Abstract

We investigated the expression of the mRNA encoding the oxytocin receptor (OTR) in rat myometrium throughout gestation and its regulation by progesterone and mechanical stretch. Using a semiquantitative reverse transcription-polymerase chain reaction approach, OTR mRNA was found to increase abruptly at the onset of spontaneous labor at term. Progesterone (4 mg/day) starting on Day 20 of gestation blocked this increase. Ovariectomy on Day 17 induced preterm labor 96 h after surgery and a significant increase in myometrial OTR mRNA levels 48 and 96 h after surgery. Both preterm labor and the rise in myometrial OTR expression were blocked by progesterone. To investigate the effects of stretch on myometrial OTR mRNA expression, unilaterally pregnant rats underwent either sham operation or placement of a tube in the nongravid uterine horn to distend the myometrium. On Day 20, stretch had no effect on OTR expression in the nongravid horns. During labor, OTR mRNA was highly expressed in the gravid horns as well as the nongravid stretched horns. In contrast, the level remained low in the nongravid unstretched horns. These results indicate that expression of rat myometrial OTR mRNA during pregnancy and labor is regulated by coordinated interactions between mechanical and endocrine signals.

Introduction

Despite the tremendous medical, social, and economic impacts that result from the preterm birth of an immature baby, our knowledge of the mechanisms that regulate the contractile activity of the myometrium and the onset of preterm or even term labor is very limited. Clearly the myometrium undergoes a major transformation in its contractile characteristics near term. Throughout the majority of pregnancy the uterus is quiescent, unexcitable, and relatively unresponsive to stimulants, and those contractions that do occur are poorly coordinated. In contrast, at term, there is a switch to a state characterized by the development of intense, frequent, and highly coordinated spontaneous contractions and increased responsiveness to uterotonic agonists (such as oxytocin and stimulatory prostaglandins). We propose that this transformation or “activation” of the myometrium can be defined biochemically as an increase in the expression of a cassette of genes encoding “contraction-associated” proteins (CAPs) [1]. The oxytocin receptor (OTR) is an excellent example of a putative CAP. Oxytocin is a potent uterotonin that stimulates uterine contractions during labor, and the number of its binding sites in the rat myometrium increases dramatically with the onset of labor [2, 3]. The rat OTR has been cloned, and the levels of mRNA encoding the OTR increase abruptly in the rat myometrium during labor [4].

The mechanisms underlying myometrial activation have yet to be determined; however, we hypothesize that an integration of fetal endocrine and growth signals that provide hormonal and mechanical inputs to the myometrium regulates the timely expression of the CAPs. We and others have provided evidence that the fetal-maternal endocrine systems might regulate myometrial gene expression, primarily through engineering an increase in the estrogen:progesterone (E:P) ratio in the maternal compartment [1]. In comparison, data on the mechanical inputs to the regulation of myometrial contractility are relatively limited. In unilaterally pregnant rats, the gravid uterine horns showed a relative depolarization of the resting membrane potential and an increased contractile force compared to the nongravid horns [5]. Recently, Kasai et al. [6] reported that in vitro stretch of the rat uterus caused transient smooth muscle contractions and a Ca2+ influx. Furthermore, clinical data suggest a much greater incidence of preterm birth and perinatal mortality in human pregnancies complicated by conditions in which intrauterine volume is increased (e.g., multifetal pregnancies or polyhydramnios) [7, 8]. Since there is no evidence of increased amniotic pressure in these pregnancies [9], it is likely that the increased intrauterine volume imposes an increase in tension in the uterine wall and hence a stretch of the myometrial smooth muscle. It is possible that this may contribute to the increased incidence of preterm labor in these pregnancies.

The regulation of expression of OTR mRNA in the myometrium has been partially studied. While data on the hormonal control of OTR mRNA during pregnancy and labor remain to be clarified, studies in nonpregnant rats suggest that estrogen acts as a positive regulator of myometrial oxytocin binding [10] and OTR mRNA levels [1113]. There is, however, little information as to whether expression of the OTR gene is regulated by mechanical inputs. In light of our previous data that the expression of another candidate CAP, connexin-43 (Cx-43), which encodes the major myometrial gap junction protein, is regulated by both hormonal and mechanical signals [14, 15], we postulate that myometrial OTR expression also follows a similar pattern. The specific objectives of this study were, therefore, to investigate the contribution of the endocrine and mechanical inputs to the regulation of OTR myometrial expression in the rat during pregnancy and labor.

