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Biology of Reproduction 60, 814-820 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Effect of Labor Induction on the Expression of Oxytocin Receptor, Cytochrome P450 Aromatase, and Estradiol Receptor in the Reproductive Tract of the Late-Pregnant Ewe1

S. Tong Leunga, D. Claire Wathes2,a, I. Ross Youngb, and Graham Jenkinb

a Department of Veterinary Basic Sciences, The Royal Veterinary College, Potters Bar, Hertford, EN6 1NB, United Kingdom b Department of Physiology, Monash University, Clayton, Victoria 3168, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the timing of changes in aromatase, estradiol receptor, and oxytocin receptor expression in ovine uterine and placental tissues before parturition. Labor was induced by betamethasone injection into the fetus on Days 130–132 of pregnancy. Tissue samples were collected at injection and then every 14 h until labor (56 h) from four ewes at each time point. Samples were analyzed for aromatase, estradiol receptor, and oxytocin receptor expression by in situ hybridization; for oxytocin binding to its receptor using a specific antagonist; and for estradiol receptor quantitation by immunocytochemistry. Aromatase mRNA expression increased by 14 h postinjection (p < 0.02) in the fetal villi and remained high until labor. Expression of estradiol and oxytocin receptor mRNAs was unchanged in myometrium but increased in the endometrial luminal epithelium by 28 h (p < 0.05) and remained high until labor. Estradiol receptor protein concentration increased modestly at labor while oxytocin receptor binding in the luminal epithelium changed in parallel to the mRNA concentration. In conclusion: 1) induction of aromatase may facilitate the expression of endometrial estradiol and oxytocin receptors in the placentome, 2) changes in endometrial rather than myometrial oxytocin receptor may be important in inducing parturition, and 3) the transcription of estradiol receptor and oxytocin receptor in the uterine epithelium are positively correlated during parturition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful delivery of the fetus involves precise timing of the events that initiate the onset of parturition, cervical softening, and fetal expulsion. Before the onset of labor in sheep, an increase in adrenocorticotropic hormone secretion is associated with a significant elevation in the fetal adrenal cortisol concentration [1]. The latter is a prerequisite for successful labor since fetal adrenalectomy prevents parturition [2]. The rise in fetal cortisol stimulates the conversion of androgen to estrogen in the placenta through increasing aromatase activity, resulting in an increase in the estrogen:progesterone ratio [3, 4]. The expression of oxytocin receptor (OTR) and the production of prostaglandin (PG) F2{alpha} are also significantly up-regulated at this time, resulting in uterine contractions and delivery [5].

The rate of conversion from androstenedione to estrogen, catalyzed by aromatase, has been analyzed in fetal cotyledonary tissues in late pregnancy and at both dexamethasone-induced and natural parturition in vivo. Aromatase activity remained at basal levels from Day 118 to Day 140 of pregnancy and increased significantly during parturition in sheep [6]. The aromatase mRNA during this period, however, has not been quantitated or localized.

Estradiol receptor mRNA concentrations in sheep measured by Northern blotting remain low from Day 100 to Day 145 of pregnancy and increase significantly in the myometrium and endometrium in both spontaneous and cortisol-induced labor, but not in fetal tissues such as amnion and chorion [7, 8]. A similar measurement of OTR protein in the endometrial luminal epithelium (LE) and myometrium has also been observed [9–12], suggesting that the expression of OTR and estradiol receptor may be related in these tissues. The development of OTR in the endometrium plays an important role in determining the timing of luteolysis in cyclic ewes via stimulation of PGF2{alpha} [13, 14]. At parturition, oxytocin and OTR have been associated with the increase in uterine activity that occurs at this time. Therefore, the timing, localization, and level of expression of OTR in the reproductive tract of late-pregnant ewes may have a profound effect on the mechanism leading to the delivery of the fetus.

It has not been established whether the surge of maternal free estradiol in late pregnancy in cattle and sheep [15–17] occurs before or after the increase in expression of endometrial OTR during parturition. To investigate the regulatory effect of the previously published parturient changes in estradiol [18] on the expression of OTR and its possible role in parturition, it is necessary to determine the time at which OTR expression becomes elevated during late pregnancy and parturition in sheep. To achieve this aim, we have used a previously characterized glucocorticoid-induced labor model [19], in which the timing of the onset of labor can be closely controlled.

