness to ET-1 was somehow related to an altered expression of ET receptors or of their natural ligands. The results are summarized in Table 3. Neither triptorelin-induced hypogonadism nor sex steroid replacement significantly affected the mRNA expression of the ECE-1 and ET-1 and did not result in any significant alteration in the protein content of ET-like immunoreactivity. In addition, changing sex steroid milieu did not modify the ET receptor gene (ETA + ETB) and protein ([125I]ET-1 binding) expression.
TABLE 3. Lack of effect of hypogonadism (triptorelin) and sex steroid (testosterone or estrogen) replacement on the expression of ET-1, its converting enzyme (ECE-1), and receptors (ETA + ETB) in rabbit epididymis at both mRNA and protein level
OT partially mediates ET-1 induced contractility
In preliminary experiments, we demonstrated that two chemically distinct OTR antagonists did not compete for [125I]ET-1 binding in epididymal homogenates (Fig. 2A) but counteracted, in a dose-dependent manner, the contractility induced by ET-1 (100 nM), with similar Imax values (73.8 ± 15.1%) and IC50 values (299 ± 148 nM; Fig. 2B). Interestingly, these IC50 values for the ET-1-induced contractility are compatible with (although slightly higher than) those of both antagonists for OT-induced contractility (5, 33, 44). In epididymal strips from control rabbits, preincubation with OTA (1 μM) significantly decreased Emax for ET-1 (control, Emax = 130.4 ± 14%; OTA-pretreated, Emax = 60.7 ± 7.6%; P < 0.0001; percentage inhibition = 114.8%), without affecting the EC50 values (shared EC50 = 60.7 ± 2.8 nM; Fig. 2C). Similar results were obtained preincubating the epididymis from untreated rabbits with atosiban (1 μM): decreased responsiveness to ET-1 (control, Emax = 133.4 ± 13%; atosiban-pretreated, Emax = 62.9 ± 7.8%; P < 0.0001; percentage inhibition = 111%), although sensitivity to ET-1 was similar (shared EC50 = 61.4 ± 2.1 nM; Fig. 2D). Because the results with OTA and atosiban were virtually identical, further studies were performed using only OTA. In hypogonadal rabbits, OTA decreased the already negligible response to ET-1 (control Emax = 37.5 ± 0.1%; OTA-pretreated Emax = 28.6 ± 0.1%; P < 0.0001; percentage inhibition = 31.57%, not shown) only slightly. In contrast, in estrogen-replaced hypogonadal rabbits, OTA substantially hampered responsiveness to ET-1 but not ET-1 EC50 values (control Emax = 104.4 ± 8.8%; OTA-pretreated Emax = 29.6 ± 2.6%; P < 0.0001; percentage inhibition = 252.7%; shared EC50 = 78.8 ± 3.2 nM; Fig. 2E). To test whether the cooperation between ET-1 and OT in stimulating epididymal contractility was also evident in humans, we repeated the in vitro experiment using human epididymal strips. The results are reported in Fig. 2F. In human strips, ET-1 induced the previously described (3, 5) sustained increase in contractility (Emax = 191.6 ± 3.9%), with EC50 = 13.8 ± 1.4 nM. Preincubation with OTA strongly decreased maximal responsiveness (Emax = 43.5 ± 2.16%; P < 0.0001; percentage inhibition = 340.4%), without affecting the EC50 (P = 0.91).
FIG. 2. Effect of different OTR antagonists on ET-1 responsiveness in rabbit epididymis. A, Competition curves for [125I]ET-1 binding to rabbit epididymis microsomes by ET-1 (n = 3), OTA (n = 3), and 1-deamino-2-D-Tyr(OEt)-4-Thr-8-Orn-OT (atosiban; n = 3). Ordinate, Bound [125I]ET-1 expressed as a percentage of the total ligand added; abscissa, total concentration (molar) of unlabeled ligands. B, Effect of increasing concentrations of OTA (n = 4 in three separate experiments) and atosiban (n = 3 in three separate experiments) on the tone induced by a fixed concentration of ET-1 (100 nM) in rabbit epididymis. Relative IC50 values are reported in the text. Ordinate: Response, expressed as percentage of the maximal response obtained with ET-1 (100 nM); abscissa, concentration of the effectors. C, Effect of increasing concentrations of ET-1 in rabbit epididymis in the absence (n = 5 in four separate experiments) or presence of the specific OTR antagonist OTA (n = 4 in three separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of the agonist. The relative IC50 values are reported in the text. D, Effect of increasing concentrations of ET-1 in rabbit epididymis in the absence (n = 3 in three separate experiments) or presence of the specific OTR antagonist, atosiban (n = 3 in three separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa: concentration of the agonist. The relative IC50 values are reported in the text. E, Effect of increasing concentrations of ET-1 on the basal tone of epididymal preparations in hypogonadal rabbits supplemented with weekly administration of E2v in the absence (n = 3 in three separate experiments) or presence of the specific OTR antagonist, OTA (n = 3 in three separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of the agonist. The relative IC50 values are reported in the text. F, Effect of increasing concentrations of ET-1 in human epididymis in the absence (n = 5 in five separate experiments) or presence of the specific OTR antagonist, OTA (n = 4 in four separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of the agonist. The relative IC50 values are reported in the text. Data are expressed as the means ± SEM.
OT and neurophysin I (NpI) are expressed in epididymis
Because the OTR antagonists did not displace [125I]ET-1 binding but blocked ET-1 action, in an estrogen-sensitive manner, we hypothesized that ET-1 might mediate part of its contractile activity, in both rabbit and human epididymis, by activating the previously described estrogen-sensitive OTR signaling (11) through the release of locally produced OT. Because no information is available on the epididymal presence of OT in rabbit and humans, we performed immunohistochemical studies, using specific antibodies against the peptide and its synthesis-associated protein NpI. Positive staining for both OT and NpI was detected in the majority of epithelial cells from human (Figs. 3 and 5) and rabbit (Figs. 4 and 6) epididymis and localized at the abluminal site, facing the smooth muscle layer. Only a few cells also displayed labeling in the apical portion, facing the epididymal lumen. Almost identical subcellular distribution of OT and NpI labeling indicates, at least indirectly, local synthesis and not uptake from extraepididymal sources, such as blood or testicular fluid (45, 46). To further verify the hypothesis of a local OT synthesis, we analyzed OT gene expression in epididymis using RT-PCR. To quantitate OT expression in human epididymis, we performed real-time RT-PCR. The results are reported as a histogram in Fig. 7A. Human epididymis showed a high abundance of OT mRNA. Because the sequence of rabbit OT gene has, to date, not been elucidated, primer design was based on the alignment of the published bovine, rat, mouse, and human OT sequences. RT-PCR revealed a transcript of an expected 193 bp in rabbit epididymis as well as in rabbit uterus and human brain used as positive controls (Fig. 7B). Sequence analysis of the RT-PCR products from rabbit epididymis confirmed its specificity and produced a partial sequence of rabbit OT cDNA of 156 bp corresponding to 108–264 nucleotides of the published human OT-specific sequence (Fig. 7C). The homology between rabbit and human OT-specific sequences was 95% and 92% at the nucleotide and amino acid levels, respectively. Taken together, these results strongly suggest a local epididymal production of OT and NpI in both rabbit and human epididymis.
FIG. 3. OT immunolocalization in human epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for OT in transversal sections of the epididymal head. The middle and right panels represent transversal sections of the epididymal body with different magnifications (B, E, and F, x40, x120, and x200, respectively). Immunopositivity for OT is detected in the majority of cells from the epithelial layer of the epididymal duct. Specific staining was in the abluminal cellular compartment, facing the smooth muscle layer. C, Control section, where the primary monoclonal antibody against OT was replaced by pure IgM antiserum (x40).
FIG. 5. Immunolocalization of OT-associated NpI in human epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for NpI in transversal sections of the epididymal head. The middle and right panels represent transversal sections of the epididymal body with different magnifications (B, E, and F, x40, x120, and x200, respectively). A diffuse immunopositivity for NpI is localized in the epithelial cells of human epididymis. In the epithelial cells of the tubules, specific staining for NpI was mostly localized in the abluminal cellular compartment, facing the smooth muscle layer. C, Control section, where the primary polyclonal antibody against NpI was replaced by pure IgM antiserum (x40).
FIG. 4. OT immunolocalization in rabbit epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for OT in transversal sections of epididymal head. In A, the arrowhead shows a testicular portion in close touch to the epididymal head (arrow). Within the testis, either Leydig cells or Sertoli cells are immunopositive for OT. The middle and right panels represent transversal sections of the epididymal tail with different magnification (B, E, F, and G, x40, x120, x200, and x400, respectively). In the epithelial cells of the tubules, specific staining for OT was mostly localized in the abluminal cellular compartment, facing the smooth muscle layer. C, Control section, where the primary monoclonal antibody against OT was replaced by pure IgM antiserum (x40). D, OT immunolocalization in rabbit epididymis.
