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Biology of Reproduction 62, 427-431 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Oxytocin Receptors in Guinea Pig Myometrium Near Term and During Labor1

Jean-Claude Schellenberg2,a, Vladimir Pliskab, and Rudolf A. Lutzc

a Department of Obstetrics & Gynaecology, Research Centre for Reproductive Medicine, University of Auckland, Auckland, New Zealand b Department of Animal Science, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland c Institute of Clinical Chemistry, Faculty of Medicine, University Hospital, CH-8091 Zürich, Switzerland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxytocin receptors in myometrium of women, rats, and rabbits rise markedly before the onset of labor, suggesting a role in the initiation of labor. In guinea pigs, a previous study reported no such rise by one-point determination of oxytocin binding. The purpose of this study was to use a more rigorous method to determine whether the binding characteristics of myometrial oxytocin receptors change in relation to labor in guinea pigs. Competitive binding studies were carried out in microsomes from inner and outer myometrium between 42 days of gestation and labor. Binding to analogs was also tested. Data were analyzed with affinity spectra and LIGAND. Oxytocin bound to one site with a dissociation constant of 6.3 ± 0.65 x 10-9 M. Binding capacity was 1.0 ± 0.1 x 10-12 mol/mg protein. The Hill coefficient was near unity. No significant changes occurred with gestation or labor in dissociation constant, binding capacity, or Hill coefficient (all P >= 0.2, nested ANOVA). Binding capacity was higher in the outer than in the inner layer (1.2 ± 0.2 vs. 0.8 ± 0.1 x 10-12 mol/mg protein, P = 0.02), but the dissociation constants were similar. Differences existed in the dissociation constants of the analogs tested. The main conclusion is that oxytocin receptors are unlikely to have a regulatory role in the initiation of labor in guinea pigs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxytocin has been implicated in a number of reproductive functions [1–4]. During labor, oxytocin promotes the expulsion of the conceptus. Postpartum, oxytocin assists haemostasis in the placental bed by supporting tonic uterine contraction. Whether oxytocin plays a role in the initiation of labor is not clear; differences between species may exist [2]. In guinea pigs, the timing of the onset of labor is not affected by oxytocin receptor blockade (term is 68 days) although labor is substantially prolonged leading to increased fetal mortality and maternal morbidity [5]. Oxytocin receptors have been identified in myometrium, cervix, endometrium, and fetal membranes, and in a number of extrauterine tissues in all mammalian species studied [1,3,4,6]. In addition to stimulating uterine contractions by a direct effect on the myometrium, oxytocin stimulates the production of prostaglandins in human decidua [7] and rabbit amnion [8]. The binding capacity of oxytocin receptors in myometrium, endometrium, and fetal membranes increases several-fold during pregnancy in all species studied, while the affinity of the receptors to oxytocin does not change significantly [1,3,6]. In rats, rabbits, and women, receptor capacity increases substantially before the onset of labor [9–12]. This has led to the suggestion that the rise in oxytocin receptors may be crucial for the initiation of labor in women [13]; recent work on a larger number of women has rendered this speculation less likely [14]. In rats, oxytocin receptor blockade does not prolong pregnancy [15].

In guinea pigs, one-point determinations of oxytocin binding to myometrial microsomes have suggested that the binding capacity during labor is similar to the capacity at 40 days of gestation (or at 60 days of gestation if binding capacity is related to DNA instead of protein content) [16]. As the dissociation constant (Kd) of myometrial oxytocin receptors was not determined in that study [16], the biological significance of these findings remains uncertain. Dissociation constants of 1.9 x 10-9 M [17] and of 6.9 ± 0.3 x 10-9 M [18] at 60 days of gestation and of 2.6 ± 0.2 x 10-9 M at 65 days [18] were reported in later studies in which binding of oxytocin was studied over a limited range of oxytocin concentrations (10-10 to 10-7 M). This range did not permit the investigators to determine whether binding sites with Kds > 10-8 M exist. In experiments that measured binding over an extended range of oxytocin concentrations (10-10 to 10-5 M) in myometrium and endometrium of sheep, rats, and cattle, binding sites with Kds of approximately 5 x 10-9 M, 4 x 10-7 M (both saturable), and > 10-5 M (possibly not fully saturable) were identified [19,20]. In pregnant women near term, oxytocin saturation studies and homologous competition experiments over a similar range of oxytocin concentrations (10-11 to 10-5 M) suggested that only one class of binding sites exists (Kd = 6 x 10-10 M), while self- and cross-displacement studies indicated the existence of 2 binding sites for oxytocin with Kds of 4.5 x 10-10 M and 8.4 x 10-10 M [11].