Materials and Methods

Animals

Wistar rats (Charles River Co., St. Constant, PQ, Canada) were individually housed under standard environmental conditions (14L:10D) and fed a Purina diet (Ralston-Purina, St. Louis, MO) and water ad libitum. All experiments were approved by the institutional animal care committee. Virgin female rats (~225 g) were mated, and the day that vaginal plugs were observed was designated Day 1 postcoitum. The average time of delivery under these standard conditions in our colony was the morning of Day 23. Our criteria for the presence of labor were based on delivery of at least two pups with at least one pup remaining in utero at the time the rats were killed.

Experimental Design

A. Normal pregnancy and spontaneous term labor

Rats were killed on Days 15, 17, 19, 20, 21, 22, and 23 (during labor) and 1 day postpartum, and the presence of OTR mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from the myometrium of these animals. Quantitative assessment of OTR mRNA expression, however, was conducted using RNA collected from rats only on Days 15, 22, and 23 and 1 day postpartum (n = 3 at each time point).

B. Progesterone-delayed labor

To examine the effects of maintenance of elevated progesterone at term, rats were randomized on Day 20 postcoitum to receive daily subcutaneous injections of either 0.2 ml of vehicle (90% corn oil and 10% ethanol) or 4 mg of progesterone (in 0.2 ml of vehicle). Animals (n = 3 at each time point for each treatment) were killed on Day 21, 22, or 23. On Day 23, control rats were killed during labor, usually around 1000 h, while progesterone-treated rats were killed in the afternoon and showed no signs of labor. In addition to our own data, evidence has been provided by Hendrix et al. [16] that this progesterone administration protocol delays labor at least 24 h in rats. We have previously reported that the same doses of progesterone resulted in maintenance of elevated plasma progesterone levels (68 ± 7 ng/ml on Day 23) as opposed to the normal fall in progesterone in control animals (14 ± 4 ng/ml on Day 23) [14].

C. Ovariectomy (OVX)-induced preterm labor

This part of the experiments entailed three groups of rats.

1. Preterm labor: On Day 17 of gestation, six rats underwent bilateral OVX through bilateral flank incisions under general anesthesia with intraperitoneal injection of a mixture of ketamine (70 mg/kg) and xylazine (7 mg/kg). These rats then received daily injections of 0.2 ml of vehicle and were killed at 48 (n = 3) or 96 (n = 3) h after surgery. These times were chosen based on a pilot study in which preterm delivery occurred 96 h after OVX on Day 17. In the present study all rats killed at 96 h were either in the process of delivery or exhibited vaginal bleeding indicative of labor. Rats that had completed delivery before 96 h were excluded from this study.

2. Progesterone blockade of preterm labor: Six rats ovariectomized on Day 17 were treated with progesterone (4 mg in 0.2 ml of vehicle) daily from the day of OVX. Rats were killed at 48 or 96 h (n = 3 at each time point) after surgery.

3. Controls: Six rats underwent sham surgery (the uterus was externalized and then placed back into the peritoneal cavity through bilateral flank incisions, but the ovaries were not removed) on Day 17 of gestation and received daily injections of 0.2 ml of vehicle from the day of surgery. Rats were killed at 48 or 96 h (n = 3 at each time point) after surgery.

D. Unilaterally pregnant rats

The effects of uterine stretch on the expression of OTR mRNA in the myometrium was investigated using unilaterally pregnant rats. The experimental procedures have been previously described [15]. Under general anesthesia, female virgin rats (~225 g) underwent unilateral tubal ligation through a flank incision to ensure that they subsequently became pregnant in only one uterine horn. Tubal ligation was randomized to left and right sides. After at least 5 days of recovery, these rats were mated and then randomized into 4 groups.

Group 1 (n = 7): On Day 15, sham operation was performed (i.e., midline abdominal incision was made but no further procedures were performed to the nongravid horn) under general anesthesia, and rats were killed on Day 20.

