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Original Studies |
Endocrinology (A.P., C.P., M.S.) and Andrology (G.F., S.G., G.F., M.M.) Units, Departments of Clinical Physiopathology, Human Anatomy (G.B.V., T.B.), Pharmacology (S.A.), and Urology (G.B.), University of Florence, 50139 Florence, Italy
Address all correspondence and requests for reprints to: Mario Maggi, M.D., Andrology Unit, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. E-mail: m.maggi{at}mednuc2.dfc.unifi.it
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Abstract |
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Introduction |
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Sperm transport from the testis into the epididymis is also assisted by ciliary activity of the luminal epithelium and by contraction of mioid elements of the efferent duct walls (6). In the rat, regular pendular and peristaltic contractions take place about once every 6–10 s and propel sperm from the caput to the cauda epididymis (7). The amplitude of spontaneous contractions increases from the caput to the proximal cauda, whereas the frequency progressively decreases (8). The fact that the rate of transport through this organ is not influenced by ligation of the vasa efferentia (9, 10) reinforces the hypothesis that sperm transport through the epididymis is mostly due to local contractile activity. That maturation of the potential for vigorous progressive sperm motility in the epididymis is of pivotal importance in providing fertile ejaculated spermatozoa is pointed out in several studies. It has been demonstrated in a group of patients presenting severely degenerated ejaculated spermatozoa (epididymal necrospermia), that improved sperm motility and viability occurred after frequent ejaculation, which reduced the time spent in the epididymis (11). The increased age of the ejaculated spermatozoa due to prolonged transit through the epididymis may at least partially explain their reduced viability (3).
It is known that in mammals the epididymis is innervated by cholinergic and adrenergic fibers, that are more represented in the distal tract of this organ (12), where the contractility appears to be mostly dependent on the adrenergic system (13). Smooth muscle cells are also more prominent distally in the epididymis (14). However, direct evidence of the presence of one or more local factors eliciting the contractile activity of the epididymis is still lacking. We have previously shown the presence of endothelin-1 (ET-1) and its receptors in the human testis (15). In particular, we have demonstrated that Sertoli cells produce ET-1, whereas specific receptors are present on Leydig, peritubular myoid, and germ cells. In that study we postulated that ET-1 might represent a new Sertoli cell-derived paracrine factor regulating steroidogenesis and spermatogenesis. In the present study we extended our observations on the presence of ET-1, ET-converting enzyme-1 (ECE-1), and ET receptors to the human epididymis to determine whether ET-1 can be regarded as a paracrine factor involved in sperm transport through this organ.
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Materials and Methods |
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[125I]ET-1 (2000 Ci/mmol),
[125I]ET-3 (2000 Ci/mmol), and
[
-32P]CTP (3000 Ci/mmol) were purchased from
Amersham (Amity, Milan, Italy). [-35S-Thio]UTP (1300
mCi/mmol) was obtained from New England Nuclear-DuPont (Paris, France).
ET-1, ET-3, the relatively ETB-selective agonist
sarafotoxin 6C (SRTX 6C), and the ETA-selective antagonist
cyclo-[D-Trp-D-Asp-Pro-D-Val-Leu]
(BQ123) were obtained from NovaBiochem (Laufelfingen, Switzerland). The
ETB-selective agonist IRL 1620 was purchased from Alexis
(Laufelfingen, Switzerland), and the nonselective ET receptors
antagonist SB 209670 was obtained from SmithKline Beecham (King of
Prussia, PA). The polyclonal antibody to ET-1 (RAS 6901) was purchased
from Peninsula Laboratories (San Carlos, CA). The monoclonal antibody
to ET-1 (clone TR.ET.48.5) was purchased from Affinity Bioreagents
(Nashanic Station, NJ). The antibody to ECE-1 was provided by Dr. M.