The objective of the present study was to carry out binding studies in myometrium of guinea pigs over an extended range of oxytocin concentrations to determine whether oxytocin binding characteristics change between 42 days of gestation and labor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

[Tyrosyl-2,6-3H]oxytocin (specific activity 1.4 TBq/mmol) and POPOP (p-bis-[2-(5-phenyloxazolyl)]-benzene were provided by New England Nuclear (DuPont NEN Research Products, Boston, MA). Trifluroacetate-oxytocin was provided by Bachem (Torrance, CA). NCS tissue solubilizer was provided by Amersham Life Sciences Australia (Auckland, New Zealand). Triton X-100 was provided by BDF Ltd. (Poole, UK). Deamino-oxytocin, mesotocin, [Arg8]vasotocin, and [Lys8]vasopressin were donated by Dr. W. Watkins, Department of Obstetrics and Gynaecology, University of Auckland, New Zealand. Des-Gly8-[D-Trp2, Thr4, Orn8]deamino-carba6-oxytocin (F372) was a donation from Dr. P. Melin, Ferring AB, Malmö, Sweden. All solvents and reagents except toluene for scintillation counting were at least analytical grade.

Animals

Permission for this study was obtained from the institutional Animal Ethics Committee, which observes the International Guiding Principles for Biomedical Research Involving Animals. Guinea pigs were of mixed breed (n = 21), and all but three (laboring) guinea pigs were date-mated [21]. The guinea pigs were arbitrarily divided into five groups: four based on days of gestation—1) 42–44 days (n = 5), 2) 50–56 days (n = 5), 3) 60 days (n = 3), 4) 67–69 days (n = 3 at 67 days and n = 1 at 69 days)—and 5) a laboring group (n = 4, all killed after at least one fetus had been delivered). One guinea pig of the group at 67–69 days gestation was fitted with a uterine EMG electrode array and an abdominal catheter to allow tissue harvesting when uterine activity indicated imminent labor (this occurred on Day 69 when uterine activity had doubled within 60 h) [5]. The guinea pigs were anesthetized with 2–4% halothane in oxygen, the uterus was removed under aseptic conditions, and the animals were killed with nitrous oxide. The outer, longitudinal layer of the uterine horns was separated from the inner, circular layer. The endometrium, which is tightly attached to the inner layer, was removed by scraping, and the two myometrial layers were then snap frozen in liquid nitrogen and stored at -85°C until assayed. Histology indicated that only approximately 50% of the endometrium was removed, presumably due to the undulating surface caused by myometrial contraction. Tissue for binding studies of oxytocin analogs was from some of the above listed animals (n = 2 guinea pigs at 67 days gestation, outer and inner layers separate; n = 1 guinea pig in labor, outer and inner layer pooled).