FIG. 6. Immunolocalization of OT-associated NpI in rabbit epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for NpI in transversal sections of the epididymal head. Note that in A, both the testis (arrowhead) and the proximal part of the epididymal head (arrow) are shown. In the testis, either Leydig cells or Sertoli cells are immunopositive for NpI. The middle and right panels represent transversal sections of the epididymal tail with different magnifications (B, E, and F, x40, x120, and x200, respectively). In the epithelial cells of the tubules, specific staining for NpI is mostly localized in the abluminal cellular compartment, facing the smooth muscle layer. C shows a control section, where the primary polyclonal antibody against NpI was replaced by pure IgM antiserum (x40).
FIG. 7. Expression of OT gene in the epididymis. A, Quantitative detection of mRNA for OT in human tissues by real-time RT-PCR. Data were normalized over glyceraldehyde-3-phosphate dehydrogenase mRNA expression according to the comparative cycle threshold method and are expressed as the mean ± SEM. B, Ethidium bromide-stained agarose gel from the RT-PCR products obtained with primers for OT cDNA. Human brain and rabbit uterus were used as positive controls. NC, Negative control (RT-PCR without template); MW, molecular weight marker. C, Alignment of nucleotide sequences of the rabbit epididymis (epi) and human brain OT, partial cDNA, from RT-PCR products shown in B. The primer sequences (sense, right arrows; antisense, left arrows) are underlined. Vertical bars indicate identity between the sequences, whereas gaps in alignment are boxed. The predicted amino acid sequence is also indicated and differences with respect to the human OT sequence (NCBI accession no. NM_000915) are reported in bold. C. cavernosum, Corpus cavernosum.
ET-1 releases OT from rEE cells
To finally investigate whether or not ET-1 interacts with OT, in isolated epididymal epithelial cells, we initiated experiments in rEE cells. Cells were prepared using a previously described protocol (5). We first performed radioligand binding studies to investigate the presence of specific receptors for ET-1 in these cells. The results are reported in Fig. 8A. Cultured rEE cells express a high density of [125I]ET-1 binding sites (53360 ± 2462 sites/cell), showing pharmacological characteristics compatible with the predominant presence of ETA receptors (Table 4). To test whether ET-1 might affect OT release, we incubated rEE cells for 24 h with increasing concentrations of ET-1. ET-1 induced a 10-fold increase in immunoreactive OT in rEE-spent medium. ET-1-induced OT release started to be significant at relatively low ET-1 concentrations (1 nM; P < 0.001; Fig. 8B).
FIG. 8. Characterization of the biological effect of ET-1 in cultured rEE cells. A, Typical family of competition curves among [125I]ET-1 and unlabeled ET-1 (x), ET-3 (), IRL1620 (), and BQ123 (+) in rEE cells. Ordinate, B/T, bound to total ratio for [125I]ET-1; abscissa, total concentration (molar) of ligands. Binding parameters are in Table 4. B, Effect of 24-h incubation with increasing concentrations of ET-1 on the release of OT in cultures of rEE cells. Ordinate, OT release (pg/106 cells) in spent medium; abscissa, concentrations of ET-1. ET-1 significantly increased OT release starting from 1 nM (*, P < 0.05; **, P < 0.01 vs. control; n = 3).
TABLE 4. Pharmacological characterization of ET-1 binding sites in cultured rEE cells
Discussion
Our results further extend the concept of epididymis as a male estrogen target, demonstrating the existence of an estrogen-driven, short paracrine loop between ET-1 and OT. We previously demonstrated that, in rabbit epididymis, OT partially mediates its contractile activity through the release of locally produced ET-1 and ETA activation (5). We now provide original evidence that, in the same tissue, ET-1 might signal through OT release and OTR activation. Hence, in our view, the spontaneous rhythmic contractions of epididymis are facilitated by a positive interactive cooperation between the OT/OTR and ET-1/ETA systems. These two systems might mutually activate each other in an estrogen-regulated feed-forward fashion, favoring fluid and sperm progression through the epididymal duct. Further studies should be conducted whether the previously described epididymal alteration in ER knockout mice (ductule dilatation and fluid overload) are due only to an impairment of fluid reabsorption (29) or to altered motility as well.
The involvement of OT in coordinating testicular and epididymal motility was first postulated by Cross (47) almost 50 yr ago. By using an abdominal chamber technique in the rabbit, he demonstrated that iv injection of OT induced "peristaltic and pendular movements in the tubules of the head and body epididymis, and segmentation contractions in the tubules of smaller calibre in the tail" (47). Subsequent experiences substantially confirmed Cross’s view: OTR is present in the epididymis and stimulates its motility (see Ref. 7 for review). However, the physiological source of the OTR ligand is still unclear. Because circulating levels of OT (picomolar range) are far from the concentrations needed to evoke a contractile response (nanomolar range), a role for posterior pituitary in driving epididymal contractility is unlikely. Hence, local production was investigated. In the early 1980s, it was found that testicular OT levels were 3 orders of magnitude higher than the circulating ones, suggesting local production (48, 49). This was confirmed later on, when specific transcripts for OT were detected in the testis of several animal species (see Ref. 50 for review), including the monkey and human (13, 51). In this study, we found, using real-time RT-PCR, that human epididymis expresses an elevated level of OT mRNA. In human epididymis, staining for OT and its synthesis-associated NpI was essentially localized in the epithelial cells, predominantly in the basal portion, facing the smooth muscle layer, although some apical labeling was also found. A similar picture was observed in rabbit (present study), ovine (52), and rat (53) epididymis. However, the final demonstration of an epididymal production of OT derives from studies on isolated epithelial cells in culture. According to a previous morphological study (24), epididymal epithelial cells express a high density of ETA receptors (more than 50,000 sites/cell). Stimulation of ETA receptors with ET-1 induced a 10-fold increase in OT release. Such a release should play a relevant role in mediating the contractile effect of ET-1, because the blocking of OTR with specific, although chemically distinct, antagonists, such as OTA and atosiban, strongly decreased ET-1 responsiveness in both rabbit and human epididymal preparations. The OTR antagonist-induced inhibition of ET-1 contractility was even more apparent in tissues derived from estrogen-treated hypogonadal rabbits. One of the main characteristics of the OT-OTR system is its estrogen dependency (50, 54). We recently demonstrated that, in a rabbit model of hypogonadotropic hypogonadism, estrogen (but not androgen) administration restored gene and protein expression of epididymal OTR and responsiveness to OT (11). However, in the present study, we were unable to demonstrate the same for ET-1, using a similar animal model. Indeed, we did not find any significant change either at gene or protein level in ET-1 and its cognate receptors, although we did find a sustained estrogen dependency in response to ET-1. It is therefore possible that the estrogen-induced change in sensitivity to ET-1 reported in the present study might depend on parallel changes in the interplay between ET-1 and the estrogen-sensitive OT-OTR system. However, the possibility that estrogens might up-regulate some contractile effectors downstream to the ET-1 receptors cannot be ruled out at present.
In conclusion, the present results indicate an estrogen-sensitive cooperation between two peptidergic systems (OT/OTR and ET-1/ETA) in the epididymis, which might help in regulating the spontaneous motility of the duct and in facilitating sperm transport through it.
Acknowledgments
We thank Paolo Ceccatelli and Mauro Beni (Centro per i Servizi di Stabulazione degli Animali di Laboratorio, University of Florence) for technical assistance in animal treatment.