Yanagisawa. This antibody was produced by immunizing rabbits with a
synthetic peptide, CPPGSPMNPHHKCEVW, corresponding to the C-terminal 16
amino acids of bovine ECE-1. BSA, bovine insulin, bacitracin,
benzamidine, soybean trypsin inhibitor, paraformaldehyde, formamide,
dithiothreitol, dextran sulfate, ethylenediamine tetraacetate, sheared
herring sperm DNA, yeast transfer ribonucleic acid (RNA), ribonuclease
A (RNase A), acetic anhydride, triethanolamine, sodium pyrophosphate,
sodium heparin, Triton X-100, levamisole,
Tris(hydroxymethyl)aminomethane, and Permount were purchased from Sigma
Chemical Co. (St. Louis, MO). 3,4,3',4'-Tetraaminodiphenylhydrochloride
(diaminobenzidine) was obtained from BDH Chemical (Poole, UK).
Universal immunoperoxidase staining kits were obtained from Cambridge
Research Laboratories (Cambridge, MA). EcoRI,
AccI, and T4 ligase were purchased from Life Technologies
(Berlin, Germany). SP6 and T7 RNA polymerase were obtained from Promega
(Madison, WI). Agarose, DNA Tailing kit, antidigoxigenin-alkaline
phosphatase-conjugated antibody, nitro blue tetrazolium, and
5-bromo-4-chloro-3-indoyl phosphate toluidinium salt were purchased
from Boehringer Mannheim Italy (Milan, Italy).
Patients and tissue preparation
Epididymes and testes were obtained at surgery, after informed consent, from 11 patients undergoing monolateral or bilateral orchidectomy for testis neoplasm (n = 5), sex reassignment (n = 4), or undescended testis (n = 2). The age of the patients ranged from 45–71 yr. Tissue specimens were fresh-frozen for in situ hybridization, Northern blot studies, autoradiography, and membrane preparation. Samples for immunohistochemistry were fixed in Bouin’s solution and embedded in paraffin. Epididymes for in vitro contractility studies were maintained in PBS containing 1 mmol/L CaCl2 at 4 C until the beginning of the experiments. Renal medulla was obtained from a patient subjected to nephrectomy for a localized renal tumor.
In situ hybridization for the detection of prepro-ET-1 messenger RNA (mRNA)
The experiments were performed as previously described (16), on frozen epididymal sections. A 35S-labeled human prepro-ET-1 RNA was synthesized as previously described (17) and was used as the probe. At the end of the experiments, the sections were analyzed using a Nikon Microphot FX microscope (Nikon, Japan). Negative controls consisted of 1) hybridization of sections to a sense RNA probe, 2) pretreatment of sections with RNase A (20 µg/mL), and 3) addition of a 100-fold excess of unlabeled antisense RNA probe to the hybridization mixture containing the antisense 35S-labeled probe.
Nonisotopic in situ hybridization for the detection of ETA and ETB mRNA
Experiments were performed as previously described, on fresh-frozen tissue specimens (18). Briefly, four different 48-mer (ETA-SP, ETA-AP, ETB-SP, and ETB-AP), synthesized by Med Probe (Oslo, Norway), were used as probes. The probes were 3'-end labeled with digoxigenin-11-deoxy-UTP using the Oligonucleotide Tailing kit (Boehringer Mannheim). The hybridized mRNAs were detected by an immunocolorimetric method (Boehringer Mannheim). The slides were examined using a Nikon Microphot FX microscope.
Northern analysis
Total RNAs extraction and Northern blot analysis were performed as previously described (15). After gel electrophoresis and membrane transfer, the RNAs were hybridized to radiolabeled complementary DNA (cDNA) probes specific for ET-1, ET-3, and ECE-1 for mRNA detection. ET-1- and ET-3-specific probes have been described previously (15). The ECE-1 probe was a 2.7-kilobase (kb) XhoI gel-purified cDNA fragment. The quality of the RNAs tested was assessed by hybridization of the same membranes using [32P]rat cyclophilin cDNA as a probe (19).
Autoradiography
Autoradiographic studies were performed as previously described (15). Briefly, frozen epididymal specimens were cut and mounted on gelatin-coated glass slides. The slides were incubated with 50 pmol/L [125I]ET-1. An excess of unlabeled ET-1 (100 nmol/L) was added to the mixture to determine nonspecific binding. Coverslips coated with nuclear emulsion Illford K5 (Illford, Mobberley, UK) were applied to the sections. The slides were exposed at 4 C in the dark for 6–8 days, then developed in Illford Phenisol, fixed in Illford Hypam, stained with hematoxylin, and observed with darkfield microscopy.
