Endothelin
in rabbit uterus during
pregnancy
ALESSANDRO PERI, GABRIELLA BARBARA VANNELLI, GUIDO FANTONI, STEFANO GIANNINI, TULLIO BARNI, CLAUDIO ORLANDO, MARIO SERIO, AND MARIO MAGGI Departments of Human Anatomy and Histology and of Clinical Physiopathology, Endocrinology Unit, University of Florence, 50134 Florence, Italy Peri, Alessandro, Gabriella Barbara Vannelli, Guido Fantoni, Stefano Giannini, Tullio Barni, Claudio Orlando, Mario Serio, and Mario Maggi. Endothelin in rabbit uterus during pregnancy. Am. J. Physiol. 263 (EndocrinoZ. Metab. 26): El58-E167, 1992.-Specific immunostaining for endothelin-1 (ET-l) was observed in the uterine mucosa of pregnant and nonpregnant rabbits. During gestation, giant cells immunopositive for ET-l were identified in either endometrium or myometrium. In the latter, they were located in closeproximity to the muscularfibers. High-affinity (dissociation constant = 0.25 nM) high-capacity [maximal binding (B,,,) = 7 pmol/mg protein] receptorsfor ET-l were presentin myometrial membranesof estrous rabbits. During pregnancy the concentration of ET-l receptors progressively decreased (B maxon day 29 = 4 pmol/mg protein), rising again at the time of spontaneousdelivery. Conversely, ET-l receptors in the aorta did not change during pregnancy and parturition. The presenceof giant cells immunopositive for ET-l in closeproximity to the myometrial cells, together with the evidence of an increasein ET-l receptorsin myometrium of parturient rabbits, suggestsa paracrine role for endometrial ET-1 during delivery. Because we previously demonstrated that oxytocin releases ET-l from endometrial cells in primary culture and that endometrial and myometrial oxytocin receptors abruptly rise at the time of parturition, we proposethat ET-1 might participate in the complex cell-to-cell interactions that occur during labor. oxytocin; labor; receptors MECHANISMS CONTROLLING uterine contractility during parturition are not completely clear. A complex array of hormones, neurotransmitters, and locally derived factors is involved in the regulation of uterine activity. Among these, oxytocin (OT) plays an important role.at the time of spontaneous delivery. Indeed, a marked increase in OT receptor density was described in the myometrium of women (10, 17) and rodents (6, 11, 18, 29, 34) at term pregnancy, and it has been attributed to the initiation of labor. OT receptors have been also identified in the endometrium of several animal species (1,22, 30, 32,33), including humans (10). We recently reported a sharp increase of OT receptors in rabbit endometrium at parturition (19). Although the function of endometrial OT receptors is not clear, they have been related to the production and release of prostaglandins (8, 30), which in turn stimulate myometrial activity and cervical ripening in a paracrine fashion (9, 16). Using immunocytochemical procedures, we recently demonstrated the presence of endothelin-1 (ET-l) in the epithelial and stromal compartment of rabbit endometrium. The immunolocalization of ET-l was greatly affected by sex steroids. In the endometrium of immature rabbits, ET-l was primarily located in the surface epithelium, whereas, in estrogen- or progesterone-primed rabbits, it was predominantly localized in the stroma, particularly in the cells surrounding the glandular epithelium (20). Furthermore, we demonstrated that endometrial cells of El58
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rabbit in primary culture are able to produce ET-l (28). Because the release of ET-l is greatly affected by OT, we proposed ET-l as a new paracrine mediator of uterine contractility. Indeed, ET- 1 stimulates uterine contractility in vitro, acting through specific receptors present in rabbit myometrium (20). The concentration of these receptors is under the control of sex steroids; estrogens dose dependently increased ET- 1 receptors, whereas progesterone blunted this effect (20). It is well known that changes in circulating sex steroids occur in pregnant rabbits, with the progesterone-to-estradiol ratio being high throughout pregnancy and low at the time of delivery (5). In the present study, we focused on the modulation of ET-l and ET-l receptor-positive cells in rabbit uterus during pregnancy. We sought to determine whether the previously described pharmacological effects of sex steroids on uterine ET-l are also physiological and whether ET-l plays a role in the process of labor. MATERIALS
AND METHODS
Animals. Nonpregnant female New Zealand White rabbits (3.3-4.0 kg, n = 23), at estrus (purple and edematousvulva),
were obtained from a commercialbreeder(Malenotti, Pratolino, Florence, Italy). Twenty animalswere mated with fertile bucks and killed by cervical dislocation at day 1 (n = 3), day 14 (n = 3), or day 29 of pregnancy (n = 8) or during parturition (n = 6). Three animals at oestruswere killed without mating. Chemicals. 12”1-labeledET-l, purchased from Amersham (Amity, Milan, Italy), was aliquoted in plastic tubes, sealed under nitrogen, and frozen at -80°C until used.ET-l and the polyclonal antibody to ET-l RAS 6901 were obtained from PeninsulaLaboratories (San Carlos,CA). Anti-mouse immunoglobulin G (IgG) peroxidaseconjugate was from Sigma Chemical (St. Louis, MO). 3,4,3’,4’-Tetraaminodiphenylhydrocloride (diaminobenzidine) wasfrom BDH Chemical.Universal immunoperoxidasestaining kits were obtained from CambridgeResearchLaboratories. Immunohistochemistry. Uterine specimensderived from rabbits at estrusbefore mating, after day 14 or day 29 of pregnancy, or during parturition were prepared as described previously (20). The tissueswere fixed in Bouin’s solution for 4 h and embeddedin paraffin (for paraffin sections) or fixed in 3.7% paraformaldeydein 0.1 M phosphate-bufferedsaline (PBS) for 20 min and stored at -20°C (for frozen sections). Deparaffinized or frozen sectionswere rinsed with PBS, pH 7.4, and immunostainedwith an indirect peroxidase technique, as described by Sternberger (35). Initial dilutions of the antiserum were carried out asrecommendedby the manufacturer and then in 0.1 M PBS. After incubation for 24 h in humidified chambers at 4°C with anti-ET-l antiserum (dilution 1:1,500),the sections were rinsed with PBS. Afterward, they were incubated with the specific IgG peroxidaseconjugate (1:1,000) for 30 min at room temperature. The sections were rinsed again with PBS and then washedwith 50 mM tris(hydroxymethyl)aminomethane