Group 2 (n = 7): On Day 15, rats underwent insertion of a polyvinyl catheter, 3 mm in outside diameter and 4 cm in length (3-mm tube), into the nongravid uterine horn through midline abdominal incision. The 3-mm tube stretched the uterine horn approximately 2-fold in both diameter and length as compared to the unstretched nongravid horn [15]. The rats were killed on Day 20.

Group 3 (n = 6): Rats underwent sham operation as described for group 1 on Day 18 and were killed during parturition on Day 23.

Group 4 (n = 8): Rats underwent insertion of a 3-mm tube into the nongravid horns as described for group 2 on Day 18 and were killed during parturition on Day 23.

We observed no difference in the timing of delivery in these unilaterally pregnant rats (with or without intrauterine tubes) as compared with untreated rats in our colony.

Tissue Collection and Total RNA Isolation

Rats were killed by carbon dioxide inhalation on the designated days. The uterus was removed, placed into ice-cold saline, and opened longitudinally. The remaining conceptuses were removed, and the endometrium was carefully removed by scraping the luminal surface of the uterus, using a scalpel blade. For the unilaterally pregnant rats, the two uterine horns were processed separately. The myometrial tissue was flash-frozen in liquid nitrogen and stored at −70°C until later analysis. The tissue was pulverized in liquid nitrogen and homogenized in 4 M guanidinium isothiocyanate at room temperature. Total RNA was extracted from the tissues using the method described by Chomczynski and Sacchi [17].

RT-PCR

Reverse transcription was performed in a volume of 10 μl reaction mixture containing 1 μg of total RNA, 5 mM MgCl2, 1 mM dNTP, 10 mM KCl, 10 mM Tris-Cl (pH 8.3), 2.5 μM random hexamers, 8.5 units of ribonuclease inhibitor (Pharmacia Biotech, Uppsala, Sweden), and 50 units of Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD) at 42°C for 30 min. PCR primers OTR-forward 5′-GCATGTTCGCCTCCATCCT-3′ and OTR-reverse 5′-CCCGTGAACAGCATGTAGATCC-3′ were used to amplify a 634-base pair (bp) fragment of the OTR cDNA (corresponding to nucleotides 365–998 in the human OTR cDNA [18]). This pair of primers defines a region containing a 12-kilobase (kb) intron [4]; therefore it theoretically does not amplify any genomic DNA. This was confirmed by PCR reaction of rat genomic DNA (data not shown). Primers β-actin forward 5′-AACCGTGAAAAGATGACCCAG-3′ and β-actin reverse 5′-CTCCTGCTTGCTGTACCACAT-3′ were used to amplify a 740-bp fragment of the β-actin cDNA (nucleotides 1671–3131 of the rat β-actin gene [19]). This pair of primers was separated by three introns and was also confirmed not to amplify any genomic DNA (data not shown). PCR was performed in a volume of 25 μl reaction mixture containing 10 μl of the cDNA, 2 mM MgCl2, 0.4 mM dNTP, 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 50 ng of each specific PCR primer, and 2.5 units of Taq DNA polymerase (Gibco BRL) for number of cycles as indicated. For OTR cDNA, each cycle consisted of 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min. For β-actin, each cycle consisted of 94°C for 1 min, 59°C for 1 min, and 72°C for 1 min. Amplification products were analyzed by electrophoresis in 1.5% agarose gel.

Subcloning and Sequencing of the OTR cDNA

The 634-bp rat OTR cDNA was generated by RT-PCR using total RNA isolated from a laboring rat myometrium. After amplification and electrophoresis, the rat OTR cDNA band was isolated from the agarose gel using Spin-x column (Corning Costar, Cambridge, MA). The cDNA fragment was purified with Geneclean Kit (Bio 101 Inc., Vista, CA) and subcloned into the TA cloning vector (Invitrogen, San Diego, CA) according to the instructions provided by the manufacturers. The sequence of this fragment was determined by the dideoxy chain-termination method [20] using M13 reverse primer and M13 (−40) forward primer. The sequence of this 634-bp product was identical to that previously reported for the rat OTR (nucleotides 2285–3671 excluding intron 2 [4]). Using the same procedures, the 740-bp fragment of the rat β-actin cDNA was also subcloned into the TA cloning vector and subsequently used as the cDNA probe for Southern hybridization.