References
El-Badawi A, Schenk EA 1967 The distribution of cholinergic and adrenergic nerves in the mammalian epididymis: a comparative histochemical study. Am J Anat 121:1–14
Nicholson HD, Parkinson TJ, Lapwood KR 1999 Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep. J Reprod Fertil 117:299–305
Peri A, 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. J Clin Endocrinol Metab 82:3797–3806
Harneit S, Ergun S, Paust HJ, Mukhopadhyay AK, Holstein AF 1997 Endothelin-1 and its receptors in the human epididymis. Adv Exp Med Biol 424:191–192
Filippi S, 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 2002 Identification, localization and functional activity of oxytocin receptors in epididymis. Mol Cell Endocrinol 193:89–100
Hess RA 2003 Estrogen in the adult male reproductive tract. Reprod Biol Endocrinol 1:52–65 (Review)
Filippi S, Vignozzi L, Vannelli GB, Ledda F, Forti G, Maggi M 2003 Role of oxytocin in the ejaculatory process. J Endocrinol Invest 26:82–86
Maggi M, 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. Endocrinology 120:986–994
Parry LJ, Bathgate RA 2000 The role of oxytocin and regulation of uterine oxytocin receptors in pregnant marsupials. Exp Physiol 85:91S–99S
Whittington K, Assinder SJ, Parkinson T, Lapwood KR, Nicholson HD 2001 Function and localization of oxytocin receptors in the reproductive tissue of rams. Reproduction 122:317–325
Filippi S, Luconi M, Granchi S, Vignozzi L, Bettuzzi S, Tozzi P, Ledda F, Forti G, Maggi M 2002 Estrogens, but not androgens, regulate expression and functional activity of oxytocin receptor in rabbit epididymis. Endocrinology 143:4271–4280
Maggi M, Baldi E, Genazzani AD, Giannini S, Natali A, Costantini A, Rodbard D, Serio M 1989 Vasopressin receptors in human seminal vesicles: identification, pharmacological characterization and comparison with the vasopressin receptors present in human kidney. J Androl 10:393–400
Einspanier A, Ivell R 1997 Oxytocin and oxytocin receptor expression in reproductive tissues of the male marmoset monkey. Biol Reprod 56:416–422
Frayne J, 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. Mol Hum Reprod 4:527–532
Hib J 1974 The in vitro effects of oxytocin and vasopressin on spontaneous contractility of the mouse cauda epididymidis. Biol Reprod 11:436–439
Hib J 1974 The contractility of the cauda epididymidis of the mouse, its spontaneous activity in vitro and the effects of oxytocin. J Reprod Fertil 36:191–193
Studdard PW, Stein JL, Cosentino MJ 2002 The effects of oxytocin and arginine vasopressin in vitro on epididymal contractility in the rat. Int J Androl 25:65–71
Cross BA, Glover TD 1958 The hypothalamus and seminal emission. J Endocrinol 16:385–395
Assinder SJ, Rezvani A, Nicholson HD 2002 Oxytocin promotes spermiation and sperm transfer in the mouse. Int J Androl 25:19–27
Yanagisawa M, Masaki T 1989 Molecular biology and biochemistry of the endothelins. Trends Pharmacol Sci 10:374–378
Rubanyi GM, Polokoff MA 1994 Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46:325–415
Masaki T, Ninomiya H, Sakamoto A, Okamoto Y 1999 Structural basis of the function of endothelin receptor. Mol Cell Biochem 190:153–156
Wong PY, Cheng-Chew SB, Leung PY, Qin DN 1991 Functional study and immunocytochemical identification of endothelin in cultured epididymal cells and intact epididymis of the rat. J Cardiovasc Pharmacol 17(Suppl 7):S242–245
Harneit S, Paust HJ, Mukhopadhyay AK, Ergun S 1997 Localization of endothelin-1 and endothelin-receptors A and B in human epididymis. Mol Hum Reprod 3:579–584
Kim SZ, Kang SY, Lee SJ, Cho KW 2000 Localization of receptors for natriuretic peptide and endothelin in the duct of the epididymis of the freshwater turtle. Gen Comp Endocrinol 118:26–38
Wong PY, Fu WO, Huang SJ 1989 Endothelin stimulates short circuit current in a cultured epithelium. Br J Pharmacol 98:1191–1196
Wang H, Sakurai K, Endoh M 2000 Pharmacological analysis by HOE642 and KB-R9032 of the role of Na(+)/H(+) exchange in the endothelin-1-induced Ca(2+) signalling in rabbit ventricular myocytes. Br J Pharmacol 131:638–644
Lei ZM, Zou W, Mishra S, Li X, Rao ChV 2003 Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy. Biol Reprod 68:888–895
Hess RA, Zhou Q, Nie R, Oliveira C, Cho H, Nakaia M, Carnes K 2001 Estrogens and epididymal function. Reprod Fertil Dev 13:273–283
Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA 2002 Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 23:870–881
Nie R, Zhou Q, Jassim E, Saunders PT, Hess RA 2002 Differential expression of estrogen receptors and ? in the reproductive tracts of adult male dogs and cats. Biol Reprod 66:1161–1168
Hess RA 2003 Estrogen in the adult male reproductive tract. Reprod Biol Endocrinol 1:52–65
Vignozzi L, 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. Endocrinology 145:1823–1834
Hess RA 2000 Oestrogen in fluid transport in efferent ducts of the male reproductive tract. Rev Reprod 5:84–92
Zhou Q, Clarke L, Nie R, Carnes K, Lai LW, Lien YH, Verkman A, Lubahn D, Fisher JS, Katzenellenbogen BS, Hess R 2001 Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proc Natl Acad Sci USA 98:14132–14137
Morelli A, 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. Endocrinology 145:2253–2263
Filippi S, Luconi M, Granchi S, Natali A, Tozzi P, Forti G, Ledda F, Maggi M 2002 Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation. Int J Impot Res 14:295–307
Smith IE 1998 Pivotal trials of letrozole: a new aromatase inhibitor. Oncology (Huntingt) 12(3 Suppl 5):41–44
Lauth M, Berger MM, Cattaruzza M, Hecker M 2000 Pressure-induced upregulation of preproendothelin-1 and endothelin B receptor expression in rabbit jugular vein in situ: implications for vein graft failure? Arterioscler Thromb Vasc Biol 20:96–103
Maggi M, Barni T, Orlando C, Fantoni G, Finetti G, Vannelli GB, Mancina R, Gloria L, Bonaccorsi L, Yanagisawa M, Forti G 1995 Endothelin-1 and its receptors in human testis. J Androl 16:213–224
Munson PJ, Rodbard D 1980 Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239
Peri A, Fantoni G, Granchi S, Vannelli GB, Barni T, 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. Steroids 63:294–298
De Lean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to biossay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97–E102
Zhang XH, Filippi S, Vignozzi L, Morelli A, Mancina R, Donati S, Luconi M, Marini M, Vannelli GB, Forti G, Maggi M 2005 Identification, localization and functional "in vitro" and "in vivo" activity of oxytocin receptor in the rat penis. J Endocrinology 184:567–576
Knickerbocker JJ, Sawyer HR, Amann RP, Tekpetey FR, Niswender GD 1988 Evidence for the presence of oxytocin in the ovine epididymis. Biol Reprod 39:391–397
Veeramachaneni DN, Amann RP 1990 Oxytocin in the ovine ductuli efferentes and caput epididymidis: immunolocalization and endocytosis from the luminal fluid. Endocrinology 126:1156–1164
Cross BA 1955 The posterior pituitary gland in relation to reproduction and lactation. Br Med Bull 11:151–155[Free Full Text]
Wathes DC, Swann RW, Hull MG, Drife JO, Porter DG, Pickering BT 1983 Gonadal sources of the posterior pituitary hormones. Prog Brain Res 60:513–519
Kasson BG, Adashi EY, Hsueh AJ 1986 Arginine vasopressin in the testis: an intragonadal peptide control system. Endocr Rev 7:156–168
Gimpl G, Fahrenholz F 2001 The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81:629–683
Ivell R, Balvers M, Rust W, Bathgate R, Einspanier A 1997 Oxytocin and male reproductive function. Adv Exp Med Biol 424:253–264
Assinder SJ, Carey M, Parkinson T, Nicholson HD 2000 Oxytocin and vasopressin expression in the ovine testis and epididymis: changes with the onset of spermatogenesis. Biol Reprod 63:448–456
Harris GC, Frayne J, Nicholson HD 1996 Epididymal oxytocin in the rat: its origin and regulation. Int J Androl 19:278–286
Zingg HH, Laporte SA 2003 The oxytocin receptor. Trends Endocrinol Metab 14:222–227(Sandra Filippi, Annamaria)
Address all correspondence and requests for reprints to: Mario Maggi, M.D., Andrology Unit, Department of Clinical Physiopathology, University of Florence, Viale G. Pieraccini, 6, 50139 Florence Italy. E-mail: m.maggi@dfc.unifi.it.
Abstract
Epididymis is a sex steroid (androgen + estrogen)-sensitive duct provided with spontaneous motility, allowing sperm transport. We previously reported that the oxytocin (OT) receptor (OTR) mediates an estrogen-dependent increase in epididymal contractility. Because endothelin (ET)-1 also regulates epididymal motility, we tested its sex steroid dependence in a rabbit model. We demonstrated that estrogens up-regulate responsiveness to ET-1, which is reduced by blocking aromatase activity (letrozole, 2.5 mg/kg) or by triptorelin (2.9 mg/kg)-induced hypogonadism, whereas it is fully restored by estradiol valerate (3.3 mg/kg weekly) but not by testosterone enanthate (30 mg/kg weekly). However, changing sex steroid milieu did not affect either ET-1, its receptor gene, or protein expression. Two structurally distinct OTR-antagonists [(d(CH2)5 1, Tyr(Me)2, Orn8)-OT and atosiban] almost completely abolished ET-1 contractility, without competing for [125I]ET-1 binding, suggesting that OT/OTR partially mediates ET-1 action. Immunohistochemical studies in human and rabbit epididymis demonstrated that both OT and its synthesis-associated protein, neurophysin I, are expressed in the epithelial cells facing the muscular layer, suggesting local OT production. Quantitative RT-PCR demonstrated a high abundance of OT transcripts in human epididymis. OT transcript was also originally detected and partially sequenced in rabbit epididymis. To verify whether ET-1 regulates OT release, we used rabbit epididymal epithelial cell cultures. These cells expressed a high density of [125I]ET-1 binding sites and responded to ET-1 with a dose-dependent OT release. Hence, we propose that an ET-1-induced OT/OTR system activation underlies the estrogen-dependent hyperresponsiveness to ET-1. These local sources might promote the spontaneous mo-tility necessary for sperm transport.