Copyright 0 1992 the American Physiological
Society
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(Tris) HCl buffer, pH 7.6. The peroxidase activity was developed with a solution of 225 PM diaminobenzidine in 50 mM Tris HCl buffer containing 0.01% H202. Specificity controls were carried out in uterine sectionsand included the following: 1) substitution of the first antiserum by normal rabbit serum, 2) serialdilution of the ET-l antiserum, and 3) preincubation of the anti-ET-l antiserum with synthetic ET-l (100 nM). The slideswere evaluated and photographatedusing a Nikon Microphot-FX microscope(Nikon, Kogaku, Tokyo, Japan). Membrane preparation. Membranes were prepared as describedpreviously (18). Myometrial or aortic tissueswere suspended in buffer 1 (10 mM Tris HCl, pH 7.4, containing 1.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor) and homogenized.The homogenatewascentrifuged at 1,000g for 10 min at 4°C. The supernatant containing the crude membrane fraction was then ultracentrifuged at 160,000g for 30 min at 4°C. The resulting pellets were washedone time in buffer 2 (50 mM Tris-maleate, pH 7.6, 10 mM MgS04, 1 mM benzamidine, 0.01% bacitracin, 0.002% soybeantrypsin inhibitor) and centrifuged again at 160,000g for 30 min at 4°C. The final pellets were then resuspendedin buffer 2 and divided into small aliquots. The membranepreparations were frozen in solid CO, and stored at -80°C until assayed.Protein concentration was determined using the Bio-Rad protein assaykit (Bio-Rad Laboratories, Munich, FRG). Binding studies. Binding studies were carried out as describedpreviously (20). Aliquots of membranes(0.075 mg/ml) were incubated for 120min at 4°C in buffer 2 in the presenceof 0.01% bovine serumalbumin (BSA) with increasingconcentrations (lo-70 PM) of 1251-labeled ET-l, with or without increasing concentrations of unlabeledET-l (3 x 10-l l to 10m7M). All measurementswere obtained in triplicate. After incubation, membraneswere filtered through Whatman GF/B filters (Clifton, NJ), presoakedin ice-cold 50 mM Tris, pH 7.4, in 0.1% BSA, using the Brandel M-48R 48-well cell harvester (Gaithersburg,MD). Filters were washedtwo times with 3 ml ice-cold 50 mM Tris, pH 7.4. Radioactivity retained by filters was measuredin a gammacounter at 70% efficiency. High-performance liquid chromatography. Purity and stability of 1251-labeled ET-l before and after the incubation period were assessedby high-performance liquid chromatography (HPLC). Free ligand was separatedfrom bound by rapid filtration on GF/B filters on a Millipore 1225 sampling manifold (Millipore, Bedford, MA). 1251-labeled ET-l was loaded on a Waters octadecyl-silica column-5S (150 x 3.9 mm) and was eluted with a linear gradient of acetonitrile (from 30 to 90% in 0.1% trifluoroacetic acid) for 40 min, with a flow rate of 1 ml/min. Fractions (1 ml) were collected and measuredin a gammacounter at 70% efficiency. Analysis of the experimental results. The binding data were evaluated quantitatively with nonlinear least-squarecurve fitting using the computer program LIGAND (24). Weighting, which is basedon the assumptionof a constant percent error in bound ligand concentration, was employed. This analysis provides optimal estimates of binding parameters (affinity constants, binding capacities,nonspecific binding) for any number of ligands reacting simultaneouslywith any number of classes of sites. Data were analyzed with one-way analysis of variance and Duncan’s new multiple-range test using the SAS statistical package (SAS Institute, Cary, NC), and reported as means& SE. l
w. cl
MVOMETRIUM
(Kd = 0.25*
0.01 nM)
l
RESULTS
Binding studies. HPLC analysis of lz51-labeled ET-l before or after 2 h of incubation at 4OC with membranes derived from rabbit at estrus, on days 14 and 29 of preg-
cl =AORTA 5 P < 0.05 *,f P < 0.01
(3) (3) dry 0
( Kd = 0.125
+ 0.015
nM )
vs. day 0, DLV
T
vs. day 0, DLV
(3)
(3) (3) day 14
(8) (31 day 29
(6) (3) DLV
Fig. 1. Endothelin-1 (ET-l) receptor density in myometrium (shaded bars) or aorta (open bars) of rabbits during pregnancy. Day of pregnancy indicated on abscissa. DLV, day of delivery. Binding capacity (pmol/mg protein) is indicated on ordinate. Each receptor concentration is mean k SE of 3-8 membrane preparations (no. of observations is indicated in parentheses). Kd, dissociation constant.
nancy, and during delivery demonstrated 40% degradation of the tracer. The simultaneous computer analysis using the program LIGAND of 23 homologous competition curves for ET-l indicates that specific ET-l receptors are present in the myometrium of pregnant and nonpregnant rabbits with affinity constant of 0.25 t 0.001 (SE) nM, virtually identical to that previously reported in immature and sex steroid-primed rabbits (20). Figure 1 depicts the density of ET-l receptors in myometrium (shaded bars) and aorta (open bars). In myometrium the concentration of
[125T 1ET-l
2
0
3.5
7 BOUND [Pfnoles/ mg protein]
10.5
14
Fig. 2. Equilibrium binding of lz51-labeled ET-l to myometrial membranes of rabbits on day 29 of pregnancy (closed boxes) or at parturition (closed circles). Ordinate: B/F, bound-to-free ratio; abscissa: concentration of bound ligand (pmol/mg protein). Fitted curves on Scatchard plots were derived from predicted relationship for one-site model for lz51-labeled ET-l as determined by LIGAND program (24).