Southern Hybridization

The RT-PCR products of OTR and β-actin cDNA amplified from total RNA derived from pregnant rat myometrium were electrophoresed on a 1.5% agarose gel, transferred onto a GeneScreen membrane (DuPont NEN Research Products, Boston, MA) in double-strength SSC (0.3 M sodium chloride, 0.03 M sodium citrate) over a 20-h period, and cross-linked by UV irradiation. The membrane was hybridized to the subcloned OTR and β-actin cDNA probes that were radiolabeled using random priming (multiprime DNA labeling system; Amersham, Oakville, ON, Canada) to a specific activity of 108 cpm/μg according to the manufacturer's instructions. The hybridization was carried out in a solution containing 0.5 M sodium phosphate, 1% BSA, 7% SDS, and 10 mM EDTA at 65°C for 20 h followed by washes to a final stringency of 0.1% SDS and 0.1-strength SSC at 65°C. The membrane was then exposed to an x-ray film (Reflection; DuPont NEN Research Products, Boston, MA) with the aid of an intensifying screen at −70°C for a sufficient period of time.

Semiquantitative RT-PCR Measurements of OTR mRNA

Expression of the OTR is generally very low as evidenced by previous reports that large amounts of poly(A)-enriched RNA were required to detect the mRNA by Northern blotting [4, 11]. Since generating such large quantities of poly(A)-enriched RNA is problematic, we chose to utilize a semiquantitative RT-PCR approach. Although RT-PCR has been applied in numerous studies to detect gene expression at the level of transcription, quantification can be difficult because of the sensitive nature of the PCR and a tendency for the product to reach a plateau at the later stages of the reaction. In order to compare the level of OTR expression under different experimental conditions we adapted our routine PCR methodologies to provide a semiquantitative measure of mRNA levels. We first determined whether the detection of the OTR PCR product was linear over the wide range of OTR expression that we expected to find in the myometrium during pregnancy. We empirically chose three cycle numbers (24, 29, and 34 cycles) and determined the abundance of PCR product in a sample of myometrium collected during labor, over a 16-fold dilution, at each cycle number. As seen in Figure 1, 24 cycles were insufficient to produce a readily detectable signal over the range of product dilutions. In contrast, at 29 cycles there was a linear relationship between the PCR product and the initial number of target molecules in the reaction. Moreover, differences in PCR product could be quantitatively differentiated over a wide range of dilutions (at least 2- to 16-fold dilution). At 34 cycles the yield of OTR cDNA reached a plateau, and the intensity of the PCR product became difficult to differentiate across the dilutions. A similar approach was used to determine the linear cycle range for β-actin. After this initial validation we chose 25, 27, and 29 cycles for OTR cDNA and 20, 22, and 24 cycles for β-actin cDNA analysis, unless otherwise specified. Within these ranges, the PCR products were detectable and showed a linear increase in signal intensity. Each sample underwent RT reaction in a total volume of 100 μl reaction mixture containing 10 μg of total RNA. A fraction of this RT pool (10 μl) was used for each PCR reaction. Each set of samples was electrophoresed on a single gel, stained with ethidium bromide, and photographed using Polaroid 665 Positive/Negative film (Polaroid Corp., Cambridge, MA). The PCR signal intensities were quantified by scanning the negative film using a laser scanner (Molecular Dynamics 300A; Sunnyvale, CA) and analyzing the scans using ImageQuant software. Since OTR and β-actin expression was linear within the cycle ranges used in this study, analysis of the OTR expression was conducted by calculating the average ratios of the relative optical densities (ROD) of 25-cycle OTR to 20-cycle β-actin, 27-cycle OTR to 22-cycle β-actin, and 29-cycle OTR to 24-cycle β-actin for each sample.