Immunohistochemistry
Immunohistochemical studies were carried out as described previously (20). Briefly, epididymal sections (fixed in Bouin’s solution and embedded in paraffin) were incubated with polyclonal or monoclonal ET-1 antisera (diluted 1:1500 and 1:100, respectively). The sections were then incubated with the specific IgG peroxidase conjugates for 30 min (dilution, 1:1000). Demonstration of peroxidase activity and controls for specificity of the antisera were performed as previously described (20). The slides were photographed using a Nikon Microphot-FX microscope.
Membrane preparation
Membranes from epididymal tissues were prepared as described previously (15). Protein concentration was assayed using a commercial protein assay kit (Bio-Rad Laboratories, Munich, Germany).
Binding studies
Binding studies were performed as described previously (15).
Aliquots of membranes (0.05 mg/mL) were incubated for 60 min at 22 C in
the presence of increasing concentrations (10–70 pmol/L) of
[125I]ET-1 or [125I]ET-3 with or without
increasing concentrations of unlabeled compounds
(10-11-10-4 mol/L). Self- and
cross-displacement curves were performed using ET-1, ET-3, BQ123, IRL
1620, SRTX 6C, and SB 209670. After incubation, membranes were filtered
through Whatman GF/B filters (Clifton, NJ), using the Brandel M-48R
48-well cell harvester (Gaithersburg, MD). Radioactivity retained by
filters was counted in a 
-counter at 70% efficiency.
In vitro contractility
Small strips (5 mm long, 3 mm wide, 1 mm thick) were dissected from human epididymes. The preparations were vertically mounted in an organ bath and a tension of 700 mg was applied. Isometric contraction was recorded by isometric transducers on a polygraph chart (Battaglia Rangoni, Italy). The strips were perfused with 5 mL Tyrode’s solution of the following composition: 118 mmol/L NaCl, 25 mmol/L NaHCO3, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, and 10 mmol/L glucose, and oxygenated with a gas mixture containing 95% O2 and 5% CO2. The temperature was kept constant at 37 C, and the pH of the solution was 7.4. The preparations were allowed to equilibrate for at least 90 min; during this period the bath medium was replaced every 15 min. Noradrenaline increased the tonic tension in a concentration-dependent manner, with a maximum effect obtained at 100 µmol/L. This value was taken as 100%, and the increases recorded in the presence of different concentrations of ET-1 and its analogs were referred to this value. Cumulative concentrations of ET-1 were added to the perfusion solution. One hour of preincubation with different concentrations of BQ 123 was performed to test the effect of ET-1 in the presence of an ETA receptor antagonist.
Analysis of experimental results
The binding data were evaluated quantitatively with nonlinear least squares curve fitting using the computer program Ligand (21).
The computer program Allfit (22) was used for the analysis of sigmoidal dose-response curves obtained in contractility studies. Data were analyzed by Student’s t test and one-way ANOVA. Each data point represents the mean ± SE.
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Results |
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The presence of mRNA for prepro-ET-1, prepro-ET-3, and ECE-1 in
human epididymis was assayed by Northern blot analysis. Total RNA from
epididymal tissue was hybridized to a prepro-ET-1 cDNA probe. Testis
and renal medulla were used as positive controls. The results, shown in
Fig. 1A
, indicate the presence of a
single prepro-ET-1-specific band of 2.3 kb in all tissues tested. The
same tissues were also assayed for the presence of prepro-ET-3 mRNA by
Northern blot analysis, using prepro-ET-3 cDNA as the probe. In this
case, total RNA extracted from a neuroblastoma cell line, SK-N-AS, was
used as the positive control. These cells showed a distinct, specific
2.5-kb autoradiographic signal, whereas no specific signal was detected
in human epididymis, testis, and renal medulla (not shown). mRNA
degradation, as the possible cause of the lack of prepro-ET-3-specific
signal in the tissues tested, was excluded by the presence of a
specific signal for cyclophilin in all the cases (not shown).
