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Original Studies |
Department of Clinical Physiopathology, Andrology Unit (M.M., R.M., G.Fo.) and Endocrinology Unit (A.P.), Department of Human Anatomy and Histology (T.B., G.B.V.), Department of Pharmacology (S.A., S.F.), Department of Obstetrics and Gynecology (T.S.), University of Florence, 50139 Florence; and the First Department of Internal Medicine, Division of Andrology, University of Catania (A.C., N.B.), Catania 95123, Italy
Address all correspondence and requests for reprints to: Mario Maggi, M.D., Andrology Unit, Viale Pieraccini 6, 50139 Florence, Italy.
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Abstract |
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Introduction |
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To investigate the subtype of ET receptors expressed by intact human ovary and their localization we performed self- and cross-competition binding curves among labeled ETs and selective analogs and autoradiographic studies using a blocking design aimed to selectively label ETA and ETB receptors. In addition, to evaluate the function of these receptors we also studied the effect of ETs on steroid production from purified human GCs and on contractility of ovarian blood vessels. Results were compared with those obtained in rat ovary and GCs. Our study indicates that the human ovary expresses a relative abundance of ETA receptors, mostly localized in the blood vessels, that mediate vasoconstriction.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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For radioligand binding studies ovarian tissues were obtained from 15 women who underwent hysterectomy with salpingo-oophorectomy for benign uterine diseases, including leyomyoma, genital prolapse, and intractable uterine bleeding. Their informed consent was obtained, and the study was approved by the local ethical committee of the Department of Clinical Physiopathology (University of Florence, Florence, Italy). Each subject was interviewed concerning menstrual history, date of the last menstruation, use of oral contraceptive (OC) or other hormonal agents, and chronic consumption of any kind of drug. Further information was obtained by histological evaluation of the endometrium by a gynecological pathologist. On the basis of the aforementioned elements, these women were divided into 3 groups to investigate the relationship between ET receptor levels in the ovary and the hormonal milieu of the host.
Group 1: normal cycling women. Eight women were included in this group (mean age, 45.6 yr; range 19–57 yr). They had a history of regular menses and a proliferative or secretive pattern of the endometrium. According to the age, they were further divided into two subgroups: one included women of up to 45 yr of age (group 1a), and the other included women over 45 years (group 1b).
Group 2: menopausal. Four women were included in this group (mean age, 63 yr; range, 57–71 yr). They had a history of at least 5 yr of amenorrhea, high levels of gonadotropins (FSH, >35 mIU/mL; LH, >25 mIU/mL), and/or a hypotrophic-atrophic pattern of the endometrium.
Group 3: OC. Three women were included in this group (mean age, 48.6 yr; range 46–52 yr). The OC used contained 20 µg ethinyl estradiol and desogestrel.
For autoradiographic, in situ hybridization, and contractility studies, 14 ovarian specimens from fertile women (aged 19–42 yr) were obtained after informed consent was given. Patients underwent ovarian resection or oophorectomy for benign diseases of the reproductive system.
For human granulosa-luteal cell culture, cells were obtained from women undergoing in vitro fertilization programs. All patients were treated by a long protocol with GnRH agonist (GnRH-a; 3.75 mg; D-Trp6-decapeptyl, Ipsen, Milan, Italy). GnRH-a was administered on day 21 of the menstrual cycle preceding oocyte retrieval. After attainment of pituitary desensitization, indicated by serum 17ß-estradiol (E2) levels below 125 pmol/L and a follicle diameter <10 mm, ovarian stimulation was initiated with human menopausal gonadotropin, corresponding to 150 IU FSH and 70 IU LH (Pergogreen, Serono, Milan, Italy). The daily dose of exogenous gonadotropin was continued on an individual basis, depending upon follicular growth. When the two largest follicles reached a mean diameter of 16 mm and the concentration of E2 was consistent, 10,000 IU hCG (Profasi HP, Serono) were administered. Oocytes were retrieved 36 h later.
Chemicals
[125I]ET-1 (2000 Ci/mmol),
[125I]ET-3 (2000 Ci/mmol), and [
-32P]CTP
(3000 Ci/mmol) were purchased from Amersham (Amity PG, Milan, Italy).
ET-1, ET-3, sarafotoxin 6C (SRTX6C), and
cyclo-[D-Trp-D-Asp-Pro-D-Val-Leu]
(BQ123) were obtained from NovaBiochem (Laufelfingen, Switzerland).