Fig. 1

Establishment of conditions for semiquantitative RT-PCR. Total RNA was isolated from myometrial tissue of Day 23 parturient rats, and cDNA was produced using random hexamer and reverse transcriptase. The cDNA was diluted over a 16-fold range and subjected to 24, 29, or 34 cycles of PCR using specific primers to amplify a 634-bp product encoding the rat OTR. The dilution of cDNA is shown over each lane. M, Molecular size markers.

Fig. 1

Establishment of conditions for semiquantitative RT-PCR. Total RNA was isolated from myometrial tissue of Day 23 parturient rats, and cDNA was produced using random hexamer and reverse transcriptase. The cDNA was diluted over a 16-fold range and subjected to 24, 29, or 34 cycles of PCR using specific primers to amplify a 634-bp product encoding the rat OTR. The dilution of cDNA is shown over each lane. M, Molecular size markers.

Statistical Analysis

Data are expressed as the mean ± SEM where appropriate. Data were subjected to one-way or two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method) to determine between-group differences. The statistical analysis was carried out using the SigmaStat version 1.01 software (Jandel Corporation, San Rafael, CA). The level of significance for comparisons was set at p < 0.05. Where variance was found to be heterogeneous, the data were subjected to log transformation.

Results

A. Myometrial OTR mRNA Expression during Late Pregnancy and Spontaneous Term Labor

Qualitative analysis of myometrial expression of OTR mRNA (Fig. 2) revealed a very low level at midgestation (Day 15). Levels increased slightly from Day 15 through to Day 22 of gestation; there was then a marked increase during parturition (Day 23). The levels of the β-actin transcript remained relatively constant throughout gestation. The changes in the intensities of the PCR products on the gel were confirmed by Southern hybridization using radiolabeled cDNA probes of OTR and β-actin (Fig. 2). One-way ANOVA on OTR mRNA levels analyzed by semiquantitative RT-PCR (Fig. 3) revealed a significant overall increase in OTR expression with gestational age (p < 0.001). Levels of OTR mRNA were low on Day 15 (ROD 0.27 ± 0.02 relative to β-actin). Messenger RNA levels increased slightly on Day 22 (ROD 0.73 ± 0.26) although this did not reach statistical significance; OTR mRNA levels were increased during labor on Day 23 (ROD 2.32 ± 0.11, p < 0.05 compared to all other groups) and fell rapidly 1 day after delivery (ROD 0.19 ± 0.05).

Fig. 2

PCR-amplified OTR (A) and β-actin (B) cDNA derived from rat tissues visualized by ethidium bromide staining after agarose gel electrophoresis. The cDNA was then transferred onto a GeneScreen membrane and hybridized with radiolabeled probe for OTR and β-actin, respectively (Southern). Total RNA samples were derived from rat myometrium from Day 15 through Day 23 (during labor) and 1 day postpartum (1PP). Myometrial OTR mRNA levels peaked during labor and declined markedly 1 day after delivery.

Fig. 2

PCR-amplified OTR (A) and β-actin (B) cDNA derived from rat tissues visualized by ethidium bromide staining after agarose gel electrophoresis. The cDNA was then transferred onto a GeneScreen membrane and hybridized with radiolabeled probe for OTR and β-actin, respectively (Southern). Total RNA samples were derived from rat myometrium from Day 15 through Day 23 (during labor) and 1 day postpartum (1PP). Myometrial OTR mRNA levels peaked during labor and declined markedly 1 day after delivery.

Fig. 3

Semiquantitative RT-PCR of OTR mRNA levels in myometrium obtained on Days 15, 22, during labor (D), and 1 day postpartum (1PP). A) Representative PCR agarose gel showing analysis of OTR and β-actin mRNA. PCR was performed at 25, 27, and 29 cycles for OTR and 20, 22, and 24 cycles for β-actin. B) Graph showing semiquantitative analysis of OTR mRNA expression during late pregnancy and labor. Values represent mean ± SEM (n = 3 at each time point) of the ratios of ROD of OTR to β-actin. Data were subjected to one-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