Preparation of Microsomes

The tissues were thawed to 4°C, minced with scissors, weighed, suspended in TED buffer (Tris-HCl 100 mM, Na2-EDTA 1.5 mM, dithiothreitol 0.5 mM, pH 7.4, 4 mL/g tissue), and homogenized with a glass tissue grinder. The homogenates were centrifuged in 50-mL polycarbonate tubes at 1000 x g for 10 min at 4°C using a Sorvall RC5 centrifuge fitted with an SS-30 rotor. The supernatant was centrifuged in 10-mL polycarbonate tubes at 160 000 x g for 30 min at 4°C under a normal-automatic vacuum system on a Sorvall Combi Ultracentrifuge fitted with a T-1270 rotor. The pellet was resuspended in <= 1 mL Tris maleate 50 mM pH 7.6 containing 10 mM MnCl2 and gelatin 1 g/L. This crude microsomal suspension was diluted 1:100, 1:200, and 1:500 with water to determine the protein concentration (in duplicate) using bovine serum albumin as standard [22]. The micro-well plates were read on a Bio-Tek EL 309 Microplate Autoreader and the protein concentration read by eye from the standard curve. The microsomal suspension was then diluted in Tris maleate buffer (as above) to a concentration of 0.5–1.3 mg protein in 200 µL.

Oxytocin Receptor Assay

Competitive binding assays were carried out in Tris maleate buffer pH 7.6 containing MnCl2 and gelatin as described above. The concentrations of oxytocin used for pilot experiments were usually 10-10 M to 10-7 M. For all other studies, displacement of [3H]oxytocin was measured over a range of oxytocin concentrations between 10-10 M and 10-9 M to 10-5 M. [3H]Oxytocin (10-10 M to 10-9 M) was added to cold oxytocin to obtain final oxytocin concentrations of up to 10-8 M. [3H]Oxytocin (approx. 10-8 M) was added to cold oxytocin to obtain final concentrations between 10-8 M and 10-5 M. Oxytocin standard, [3H]oxytocin (each 25 µL), and microsomal suspension (200 µL containing 0.5–1.3 mg protein) were incubated in Eppendorf centrifuge tubes (final volume 250 µL) at ambient temperature for 1 h, placed onto dry ice for 1 min, and centrifuged at 6000 x g for 5 min in a Heraeus Sepatech Biofuge A. The pellet was dispersed by vortexing in ice-cold Tris maleate buffer (1 mL, as above but without gelatin), and the suspension centrifuged at 10,000 x g for 5 min. After solubilization of the pellet by overnight incubation with NCS tissue solubilizer (100 µL) at ambient temperature, scintillation fluid (1 mL) was added and the radioactivity was determined [23].

Pilot Experiments

[Tyrosyl-2,6-3H]oxytocin eluted in one peak after 28 min and trifluroacetate-oxytocin eluted in one peak after 24 min during HPLC [24], suggesting satisfactory purity. Binding of [3H]oxytocin was unchanged after purification by HPLC [24]. Binding of oxytocin at room temperature reached a plateau between 30 and 60 min (tested: 30, 45, 60, 120 min, 16 h, n = 2 experiments). Addition of aprotinin (105 U/mL) to the TED and Tris maleate buffers did not affect Kd and decreased binding capacity by 30% and 40%, respectively (n = 2). Storage of tissue for 40 min at room temperature before snap freezing had no clear effect on Kd and binding capacity (n = 2 experiments). Doubling the amount of microsomal protein doubled binding capacity (n = 2 experiments).

Data Analysis

Affinity spectra were constructed from the binding data of individual experiments using the program STEP [19,25]. The binding parameters (dissociation constant Kd, binding capacity Bmax) were calculated from the best-fitting binding model for individual experiments using the program LIGAND [26]. If the affinity spectrum suggested the existence of two binding sites, the Kds indicated by the affinity spectrum were used as initial estimates for the LIGAND procedure. The quality of the curve-fit of the one-site model was compared with that of the two-site model by F test [26]. The Hill coefficient was calculated in the oxytocin concentration range of 10-10 to 10-6 M for the binding data obtained at 42–44 days of gestation and during labor. Nested models (animal nested within gestational group) using General Linear Model procedures were used to examine the effect of gestation and myometrial layer on Kd, Bmax, and the Hill coefficient [27]. Statistical significance was assumed at P < 0.05. Data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of Oxytocin and Analogs

In only 3 of the 41 binding experiments there was some suggestion of a second binding site under the conditions of the binding protocol used, but the improvements in fit from a one-site model to a two-site model were not significant (P > 0.05, by F test). Oxytocin bound to microsomal membranes with a dissociation constant Kd = 6.33 ± 0.65 x 10-9 M (range 1.39–18.9 x 10-9 M, n = 41). Mean binding capacity Bmax was 1.01 ± 0.11 x 10-12 mol/mg protein (range 0.06–3.05 x 10-12 mol/mg, n = 41).