Introduction
THE HUMAN EPIDIDYMIS is an elongated and convoluted single duct, continuous with the lumina of vasa efferentes in the most proximal portion to the testis (caput) and with the vas deferens in the most distal one (cauda). In the caput epididymis and in the middle portion (corpus), the contractile cells are sparse, forming a loose layer around the tubule. Conversely, in the cauda there are three distinct muscular layers with increasing thickness in close proximity to the vas deferens. The main functions of the epididymis are: 1) the reabsorption of testicular fluid, 2) the storage of released spermatozoa from the testis, and 3) the facilitation of sperm transport toward the cauda, because it is generally accepted that epididymal spermatozoa are immotile. In addition, the cauda epididymis participates in the first part of the ejaculatory process (emission), enabling stored semen to be propelled toward the farther portions of the genital tract. The caput and corpus epididymis have little innervation (1). In these regions, spontaneous rhythmic peristaltic movements are predominant, allowing a constant basal flow of sperm from the epididymis to the vas deferens, even in the absence of nervous stimulation (2). In contrast, the cauda is provided with a great number of nerve fibers that coordinate the forceful contractile activity necessary for semen emission (1).
During the last 2 decades, it has been clarified that epididymal motility is promoted not only by sympathetic and parasympathetic nerves but also by a complex interplay between locally produced peptides, such as endothelin (ET)-1 (3, 4, 5) and steroidal or peptide hormonal regulators (see Refs. 6 and 7 for reviews). In fact, receptors for the neurohypophysial hormone oxytocin (OT) have been demonstrated and localized in both the epithelial and muscular layers of the epididymis of several animal species (5, 8, 9, 10, 11), including the monkey and human (5, 12, 13, 14). An intense muscular immunolabeling for the OT receptor (OTR) has been found mostly in the cauda epididymis (5, 10, 11), although it is also present in the peritubular myoid cells of the caput and corpus (5). In these regions, OTR mediates an OT-induced in vitro (5, 7, 11, 15, 16, 17) and in vivo contractility (2, 18, 19), most probably assisting sperm progression through the duct. Conversely, epithelial OTR is more abundant in the caput (2, 5), where it is involved in functions still only partially clarified, such as ET-1 release (5). Indeed, we recently reported that OT stimulates ET-1 release from rabbit epididymal epithelial (rEE) cells in culture (5). ET-1 was originally characterized as an endothelium-derived potent constrictor peptide, which is cleaved from a bigger precursor (Big-ET) by a specific peptidase family [ET-converting enzyme (ECE)-1] and is involved in regulating vascular tone (20). Later on, it was recognized that ET-1 acts through two distinct receptor subtypes, ETA and ETB, and it is largely present in the reproductive tissues of both sexes (see Refs. 21 and 22 for reviews). Concerning the epididymal duct, ET-1 and ECE-1 are expressed by the basal epithelial cells of the lumen, facing the muscular layers (3, 5, 23, 24) where ETA receptors are present (3, 25). Exposure of human (3) or rabbit (5) epididymal strips to nanomolar concentrations of ET-1 elicited vigorous contractions, mostly through ETA receptor activation. Interestingly, ETA receptors are also present in the epithelial cells of human caput epididymis (24). The stimulation of rat epididymal epithelial cells by ET-1 induced changes in short-circuit current and anion secretion (26, 27). These findings generated the hypothesis that ET-1 plays a role in the control of water and electrolyte transport through the antiport Na+-H+ exchange (27).
Epididymal functions are tightly regulated by testicular androgens: testosterone (T) and dihydrotestosterone. However, the rudimentary epididymal phenotype derived by the LH receptor knockout in mice cannot be completely rescued by T replacement (28), suggesting that other LH-dependent testicular substances are essential for fully restoring epididymal features. Because high concentrations of estrogens are present in the epididymis and ER and ER? (estrogen receptors) are widely expressed, it has been speculated that estrogens play physiological functions (11, 29, 30, 31, 32, 33). Accordingly, mice genetically lacking the subtype of estrogen receptors (ER knockout) are subfertile, showing efferent ductule dilatation and fluid overload, due to an impaired synthesis of several proteins involved in epididymal fluid reabsorption (34), such as sodium/hydrogen exchanger-3 (35). We believe that estrogens control not only epididymal fluid reabsorption but also its contractility. In fact, we recently demonstrated that estrogens, but not androgens, up-regulate OT-induced contractility in the rabbit epididymal duct (11). The aim of the present study was to investigate whether estrogens regulate responsiveness to another potent stimulator of epididymal motility, such as ET-1, and whether the estrogen-sensitive OT-OTR system is involved in mediating ET-1 effects.
Materials and Methods
Chemicals
Noradrenaline (NA), the ETB selective agonist (N-Suc-[Glu9, AL11, 15]-ET-1 [8, 21] (IRL1620) and DMEM were purchased from Sigma (St. Louis, MO). Potassium chloride (KCl) was supplied from Merck & Co., Inc. (Darmstadt, Germany). (d(CH2)5 1, Tyr(Me)2, Orn8)-OT (OTA) was purchased from Bachem AG (Bubendorf, Switzerland). 1-Deamino-2-D-Tyr(OEt)-4-Thr-8-Orn-OT (atosiban) was supplied by Ferring AB (Limhamn, Sweden). ET-1, ET-3, and BQ123 were purchased from Calbiochem (La Jolla, CA). Testosterone enanthate (T) and estradiol valerate (E2v) were supplied by Schering AG (Berlin, Germany); letrozole was a gift from Novartis Pharmaceuticals (Basel, Switzerland). Triptorelin pamoate was supplied by Ipsen (Milan, Italy). The anti-OT polyclonal antibody and the antineurophysin polyclonal antibody were purchased from Chemicon International (Temecula, CA). [125I]-ET-1 (2000 Ci/mmol) was purchased from Amersham Biosciences (Freiburg, Germany).
Epididymal and corpora cavernosal tissue preparations
Human tissues from male genital tract (testis, vas deferens, epididymis, corpora cavernosa, and prostate) and uterus samples were collected during surgery for benign diseases, as already reported (36). All the uterine samples were from normal cycling women, in the secretive phase, according to endometrial histology. Rabbit tissues (epididymis, corpora cavernosa, placenta, and uterus) were obtained from New Zealand White rabbits. The animals were killed by a lethal dose of pentobarbital. Human and rabbit samples were prepared and stored as previously described (36). All human tissue samples were collected after the approval from the Hospital Committee for Investigation in Humans (Azienda Ospedaliera Careggi, Florence, Italy; Protocol no. 6783–04) and after receiving consent from the informed patients. All the animal experiments were performed in accordance to the Italian Ministerial Law no. 116/92 and approved by the Institutional Animal Care and Use Committee of the University of Florence.
In vitro contractility
The experiments were carried out as previously described (5, 11). Briefly, rabbit epididymal strips were vertically mounted under 700 mg resting tension in organ chambers containing 10 ml Krebs solution (5, 11). Changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, Casalecchio Reno, Bologne, Italy). NA (0.1–10 μM) increased the tonic tension in a concentration-dependent manner, with a maximum effect obtained at 10 μM (taken as 100%). ET-1 cumulative concentrations (0.01–1000 nM) were added to the bath at 7-min intervals. Preexposure to NA (10 μM) did not significantly affect ET-1 responsiveness in terms of milligrams of tension generated (not shown). Selective antagonists were added 30–60 min before the concentration-response curve for the agonist.
Experimental hypogonadism and sex steroid replacement
New Zealand White male rabbits (weighing 3 kg; n = 24) were divided into the following four groups and treated as previously described (11, 33, 36, 37): 1) controls (n = 6), 2) treated with the long-acting GnRH analog triptorelin pamoate (2.9 mg/kg; n = 18), 3) treated with the GnRH analog plus T (30 mg/kg weekly; n = 6), and 4) treated with the GnRH analog plus E2v (3.3 mg/kg; n = 6). Another group of sexually mature, intact animals (weighing 3 kg) was treated for 3 wk with letrozole (2.5 mg/kg daily; n = 4), an aromatase inhibitor (38), dissolved in drinking water, or with vehicle (n = 4) as already described (11).
Isolation of RNA
Total RNA was extracted from frozen tissues using TRIZOL (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and checked for quantity and quality as previously described (33, 36). Total RNA from human brain was purchased from Stratagene (La Jolla, CA).
Real-time RT-PCR for OT mRNA expression in human tissues
The quantitative assay was performed according to the fluorescent TaqMan method, as already reported (33). The primers and probe for OT mRNAs were gene expression products purchased from Applied Biosystems (Foster City, CA). The glyceraldehyde-3-phosphate dehydrogenase gene was chosen as the reference gene. Amplification and detection were performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Each measurement was carried out in duplicate. The mRNA quantitation was based on the comparative cycle threshold method according to the manufacturer’s instructions (Applied Biosystems) and as previously described (33).