Fig. 3

Semiquantitative RT-PCR of OTR mRNA levels in myometrium obtained on Days 15, 22, during labor (D), and 1 day postpartum (1PP). A) Representative PCR agarose gel showing analysis of OTR and β-actin mRNA. PCR was performed at 25, 27, and 29 cycles for OTR and 20, 22, and 24 cycles for β-actin. B) Graph showing semiquantitative analysis of OTR mRNA expression during late pregnancy and labor. Values represent mean ± SEM (n = 3 at each time point) of the ratios of ROD of OTR to β-actin. Data were subjected to one-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

B. Effects of Maintenance of Elevated Progesterone at Term

Two-way ANOVA showed an overall significant effect of gestational age on myometrial OTR expression (F = 12.11; df 2,17; p = 0.0013). Control rats receiving injections of vehicle exhibited a 5-fold increase in OTR transcript on Day 23 relative to Day 21 (p < 0.05) (Fig. 4). In contrast, animals treated with progesterone failed to show the expected increase in transcript level on Day 23 (Fig. 4), and none of these rats delivered or showed signs of labor (e.g., vaginal bleeding) when they were killed between 1400 and 1500 h. While there was no significant difference between the control and progesterone-treated groups on Day 21 or 22, OTR mRNA level in rats treated with progesterone was significantly lower than that of the control rats on Day 23 (ROD 0.9 ± 0.3 vs. 1.5 ± 0.2 for progesterone-treated and control groups, respectively; p < 0.05).

Fig. 4

The effects of progesterone on myometrial OTR mRNA at term. Data represent mean ± SEM (n = 3 at each time point) of myometrial OTR mRNA levels obtained from pregnant rats on Days 21, 22, and 23 that had received daily injections of either vehicle or progesterone (4 mg/day) beginning on Day 20 of gestation. Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

Fig. 4

The effects of progesterone on myometrial OTR mRNA at term. Data represent mean ± SEM (n = 3 at each time point) of myometrial OTR mRNA levels obtained from pregnant rats on Days 21, 22, and 23 that had received daily injections of either vehicle or progesterone (4 mg/day) beginning on Day 20 of gestation. Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

C. Myometrial OTR Expression during Preterm Labor

Pilot studies indicated that OVX of rats on Day 17 of gestation resulted in preterm delivery approximately 96 h later. Consequently myometrial OTR mRNA expression was determined in samples collected 48 and 96 h after OVX or sham surgery on Day 17. Two-way ANOVA revealed a significant effect of different treatments on OTR mRNA levels (F = 18.75; df 2,17; p = 0.0002). OVX resulted in a significant increase in myometrial OTR mRNA levels compared to those in the sham-operated controls (ROD 1.7 ± 0.3 vs. 0.6 ± 0.2; p < 0.05) within 48 h of surgery that was maintained through to 96 h (ROD 1.9 ± 0.4 vs. 0.8 ± 0.01; p < 0.05) (Fig. 5). These rats had either delivered one or more pups but not completed delivery or had shown signs of impending delivery (vaginal bleeding) at 96 h. The elevation in OTR mRNA levels associated with OVX-induced preterm labor was completely blocked by the administration of progesterone (ROD 0.5 ± 0.1 at both 48 and 96 h). Rats treated with progesterone did not deliver or show signs of impending delivery that were apparent in the ovariectomized animals 96 h after surgery.

Fig. 5

Myometrial expression of OTR during OVX-induced preterm labor. Rats (n = 3 at each time point with each treatment) underwent either sham operation or OVX on Day 17 of gestation and then received daily injections of either vehicle (SHAM and OVX) or progesterone (4 mg/day, OVX-P). Rats were killed at 48 or 96 h after surgery. Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

Fig. 5

Myometrial expression of OTR during OVX-induced preterm labor. Rats (n = 3 at each time point with each treatment) underwent either sham operation or OVX on Day 17 of gestation and then received daily injections of either vehicle (SHAM and OVX) or progesterone (4 mg/day, OVX-P). Rats were killed at 48 or 96 h after surgery. Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