Deamino-oxytocin (Kd = 4.94 ± 1.48 x 10-9 M, n = 5), [Arg8]vasotocin (Kd = 4.71 ± 1.0 x 11-9 M, n = 5) and the oxytocin analogue F372 (Kd = 4.49 ± 0.37 x 10-9 M, n = 6) had similar binding affinites to oxytocin (Kd = 6.33 ± 0.65 x 10-9 M, n = 41), while the affinities of [Lys8]vasopressin (35.9 ± 4.8 x 10-9 M, n = 4) and mesotocin (Kd = 19.1 ± 5.7 x 10-9 M, n = 5) were roughly three to eight times lower.

Effect of Gestation and Myometrial Layer on Oxytocin Binding

No significant difference existed between the gestational groups in regard to Kd (P = 0.18, by nested ANOVA) and Bmax (P = 0.15, Fig. 1). Binding capacity was significantly higher in the outer myometrial layer (1.20 ± 0.18 x 10-12 mol/mg protein, n = 21) than the inner layer (0.82 ± 0.13 x 10-12 mol/mg protein, n = 20, P = 0.019, by nested ANOVA). The dissociation constant Kd was similar in the two layers (6.63 ± 0.88 x 10-9 M, outer; 6.01 ± 0.97 x 10-9 M, inner, P = 0.36). In the guinea pig in which labor was imminent according to the uterine EMG recording, Bmax was 1.7 x 10-12 mol/mg in outer myometrium and 1.1 x 10-12 mol/mg in inner myometrium, while the Kds were 9.5 x 10-9 M and 8.5 x 10-9 M, respectively.



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  		FIG. 1. Dissociation constants Kd (A) and maximum binding capacities Bmax (B) during late pregnancy and labor in guinea pigs (mean ± SEM)

The Hill coefficient was close to unity in all cases investigated. No difference existed in the Hill coefficient between 42 and 44 days (0.99 ± 0.02, n = 9) and labor (1.26 ± 0.14, n = 8, P = 0.2, by nested ANOVA) or between the inner and the outer myometrial layers (P = 0.8).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
No significant changes in the binding characteristics for oxytocin were detected in myometrium of guinea pigs between 42 days of gestation (corresponding to 0.6 of mean pregnancy length) and labor. As oxytocin receptors are down-regulated during labor in women [11,14,28], we wondered whether our failure to detect any significant change in Bmax during labor might be the result of intrapartum down-regulation of receptors that were up-regulated shortly before the onset of labor. As it was not possible to determine the time between when they were killed and the onset of labor in the three guinea pigs killed at 67 days of gestation (term is 68 days), we determined Kd and Bmax in one instrumented guinea pig on Day 69 of gestation when labor was imminent. Uterine activity in this guinea pig suggested that labor was to occur between 0 and 6 h [5]. As the values for Kd and Bmax were comparable to the values obtained in the other guinea pigs near term, an increase in Bmax immediately before the onset of labor is unlikely to be an obligatory biological event.

This absence of any significant change in Bmax is in keeping with previous results from one-point measurements of oxytocin binding [16]. The absolute values for Bmax in the present study are similar to those reported by Chwalisz et al. [29] and Fuchs et al. [17], fourfold lower than those published by Fahrenholz et al. [18], and 25 times higher than those obtained by one-point determinations [16]. Values of Hill coefficients close to unity, as assessed in our experiments, are similar to published values of 1.1–1.8 at 60–65 days of gestation [18,29].