RT-PCR for OT mRNA expression in rabbit epididymis
Total RNA (250 ng) from rabbit samples (epididymis and uterus) and human brain was reverse-transcribed for 30 min at 50 C, denatured for 2 min at 95 C, and amplified for 35 cycles with the following steps: 45 sec at 95 C, 1 min at 58 C, and 1 min at 70 C. Because no rabbit OT sequence is deposited in the GenBank at the National Center for Biotechnology Information, the primer design was based on homology to the human, bovine, rat, and mouse sequences. The sense primer was 5'-CGC CTG CTA CAT CCA GAA CT-3' (corresponding to nucleotides 71–80 of the human OT cDNA sequence); the antisense primer was 5'-CGG CAG GTA GTT CTC CTC CT-3' (corresponding to nucleotides 244–263 of the human OT cDNA sequence). The amplified cDNA was run on a 2% ethidium bromide-stained agarose gel and visualized under UV light. To confirm the amplification specificity, the product of expected 193 bp was sequenced using the ABI-Prism 310 automatic sequencer (Applied Biosystems). Sequencing was performed according to the Applied Biosystems protocol and showed a homology (rabbit vs. human) of 95%.
RT-PCR for ET-1, ET-1 receptors, and ECE-1 mRNA expression in rabbit epididymis
By using the Superscript One Step RT-PCR kit (Life Technologies, Inc., Milan, Italy), total RNA from rabbit epididymis (500 ng) was reverse-transcribed and then amplified for 28 cycles (ET-1, ECE-1, ETA, and ETB) or 23 cycles (-actin) with the following steps: 45 sec at 95 C; 1 min at 55 C for ETA, ETB, and -actin or 58 C for ET-1 and ECE-1; 1 min at 70 C. RT-PCR for -actin was performed to verify the integrity of the total RNA.
Primers used are described (primer, sequence, length, source) as follows: 1) ETA, 5'-GGC TCT TCG GCT TCT ATT TC-3' (sense) and 5'-CAT CGG TTC TTG TCC ATC TC-3' (antisense), 250 bp, AF311974; 2) ETB, 5'-TAC CCA AAG AAG GGA GGA CA-3' (sense) and 5'-CAG GAA GCA ACA GCT CGA TA-3' (antisense), 400 bp, AB043704; 3) ET-1, 5'-CAA GCG GTG CTC CTG CTC CT-3' (sense) and 5'-GCT CGT GCA CTG GCA CCT GTT-3' (antisense), 193 bp, X59931; 4) ECE-1 (39), 5'-GCA CCC TCA AGT GGA TGG AC-3' (sense) and 5'-CCG GAA ACA CGA TCT CGT TC-3' (antisense), 309 bp, Z35307; and 5) -ACT, 5'-ACA TGG AGA AGA TCT GGC AC-3 (sense) and 5'-CAT GAG GTA GTC GGT CAG GT-3' (antisense), 328 bp, X60733.
Binding studies on epididymal membranes
Epididymal membrane preparations from rabbits and binding studies with [125I]ET-1 were carried out as previously described (40). Briefly, aliquots of membranes (0.075 mg/ml) were incubated at 37 C for 60 min in the presence of [125I]ET-1 with or without increasing concentrations of the following unlabeled peptides: ET-1 (0.01–100 nM), OTA (0.01 nM-100 μM), and atosiban (0.01 nM-100 μM). All measurements were performed in triplicate. Afterward, membrane suspensions were filtered through Whatman GF/B filters (Whatman International Ltd., Maidstone, UK) and the retained radioactivity was counted in a -counter. The results were simultaneously fitted using the LIGAND program (41).
Measurements of ET-like immunoreactivity in epididymal tissues
ET-like immunoreactivity was evaluated in rabbit epididymal tissues as already published (39). Briefly, samples were individually pulverized under liquid nitrogen, and 150 mg of each sample was incubated with 1 ml chloroform/methanol [2:1 (vol/vol)] 0.1% trifluoroacetic acid (TFA) for 18 h at 0 C to 4 C. After the addition of 1 ml double-distilled water containing 0.1% TFA, vigorous mixing, and brief centrifugation, the aqueous protein-containing phase was applied to a preactivated 100-mg Sep-Pak Vac C18 cartridge (Waters, Milford, MA). After washing with 10 ml H2O/0.1% TFA (vol/vol), elution from the cartridge was performed with 2 ml of an eluant mix (60% H2O, 39.9% acetonitrile, 0.1% TFA). After removal of the solvent in an Univapo 150H Speed-Vac (UniEquip, Martinsreid, Munich, Germany), the nearly dried residues were dissolved in 250 μl assay buffer. The concentration of ET-like immunoreactivity in these tissue extracts was determined by using a commercially available ELISA kit (Cayman Company, Ann Arbor) according to the manufacturer’s instructions. This assay did not discriminate between the different ET isoforms (i.e. ET-1 and ET-3) and did not recognize the biologically inactive precursor, Big-ET-1.
Immunocytochemistry
Immunohistochemical studies were carried out as previously described (3, 42). Sections were incubated in 2% fetal calf serum in PBS (1 h) and with primary antibody (anti-OT, 10 μg/ml; antineurophysin-1, 1:2500 vol/vol) overnight at 4 C. Control sections were incubated with pure mouse IgM in place of primary antibody.
OT measurement in conditioned medium from rEE
rEE cells were prepared as previously described (5). Cells were growth in DMEM. After 24 h of starvation, cells were incubated with increasing concentrations (0.1–1000 nM) of ET-1. After 24 h, the conditioned media were collected and kept frozen at –80 C until the assay was conducted. Experiments were performed in triplicate for each cell preparation. Cells in DMEM and vehicle were used as the control. OT secretion was measured using the Correlate-EIA Oxytocin Kit (Assay Designs, Inc., Ann Arbor, MI), following the manufacturer’s instruction. All the samples analyzed were above the detection limit of this method. OT release was expressed as picograms per million cells.
Binding assays of ET receptors on rEE
Cells were incubated in 200 μl of the same growth medium at room temperature, with fixed concentrations (70 pmol/liter) of [125I]ET-1 (2200 Ci/mmol) in the presence or absence of increasing concentrations of the following unlabeled ligands: ET-1 (0.1–100 nM), ET-3 (1 nM-10 μM), the ETB agonist IRL1620 (1 nM-10 μM), the ETA antagonist BQ123 (0.01–100 nM). After 60 min, cells were washed twice with ice-cold PBS, 0.1% BSA and solubilized in 0.1 N NaOH. The cell-bound radioactivity was determined in a -counter. Measurements were performed in triplicate. Cell counts between wells routinely varied by less than 10%.
Statistical analysis
The results are expressed as the mean ± SEM for n experiments. Statistical analysis was performed with the Student’s t test for paired or unpaired data, with ANOVA followed by Fisher’s test to evaluate the differences between groups, and P < 0.05 was taken as significant. Half-maximal response effective concentration (EC50) and half-maximal response inhibiting concentration (IC50) values were calculated by the computer program ALLFIT (43). The computer program ALLFIT was also used for the analysis of the curves. ALLFIT uses the constrained four-parameter logistic model to analyze families of sigmoid curves, to obtain estimates of EC50 values, and to compare them using an F test and tests of randomness of the residuals around the fitted curves. In particular, the statistical hypothesis that two or more curves share EC50s was tested by first forcing the curves to share this parameter (shared EC50) and then verifying that such constraints have only minimal (not significant) effect on several indicators of "goodness of fit". The binding data were analyzed using the computer program LIGAND (41). ALLFIT and Ligand computer programs were supplied by P. J. Munson (National Institutes of Health, Bethesda, MD).
Results
Estrogens increase responsiveness to ET-1
To verify whether the responsiveness to ET-1 was somehow affected by changing endocrine milieu, we treated adult rabbits with the long-acting GnRH agonist triptorelin (2.9 mg/kg), using a previously described protocol (11, 33, 36, 37). Subsets of rabbits were supplemented, after 2 wk, with vehicle (control), T (30 mg/kg), or E2v (3.3 mg/kg). Table 1 shows the levels of T and 17? estradiol (E2) in the peripheral blood at the time of the experiment. Both T and 17?E2 were significantly decreased by triptorelin (P < 0.01 and P < 0.05, respectively) and completely restored by administration of the corresponding sex steroid. Figure 1A shows the effects of the different pharmacological treatments on epididymal contractility. In control rabbits, increasing concentrations of ET-1 induced a dose-dependent increase in contractility (EC50 = 33 ± 5 nM). The contractile effect of ET-1 was as potent [maximal effect (Emax) = 105.7 ± 3.8%] as 10 μM NA, taken as 100%. Changing sex steroid milieu did not significantly affect the EC50 values for ET-1 (P = 0.184) but significantly changed Emax values. In fact, in hypogonadal rabbits, the maximal contractility elicited by ET-1 was dramatically reduced (Emax = 20.8 ± 3.1%; P < 0.0001) and only partially restored by T administration (Emax = 42.8 ± 3.4%; P < 0.005 vs. hypogonadism; P < 0.0001 vs. control). Conversely, estrogen administration fully restored the ET-1-induced contractility at a level not statistically different from the control (P = 0.236). Because rabbit epididymis expresses P450 aromatase (11), we tested the effect of a subacute endogenous estrogen deprivation, treating adult rabbits with the aromatase inhibitor letrozole (2.5 mg/kg daily for 3 wk), according to previously described experimental design (11, 33). Letrozole administration significantly decreased 17?E2 levels (P < 0.001; Table 2) and Emax for ET-1 (P < 0.0001; control Emax = 118 ± 6.3%; letrozole Emax = 55.8 ± 2.6%; Fig. 1B), without affecting the EC50 values (P = 0.275; shared EC50 = 39.6 ± 7.6 nM). Taken together, these results, strongly suggest that estrogens, but not androgens, regulate ET-1 sensitivity in rabbit epididymis.