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Fig. 3. Hematoxylin-counterstained micrographs from uterine mucosa of estrous rabbits. A: ET-l like immunoreactivity is present in stromal cells surrounding glands. B: immunostaining is absent in control section after preabsorption of antiserum with 100 nM ET-1 (frozen sections, ~500 magnification).
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Fig. 4. Micrograph (hematoxylin counterstained) of myometrium on day 14 of pregnancy. Giant cells deeply penetrating muscular fibers are present with intense immunostaining for ET-l (arrowheads). Smooth muscle cells are negative (arrows; paraffin section, X500 magnification).
ET-l receptors is four- to ninefold higher than in aorta and is greatly affected by the different stages of pregnancy. Indeed, ET-l receptors were elevated in rabbits at estrus (day 0 = 7,048 + 127 fmol/mg protein, n = 3) or on duy 1 of pregnancy (6,076 f 525 fmol/mg protein, n = 3) but progressively decreased (P < 0.05) throughout pregnancy (day 14 = 4,619 + 169 fmol/mg protein, n = 3; day 29 = 4,052 + 173 fmol/mg protein, n = 8). A marked increase (P c 0.01) was observed at the time of spontaneous delivery, when ET-l receptors reached the levels observed at estrus (6,909 rt 845 fmol/mg protein, n = 6). Unlike uterine tissue, ET-l receptors present in aorta did not change throughout the entire gestation. Figure 2 shows the Scatchard analysis of equilibrium binding curves for ET-l, obtained using myometrial membranes of rabbits on duy 29 of pregnancy or at the time of spontaneous delivery. Although the dissociation constant values were not different, a twofold increase in ET-l receptor density was observed at parturition. Immunohktochemistry. In this study, we found cells positive for ET-l staining in the endometrium of pregnant and nonpregnant rabbits. The immunolocalization of ET-l was in perfect agreement with our previous
report in sex steroid-primed rabbits (20). The specificity of the immunohistochemical staining was demonstrated in uterine cells by the absence of labeling in sections incubated with normal rabbit serum (data not shown) and by the complete absence of staining after preabsorption of anti-endothelin antibody with synthetic ET-l (0.1 PM; see Figs. 3B, 6B, and 8B). Immunostaining for ET-l in the endometrium of estrous rabbits was predominantly located in the stromal cells surrounding the glands (Fig. 3A), although few epithelial cells were positive (data not shown). In rabbits at estrus, myometrial cells were negative for ET-l, as previously reported in sex steroidprimed animals (Ref. 20 and unpublished observations). The immunohistochemical pattern of positivity for ET-l of the uterine mucosa was rather identical on duy 14 of pregnancy, with ET-l staining mainly localized in the stromal cells. However, at this time of pregnancy, we found some giant cells intimately associated with smooth muscle fibers showing intense positivity for ET-l (Fig. 4). Similar giant cells were also observed in the periplacental stroma of the endometrium (data not shown). On day 14 of pregnancy, we also focused on the fetal compartment. Figure 5 represents the placental villi (A). Trophoblastic cells showed an intense ET-l immunoreactivity (magnification in B).
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Fig. 5. Hematoxylin-counterstained micrographs from paraffin sections of placental villi on day I4 of pregnancy. Immunostaining for ET-l is present in many trophoblastic cells (A, ~125 magnification). Arrowhead indicates magnification of trophoblastic positive cells (B, ~750 magnification).
In rabbits on day 29 of pregnancy, uterine mucosa appeared edematous. Although some epithelial cells were positive, ET-l immunostaining was predominantly localized in the stromal cells of the endometrium (Fig. 6A). The number of giant cells intimately associated with smooth muscle cells significantly increased. According to our observation at midpregnancy, giant cells but not smooth muscle cells showed an intense ET-l immunostaining (Fig. 7). In myometrial sections of parturient rabbits a great number of giant cells positive for ET-l was observed. These cells penetrated the myometrium in a large amount so that they markedly dissociated the edematous muscle cells of either the longitudinal (Fig. 8A) or the circular layer of the myometrium (Fig. 8C). Myometrial cells were definitely negative. DISCUSSION
The present study confirms our recent observation on the sex steroid modulation of ET-l receptors in rabbit uterus. Indeed, during pregnancy, we observed a progressive decline in the myometrial concentration of ET-l receptors that is concomitant to the rise of the progesterone-to-estradiol ratio found in maternal blood (5). Because our laboratory (20) and others (4, 14, 21, 39, 38)
demonstrated that ET-l is a potent uterotonic peptide, it is likely that the decreased sensitivity of myometrium to ET-l is one of the biochemical mechanisms that facilitate smooth muscle relaxation and uterine quiescence throughout pregnancy. Conversely, at the time of spontaneous parturition, we found a twofold increase in ET-l receptor density that parallels the abrupt fall in progesterone plasma levels and the sustained uterine activity characteristic of labor. Furthermore, we confirm that the hormonal modulation of ET-l receptors is specific for the uterine smooth muscle cells, since the concentration of ET-l receptors in aorta remained relatively unchanged throughout the entire gestation. This finding is in agreement with a previous report indicating that, in rat, the vascular sensitivity to ET-l does not change during the entire pregnancy (23). Although several studies indicate that ET-l increases in maternal circulation at the end of pregnancy or during labor (2,37), other reports were contradictory (13,25,36). Furthermore, the concentration of ET-l in the maternal blood is two orders of magnitude lower than that required for the activation of myometrial receptors. Therefore, it is unlikely that maternal ET-l represents a hormonal message for the initiation of uterine contraction. Conversely, we propose that local (fetal or uterine) production of
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Fig. 6. Micrographs (hematoxylin counterstained) of endometrium from day 29 pregnant rabbit. A: intense immunostaining for ET-l is present in stromal compartment and in some epithelial cells (arrowheads). B: note complete absence of staining after preabsorption with excess ET-l (100 nM; paraffin section, ~500 magnification).