The wide range of oxytocin concentrations used in our assay allowed the detection of binding sites with dissociation constants in the range of 10-9 M to 10-6 M [19]. Only one class of binding sites was detected (Kd = 6.3 x 10-9 M), which is in keeping with results obtained in saturation and homologous competition studies in pregnant women [11,14] but contrasts with similar studies in rats, sheep and cattle [19,20]. In these studies at least two saturable binding sites were found (approx. Kd = 5 x 10-9 M and 4 x 10-7 M) [19,20]. This raised the question of the identity of the oxytocin receptor [19]. Pharmacological studies with non-competitive oxytocin antagonists suggest that both of these binding sites may be associated with the uterotonic response to oxytocin [30]. A number of studies have identified the binding site with a Kd of approximately 5 x 10-9 M as the oxytocin receptor [9,10,12,13,17,18,28,29]. Although the limited range of oxytocin concentrations used in these studies (10-9 M to 10-7 M) would not have allowed the detection of saturable binding in the 10-7 M range, the results are consistent with the present findings in guinea pigs in which only a single binding site was detected. The question arises whether these differences may be due to differences in the protocols used rather than to differences between animal species. Rinsing of the separated particles containing the bound ligand, for example, may cause a considerable loss of binding in some protocols, in particular in the low affinity binding range [31,32]. Also, it remains to be established whether only one or several binding proteins with similar dissociation constants exist in guinea pigs. In women, two distinct binding sites within a narrow Kd range (Kd = 4 x 10-10 M and Kd = 8 x 10-10 M) were demonstrated in self- and cross-displacement studies, while homologous competition and saturation studies identified only one site (Kd = 6 x 10-10 M) [11]. The ability of currently used methods to reliably identify two distinct binding sites that differ only by a factor two remains highly disputable.

Maximum binding capacity of oxytocin receptors was 50% higher in the outer than in the inner myometrial layer. This difference cannot be accounted for by contamination of the inner myometrial layer with endometrium, as the endometrium remaining after scraping (approximately 50%) would have constituted only approximately 5% of the mass of the tissue used for receptor analysis [23]. The functional correlate of the higher Bmax in the outer myometrial layer is unknown. In rats, the maximum contractile response to oxytocin is greater in the outer than the inner myometrium near term in vitro [33], but the relationship with oxytocin binding capacity is not known. In guinea pigs treated with a progesterone antagonist at 43 days of gestation the uterine response to oxytocin in vivo changed from a tonic to a phasic pattern, with a concomitant decrease of approximately 30% in oxytocin binding capacity [29]. A causal relationship between the observed change in binding capacity and the changes in the contractile pattern appears unlikely.

Oxytocin and vasopressin analogs showed definite differences in their affinity for the oxytocin receptor. Compared to the affinity of oxytocin, the affinities of [Lys8]vasopressin and mesotocin were several-fold lower, while the affinities of other analogs tested in this and a previous study [18] tended to be higher.

In summary, this study found only a single oxytocin binding site in myometrium of pregnant guinea pigs (approx. Kd = 6 x 10-9 M). The dissociation constant Kd, maximum binding capacity Bmax, and the Hill coefficient did not change significantly between 42 days of gestation and labor. Hence, a role of oxytocin receptors in the initiation of labor is unlikely in guinea pigs. Maximum binding capacity was 50% higher in the outer myometrial layer than in the inner layer, but the functional correlate is unknown.


   ACKNOWLEDGMENTS
 
The authors thank Dr. V. Choy for HPLC analysis and receptor binding work; Mrs. S. Ferguson, Mr. P. Johnson, Mr. W. Kirkby, and the personnel of the Surgical Laboratory, National Women's and Greenlane Hospitals, Auckland, New Zealand, for technical assistance; and Mr. A.W. Stewart, Department of Community Health, University of Auckland, for advice regarding statistics.


   FOOTNOTES
 
First decision: 6 August 1999.

1 This research was supported by the New Zealand Health Research Council. Back

2 Correspondence: J-C. Schellenberg, Department of Obstetrics & Gynaecology, Hôpital Cantonal Universitaire, 32 Boulevard de la Cluse CH-1211 Geneva 14, Switzerland. FAX: +41 22 382 4030;jean-claude.schellenberg{at}hcuge.ch Back

Accepted: September 15, 1999.

Received: June 17, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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