D. Effects of Uterine Stretch on Myometrial OTR Expression in Unilaterally Pregnant Rats

Two-way ANOVA revealed an overall significant effect of gestational age on myometrial OTR mRNA expression (F = 31.9; df 1,55; p < 0.0001) and a significant interaction between gestational age and treatments (F = 2.87; df 3,55; p = 0.046). On Day 20 of gestation, OTR mRNA levels in the nongravid unstretched control horns, the nongravid horns stretched with 3-mm tubes, and the normal gravid horns were basal (Fig. 6). During labor, as anticipated, the OTR mRNA levels in the gravid horns increased significantly in comparison to the levels in their counterparts on Day 20 of pregnancy. Interestingly, the level in the nongravid, unstretched horns remained basal, even though these horns were exposed to the same systemic endocrine changes associated with labor as the contralateral gravid horns. In contrast, the nongravid horns stretched with tubes, under the same endocrine environments as the contralateral gravid horns, expressed a high level of OTR mRNA, similar to the gravid horns (Fig. 6).

Fig. 6

The effects of mechanical stretch on myometrial expression of OTR mRNA in unilaterally pregnant rats. Unilaterally pregnant rats underwent either sham operation or placement of a 3-mm tube in the nongravid uterine horn on Day 15 (n = 7 in both sham-operated and tube-stretched groups) or Day 18 (n = 6 in the sham-operated group and n = 8 in the tube-stretched group) and were killed 5 days after surgery. Data represent mean ± SEM of myometrial OTR mRNA levels from nongravid unstretched horns (C), nongravid stretched horns (T), and normal gravid horns (P). Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

Fig. 6

The effects of mechanical stretch on myometrial expression of OTR mRNA in unilaterally pregnant rats. Unilaterally pregnant rats underwent either sham operation or placement of a 3-mm tube in the nongravid uterine horn on Day 15 (n = 7 in both sham-operated and tube-stretched groups) or Day 18 (n = 6 in the sham-operated group and n = 8 in the tube-stretched group) and were killed 5 days after surgery. Data represent mean ± SEM of myometrial OTR mRNA levels from nongravid unstretched horns (C), nongravid stretched horns (T), and normal gravid horns (P). Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (p < 0.05).

Discussion

The onset of labor requires activation of the myometrium, a process resulting from an increased expression of CAPs of which the OTR is one example [1]. The primary stimulus to the onset of labor is believed to be an increase in the E:P ratio in the maternal circulation, which in most species is induced by activation of the fetal hypothalamic-pituitary-adrenal axis [1]. Our data suggest that this endocrine axis, while necessary, is not sufficient to bring about the increased expression of CAP genes and that mechanical signals are required to activate the myometrium.

Our demonstration that the endocrine environment is critical for OTR mRNA expression in the pregnant uterus supports studies showing abrupt increases in oxytocin binding before the onset of labor in the rat [2, 3] and the human [21, 22]. Estrogen is an important positive regulator of OTR expression. Not only does it increase the level of OTR mRNA and the number of OTR in the myometrium of nonpregnant rats [1013]; the increase in myometrial OTR expression during labor is also closely associated with an increase in plasma E:P ratio [2, 3]. The rat OTR gene possesses several half palindromic estrogen-response elements and a functional full estrogen-response element within its 5′ flanking region [4, 23] that could mediate the effects of this steroid hormone.

Our data also suggest that progesterone is a major negative regulator of OTR mRNA levels in the pregnant rat myometrium. Administration of progesterone blocked both the increase in OTR mRNA levels and labor in rats both at term and following preterm OVX. These results are consistent with data showing that progesterone blocked the increase in OTR concentration in the myometrium of pregnant ovariectomized rats [24] and with the report of Fang et al. [25], who found that treatment with RU-486 induced a premature increase in OTR mRNA levels. The mechanisms by which progesterone down-regulates the expression of OTR in the myometrium remain obscure, since the promoter of the rat OTR gene lacks a consensus progesterone-response element [4, 23]. It is possible that during labor progesterone antagonizes the effects of estrogen, or that by promoting uterine growth progesterone reduces stretch-induced myometrial tension and hence the mechanical stimulus to OTR expression.

Surprisingly, in nonpregnant rats progesterone does not appear to suppress OTR gene expression, although it did block the increase in oxytocin binding [11, 12]. This discrepancy may suggest differences between nonpregnant and pregnant rats or may reflect analysis using whole uterus rather than myometrium alone (as was the case in our study). OTR expression in the endometrium and the myometrium is known to be differentially regulated during the estrous cycle, during pregnancy, and after treatment with gonadal steroids [13, 2628].