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Fig. 7. Hematoxylin-counterstained micrograph of myometrium of rabbit on day 29 of pregnancy. Note intense mitotic activity of muscle cells (thin arrows). Endothelin is found in giant cells (arrowheads), while smooth muscle cells are negative (thick arrows; paraffin section, ~500 magnification). -
ET-l might play a relevant role in the communication system that culminates in labor. Indeed, either the ET-l (12) or ET-3 (27) genes are expressed in human placenta, and the levels of ET-l are higher in fetal blood or amniotic fluid than in the maternal circulation (26, 37). Furthermore ET-l mRNA is relatively abundant in rat uterine tissue (31), and both rabbit (28) and human (15) endometrial cells in primary culture are able to produce ET-l. In the present study, we confirm our previous report on the presence of ET-l in the endometrium of the nonpregnant rabbit (20), and we extend our observations to the uterus and placenta of the pregnant rabbit. In the fetal compartment of the placenta, ET-l-positive cells were found in the trophoblastic cells of the chorionic villi, whereas the stroma was negative. In the endometrium of both estrous and pregnant rabbit, immunoreactive ET-l was predominantly located in the stromal cells surrounding the uterine glands, although some luminal and glandular epithelial cells were also positive for ET-l. In addition, at midpregnancy, we found giant cells either in the uterine mucosa or in the underlying myometrium that showed an intense staining for ET-l and displayed several similarities with the obplacental giant cells, a quite mysterious cell population with unknown physiological
functions. These giant cells appeared in myometrium in such an intimate relationship with smooth muscle cells that a myogenic origin has been proposed (7), although a more recent study demonstrates that these cells are derived from trophoblastic knobs (3). As gestation proceeds, the concentration of ET-l-positive giant cells increases progressively, reaching a maximum at parturition. At that time the number of immunostained giant cells penetrating the myometrium was so high that a marked dissociation between muscle cells of either longitudinal or circular layer was observed. In conclusion, we suggest that uterine ET-1 represents, as prostaglandins, an interface among the different components of the endocrine system (sex steroids, OT) and the uterine smooth muscle cells. Consistent with this concept is the evidence that OT is able to release ET-l from rabbit endometrial cells in primary culture (28) and that the density of OT receptors increases dramatically at the time of spontaneous parturition not only in the myometrium (18) but also in the endometrium (19). Thus we hypothesize that uterine contractions and therefore labor in rabbit may begin when progesterone withdrawal sufficiently increases the sensitivity of myometrium and endometrium to OT to respond to basal or slightly elevated levels of maternal and/or fetal OT. The stimulation of OT
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Fig. 8. Longitudinal (A and B, ~500 magnification) or circular (C, ~250 magnification) layer of myometrium of parturient rabbits. Giant cells immunostained for ET-l (arrowheads) increase and are found between muscular fibers of either longitudinal (A) or circular (C) layer of myometrium. Muscle cells are negative (arrows). Immunostaining was blocked by preabsorption of antiserum with 100 nM ET-1 (B; paraffin sections).
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receptors, in turn, causes myometrial contraction and, probably, the release of uterine ET-l that amplifies the action of OT by binding to the upregulated ET-l myometrial receptors. Because ET-l receptors are also present in endometrial cells in ,primary culture (unpublished observation), an autocrine loop for ET-l must be also considered. We thank Dr. Vincenzo Guardabasso (Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy) for the excellent assistance with statistical analysis. We thank Dr. Giovanna Danza (Department of Clinical Physiopathology, Endocrinology Unit, University of Florence, Florence, Italy) for HPLC assistance. This paper was supported by a grant from the University of Florence and by a grant from Centro Nazionale delle Ricerche. These data were presented in part at the 73th Annual Meeting of the Endocrine Society in Washington, DC, in June 1991. Address for reprints requests: M. Maggi, Endocrinology Unit, Viale Morgagni 85, 50134 Florence, Italy. Received 17 September 1991; accepted in final form 6 February 1992.
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Kubota, T., S. Kamada, M. Taguchi, and T. Aso. Study of endothelin-1 release from human decidual cells in early pregnancy (Abstract). Proc. Annu. Meet. Endocr. Sot. 73rd Washington DC 1991, p. 63. 16. Liggins, G. C., C. S. Forster, S. A. Grieves, and A. L. Schwartz. Control of parturition in man. Biol. Reprod. 16: 39-56, 15.
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Maggi, M., A. D. Genazzani, S. Giannini, C. Torrisi, E. Baldi, M. Di Tommaso, P. J. Munson, D. Rodbard, and M. Serio. Vasopressin and oxytocin receptors in vagina, myometrium, and oviduct of rabbits. Endocrinology 122: 2970-2980,
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Maggi, M., G. B. Vannelli, A. Peri, M. L. Brandi, G. Fantoni, S. Giannini, C. Torrisi, V. Guardabasso, T. Barni, V. Toscano, G. Massi, and M. Serio. Immunolocalization, binding, and biological activity of endothelin in rabbit uterus: effect of ovarian steroids. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E292-E305, 1991. 21. Maher, E., A. Bardequez, J. P. Gardner, L. Goldsmith, G. Weiss, M. Mascarina, and A. Aviv. Endothelin- and oxytocin-induced calcium signaling in cultured human myometrial cells. J. Clin. Inuest. 87: 1251-1258, 1991. 22. Meyer, H. H. D., T. Mittermeier, and D. Schams. Dynamic of oxytocin, estrogen and progestin receptors in the bovine endometrium during the estrous cycle. Acta EndocrinoZ. 118: 96-104, 20.
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Taylor, R. N., M. Varma, N. N. H. Teng, and J. M. Roberts. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J. Clin. Endocrinol. Metab.