The aims of this experiment were to determine the timing, localization, and level of expression of OTR mRNA, aromatase mRNA, and estradiol receptor mRNA in the endometrium, myometrium, cervix, and placentome in late-pregnant ewes immediately before and during parturition. OTR binding activity and estradiol receptor protein concentration in the endometrium and myometrium were also investigated in the same animals.


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

Twenty ewes (Border Leicester x Merino; normal term 145 days) were treated with betamethasone (1 ml of a 5.7 mg/ml solution, diluted in aqueous vehicle; Celestone Chronodose; Schering Plough Pharmaceuticals, Australia) to induce labor. Injection of the betamethasone was performed by maternal transabdominal insertion of a needle into skeletal muscle of the fetus on Days 130–132 of pregnancy using ultrasound guidance to locate the site of injection. The site of injection was confirmed by the localization of Indian ink (mixed with the injected betamethasone) in the fetus at postmortem. Only ewes in which the injected solution was visualized in the fetal compartment were included in this study.

The ewes were divided into 5 groups each containing 4 animals and were killed by lethal injection of 20 ml of 325 mg/ml sodium pentobarbitone (Euthatal; Arnold of Reading, Peakhurst, Australia) into the jugular vein. The time from injection to parturition was established as 56.6 ± 0.8 h (mean ± SEM, n = 5) in a previous study using an identical protocol and undertaken by us, with an increase and change in uterine EMG activity from contractures to contractions occurring within 48.6 h and 50.6 h of betamethasone injection (Fig. 1) [19, 20]. Tissues were collected within 10 min of slaughter at 0 h (control), 14 h, 28 h, 42 h, and 56 ± 2 h (labor) after betamethasone injection. The final collection was made when the fetus was visible in the birth canal. Pieces of placentome, intercotyledonary endometrium, myometrium, and cervices were wrapped in aluminum foil, frozen in isopentane, cooled in liquid nitrogen, and stored at -80°C until processed. The protocol was approved by the Animal Ethics Committee of the Department of Physiology as conforming to the NH & MRC/CSIRO/AAC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 1997, Approval No. P418: PHYS 93/133.



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  		FIG. 1. A) EMG activity recorded in a previous study [19] during 1 h immediately before administration of betamethasone, indicative of low-frequency uterine contractures that occur throughout late gestation. B) EMG activity recorded between 46 h and 50 h after administration of betamethasone, in which contractures are breaking up into high-frequency contractions of relatively short duration, indicative of labor onset [20]. C) EMG activity recorded between 50 h and delivery at 54 h after administration of betamethasone, in which contractions finally coalesce to result in delivery of the fetus. In this study, all animals killed at 56 h were either delivering a fetus (fetal body part visible at vagina) or exhibiting labor contractions as shown in C above.

Reagents

Chemicals were purchased from Sigma Chemical Co. (Poole, Dorset, UK) or Merck (Poole, Dorset, UK) unless otherwise specified.

Section Preparation

Cross sections (10 µm thick) were cut and thaw-mounted on poly-L-lysine (0.1 mg/ml)-coated slides. The sections were fixed in 4% w:v paraformaldehyde in PBS (0.13 M NaCl, 0.007 M Na2HPO4) for 5 min, washed in PBS for 2 min (3 times), and dehydrated in 75% followed by 95% ethanol, for 5 min each. The sections were then stored in 95% absolute ethanol at 4°C until used.

In Situ Hybridization

The in situ hybridization procedures were applied as previously described [11]. Briefly, the mRNA specific probes (antisense; 45 mer synthetic oligonucleotide), were end-labeled with [35S]dATP (NEN Research Products, Stevenage, Hertford, UK). The reaction mix containing the labeled probes in hybridization buffer (100 000 cpm per 100 µl of hybridization buffer per slide) was added to the sections, covered with a Parafilm (Merck) coverslip, and incubated at 43°C overnight. After incubation, the sections were washed at room temperature for 30 min followed by 1 h at 55°C in single-strength sodium saline citrate (15 mM sodium chloride, 15 mM sodium citrate, pH 7.0) containing 0.2% (w:v) sodium thiosulfate-5 hydrate. The slides were then dehydrated in a gradient of ethanol, air-dried, and exposed to hyperfilm-ßmax (Amersham International plc, Amersham, Bucks, UK) for a defined period (see below). The sense sequence of the respective probe was used as the negative control. The uterus of a ewe at estrus was used as the positive control for the estradiol receptor and OTR probes. All slides using the same probe that was later used for quantitation were processed in the same batch.