Our results clearly demonstrate an important role of mechanical inputs in addition to endocrine factors in the regulation of myometrial OTR expression during parturition. The finding that OTR mRNA level remained low in the nongravid, sham-operated uterine horns during labor indicates that the mere presence of an increased E:P ratio was not sufficient to induce an increase in the expression of this receptor. The high level of expression of OTR mRNA in the nongravid horns that were stretched with an inert mechanical device suggested that myometrial distention at term was also a prerequisite for the full activation of OTR expression. This effect of stretch is unlikely to be due to the presence of a foreign body inside the uterine cavity, since on Day 20 of gestation the presence of the 3-mm tube did not increase OTR mRNA levels. Furthermore, we have previously demonstrated that in nonpregnant rats, distention with a 3-mm tube increased myometrial Cx-43 mRNA expression while the presence of a smaller tube, 1 mm in diameter, failed to induce similar changes [15]. These observations, together with the results that progesterone abolished the increase in myometrial OTR mRNA expression in term or OVX-induced preterm labor, suggest that the high circulating level of progesterone—a hormone believed to maintain myometrial quiescence during pregnancy—might suppress the stretch-induced effects at this time of gestation. Our studies are in agreement with the data of Csapo and Lloyd-Jacob [29] showing that in pregnant rabbits with declining progesterone levels, intrauterine volume became a predominant factor that determined uterine contractile activity and the timing of parturition.

In contrast to our data on OTR mRNA, studies by Alexandrova and Soloff [30] and Fuchs et al. [31] failed to show increased OTR binding relative to DNA content in gravid versus nongravid horns of rats at term. The reasons for the discrepancies between our data and these reports are not clear. Our data clearly show that expression of the OTR mRNA is regulated by mechanical signals; however, it is possible that translational or posttranslational events might also influence the apparent number of binding sites per cell. It may also be that the effects of stretch are primarily exerted during labor. In our study, myometrial samples were collected during delivery of the fetuses, whereas the other investigators collected samples either 1 day before labor [31] or from a mixed population of rats before, during, or after labor [30]. Data also suggest that there may be tissue-specific influences on the differential expression of OTR in gravid and nongravid horns. Lamming et al. [28], using unilaterally pregnant ewes at Day 16 of pregnancy, reported a down-regulation of endometrial OTR mRNA levels and OT binding only in gravid horns, with no reduction in OTR expression in nongravid horns. The complexities surrounding the temporal and tissue-specific regulation of OTR expression remain to be resolved.

The patterns of OTR mRNA expression during term and preterm labor, and its regulation by hormonal and mechanical factors, are strikingly similar to those we have previously reported for Cx-43 [14, 15, 32, 33]. These data suggest that their transcription is coregulated and support our hypothesis that these two genes contribute to a cassette of genes encoding CAPs that must be activated in order for labor to occur. Our previous studies with Cx-43 provided evidence that the product of the proto-oncogene, c-fos, might be involved in the mechanisms mediating myometrial Cx-43 expression during labor and after gonadal steroid treatment [33, 34]. Interestingly the rat OTR gene also contains an AP-1 sequence within the 5′-flanking region [4, 23], raising the possibility that the same nuclear signals may regulate transcription of both the Cx-43 and OTR genes.

In summary, we have demonstrated that the level of mRNA encoding the OTR is increased in the rat myometrium in both term and preterm labor and that progesterone can block the increase in OTR expression at these times as well as the occurrence of labor. We have further demonstrated that both hormonal and mechanical signals are critical in regulating OTR mRNA levels during pregnancy and parturition. Taking these findings together with our recent report of a similar pattern of regulation of Cx-43 expression in the myometrium [15], we conclude that integration of endocrine and mechanical inputs may provide a general mechanism to activate the myometrium by inducing the expression of CAPs that are essential for the development of coordinated intense contractions of labor.

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Author notes

1
This work was supported by grants from the Hospital for Sick Children Foundation, Toronto, and the Medical Research Council of Canada (Group Grant in Development and Fetal Health).