IRL-1620 was purchased from Alexis (Laufelfingen, Switzerland), and
SB209670 was obtained from SmithKline Beecham (King of Prussia,
PA). BSA, bovine insulin, bacitracin, benzamidine, diethylstilbestrol
(DES), methylisobutylxanthine, and soybean trypsin inhibitor were
obtained from Sigma Chemical Co. (St. Louis, MO). McCoy’s 5a medium,
L-glutamine, and fungizone were obtained from Life
Technologies (Paisley, UK). Insulin was purchased from Eli Lilly Co.
(Indianapolis, IN).
Binding studies
Ovarian samples obtained at surgery were stored in liquid
nitrogen. Membranes were prepared as described previously (19). The
protein concentration was determined using the Bio-Rad protein assay
kit (Bio-Rad Laboratories, Munich, Germany). Binding studies on ovarian
membranes were carried out as described previously (19). Aliquots of
membranes (0.075 mg/mL) were incubated for 60 min at 22 C with
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) in
buffer 2 in the presence of 0.1% BSA. Self- and cross-displacement
curves using ET-1, ET-3, BQ123, IRL-1620, SB209670, and SRTX6C were
performed using pooled membranes. All measurements were obtained in
triplicate. After incubation, membranes or cells were filtered through
Whatman GF/B (membranes) or GF/C (cells) filters (Whatman, Clifton,
NJ), presoaked in ice-cold 50 mmol/L Tris, pH 7.4, in 0.1% BSA, using
the Brandel M-48R 48-well cell harvester (Gaithersburg, MD). Filters
were washed twice with 3 mL ice-cold 50 mmol/L Tris, pH 7.4.
Radioactivity retained by filters was measured in a 
-counter at 70%
efficiency.
Autoradiography
Autoradiographic studies in human or rat ovary were carried out according to the method of Maggi et al. (9). For studies in rat ovary, intact female Sprague-Dawley rats (Charles River, Calco, Italy) were used. Ovarian specimens were quickly frozen in isopentane cooled with liquid nitrogen. Serial sections (6–8 µm thick) were obtained using a cryostat at -18 C and mounted on gelatin-coated glass slides. The slides were air-dried at room temperature for 10 min and then incubated with 50 pmol/L [125I]ET-1 (in the presence of 100 nmol/L IRL-1620 to mask ETB receptors) or [125I]ET-3 (in the presence of 100 nmol/L BQ123 to mask ETA receptors) in buffer 2 and 0.2% BSA for 60 min at 22 C. To determine nonspecific binding, an excess (100 nmol/L) of unlabeled ET-1 or ET-3, respectively, was added to the mixture. The sections were washed three times with Tris-HCl buffer, pH 7.4; rinsed in deionized water; air-dried; and fixed by exposure to formaldehyde vapor at 80 C for 60 min. Coverslips coated with nuclear emulsion Illford K5 (Illford, Mobberley, UK) diluted 1:1 with distilled water were applied to the sections, which were exposed at 4 C in the dark for 6–8 days. The slides were then developed in Illford Phenisol, fixed in Illford Hypam, stained with hematoxylin, and observed with dark- and brightfield microscopy.
Granulosa cell culture
Rat and human granulosa cells were cultured as previously described (20, 21). For rat GCs, intact female immature (25 days old) Sprague-Dawley rats (Charles River, Calco) were implanted with a 10-mm SILASTIC brand capsules (Dow Corning, Midland, MI) filled with DES. Four days after implantation, rats were killed by cervical dislocation, ovaries were punctured with 27-gauge hypodermic needles, and GCs were carefully expressed into McCoy’s 5a medium. An aliquot was diluted with trypan blue stain, and viable cells were counted with a hemacytometer. Cells were plated into 16-mm, 24-multiwell plates (Corning, Milan, Italy) at a density of 50,000 cells/well·0.5 mL at 37 C in a humidified, 95% air-5% CO2 water-jacketed incubator in the presence of graded concentrations of ET-1, ET-3, or SRTX6C, for 6 days. Each experiment was run in triplicate in at least three different cultures. Incubation was carried out using McCoy’s 5a medium supplemented with 2 mmol/L L-glutamine, 200 U/mL penicillin, 200 µg/mL streptomycin sulfate, 0.5 µg/mL fungizone, 1 µmol/L androstenedione, 0.1 µmol/L DES, 37.5 ng/mL hCG (NIDDK, CR-127), 35 µg/mL methylisobutylxanthine, and 1.25 µg/mL insulin. At the end of the culture period, medium samples were frozen at -20 C until assayed for estrogen.