TABLE 1. Effect of a GnRH analog (triptorelin), with or without T/E2v replacement, on sex steroid plasma levels
FIG. 1. Effect of changing sex steroid milieu on epididymal responsiveness to ET-1. A, Effect of increasing concentrations of ET-1 on the basal tone of preparations from sexually mature rabbits: untreated (n = 8 in six separate experiments); hypogonadal (GnRH analog) without (n = 7 in six separate experiments) or with hormonal replacement by weekly administration of T (n = 8 in six separate experiments) or E2v (n = 6 in six separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of ET-1. Data were expressed as the means ± SEM. B, Effect of increasing concentrations of ET-1 on the basal tone of preparations from sexually mature intact rabbits, untreated (n = 6, in four separate experiments) or chronically treated with the aromatase inhibitor letrozole (n = 6 in four separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of ET-1. Data are expressed as means ± SEM. The relative EC50 and Emax values are reported in the text.
TABLE 2. Effect of an aromatase inhibitor (letrozole) on sex steroid plasma levels
Sex steroids do not affect ET-1 and its receptor gene and protein expression
We next investigated whether the estrogen-induced change in responsiveness to ET-1 was somehow related to an altered expression of ET receptors or of their natural ligands. The results are summarized in Table 3. Neither triptorelin-induced hypogonadism nor sex steroid replacement significantly affected the mRNA expression of the ECE-1 and ET-1 and did not result in any significant alteration in the protein content of ET-like immunoreactivity. In addition, changing sex steroid milieu did not modify the ET receptor gene (ETA + ETB) and protein ([125I]ET-1 binding) expression.
TABLE 3. Lack of effect of hypogonadism (triptorelin) and sex steroid (testosterone or estrogen) replacement on the expression of ET-1, its converting enzyme (ECE-1), and receptors (ETA + ETB) in rabbit epididymis at both mRNA and protein level
OT partially mediates ET-1 induced contractility
In preliminary experiments, we demonstrated that two chemically distinct OTR antagonists did not compete for [125I]ET-1 binding in epididymal homogenates (Fig. 2A) but counteracted, in a dose-dependent manner, the contractility induced by ET-1 (100 nM), with similar Imax values (73.8 ± 15.1%) and IC50 values (299 ± 148 nM; Fig. 2B). Interestingly, these IC50 values for the ET-1-induced contractility are compatible with (although slightly higher than) those of both antagonists for OT-induced contractility (5, 33, 44). In epididymal strips from control rabbits, preincubation with OTA (1 μM) significantly decreased Emax for ET-1 (control, Emax = 130.4 ± 14%; OTA-pretreated, Emax = 60.7 ± 7.6%; P < 0.0001; percentage inhibition = 114.8%), without affecting the EC50 values (shared EC50 = 60.7 ± 2.8 nM; Fig. 2C). Similar results were obtained preincubating the epididymis from untreated rabbits with atosiban (1 μM): decreased responsiveness to ET-1 (control, Emax = 133.4 ± 13%; atosiban-pretreated, Emax = 62.9 ± 7.8%; P < 0.0001; percentage inhibition = 111%), although sensitivity to ET-1 was similar (shared EC50 = 61.4 ± 2.1 nM; Fig. 2D). Because the results with OTA and atosiban were virtually identical, further studies were performed using only OTA. In hypogonadal rabbits, OTA decreased the already negligible response to ET-1 (control Emax = 37.5 ± 0.1%; OTA-pretreated Emax = 28.6 ± 0.1%; P < 0.0001; percentage inhibition = 31.57%, not shown) only slightly. In contrast, in estrogen-replaced hypogonadal rabbits, OTA substantially hampered responsiveness to ET-1 but not ET-1 EC50 values (control Emax = 104.4 ± 8.8%; OTA-pretreated Emax = 29.6 ± 2.6%; P < 0.0001; percentage inhibition = 252.7%; shared EC50 = 78.8 ± 3.2 nM; Fig. 2E). To test whether the cooperation between ET-1 and OT in stimulating epididymal contractility was also evident in humans, we repeated the in vitro experiment using human epididymal strips. The results are reported in Fig. 2F. In human strips, ET-1 induced the previously described (3, 5) sustained increase in contractility (Emax = 191.6 ± 3.9%), with EC50 = 13.8 ± 1.4 nM. Preincubation with OTA strongly decreased maximal responsiveness (Emax = 43.5 ± 2.16%; P < 0.0001; percentage inhibition = 340.4%), without affecting the EC50 (P = 0.91).
FIG. 2. Effect of different OTR antagonists on ET-1 responsiveness in rabbit epididymis. A, Competition curves for [125I]ET-1 binding to rabbit epididymis microsomes by ET-1 (n = 3), OTA (n = 3), and 1-deamino-2-D-Tyr(OEt)-4-Thr-8-Orn-OT (atosiban; n = 3). Ordinate, Bound [125I]ET-1 expressed as a percentage of the total ligand added; abscissa, total concentration (molar) of unlabeled ligands. B, Effect of increasing concentrations of OTA (n = 4 in three separate experiments) and atosiban (n = 3 in three separate experiments) on the tone induced by a fixed concentration of ET-1 (100 nM) in rabbit epididymis. Relative IC50 values are reported in the text. Ordinate: Response, expressed as percentage of the maximal response obtained with ET-1 (100 nM); abscissa, concentration of the effectors. C, Effect of increasing concentrations of ET-1 in rabbit epididymis in the absence (n = 5 in four separate experiments) or presence of the specific OTR antagonist OTA (n = 4 in three separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of the agonist. The relative IC50 values are reported in the text. D, Effect of increasing concentrations of ET-1 in rabbit epididymis in the absence (n = 3 in three separate experiments) or presence of the specific OTR antagonist, atosiban (n = 3 in three separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa: concentration of the agonist. The relative IC50 values are reported in the text. E, Effect of increasing concentrations of ET-1 on the basal tone of epididymal preparations in hypogonadal rabbits supplemented with weekly administration of E2v in the absence (n = 3 in three separate experiments) or presence of the specific OTR antagonist, OTA (n = 3 in three separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of the agonist. The relative IC50 values are reported in the text. F, Effect of increasing concentrations of ET-1 in human epididymis in the absence (n = 5 in five separate experiments) or presence of the specific OTR antagonist, OTA (n = 4 in four separate experiments). Ordinate, Contractile activity, expressed as a percentage of the maximal response obtained with NA (10 μM); abscissa, concentration of the agonist. The relative IC50 values are reported in the text. Data are expressed as the means ± SEM.
OT and neurophysin I (NpI) are expressed in epididymis
Because the OTR antagonists did not displace [125I]ET-1 binding but blocked ET-1 action, in an estrogen-sensitive manner, we hypothesized that ET-1 might mediate part of its contractile activity, in both rabbit and human epididymis, by activating the previously described estrogen-sensitive OTR signaling (11) through the release of locally produced OT. Because no information is available on the epididymal presence of OT in rabbit and humans, we performed immunohistochemical studies, using specific antibodies against the peptide and its synthesis-associated protein NpI. Positive staining for both OT and NpI was detected in the majority of epithelial cells from human (Figs. 3 and 5) and rabbit (Figs. 4 and 6) epididymis and localized at the abluminal site, facing the smooth muscle layer. Only a few cells also displayed labeling in the apical portion, facing the epididymal lumen. Almost identical subcellular distribution of OT and NpI labeling indicates, at least indirectly, local synthesis and not uptake from extraepididymal sources, such as blood or testicular fluid (45, 46). To further verify the hypothesis of a local OT synthesis, we analyzed OT gene expression in epididymis using RT-PCR. To quantitate OT expression in human epididymis, we performed real-time RT-PCR. The results are reported as a histogram in Fig. 7A. Human epididymis showed a high abundance of OT mRNA. Because the sequence of rabbit OT gene has, to date, not been elucidated, primer design was based on the alignment of the published bovine, rat, mouse, and human OT sequences. RT-PCR revealed a transcript of an expected 193 bp in rabbit epididymis as well as in rabbit uterus and human brain used as positive controls (Fig. 7B). Sequence analysis of the RT-PCR products from rabbit epididymis confirmed its specificity and produced a partial sequence of rabbit OT cDNA of 156 bp corresponding to 108–264 nucleotides of the published human OT-specific sequence (Fig. 7C). The homology between rabbit and human OT-specific sequences was 95% and 92% at the nucleotide and amino acid levels, respectively. Taken together, these results strongly suggest a local epididymal production of OT and NpI in both rabbit and human epididymis.