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Usuki, S., T. Saitoh, T. Sawamura, N. Suzuki, S. Shigemitsu, M. Yanagisawa, K. Goto, H. Onda, M. Fujino, and T. Masaki. Increased maternal plasma concentration of endothelin-1 during labor pain or on delivery and the existence of a large amount of endothelin-1 in amniotic fluid. GynecoL. Endocrinot. 4:
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Word, R. A., M. L. Casey, K. E. Kamm, and J. T. Stull. Effects of cGMP on [Ca*+]i, myosin light chain phosphorylation, and contraction in human myometrium. Am. J. Physiol. 260 (Cell
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Soloff, M. S., and M. J. Fields. Changes in uterine oxytocin receptor concentrations throughout the estrous cycle of the cow. Biol.
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Soloff, M. S., B. T. Schroeder, J.Chakraborty, and A. F. Pearlmutter. Characterization of oxytocin receptors in the uterus and mammary gland. Federation Proc. 36: 1861-1866,1977. 35. Sternberger, L. A. The unlabeled antibody peroxidase-antiperoxidase (PAP) method. In: Immunocitochemistry. New York: Wiley, 1979, p. 104-115.
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1990.
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in rabbit uterus during
pregnancy
ALESSANDRO PERI, GABRIELLA BARBARA VANNELLI, GUIDO FANTONI, STEFANO GIANNINI, TULLIO BARNI, CLAUDIO ORLANDO, MARIO SERIO, AND MARIO MAGGI Departments of Human Anatomy and Histology and of Clinical Physiopathology, Endocrinology Unit, University of Florence, 50134 Florence, Italy Peri, Alessandro, Gabriella Barbara Vannelli, Guido Fantoni, Stefano Giannini, Tullio Barni, Claudio Orlando, Mario Serio, and Mario Maggi. Endothelin in rabbit uterus during pregnancy. Am. J. Physiol. 263 (EndocrinoZ. Metab. 26): El58-E167, 1992.-Specific immunostaining for endothelin-1 (ET-l) was observed in the uterine mucosa of pregnant and nonpregnant rabbits. During gestation, giant cells immunopositive for ET-l were identified in either endometrium or myometrium. In the latter, they were located in closeproximity to the muscularfibers. High-affinity (dissociation constant = 0.25 nM) high-capacity [maximal binding (B,,,) = 7 pmol/mg protein] receptorsfor ET-l were presentin myometrial membranesof estrous rabbits. During pregnancy the concentration of ET-l receptors progressively decreased (B maxon day 29 = 4 pmol/mg protein), rising again at the time of spontaneousdelivery. Conversely, ET-l receptors in the aorta did not change during pregnancy and parturition. The presenceof giant cells immunopositive for ET-l in closeproximity to the myometrial cells, together with the evidence of an increasein ET-l receptorsin myometrium of parturient rabbits, suggestsa paracrine role for endometrial ET-1 during delivery. Because we previously demonstrated that oxytocin releases ET-l from endometrial cells in primary culture and that endometrial and myometrial oxytocin receptors abruptly rise at the time of parturition, we proposethat ET-1 might participate in the complex cell-to-cell interactions that occur during labor. oxytocin; labor; receptors MECHANISMS CONTROLLING uterine contractility during parturition are not completely clear. A complex array of hormones, neurotransmitters, and locally derived factors is involved in the regulation of uterine activity. Among these, oxytocin (OT) plays an important role.at the time of spontaneous delivery. Indeed, a marked increase in OT receptor density was described in the myometrium of women (10, 17) and rodents (6, 11, 18, 29, 34) at term pregnancy, and it has been attributed to the initiation of labor. OT receptors have been also identified in the endometrium of several animal species (1,22, 30, 32,33), including humans (10). We recently reported a sharp increase of OT receptors in rabbit endometrium at parturition (19). Although the function of endometrial OT receptors is not clear, they have been related to the production and release of prostaglandins (8, 30), which in turn stimulate myometrial activity and cervical ripening in a paracrine fashion (9, 16). Using immunocytochemical procedures, we recently demonstrated the presence of endothelin-1 (ET-l) in the epithelial and stromal compartment of rabbit endometrium. The immunolocalization of ET-l was greatly affected by sex steroids. In the endometrium of immature rabbits, ET-l was primarily located in the surface epithelium, whereas, in estrogen- or progesterone-primed rabbits, it was predominantly localized in the stroma, particularly in the cells surrounding the glandular epithelium (20). Furthermore, we demonstrated that endometrial cells of El58
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rabbit in primary culture are able to produce ET-l (28). Because the release of ET-l is greatly affected by OT, we proposed ET-l as a new paracrine mediator of uterine contractility. Indeed, ET- 1 stimulates uterine contractility in vitro, acting through specific receptors present in rabbit myometrium (20). The concentration of these receptors is under the control of sex steroids; estrogens dose dependently increased ET- 1 receptors, whereas progesterone blunted this effect (20). It is well known that changes in circulating sex steroids occur in pregnant rabbits, with the progesterone-to-estradiol ratio being high throughout pregnancy and low at the time of delivery (5). In the present study, we focused on the modulation of ET-l and ET-l receptor-positive cells in rabbit uterus during pregnancy. We sought to determine whether the previously described pharmacological effects of sex steroids on uterine ET-l are also physiological and whether ET-l plays a role in the process of labor. MATERIALS
AND METHODS
Animals. Nonpregnant female New Zealand White rabbits (3.3-4.0 kg, n = 23), at estrus (purple and edematousvulva),