Subsequently, to determine the active processing of pro-ET-1 in the
human epididymis, total RNA from this tissue was hybridized to a cDNA
probe specific for ECE-1. This enzyme converts the precursor pro-ET-1
into the active peptide ET-1. Total RNAs from human testis and renal
medulla were tested at the same time for ECE-1 expression, as positive
controls. As shown in Fig. 1B, a specific 4.7-kb signal was obtained in
the three tissues studied. Two less intense signals, of about 4 and 3.1
kb, respectively, were detected. The different sizes of ECE-1 mRNA have
been previously described and could be generated by alternative
polyadenylation of the 3'-noncoding region (23).
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, thin arrows), but not in
the muscle cells. A blood vessel showing endothelial cells positive for
ET-1 hybridization signal is indicated by the arrowheads in
the same section. There was no specific hybridization signal detectable
in the negative control sections hybridized with the sense probe (Fig. 2B) or with the radiolabeled antisense probe in the presence of an
excess of unlabeled antisense probe (not shown) or when treated with
RNase before hybridization (not shown).
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, thin arrows). The
positivity was observed in both the columnar principal cells and the
basal cells. When the anti-ET-1 antibody was further diluted
(1:10,000), no specific immunostaining was observed (not shown).
Similarly, immunoabsorption of the antiserum with synthetic ET-1 (100
nmol/L; Fig. 3B) or replacement of the first antibody with preimmune
serum (not shown) completely prevented any positive staining. Also, the
presence of ECE-1 in human epididymis was assayed by means of
immunohistochemistry, using an anti-ECE-1 polyclonal antibody (working
dilution, 1:2,500) provided by Dr. Yanagisawa (see Materials and
Methods). The results of a typical experiment (n = 4) are
showed in Fig. 4A. As in the case of
ET-1, ECE-1-specific staining appeared limited to the epithelial cells
(thin arrows). No positivity was observed when the
anti-ECE-1 antibody was further diluted (1:10,000; not shown) or when
the first antibody was replaced by preimmune rabbit serum (Fig. 4B).
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To identify and pharmacologically characterize ET-binding sites in
human epididymis, binding studies in pooled epididymal membranes were
performed. Preliminary results indicated that [125I]ET-1
and [125I]ET-3 binding to epididymal membranes reached an
apparent equilibrium within 60 min at 22 C (data not shown). Therefore,
subsequent studies were performed using these experimental conditions.
Mathematical analysis of multiple groups of competition curves for
125I-labeled ET-1 and 125I-labeled ET-3, the
corresponding unlabeled peptides, and other ET isopeptides strongly
indicated the presence of a heterogeneity of ET receptors in human
epididymis. In three consecutive experiments performed using three
different epididymal membrane preparations, the fit for the two-site
model was consistently better than that for the one-site model
(P < 0.00001). Table 1
summarizes the Kd and binding capacity for the best-fitting
model. Figure 5 shows a typical group of
competition curves for [125I]ET-1 (upper
panel) or [125I]ET-3 (lower panel) in
human epididymis. The high capacity site (R1) bound with high affinity
ET-1, BQ123, and SB209670, whereas ET-3, IRL1620, and SRTX 6C showed
lower specificity for this site. This site appears to correspond to the
receptor cloned by Arai et al. (24) from bovine lung, termed
ETA. The low capacity site (R2) bound with high affinity
ET-1, ET-3, IRL1620, and SB209670. This site, therefore, corresponds to
the ET nonselective receptor cloned from rat lung (25), termed
ETB.
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, thin arrows). The
positivity was extended to the longitudinal and circular smooth muscle
layers. As a negative control, some samples were hybridized to a 48-mer
probe complementary to ETA/AP, named ETA/SP. In
this case, no positive staining was detectable in the muscle cells of
the epididymis (Fig. 6B). A specific staining in the epididymis was
detected when the sections were incubated in the presence of a probe
specific for ETB receptors (ETB/AP; Fig. 6C).
The intensity of the staining was comparable to that observed for
ETA. However, ETB receptor expression appeared
limited to the inner layer of the muscular wall of the epididymis
(thin arrows). Also in this case, to test the specificity of
the staining, some specimens were hybridized to the sense probe
(ETB/SP). An example, which demonstrated the absence of any
positive immunoenzymatic reaction, is shown in Fig. 6D.
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, thin arrows). Silver
grains were absent in adjacent sections incubated in the presence of
100 nmol/L unlabeled ET-1 (Fig. 7B).