FIG. 3. OT immunolocalization in human epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for OT in transversal sections of the epididymal head. The middle and right panels represent transversal sections of the epididymal body with different magnifications (B, E, and F, x40, x120, and x200, respectively). Immunopositivity for OT is detected in the majority of cells from the epithelial layer of the epididymal duct. Specific staining was in the abluminal cellular compartment, facing the smooth muscle layer. C, Control section, where the primary monoclonal antibody against OT was replaced by pure IgM antiserum (x40).
FIG. 5. Immunolocalization of OT-associated NpI in human epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for NpI in transversal sections of the epididymal head. The middle and right panels represent transversal sections of the epididymal body with different magnifications (B, E, and F, x40, x120, and x200, respectively). A diffuse immunopositivity for NpI is localized in the epithelial cells of human epididymis. In the epithelial cells of the tubules, specific staining for NpI was mostly localized in the abluminal cellular compartment, facing the smooth muscle layer. C, Control section, where the primary polyclonal antibody against NpI was replaced by pure IgM antiserum (x40).
FIG. 4. OT immunolocalization in rabbit epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for OT in transversal sections of epididymal head. In A, the arrowhead shows a testicular portion in close touch to the epididymal head (arrow). Within the testis, either Leydig cells or Sertoli cells are immunopositive for OT. The middle and right panels represent transversal sections of the epididymal tail with different magnification (B, E, F, and G, x40, x120, x200, and x400, respectively). In the epithelial cells of the tubules, specific staining for OT was mostly localized in the abluminal cellular compartment, facing the smooth muscle layer. C, Control section, where the primary monoclonal antibody against OT was replaced by pure IgM antiserum (x40). D, OT immunolocalization in rabbit epididymis.
FIG. 6. Immunolocalization of OT-associated NpI in rabbit epididymis. The left panels (A and D, x40 and x80, respectively) show immunopositivity for NpI in transversal sections of the epididymal head. Note that in A, both the testis (arrowhead) and the proximal part of the epididymal head (arrow) are shown. In the testis, either Leydig cells or Sertoli cells are immunopositive for NpI. The middle and right panels represent transversal sections of the epididymal tail with different magnifications (B, E, and F, x40, x120, and x200, respectively). In the epithelial cells of the tubules, specific staining for NpI is mostly localized in the abluminal cellular compartment, facing the smooth muscle layer. C shows a control section, where the primary polyclonal antibody against NpI was replaced by pure IgM antiserum (x40).
FIG. 7. Expression of OT gene in the epididymis. A, Quantitative detection of mRNA for OT in human tissues by real-time RT-PCR. Data were normalized over glyceraldehyde-3-phosphate dehydrogenase mRNA expression according to the comparative cycle threshold method and are expressed as the mean ± SEM. B, Ethidium bromide-stained agarose gel from the RT-PCR products obtained with primers for OT cDNA. Human brain and rabbit uterus were used as positive controls. NC, Negative control (RT-PCR without template); MW, molecular weight marker. C, Alignment of nucleotide sequences of the rabbit epididymis (epi) and human brain OT, partial cDNA, from RT-PCR products shown in B. The primer sequences (sense, right arrows; antisense, left arrows) are underlined. Vertical bars indicate identity between the sequences, whereas gaps in alignment are boxed. The predicted amino acid sequence is also indicated and differences with respect to the human OT sequence (NCBI accession no. NM_000915) are reported in bold. C. cavernosum, Corpus cavernosum.
ET-1 releases OT from rEE cells
To finally investigate whether or not ET-1 interacts with OT, in isolated epididymal epithelial cells, we initiated experiments in rEE cells. Cells were prepared using a previously described protocol (5). We first performed radioligand binding studies to investigate the presence of specific receptors for ET-1 in these cells. The results are reported in Fig. 8A. Cultured rEE cells express a high density of [125I]ET-1 binding sites (53360 ± 2462 sites/cell), showing pharmacological characteristics compatible with the predominant presence of ETA receptors (Table 4). To test whether ET-1 might affect OT release, we incubated rEE cells for 24 h with increasing concentrations of ET-1. ET-1 induced a 10-fold increase in immunoreactive OT in rEE-spent medium. ET-1-induced OT release started to be significant at relatively low ET-1 concentrations (1 nM; P < 0.001; Fig. 8B).
FIG. 8. Characterization of the biological effect of ET-1 in cultured rEE cells. A, Typical family of competition curves among [125I]ET-1 and unlabeled ET-1 (x), ET-3 (), IRL1620 (), and BQ123 (+) in rEE cells. Ordinate, B/T, bound to total ratio for [125I]ET-1; abscissa, total concentration (molar) of ligands. Binding parameters are in Table 4. B, Effect of 24-h incubation with increasing concentrations of ET-1 on the release of OT in cultures of rEE cells. Ordinate, OT release (pg/106 cells) in spent medium; abscissa, concentrations of ET-1. ET-1 significantly increased OT release starting from 1 nM (*, P < 0.05; **, P < 0.01 vs. control; n = 3).
TABLE 4. Pharmacological characterization of ET-1 binding sites in cultured rEE cells
Discussion
Our results further extend the concept of epididymis as a male estrogen target, demonstrating the existence of an estrogen-driven, short paracrine loop between ET-1 and OT. We previously demonstrated that, in rabbit epididymis, OT partially mediates its contractile activity through the release of locally produced ET-1 and ETA activation (5). We now provide original evidence that, in the same tissue, ET-1 might signal through OT release and OTR activation. Hence, in our view, the spontaneous rhythmic contractions of epididymis are facilitated by a positive interactive cooperation between the OT/OTR and ET-1/ETA systems. These two systems might mutually activate each other in an estrogen-regulated feed-forward fashion, favoring fluid and sperm progression through the epididymal duct. Further studies should be conducted whether the previously described epididymal alteration in ER knockout mice (ductule dilatation and fluid overload) are due only to an impairment of fluid reabsorption (29) or to altered motility as well.
The involvement of OT in coordinating testicular and epididymal motility was first postulated by Cross (47) almost 50 yr ago. By using an abdominal chamber technique in the rabbit, he demonstrated that iv injection of OT induced "peristaltic and pendular movements in the tubules of the head and body epididymis, and segmentation contractions in the tubules of smaller calibre in the tail" (47). Subsequent experiences substantially confirmed Cross’s view: OTR is present in the epididymis and stimulates its motility (see Ref. 7 for review). However, the physiological source of the OTR ligand is still unclear. Because circulating levels of OT (picomolar range) are far from the concentrations needed to evoke a contractile response (nanomolar range), a role for posterior pituitary in driving epididymal contractility is unlikely. Hence, local production was investigated. In the early 1980s, it was found that testicular OT levels were 3 orders of magnitude higher than the circulating ones, suggesting local production (48, 49). This was confirmed later on, when specific transcripts for OT were detected in the testis of several animal species (see Ref. 50 for review), including the monkey and human (13, 51). In this study, we found, using real-time RT-PCR, that human epididymis expresses an elevated level of OT mRNA. In human epididymis, staining for OT and its synthesis-associated NpI was essentially localized in the epithelial cells, predominantly in the basal portion, facing the smooth muscle layer, although some apical labeling was also found. A similar picture was observed in rabbit (present study), ovine (52), and rat (53) epididymis. However, the final demonstration of an epididymal production of OT derives from studies on isolated epithelial cells in culture. According to a previous morphological study (24), epididymal epithelial cells express a high density of ETA receptors (more than 50,000 sites/cell). Stimulation of ETA receptors with ET-1 induced a 10-fold increase in OT release. Such a release should play a relevant role in mediating the contractile effect of ET-1, because the blocking of OTR with specific, although chemically distinct, antagonists, such as OTA and atosiban, strongly decreased ET-1 responsiveness in both rabbit and human epididymal preparations. The OTR antagonist-induced inhibition of ET-1 contractility was even more apparent in tissues derived from estrogen-treated hypogonadal rabbits. One of the main characteristics of the OT-OTR system is its estrogen dependency (50, 54). We recently demonstrated that, in a rabbit model of hypogonadotropic hypogonadism, estrogen (but not androgen) administration restored gene and protein expression of epididymal OTR and responsiveness to OT (11). However, in the present study, we were unable to demonstrate the same for ET-1, using a similar animal model. Indeed, we did not find any significant change either at gene or protein level in ET-1 and its cognate receptors, although we did find a sustained estrogen dependency in response to ET-1. It is therefore possible that the estrogen-induced change in sensitivity to ET-1 reported in the present study might depend on parallel changes in the interplay between ET-1 and the estrogen-sensitive OT-OTR system. However, the possibility that estrogens might up-regulate some contractile effectors downstream to the ET-1 receptors cannot be ruled out at present.
In conclusion, the present results indicate an estrogen-sensitive cooperation between two peptidergic systems (OT/OTR and ET-1/ETA) in the epididymis, which might help in regulating the spontaneous motility of the duct and in facilitating sperm transport through it.