were obtained from a commercialbreeder(Malenotti, Pratolino, Florence, Italy). Twenty animalswere mated with fertile bucks and killed by cervical dislocation at day 1 (n = 3), day 14 (n = 3), or day 29 of pregnancy (n = 8) or during parturition (n = 6). Three animals at oestruswere killed without mating. Chemicals. 12”1-labeledET-l, purchased from Amersham (Amity, Milan, Italy), was aliquoted in plastic tubes, sealed under nitrogen, and frozen at -80°C until used.ET-l and the polyclonal antibody to ET-l RAS 6901 were obtained from PeninsulaLaboratories (San Carlos,CA). Anti-mouse immunoglobulin G (IgG) peroxidaseconjugate was from Sigma Chemical (St. Louis, MO). 3,4,3’,4’-Tetraaminodiphenylhydrocloride (diaminobenzidine) wasfrom BDH Chemical.Universal immunoperoxidasestaining kits were obtained from CambridgeResearchLaboratories. Immunohistochemistry. Uterine specimensderived from rabbits at estrusbefore mating, after day 14 or day 29 of pregnancy, or during parturition were prepared as described previously (20). The tissueswere fixed in Bouin’s solution for 4 h and embeddedin paraffin (for paraffin sections) or fixed in 3.7% paraformaldeydein 0.1 M phosphate-bufferedsaline (PBS) for 20 min and stored at -20°C (for frozen sections). Deparaffinized or frozen sectionswere rinsed with PBS, pH 7.4, and immunostainedwith an indirect peroxidase technique, as described by Sternberger (35). Initial dilutions of the antiserum were carried out asrecommendedby the manufacturer and then in 0.1 M PBS. After incubation for 24 h in humidified chambers at 4°C with anti-ET-l antiserum (dilution 1:1,500),the sections were rinsed with PBS. Afterward, they were incubated with the specific IgG peroxidaseconjugate (1:1,000) for 30 min at room temperature. The sections were rinsed again with PBS and then washedwith 50 mM tris(hydroxymethyl)aminomethane
Copyright 0 1992 the American Physiological
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(Tris) HCl buffer, pH 7.6. The peroxidase activity was developed with a solution of 225 PM diaminobenzidine in 50 mM Tris HCl buffer containing 0.01% H202. Specificity controls were carried out in uterine sectionsand included the following: 1) substitution of the first antiserum by normal rabbit serum, 2) serialdilution of the ET-l antiserum, and 3) preincubation of the anti-ET-l antiserum with synthetic ET-l (100 nM). The slideswere evaluated and photographatedusing a Nikon Microphot-FX microscope(Nikon, Kogaku, Tokyo, Japan). Membrane preparation. Membranes were prepared as describedpreviously (18). Myometrial or aortic tissueswere suspended in buffer 1 (10 mM Tris HCl, pH 7.4, containing 1.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM benzamidine, 0.01% bacitracin, and 0.002% soybean trypsin inhibitor) and homogenized.The homogenatewascentrifuged at 1,000g for 10 min at 4°C. The supernatant containing the crude membrane fraction was then ultracentrifuged at 160,000g for 30 min at 4°C. The resulting pellets were washedone time in buffer 2 (50 mM Tris-maleate, pH 7.6, 10 mM MgS04, 1 mM benzamidine, 0.01% bacitracin, 0.002% soybeantrypsin inhibitor) and centrifuged again at 160,000g for 30 min at 4°C. The final pellets were then resuspendedin buffer 2 and divided into small aliquots. The membranepreparations were frozen in solid CO, and stored at -80°C until assayed.Protein concentration was determined using the Bio-Rad protein assaykit (Bio-Rad Laboratories, Munich, FRG). Binding studies. Binding studies were carried out as describedpreviously (20). Aliquots of membranes(0.075 mg/ml) were incubated for 120min at 4°C in buffer 2 in the presenceof 0.01% bovine serumalbumin (BSA) with increasingconcentrations (lo-70 PM) of 1251-labeled ET-l, with or without increasing concentrations of unlabeledET-l (3 x 10-l l to 10m7M). All measurementswere obtained in triplicate. After incubation, membraneswere filtered through Whatman GF/B filters (Clifton, NJ), presoakedin ice-cold 50 mM Tris, pH 7.4, in 0.1% BSA, using the Brandel M-48R 48-well cell harvester (Gaithersburg,MD). Filters were washedtwo times with 3 ml ice-cold 50 mM Tris, pH 7.4. Radioactivity retained by filters was measuredin a gammacounter at 70% efficiency. High-performance liquid chromatography. Purity and stability of 1251-labeled ET-l before and after the incubation period were assessedby high-performance liquid chromatography (HPLC). Free ligand was separatedfrom bound by rapid filtration on GF/B filters on a Millipore 1225 sampling manifold (Millipore, Bedford, MA). 1251-labeled ET-l was loaded on a Waters octadecyl-silica column-5S (150 x 3.9 mm) and was eluted with a linear gradient of acetonitrile (from 30 to 90% in 0.1% trifluoroacetic acid) for 40 min, with a flow rate of 1 ml/min. Fractions (1 ml) were collected and measuredin a gammacounter at 70% efficiency. Analysis of the experimental results. The binding data were evaluated quantitatively with nonlinear least-squarecurve fitting using the computer program LIGAND (24). Weighting, which is basedon the assumptionof a constant percent error in bound ligand concentration, was employed. This analysis provides optimal estimates of binding parameters (affinity constants, binding capacities,nonspecific binding) for any number of ligands reacting simultaneouslywith any number of classes of sites. Data were analyzed with one-way analysis of variance and Duncan’s new multiple-range test using the SAS statistical package (SAS Institute, Cary, NC), and reported as means& SE. l
w. cl
MVOMETRIUM
(Kd = 0.25*
0.01 nM)
l
RESULTS
Binding studies. HPLC analysis of lz51-labeled ET-l before or after 2 h of incubation at 4OC with membranes derived from rabbit at estrus, on days 14 and 29 of preg-
cl =AORTA 5 P < 0.05 *,f P < 0.01
(3) (3) dry 0
( Kd = 0.125
+ 0.015
nM )
vs. day 0, DLV
T
vs. day 0, DLV
(3)
(3) (3) day 14
(8) (31 day 29
(6) (3) DLV
Fig. 1. Endothelin-1 (ET-l) receptor density in myometrium (shaded bars) or aorta (open bars) of rabbits during pregnancy. Day of pregnancy indicated on abscissa. DLV, day of delivery. Binding capacity (pmol/mg protein) is indicated on ordinate. Each receptor concentration is mean k SE of 3-8 membrane preparations (no. of observations is indicated in parentheses). Kd, dissociation constant.