For L-HGCs, after identification and removal of the oocyte, the follicular fluid containing L-HGCs was centrifuged in 15-mL conical tubes at 300 x g for 5 min, and the pellet was resuspended in 1 mL McCoy’s 5a medium supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, 0.5 µg/mL fungizone, 25 mmol/L HEPES, and 0.1% BSA. This cell suspension was layered onto a 3-mL 50% Percoll gradient diluted with the above medium and centrifuged at 400 x g for 20 min to pellet the red cells. The band of granulosa-luteal cells was collected by aspiration and washed in 10 mL tissue culture medium, and the pellet was resuspended in 1 mL tissue culture medium for cell counting; cells were plated into 11-mm 48-multiwell plates (Costar, Cambridge, MA) at a density of 20,000 cells/well·0.2 mL and incubated at 37 C in a humidified, 95% air-5% CO2 water-jacketed incubator for 2 days in the presence of graded concentrations of ET-1, ET-3, and SRTX6C. Androstenedione (1 µmol/L) was added to the incubation medium. The effects of ETs were not tested on FSH-stimulated estrogen production because of the inconsistent response of these cells to FSH. Each experiment was performed in triplicate in at least three different cultures.
Estrogen RIA
The concentration of estrogen in the incubation medium was
measured directly, without extraction, by RIA, as previously described
(21). Aliquots (200 µL) of medium, diluted 1:40 to 1:100, or standard
solutions were incubated with 100 µL antiserum (final dilution,
1:10,000) and 100 [3H]E2 (
12,000 cpm) at 4
C for 18–20 h. Ice-cold charcoal-dextran was added to achieve
separation of bound from free labeled hormone. Tubes were centrifuged
at 1,500 x g at 4 C for 11 min, and the supernatants
were collected and counted in a ß-counter after the addition of
scintillation fluid. Total and nonspecific binding values were
36.5 ± 2.3% and 2.4 ± 0.07%, respectively. Intra- and
interassay coefficients of variation were 4.33 ± 0.29% and
15.5%, respectively.
P4 RIA
The concentration of P4 in the incubation medium was
measured directly, without extraction, by RIA, as previously described
(22). Aliquots (100 µL) of medium (diluted 1:10) or standard
solutions were incubated with 100 µL anti-P4 serum (CU
413/1; Analytical Antibodies, Segrate, Milan, Italy; final dilution,
1:6000) and 100 µL [3H]P4 (6000 cpm) at
4 C for 18–20 h. Ice-cold charcoal-dextran was added to achieve
separation of bound from free labeled hormone. Tubes were centrifuged
at 1500 x g at 4 C for 11 min, and the supernatants
were collected and counted in a ß-counter after the addition of
scintillation fluid. Total and nonspecific binding were 42.5% and
6.7%, respectively. Intra- and interassay coefficients of variation
were 8.9 ± 1.2% and 12.4%, respectively.
In vitro contractility of ovarian artery
At the time of surgery, the artery entering the hilum of the ovary was carefully dissected and cut into four or five rings (5 mm long). Ring segments were vertically mounted under 1.5 g resting tension in organ chambers containing 10 mL Tyrode’s solution (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, 10 mmol/L glucose) at 37 C, gassed with 95% O2 and 5% CO2, pH 7.4. The rings were allowed to equilibrate for at least 90 min; during this period the bath medium was replaced every 15 min. Changes in isometric tension was recorded on a chart polygraph. Noradrenaline (NA) increased the tonic tension in a concentration-dependent manner. The maximal effect was obtained at 100 µmol/L. The response to ETs was expressed as a percentage of the maximal effect of NA to normalize the data.
Analysis of experimental results
The binding data were evaluated quantitatively with nonlinear least squares curve fitting using the computer program Ligand (23). The computer program Allfit (24) was used for the analysis of sigmoidal dose-response curves obtained in estrogen accumulation and contractility studies.
Data were analyzed by one-way ANOVA followed by Duncan’s multiple range test. Each data point represents the mean ± SE.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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graphically
represents binding curves and the predicted relationships for the
two-site model. Binding parameters are given in Table 1. ET-1 and SB209670 bound with virtually
equal affinities to both sites. Conversely, ET-3, and SRTX6c were
relatively selective for the lowest capacity site. BQ123 and IRL-1620
showed high selectivity for the highest and the lowest capacity sites,
respectively. Hence, the high capacity, BQ123-selective site
corresponds to the ETA receptor, whereas the very low
capacity, IRL-1620-selective site corresponds to the ETB
receptor.