Probes (Antisense Sequence)

The probe sequences used were based on published cDNA sequences. Oligonucleotide 45 mers were synthesized (Babraham Institute, Cambridge, UK) after specific sequences were chosen. Aromatase (exposure time 2 wk) [21]: 5'-TCA CCG GGT AGC CAT CGA TGA CAT CAT CCT CTA AGG CTT TGC GCA-3'. Estradiol receptor (exposure time 3 wk) [22]: 5' TGG CCT GTA GTA GGC GGG AGG GCC GGC TTC GCG CAC CGC ATA GCC-3'. OTR (exposure time 2 wk) [23]: 5'-TTC CTT GGG CGC ATC GGC ATC CCA GAC ACT CCA CAT CTG CAG GAA-3'.

Autoradiography

The method was based on that described by Ayad et al. [24]. Cross sections (20 µm thick) were cut from frozen samples and thaw-mounted on chrome alum (0.05% w:v; BDH, Toronto, ON)-gelatine (5% w:v; BDH)-coated slides. The slides were washed in ice-cold phosphate buffer (100 mM, pH 7.4, 0.1% BSA) without magnesium chloride (MgCl2), with orbital shaking to remove endogenous bound oxytocin. The slides were then incubated with 300 µl of iodinated OTR antagonist (OTA; 125I-OTA, D-(CH2)5[Tyr(Me)2Thr4TyrNH29]-vasotocin; 1250 cpm per µl in buffer containing MgCl2 (2.04 g/l); kindly supplied by Professor M. Manning, Medical College of Ohio, Toledo, OH) to give the maximum binding (total counts). The nonspecific binding solution containing excess unlabeled oxytocin (10 µg/ml; Bachem UK Ltd., Saffron Walden, Essex, UK) was added to the control slides. The slides were then covered by a Parafilm coverslip and incubated at room temperature for 1 h. After incubation, the coverslips were removed, and the slides were washed vigorously in fresh ice-cold PBS containing MgCl2 for 1 min (3 times). Finally, the slides were dipped in ice-cold distilled water and then air-dried at room temperature. The slides were then exposed to a hyperfilm-ßmax (Amersham International) for 48 h.

Photographic Emulsions

The procedures were similar to the instructions provided by the manufacturing company (LM-1; Amersham International). Briefly, completely dried slides were dipped into the emulsion vertically for 5 sec at 43°C and allowed to dry horizontally at room temperature and then on a metal plate precooled with dry-ice for 10 min each. The slides were then placed into a light-tight box with anhydrous silica gel in the base of the box, sealed, and incubated at 4°C until developed. The incubation time depended on the probe used: OTR for 3 wk, aromatase for 3 wk, and estradiol receptor for 4 wk. After incubation, the slides were dipped into developer (Phenisol; Ilford Limited, Ilford, UK) for 5 min, stop bath (0.5% acetic acid v:v) for 1 min, fixative (47% w:v, sodium thiosulfate pentahydrate) for 10 min, and then distilled water for at least 10 min before counterstaining with Harris' hematoxylin (BDH) and eosin (Sigma) to identify the cell types.

Immunohistochemistry for Estradiol Receptor

The procedures were applied as described previously [25]. Cross sections (5 µm) from frozen samples were cut and thaw-mounted onto 3-aminopropyltriethoxysilane (2% v:v; BDH)-coated slides and fixed in formaldehyde (3.7% v:v; BDH) for 10 min. Then the sections were treated with acetone (100%) and methanol (100%) at room temperature for 5 sec each and then washed twice in single-strength PBS for 5 min. All subsequent incubations were in a humidified box at room temperature unless otherwise specified. After being washed, the sections were incubated with normal rabbit serum (NRS, 2.5%; Sigma) for 10 min. The NRS was then blotted off, and the sections were incubated with 100 µl of mouse estradiol receptor antiserum (1:80 diluted in PBS; DAKO, Ely, Cambridge, UK) at 4°C overnight. For control sections, mouse IgG (1 µg/ml; Sigma) was added in place of the estradiol receptor antiserum.