nancy, and during delivery demonstrated 40% degradation of the tracer. The simultaneous computer analysis using the program LIGAND of 23 homologous competition curves for ET-l indicates that specific ET-l receptors are present in the myometrium of pregnant and nonpregnant rabbits with affinity constant of 0.25 t 0.001 (SE) nM, virtually identical to that previously reported in immature and sex steroid-primed rabbits (20). Figure 1 depicts the density of ET-l receptors in myometrium (shaded bars) and aorta (open bars). In myometrium the concentration of
[125T 1ET-l
2
0
3.5
7 BOUND [Pfnoles/ mg protein]
10.5
14
Fig. 2. Equilibrium binding of lz51-labeled ET-l to myometrial membranes of rabbits on day 29 of pregnancy (closed boxes) or at parturition (closed circles). Ordinate: B/F, bound-to-free ratio; abscissa: concentration of bound ligand (pmol/mg protein). Fitted curves on Scatchard plots were derived from predicted relationship for one-site model for lz51-labeled ET-l as determined by LIGAND program (24).
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Fig. 3. Hematoxylin-counterstained micrographs from uterine mucosa of estrous rabbits. A: ET-l like immunoreactivity is present in stromal cells surrounding glands. B: immunostaining is absent in control section after preabsorption of antiserum with 100 nM ET-1 (frozen sections, ~500 magnification).
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Fig. 4. Micrograph (hematoxylin counterstained) of myometrium on day 14 of pregnancy. Giant cells deeply penetrating muscular fibers are present with intense immunostaining for ET-l (arrowheads). Smooth muscle cells are negative (arrows; paraffin section, X500 magnification).
ET-l receptors is four- to ninefold higher than in aorta and is greatly affected by the different stages of pregnancy. Indeed, ET-l receptors were elevated in rabbits at estrus (day 0 = 7,048 + 127 fmol/mg protein, n = 3) or on duy 1 of pregnancy (6,076 f 525 fmol/mg protein, n = 3) but progressively decreased (P < 0.05) throughout pregnancy (day 14 = 4,619 + 169 fmol/mg protein, n = 3; day 29 = 4,052 + 173 fmol/mg protein, n = 8). A marked increase (P c 0.01) was observed at the time of spontaneous delivery, when ET-l receptors reached the levels observed at estrus (6,909 rt 845 fmol/mg protein, n = 6). Unlike uterine tissue, ET-l receptors present in aorta did not change throughout the entire gestation. Figure 2 shows the Scatchard analysis of equilibrium binding curves for ET-l, obtained using myometrial membranes of rabbits on duy 29 of pregnancy or at the time of spontaneous delivery. Although the dissociation constant values were not different, a twofold increase in ET-l receptor density was observed at parturition. Immunohktochemistry. In this study, we found cells positive for ET-l staining in the endometrium of pregnant and nonpregnant rabbits. The immunolocalization of ET-l was in perfect agreement with our previous
report in sex steroid-primed rabbits (20). The specificity of the immunohistochemical staining was demonstrated in uterine cells by the absence of labeling in sections incubated with normal rabbit serum (data not shown) and by the complete absence of staining after preabsorption of anti-endothelin antibody with synthetic ET-l (0.1 PM; see Figs. 3B, 6B, and 8B). Immunostaining for ET-l in the endometrium of estrous rabbits was predominantly located in the stromal cells surrounding the glands (Fig. 3A), although few epithelial cells were positive (data not shown). In rabbits at estrus, myometrial cells were negative for ET-l, as previously reported in sex steroidprimed animals (Ref. 20 and unpublished observations). The immunohistochemical pattern of positivity for ET-l of the uterine mucosa was rather identical on duy 14 of pregnancy, with ET-l staining mainly localized in the stromal cells. However, at this time of pregnancy, we found some giant cells intimately associated with smooth muscle fibers showing intense positivity for ET-l (Fig. 4). Similar giant cells were also observed in the periplacental stroma of the endometrium (data not shown). On day 14 of pregnancy, we also focused on the fetal compartment. Figure 5 represents the placental villi (A). Trophoblastic cells showed an intense ET-l immunoreactivity (magnification in B).
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Fig. 5. Hematoxylin-counterstained micrographs from paraffin sections of placental villi on day I4 of pregnancy. Immunostaining for ET-l is present in many trophoblastic cells (A, ~125 magnification). Arrowhead indicates magnification of trophoblastic positive cells (B, ~750 magnification).
In rabbits on day 29 of pregnancy, uterine mucosa appeared edematous. Although some epithelial cells were positive, ET-l immunostaining was predominantly localized in the stromal cells of the endometrium (Fig. 6A). The number of giant cells intimately associated with smooth muscle cells significantly increased. According to our observation at midpregnancy, giant cells but not smooth muscle cells showed an intense ET-l immunostaining (Fig. 7). In myometrial sections of parturient rabbits a great number of giant cells positive for ET-l was observed. These cells penetrated the myometrium in a large amount so that they markedly dissociated the edematous muscle cells of either the longitudinal (Fig. 8A) or the circular layer of the myometrium (Fig. 8C). Myometrial cells were definitely negative. DISCUSSION
The present study confirms our recent observation on the sex steroid modulation of ET-l receptors in rabbit uterus. Indeed, during pregnancy, we observed a progressive decline in the myometrial concentration of ET-l receptors that is concomitant to the rise of the progesterone-to-estradiol ratio found in maternal blood (5). Because our laboratory (20) and others (4, 14, 21, 39, 38)
demonstrated that ET-l is a potent uterotonic peptide, it is likely that the decreased sensitivity of myometrium to ET-l is one of the biochemical mechanisms that facilitate smooth muscle relaxation and uterine quiescence throughout pregnancy. Conversely, at the time of spontaneous parturition, we found a twofold increase in ET-l receptor density that parallels the abrupt fall in progesterone plasma levels and the sustained uterine activity characteristic of labor. Furthermore, we confirm that the hormonal modulation of ET-l receptors is specific for the uterine smooth muscle cells, since the concentration of ET-l receptors in aorta remained relatively unchanged throughout the entire gestation. This finding is in agreement with a previous report indicating that, in rat, the vascular sensitivity to ET-l does not change during the entire pregnancy (23). Although several studies indicate that ET-l increases in maternal circulation at the end of pregnancy or during labor (2,37), other reports were contradictory (13,25,36). Furthermore, the concentration of ET-l in the maternal blood is two orders of magnitude lower than that required for the activation of myometrial receptors. Therefore, it is unlikely that maternal ET-l represents a hormonal message for the initiation of uterine contraction. Conversely, we propose that local (fetal or uterine) production of
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Fig. 6. Micrographs (hematoxylin counterstained) of endometrium from day 29 pregnant rabbit. A: intense immunostaining for ET-l is present in stromal compartment and in some epithelial cells (arrowheads). B: note complete absence of staining after preabsorption with excess ET-l (100 nM; paraffin section, ~500 magnification).