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To localize the ET-binding sites in the human ovary we performed
in situ binding studies using a blocking experimental
design. We employed 50 pmol/L [125I]ET-1 (in the presence
of 100 nmol/L IRL-1620 to mask ETB receptors) to study the
ETA site and 50 pmol/L [125I]ET-3 (in the
presence of 100 nmol/L BQ123 to mask ETA receptors) to
study the ETB site. Experiments were carried out in five
different ovarian specimens from normal cycling women at different
stages of the menstrual cycle. Rat ovary was analyzed as a control. In
all experiments a positive signal for ETB and
ETA receptors was found in the largest ovarian blood
vessels (Fig. 2, A and B). In particular,
ETB sites were predominantly located in the endothelial
cells, whereas ETA sites were more abundant in the muscular
wall. As expected, in the rat ovary, an intense positivity for
ETB receptors was found in GCs (data not shown).
Conversely, in human ovary, GCs that do express ET-1 mRNA (10) were
absolutely negative for either ETA- or
ETB-binding sites. Figure 3
shows results obtained in a tertiary follicle. Although an evident
hybridization signal of ET-1 mRNA was localized in the majority of GCs
and in some endothelial cells of the follicular vessels (Fig. 3A), GCs
were definitively negative for ETA (Fig. 3B) or
ETB (Fig. 3D) receptors. On the other hand, almost all the
smooth muscle cells of the ovarian vessels were positive for
ETA receptors (Fig. 3B). Silver grains for ETA
receptors were virtually absent in control sections incubated in the
presence of an excess (100 nmol/L) of ET-1 (Fig. 3C). Similar results
were obtained in follicles at different stages of development (not
shown).
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shows the effect of ETs on estrogen
accumulation. We found that both ET-1 and ET-3 significantly inhibited
estrogen production in rat GC (P < 0.05), with
half-maximal inhibitory concentrations (IC50) of 74 and 2.2
pmol/L, respectively. Analysis of the sigmoidal dose-response
relationships using the program Allfit indicated that constraining both
curves to share identical IC50 values had no deleterious
effect on the goodness of fit (P = 0.961). This finding
indicates that rat GCs are equally responsive to ET-1 and ET-3,
suggesting expression of ETB receptors. Conversely, in
L-HGCs both ETs as well as SRTX6c (data not shown) failed to affect
estrogen accumulation. As a recent report (11) indicated that in
L-HGCs, 100 nmol/L ET-1, ET-3, and IRL-1620 did not affect estrogen
secretion, but slightly inhibited P4 accumulation (25%),
we tested the effects of different concentration of ET-1, ET-3, and
SRTX6c on P4 accumulation; the results are reported in
Table 2. As shown, basal P4
production from L-HGCs was not affected by any of the peptides
tested.
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shows
traces of the dose-response relationship for cumulative applied ET-1
(upper panel) and IRL-1620 (lower panel) in
isolated ovarian artery rings in a typical experiment. Results from
four independent experiments are reported in Table 3. ET-1 caused dose-dependent
vasoconstriction, with an ED50 of 3.9 ± 1.3 nmol/L.
This value is in good agreement with the affinity constant for ET-1
derived from binding studies (0.8 nmol/L). The maximum response to ET-1
was 2.7 ± 0.16-fold greater than that to NA (100 µmol/L),
indicating that ET-1 is one of the most potent vasoconstrictors of the
ovarian artery. The increased tone of the arterial rings was hardly
reduced and did not return to the resting state even after repeated
washings with fresh solution. Mathematical analysis, using the program
Allfit, of families of ET-1 dose-response curves in the presence or
absence of different concentrations of the ETA antagonist
BQ123 indicates that BQ123 significantly increased the ED50
for ET-1 (P < 0.0001; see Table 3), without affecting
the maximum response (P = 0.96). These findings
indicate that the majority of ET receptors present in human ovarian
artery belong to the ETA subtype and that BQ123 is a
competitive antagonist for these receptors. Accordingly, increasing
concentrations of the selective ETB agonist IRL-1620 were
almost ineffective in stimulating ovarian artery contractility compared
to ET-1 (see Fig. 5, lower panel).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our autoradiographic studies indicate that in rat ovary, which we used as a control, ETB receptors are present in the developing follicular wall. Hence, the ETB receptor protein is expressed in the same cells where previous in situ hybridization studies found specific signal for ETB mRNA (12). Based on previous reports (13, 26) and the present study, it is conceivable that in the rat, ETB receptor negatively regulates steroidogenesis. A recent report indicates that this is the case also in L-HGCs (11). Indeed, a slight (25%), yet significant, inhibition of basal P4 secretion was found with 100 nmol/L ET-1, ET-3, and IRL-1620 (11). However, in a Percoll-purified preparation of L-HGCs (20), we were unable to demonstrate any effect of a wide range of concentrations of SRTX6c and ET-3 (selective ETB agonists) or ET-1 (unselective ETA/ETB agonist) on estrogen or P4 accumulation, whereas the same preparation of L-HGCs was still responsive to other physiological stimuli (21). These results are in good agreement with those derived from in situ binding on intact human ovary. Indeed, we did not find any specific binding sites for [125I]ET-3 (in the presence of 100 nmol/L BQ123) in human GCs. By using this tracer, binding sites were only identified in the endothelial cells and the muscular wall of large ovarian vessels. Accordingly, quantitative analysis of self- and cross-competition curves among [125I]ET-1, [125I]ET-3, and several selective analogs indicates that the concentration of ETB sites in intact human ovary is 100-fold lower than the estimated density of ETA sites. In conclusion, we do not believe that ETB receptors play any important role in human GCs, at variance with the situation in rat ovary. A similar discrepancy between primate and rat follicular physiology has been reported for estrogen receptor. Indeed, although in rodents estrogens regulate preantral follicle growth and sensitivity to FSH, compelling evidence that this is also the case in the primate follicle is lacking (27).