After overnight incubation, the slides were washed in single-strength PBS for 5 min (2 times) before incubation with: 1) rabbit anti-mouse IgG antiserum (1:100; Sigma) for 30 min, then 2) 100 µl of peroxidase anti-peroxidase (1:50; Sigma) for 30 min, and 3) 100 µl of activated diaminobenzidine tetrahydrochloride (DAB; 0.5 mg/ml; Sigma) for 10 min in the dark. After incubation, the excess DAB was washed away with distilled water, and the slides were mounted after serial dehydration in 75% ethanol, 100% ethanol, and 100% xylene for 2 min each.

Data Analysis

After exposure, the images of the in situ hybridization and the 125I-OTA autoradiographs on the film were quantified using an image analysis system (Seescan plc, Cambridge, UK) by measuring the optical density of specific areas identified on the corresponding slides by hematoxylin and eosin staining. Emulsion-coated slides were also used to confirm the cellular localization of the signals but were not quantified. The results from the autoradiographs were expressed as arbitrary optical density (OD) units (means ± SEM) with a linear range from 0.01 to 2.1. Three readings per section and four sections per sample were taken from the probed slides. The sense/nonspecific binding values were subtracted from the antisense/total counts values to produce a mean value of specific hybridization/specific binding in each region. The intraassay coefficients of variation for OD measurements from duplicate pairs of slides were 11.7% for OTR mRNA, 20.1% for aromatase mRNA, 27.1% for estradiol receptor mRNA, and 12.0% for 125I-OTA, respectively. The immunohistochemistry results for measuring estradiol receptor protein were quantified by giving a score of 0 (no staining) to 3 (very strong) by two independent assessors. The final results were analyzed by one-way ANOVA with time as factor followed by the Neuman-Keuls procedure. Significance is defined as a p value < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Localization of Aromatase mRNA in the Placentome

Aromatase mRNA was detected in the fetal villi of the placentomes. There was a significant increase in concentration by 14 h after the administration of betamethasone to the fetus. The aromatase mRNA level then remained high until labor (Figs. 2 and 3).



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  		FIG. 2. The expression of aromatase mRNA in the placentome (P) was investigated using in situ hybridization. a) An antisense ovine specific probe, end-labeled with [35S]dATP, was hybridized with a cross section of the placentome in labor (56 h postinjection of betamethasone). c) The site of expression was localized using photographic emulsion; the results showed that aromatase was strongly expressed in the fetal villi (FV) of the placentome. Note that in a, two serial sections are illustrated. b, d) The sense sequence was used as the negative control. e) The structure of the uterine tissue samples investigated showing the placentome (P), endometrium (Endo), LE, and myometrium (Myo) was illustrated by hematoxylin and eosin staining. f) The region outlined in the box is shown at a higher magnification. The scale bars represent 12.5 mm (a, b), 10 µm (c, d), 30 mm (e), and 2.5 mm (f), respectively.



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  		FIG. 3. The expression of aromatase mRNA in the placenta during the labor induction period following an injection of betamethasone to the fetus at 0 h was investigated. The results are expressed as the mean ± SEM OD unit at each time point (n = 4). Aromatase mRNA was present at 0 h in the fetal villi within the placentome, increased significantly by 14 h (d > c; p < 0.02), and remained high until labor.

Localization of Estradiol Receptor in the Placentome, Uterus, and Cervix

No estradiol receptor mRNA was detected in the center of the placentome during the 56-h induction period. It was present, however, in the maternal LE at the edge of the placentome (Fig. 2), first appearing at 28 h (p < 0.05), and being maintained at this level at 56 h after the injection of betamethasone (Figs. 4 and 5). Quantification of these data was from the autoradiographs. Examination of the emulsions indicated that most of the specific hybridization was to the maternal LE, although there may also have been some low expression in the underlying stromal tissue. There was no significant increase in estradiol receptor mRNA observed in the myometrium between 0 h (not detectable, OD < 0.01) and 56 h (OD = 0.02 ± 0.001). Estradiol receptor mRNA in the cervix was below the detection limit (OD < 0.01) throughout the period studied. The immunohistochemistry results showed slight positive nuclear staining (score 0–1) for the estradiol receptor protein in the uterine LE, myometrium, and cervical stroma cells adjacent to the LE at labor (56 h) but not at 0 h (data not shown). These concentrations were much lower than those found in the uterus at estrus, which scored 2 and 3 in the uterine LE and myometrium, respectively.