Acknowledgments
We thank Paolo Ceccatelli and Mauro Beni (Centro per i Servizi di Stabulazione degli Animali di Laboratorio, University of Florence) for technical assistance in animal treatment.
References
El-Badawi A, Schenk EA 1967 The distribution of cholinergic and adrenergic nerves in the mammalian epididymis: a comparative histochemical study. Am J Anat 121:1–14
Nicholson HD, Parkinson TJ, Lapwood KR 1999 Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep. J Reprod Fertil 117:299–305
Peri A, 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. J Clin Endocrinol Metab 82:3797–3806
Harneit S, Ergun S, Paust HJ, Mukhopadhyay AK, Holstein AF 1997 Endothelin-1 and its receptors in the human epididymis. Adv Exp Med Biol 424:191–192
Filippi S, 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 2002 Identification, localization and functional activity of oxytocin receptors in epididymis. Mol Cell Endocrinol 193:89–100
Hess RA 2003 Estrogen in the adult male reproductive tract. Reprod Biol Endocrinol 1:52–65 (Review)
Filippi S, Vignozzi L, Vannelli GB, Ledda F, Forti G, Maggi M 2003 Role of oxytocin in the ejaculatory process. J Endocrinol Invest 26:82–86
Maggi M, 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. Endocrinology 120:986–994
Parry LJ, Bathgate RA 2000 The role of oxytocin and regulation of uterine oxytocin receptors in pregnant marsupials. Exp Physiol 85:91S–99S
Whittington K, Assinder SJ, Parkinson T, Lapwood KR, Nicholson HD 2001 Function and localization of oxytocin receptors in the reproductive tissue of rams. Reproduction 122:317–325
Filippi S, Luconi M, Granchi S, Vignozzi L, Bettuzzi S, Tozzi P, Ledda F, Forti G, Maggi M 2002 Estrogens, but not androgens, regulate expression and functional activity of oxytocin receptor in rabbit epididymis. Endocrinology 143:4271–4280
Maggi M, Baldi E, Genazzani AD, Giannini S, Natali A, Costantini A, Rodbard D, Serio M 1989 Vasopressin receptors in human seminal vesicles: identification, pharmacological characterization and comparison with the vasopressin receptors present in human kidney. J Androl 10:393–400
Einspanier A, Ivell R 1997 Oxytocin and oxytocin receptor expression in reproductive tissues of the male marmoset monkey. Biol Reprod 56:416–422
Frayne J, 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. Mol Hum Reprod 4:527–532
Hib J 1974 The in vitro effects of oxytocin and vasopressin on spontaneous contractility of the mouse cauda epididymidis. Biol Reprod 11:436–439
Hib J 1974 The contractility of the cauda epididymidis of the mouse, its spontaneous activity in vitro and the effects of oxytocin. J Reprod Fertil 36:191–193
Studdard PW, Stein JL, Cosentino MJ 2002 The effects of oxytocin and arginine vasopressin in vitro on epididymal contractility in the rat. Int J Androl 25:65–71
Cross BA, Glover TD 1958 The hypothalamus and seminal emission. J Endocrinol 16:385–395
Assinder SJ, Rezvani A, Nicholson HD 2002 Oxytocin promotes spermiation and sperm transfer in the mouse. Int J Androl 25:19–27
Yanagisawa M, Masaki T 1989 Molecular biology and biochemistry of the endothelins. Trends Pharmacol Sci 10:374–378
Rubanyi GM, Polokoff MA 1994 Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46:325–415
Masaki T, Ninomiya H, Sakamoto A, Okamoto Y 1999 Structural basis of the function of endothelin receptor. Mol Cell Biochem 190:153–156
Wong PY, Cheng-Chew SB, Leung PY, Qin DN 1991 Functional study and immunocytochemical identification of endothelin in cultured epididymal cells and intact epididymis of the rat. J Cardiovasc Pharmacol 17(Suppl 7):S242–245
Harneit S, Paust HJ, Mukhopadhyay AK, Ergun S 1997 Localization of endothelin-1 and endothelin-receptors A and B in human epididymis. Mol Hum Reprod 3:579–584
Kim SZ, Kang SY, Lee SJ, Cho KW 2000 Localization of receptors for natriuretic peptide and endothelin in the duct of the epididymis of the freshwater turtle. Gen Comp Endocrinol 118:26–38
Wong PY, Fu WO, Huang SJ 1989 Endothelin stimulates short circuit current in a cultured epithelium. Br J Pharmacol 98:1191–1196
Wang H, Sakurai K, Endoh M 2000 Pharmacological analysis by HOE642 and KB-R9032 of the role of Na(+)/H(+) exchange in the endothelin-1-induced Ca(2+) signalling in rabbit ventricular myocytes. Br J Pharmacol 131:638–644
Lei ZM, Zou W, Mishra S, Li X, Rao ChV 2003 Epididymal phenotype in luteinizing hormone receptor knockout animals and its response to testosterone replacement therapy. Biol Reprod 68:888–895
Hess RA, Zhou Q, Nie R, Oliveira C, Cho H, Nakaia M, Carnes K 2001 Estrogens and epididymal function. Reprod Fertil Dev 13:273–283
Zhou Q, Nie R, Prins GS, Saunders PT, Katzenellenbogen BS, Hess RA 2002 Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J Androl 23:870–881
Nie R, Zhou Q, Jassim E, Saunders PT, Hess RA 2002 Differential expression of estrogen receptors and ? in the reproductive tracts of adult male dogs and cats. Biol Reprod 66:1161–1168
Hess RA 2003 Estrogen in the adult male reproductive tract. Reprod Biol Endocrinol 1:52–65
Vignozzi L, 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. Endocrinology 145:1823–1834
Hess RA 2000 Oestrogen in fluid transport in efferent ducts of the male reproductive tract. Rev Reprod 5:84–92
Zhou Q, Clarke L, Nie R, Carnes K, Lai LW, Lien YH, Verkman A, Lubahn D, Fisher JS, Katzenellenbogen BS, Hess R 2001 Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function. Proc Natl Acad Sci USA 98:14132–14137
Morelli A, 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. Endocrinology 145:2253–2263
Filippi S, Luconi M, Granchi S, Natali A, Tozzi P, Forti G, Ledda F, Maggi M 2002 Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation. Int J Impot Res 14:295–307
Smith IE 1998 Pivotal trials of letrozole: a new aromatase inhibitor. Oncology (Huntingt) 12(3 Suppl 5):41–44
Lauth M, Berger MM, Cattaruzza M, Hecker M 2000 Pressure-induced upregulation of preproendothelin-1 and endothelin B receptor expression in rabbit jugular vein in situ: implications for vein graft failure? Arterioscler Thromb Vasc Biol 20:96–103
Maggi M, Barni T, Orlando C, Fantoni G, Finetti G, Vannelli GB, Mancina R, Gloria L, Bonaccorsi L, Yanagisawa M, Forti G 1995 Endothelin-1 and its receptors in human testis. J Androl 16:213–224
Munson PJ, Rodbard D 1980 Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239
Peri A, Fantoni G, Granchi S, Vannelli GB, Barni T, 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. Steroids 63:294–298
De Lean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to biossay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97–E102
Zhang XH, Filippi S, Vignozzi L, Morelli A, Mancina R, Donati S, Luconi M, Marini M, Vannelli GB, Forti G, Maggi M 2005 Identification, localization and functional "in vitro" and "in vivo" activity of oxytocin receptor in the rat penis. J Endocrinology 184:567–576
Knickerbocker JJ, Sawyer HR, Amann RP, Tekpetey FR, Niswender GD 1988 Evidence for the presence of oxytocin in the ovine epididymis. Biol Reprod 39:391–397
Veeramachaneni DN, Amann RP 1990 Oxytocin in the ovine ductuli efferentes and caput epididymidis: immunolocalization and endocytosis from the luminal fluid. Endocrinology 126:1156–1164
Cross BA 1955 The posterior pituitary gland in relation to reproduction and lactation. Br Med Bull 11:151–155[Free Full Text]
Wathes DC, Swann RW, Hull MG, Drife JO, Porter DG, Pickering BT 1983 Gonadal sources of the posterior pituitary hormones. Prog Brain Res 60:513–519
Kasson BG, Adashi EY, Hsueh AJ 1986 Arginine vasopressin in the testis: an intragonadal peptide control system. Endocr Rev 7:156–168
Gimpl G, Fahrenholz F 2001 The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81:629–683
Ivell R, Balvers M, Rust W, Bathgate R, Einspanier A 1997 Oxytocin and male reproductive function. Adv Exp Med Biol 424:253–264
Assinder SJ, Carey M, Parkinson T, Nicholson HD 2000 Oxytocin and vasopressin expression in the ovine testis and epididymis: changes with the onset of spermatogenesis. Biol Reprod 63:448–456
Harris GC, Frayne J, Nicholson HD 1996 Epididymal oxytocin in the rat: its origin and regulation. Int J Androl 19:278–286
Zingg HH, Laporte SA 2003 The oxytocin receptor. Trends Endocrinol Metab 14:222–227(Sandra Filippi, Annamaria)