Although the concentration of ETB receptor in intact human ovary is negligible, the density of ETA receptor is relatively high (i.e. 5-fold higher than that in human testis) (9). However, we were unable to demonstrate significant differences in the concentration of [125I]ET-1-binding sites in ovarian membranes derived from normal cycling, postmenopausal, or OC-treated women. In addition, we did not find specific signal for ETA receptor in the large majority of GCs examined by in situ binding. Similar results were reported in porcine ovary (17). In particular, we did not find ET receptors in GCs expressing a positive signal for ET-1 mRNA. Our results are in apparent contrast with those of Kamada et al. (11), showing that ETA receptor is involved in the regulation of L-HGC proliferation and calcium mobilization. However, their data have been generated in cultured luteinized GCs, whereas our morphological data reflect the scenario of the intact human follicles. It is indeed possible that the cell culture conditions altered the number and subtype of ET receptors expressed, as we have recently demonstrated for human hepatic stellate cells (28).
As human GCs contain ET-1 mRNA and protein (10) without expressing an abundance of the corresponding receptor (present study), an autocrine action for follicular ET-1 is unlikely. Hence, we sought other ovarian targets. By autoradiography, we found an intense positivity for ETA (but not ETB) receptors in the vessels of the innermost part of the theca, immediately adjacent to the granulosa layer. This finding was more apparent in the thecal compartment of the ovulatory follicles and suggests a possible role for ET-1 in the regulation of ovarian vasomotion. To investigate the function of ETA and ETB receptors in ovarian vessels, we performed in vitro contractility studies using arterial rings prepared from the arterial vessels at the hilum of the ovary, where in situ binding demonstrated the presence of both subtypes of receptors. We found that ET-1 stimulated vasoconstriction (ED50 = 4 nmol/L) acting through ETA receptors. As the effect of ET-1 was 2.5-fold more potent than that of NA, this peptide should be considered the most important vasoconstrictor for ovarian vessels. Hence, we speculate that ET-1 produced by granulosa cells (10) may act in a paracrine fashion on the theca interna, which is extremely rich in blood vessels. Studies with radioactive microspheres in the ewe showed great variations in the follicular blood supply near the time of ovulation (29, 30). In particular, Murdoch reported that the LH surge induced an initial phase of intense hyperemia in the ovulatory follicle, followed by vasoconstriction. As we found higher levels of ET-1 in the follicular fluid of gonadotropin-stimulated than in spontaneous in vitro fertilization protocols (10), it is possible that the LH surge at midcycle stimulates ET-1 production from GCs, which, in turn, plays a role in regulating blood supply to the ovulatory follicle. Similar results have been recently provided for rat testis by Collins et al. (31). These researchers demonstrated that in the testis, hCG induced a time-dependent increase in ET-1 accumulation and that relatively low concentrations of ET-1 regulate testicular blood flow, acting through the ETA receptors (31).
In conclusion, we demonstrated that human ovary expresses an elevated concentration of ETA receptors (4–5 pmol/mg protein), mostly located on the muscular wall of the ovarian vessels. As human GCs produce ET-1, and receptors are present in arterioles of theca interna in close proximity to the granulosa layer, a paracrine role for follicular ET-1 is suggested.
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Acknowledgments |
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Footnotes |
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Received March 14, 1997.
Revised August 14, 1997.
Accepted August 21, 1997.
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-induced antisteroidogenic action. Endocrinology. 137:1306–1312.[Abstract]
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