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Finally, to determine whether ET receptors mediate the
contractility of the epididymal duct wall, in vitro
contractility studies were performed. Small strips, dissected from
human epididymis, were first tested with 100 µmol/L noradrenaline,
which induced an increase in tonic tension of 82.5 ± 13.5 mg
(n = 8; data not shown). The contractile activity induced by ET-1
and analogs in each strip was normalized by the maximal effect of
noradrenaline, considered as 100%. ET-1, in a concentration-dependent
manner, induced a slow increase in tonic tension in strips either from
caput (n = 5) or cauda epididymis, with the concomitant appearance
of occasional phasic contractions (n = 2; data not shown). The
frequency and amplitude of phasic contractions increased as a function
of the concentration of ET-1 tested. A concentration-response curve for
ET-1 obtained from five experiments in human epididymal strips is
reported in Fig. 8. The maximum effect
for ET-1 was observed at 0.3 µmol/L, and it was 2-fold greater than
the effect observed with 100 µmol/L noradrenaline. Hence, ET-1
appears to be more active and more potent than noradrenaline in
eliciting epididymal contractility. The ED50 value for
ET-1-induced contractions was 20 ± 2.7 nmol/L. Pretreatment with
different BQ123 concentrations (3, 100, and 3000 nmol/L) did not modify
the basal tone, but dose-dependently blunted the ET-1 effect (Fig. 8).
In the inset in Fig. 8, the antagonistic effect of different
BQ 123 concentrations in the presence of 0.01 µmol/L ET-1 is
reported. The IC50 value for BQ 123 was 40 ± 17
nmol/L. Furthermore, the effect of IRL 1620, a selective agonist of
ETB receptors, was evaluated (not shown). An increase in
tonic tension was recorded only at high concentrations (0.3–10
µmol/L), and the appearance of phasic contractions was never
observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we extended the concept of a paracrine circuit mediated by ET-1 in the human epididymis. The presence of prepro-ET-1 mRNA was assayed by Northern blot analysis and in situ hybridization. Our results indicate that prepro-ET-1-specific transcripts are readily detectable in the human epididymis and are located in the epithelial compartment. mRNAs from testis and renal medulla were included in Northern analysis as positive controls, as we have previously shown the presence of prepro-ET-1 mRNA in human testis (15) and renal medulla (15, 16). In addition, we demonstrated the presence of mRNA for ECE-1 in the epididymis. ECE-1 is the enzyme that converts the 38-amino acid residue precursor pro-ET-1 (big ET-1), whose biological activity is negligible (30), into the active 21-amino acid residue peptide ET-1 through a proteolytic cleavage between Trp21 and Val22 (30). Therefore, detectable expression of this gene indicates an active processing of the prohormone. ECE-1 has been biochemically characterized (31, 32, 33) and its cDNA cloned in different animal species (23, 34, 35, 36, 37). This enzyme is a membrane-bound neutral metalloprotease structurally related to neutral endopeptidase 24.11 and Kell blood group protein (23). ECE-1 cleaves big ET-1 more efficiently than big ET-2 and big ET-3 (32, 36), and appears to be functioning either on the cell surface and or intracellularly. In our study we also demonstrated the presence of immunoreactive ET-1 as well as ECE-1 in the epididymis and confirmed that the positivity was specifically located in the epithelial cells. In contrast to ET-1 and ECE-1, no ET-3 gene expression could be detected in human epididymis. This observation is in agreement with our previous data on human testis (15), in which no specific-ET-3 transcript was detected.