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Fig. 7. Hematoxylin-counterstained micrograph of myometrium of rabbit on day 29 of pregnancy. Note intense mitotic activity of muscle cells (thin arrows). Endothelin is found in giant cells (arrowheads), while smooth muscle cells are negative (thick arrows; paraffin section, ~500 magnification). -
ET-l might play a relevant role in the communication system that culminates in labor. Indeed, either the ET-l (12) or ET-3 (27) genes are expressed in human placenta, and the levels of ET-l are higher in fetal blood or amniotic fluid than in the maternal circulation (26, 37). Furthermore ET-l mRNA is relatively abundant in rat uterine tissue (31), and both rabbit (28) and human (15) endometrial cells in primary culture are able to produce ET-l. In the present study, we confirm our previous report on the presence of ET-l in the endometrium of the nonpregnant rabbit (20), and we extend our observations to the uterus and placenta of the pregnant rabbit. In the fetal compartment of the placenta, ET-l-positive cells were found in the trophoblastic cells of the chorionic villi, whereas the stroma was negative. In the endometrium of both estrous and pregnant rabbit, immunoreactive ET-l was predominantly located in the stromal cells surrounding the uterine glands, although some luminal and glandular epithelial cells were also positive for ET-l. In addition, at midpregnancy, we found giant cells either in the uterine mucosa or in the underlying myometrium that showed an intense staining for ET-l and displayed several similarities with the obplacental giant cells, a quite mysterious cell population with unknown physiological
functions. These giant cells appeared in myometrium in such an intimate relationship with smooth muscle cells that a myogenic origin has been proposed (7), although a more recent study demonstrates that these cells are derived from trophoblastic knobs (3). As gestation proceeds, the concentration of ET-l-positive giant cells increases progressively, reaching a maximum at parturition. At that time the number of immunostained giant cells penetrating the myometrium was so high that a marked dissociation between muscle cells of either longitudinal or circular layer was observed. In conclusion, we suggest that uterine ET-1 represents, as prostaglandins, an interface among the different components of the endocrine system (sex steroids, OT) and the uterine smooth muscle cells. Consistent with this concept is the evidence that OT is able to release ET-l from rabbit endometrial cells in primary culture (28) and that the density of OT receptors increases dramatically at the time of spontaneous parturition not only in the myometrium (18) but also in the endometrium (19). Thus we hypothesize that uterine contractions and therefore labor in rabbit may begin when progesterone withdrawal sufficiently increases the sensitivity of myometrium and endometrium to OT to respond to basal or slightly elevated levels of maternal and/or fetal OT. The stimulation of OT
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Fig. 8. Longitudinal (A and B, ~500 magnification) or circular (C, ~250 magnification) layer of myometrium of parturient rabbits. Giant cells immunostained for ET-l (arrowheads) increase and are found between muscular fibers of either longitudinal (A) or circular (C) layer of myometrium. Muscle cells are negative (arrows). Immunostaining was blocked by preabsorption of antiserum with 100 nM ET-1 (B; paraffin sections).
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receptors, in turn, causes myometrial contraction and, probably, the release of uterine ET-l that amplifies the action of OT by binding to the upregulated ET-l myometrial receptors. Because ET-l receptors are also present in endometrial cells in ,primary culture (unpublished observation), an autocrine loop for ET-l must be also considered. We thank Dr. Vincenzo Guardabasso (Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy) for the excellent assistance with statistical analysis. We thank Dr. Giovanna Danza (Department of Clinical Physiopathology, Endocrinology Unit, University of Florence, Florence, Italy) for HPLC assistance. This paper was supported by a grant from the University of Florence and by a grant from Centro Nazionale delle Ricerche. These data were presented in part at the 73th Annual Meeting of the Endocrine Society in Washington, DC, in June 1991. Address for reprints requests: M. Maggi, Endocrinology Unit, Viale Morgagni 85, 50134 Florence, Italy. Received 17 September 1991; accepted in final form 6 February 1992.
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Kubota, T., S. Kamada, M. Taguchi, and T. Aso. Study of endothelin-1 release from human decidual cells in early pregnancy (Abstract). Proc. Annu. Meet. Endocr. Sot. 73rd Washington DC 1991, p. 63. 16. Liggins, G. C., C. S. Forster, S. A. Grieves, and A. L. Schwartz. Control of parturition in man. Biol. Reprod. 16: 39-56, 15.
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Maggi, M., P. Del Carlo, G. Fantoni, S. Giannini, C. Torrisi, D. Casparis, G. Massi, and M. Serio. Human myometrium during pregnancy contains and responds to Vl vasopressin receptors as well as oxytocin receptors. J. Clin. Endocrinol.
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Maggi, M., A. D. Genazzani, S. Giannini, C. Torrisi, E. Baldi, M. Di Tommaso, P. J. Munson, D. Rodbard, and M. Serio. Vasopressin and oxytocin receptors in vagina, myometrium, and oviduct of rabbits. Endocrinology 122: 2970-2980,
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