To determine whether target cells for ET-1 could be identified in the epididymis itself, additional in situ hybridization studies, binding studies, and autoradiography were performed. In situ binding studies clearly demonstrated that ET-specific binding sites are present in this organ and are localized in the muscle cells of the duct wall. The paracrine mode of action of ET-1 in the epididymis we postulated in the present work is in good agreement with the localization of ET-1 staining at the basal pole of either columnar principal or basal epithelial cells, in close proximity to muscle cells. The active expression of ECE-1 supports this hypothesis. In fact, the conversion of big ET-1 into the active ET-1 either inside the epithelial cells or on the portion of the cell surface next to the target cells might result in high amounts of ET-1 locally released that are readily available for binding to ET receptors. The pharmacological characterization of these sites indicated the presence of two distinct sites that correspond, in terms of affinity constants, to the previously characterized ETA and ETB receptors. As in the human testis (15), ETA appeared severalfold more represented than ETB in the human epididymis. This finding was confirmed by in vitro contractility studies in which we showed that a specific antagonist of ETA (BQ 123) markedly decreased the ET-1-induced contractile activity of epididymal strips in a concentration-dependent manner, whereas a specific agonist of ETB (IRL 1620) only partially mimicked the effect of ET-1. Our data from in situ hybridization studies, using specific oligonucleotide probes for the two receptor subtypes, may support the above-mentioned finding, namely that ETA receptors are more represented than ETBs in the human epididymis. Indeed, although the intensity of the staining for ETA and ETB mRNA was substantially the same, we noted a different pattern of localization of the staining. In particular, ETA appeared widely represented throughout the muscle layer of the epididymis, whereas ETB was limited to the inner portion. Hence, it is possible that the wider distribution of ETA receptors accounts at least in part for the higher capacity observed in binding studies.
In view of the present results, it appears that ET-1 may be one of the still unknown factors that promote contractility in the epididymis. To our knowledge, this is the first report on epididymal contractility elicited through a paracrine mode of action. It has been previously shown that both adrenergic and cholinergic fibers innervate the muscle cells in the epididymis (12). However, to date there has been no experimental evidence that muscle cells are the target of locally released humoral factors that stimulate contraction. The physiological significance of our results is substantiated by the fact that ET-1 appeared 2-fold more potent than noradrenaline in inducing contractile activity in in vitro contractility studies. Although we do not have information on the concentration of ET-1 released at the epithelial-smooth muscle border, previous studies indicated that the concentrations of this peptide and its precursor in seminal fluid are definitively higher than those in peripheral blood (15, 38). As in the present report we demonstrated that epithelial cells of the epididymis contain not only ET-1 mRNA and protein, but also the enzyme ECE-1 that is functioning intracellularly as well as at the cell surface level, it is possible that these cells actively convert big ET-1 of seminal origin into bioactive ET-1.
The findings we presented in this paper add new information to understanding the functions of the epididymis, which are still rather obscure at the molecular level. In addition to its direct contribution to sperm maturation (1, 39), the epididymis is involved in other physiological processes, such as sperm transport, concentration, and storage, that contribute to provide fertile ejaculated sperms. Thus, a morphological or functional abnormality in the epididymis can be envisaged as the cause of or a contributing factor in cases of infertility due to the male partner. In view of the results presented in this paper, it is tempting to speculate that altered production or sensitivity to ET-1 might impair sperm progression through the genital tract and the viability of fertile germ cells in the ejaculate.
The release of ET-1 from the epithelial cells could be regulated by neuronal or hormonal control. Oxytocin (OT) could be a candidate hormone for playing such a role. In fact, in previous studies we demonstrated that receptors for OT are present in the epididymis (40). It has been shown that the OT level increases in human blood at the time of sexual arousal and ejaculation (41, 42). As we previously demonstrated that OT mediates ET-1 release from endometrial cells (29), it is possible that this phenomenon also occurs in the epididymis during ejaculation. A combined action of the two peptides might induce effective muscle cell contraction at the time of ejaculation, leading to evacuation of spermatozoa. However, additional studies are needed to establish whether an OT-regulated control of ET-1 secretion in the epididymis takes place.
In conclusion, in this study we provided the first evidence that the human epididymis contains and responds to ET-1 in a paracrine fashion. The presence of such an ET-1-driven paracrine loop could be responsible for the autonomous motility of the human epididymis.
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Acknowledgments |
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Footnotes |
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Received February 10, 1997.
Revised July 9, 1997.
Accepted July 17, 1997.
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) or presence of
different concentrations of BQ 123 (•, 
, and 
).
Abscissa, Molar concentration of ET-1.
Ordinate, Contractile activity, expressed as percentage
of the ratio between the maximal response obtained with ET-1 and that
obtained with noradenaline (NA; 100 µmol/L). In the
inset, the inhibitory effect of increasing
concentrations of BQ 123 on the contractile activity induced by 0.01
µmol/L ET-1 is reported.