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  		FIG. 4. The expression of estradiol receptor mRNA in the placentome was investigated using in situ hybridization. An antisense ovine specific probe, end-labeled with [35S]dATP, was hybridized with a cross section of the placentome in labor (56 h postinjection of betamethasone). The site of expression was localized using photographic emulsion. The results show that estradiol receptor was expressed in the LE at the edge of the placentome (a). The sense sequence was used as the negative control (b). The scale bar represents 10 µm.



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  		FIG. 5. The expression of estradiol receptor mRNA in the LE at the edge of the placentome during the labor induction period following an injection of betamethasone to the fetus at 0 h was investigated. The results are expressed as the mean ± SEM OD unit at each time point (n = 4). The results show that a significant increase in estradiol receptor mRNA was observed in the LE by 28 h and remained high until labor (b > a, p < 0.05).

Localization of OTR mRNA in the Placentome, Uterus, and Cervix

The endometrial LE showed a significant increase in OTR mRNA concentration by 28 h (p < 0.05; Figs. 6 and 7a). No other region in the endometrium or placentome showed any significant variation in OTR mRNA concentration. In contrast, although the OTR mRNA in the myometrium increased slightly from 42 h to 56 h (labor) after the injection of betamethasone, there was no significant difference between the control and labor groups (p > 0.05, Figs. 6 and 7b). No significant change in OTR mRNA expression was observed in the cervix, in which concentrations remained basal (OD < 0.01) throughout the period studied.



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  		FIG. 6. The expression of OTR mRNA in the uterus and placentome during the labor induction period was investigated using in situ hybridization. An antisense ovine-specific probe, end-labeled with [35S]dATP, was hybridized with a cross section of the intercotyledonary uterine tissues (a) and placentome (c) in labor (56 h postinjection of betamethasone). c) The site of expression was localized using photographic emulsion; the results show that OTR was strongly expressed in the LE at the edge of the placentome. b, d) The sense sequence was used as the negative control. e, g) The expression of OTR protein was also investigated using 125I-OTA autoradiography; the results showed that strong 125I-OTA binding was observed in the LE at the edge of the placentome (e). Weaker binding was observed in the myometrium (Myo; g). The nonspecific binding, containing excess unlabeled peptide to compete with the 125I-OTA, was used as the control for the autoradiography (f, h). The scale bars represent 20 mm (a, b, e–h), 10 µm (c, d).



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  		FIG. 7. OTR mRNA and protein in the LE covering the placentome (a) and myometrium (b) were localized using in situ hybridization and 125I-OTA binding after the induction of labor by fetal injection of betamethasone at 0 h. The results are expressed as the mean ± SEM OD units at each time point (n = 4). A significant elevation of the concentrations of both OTR mRNA and protein was observed in the LE by 28 h (b > a, e > c, p < 0.05), and concentrations remained high until labor. In addition, a significant fall in the 125I-OTA binding was noted between 0 h and 14 h (d < c, p < 0.02). No significant increases in either OTR mRNA or protein were observed in the myometrium (p > 0.05) throughout the induction period although both demonstrated a small rise between 42 h and 56 h (labor).

There was a significant increase in 125I-OTA binding at 28 h in the LE (p < 0.05), which remained elevated to 56 h after the injection of betamethasone. A significant decrease in OTA binding was observed in the endometrial LE at 14 h (p < 0.02) (Figs. 6 and 7a). In contrast, there was no significant increase in 125I-OTA binding in the myometrium throughout the induction period (p > 0.05, Figs. 6 and 7b).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
All of the animals designated for the final 56-h group were in active labor when the tissue was collected, with the fetus visible within the birth canal. This confirms that the treatment protocol was effective in inducing labor. A previous study using the same procedure showed that uterine electromyographic activity increased and changed from contracture to contraction-like activity consistently 6–8 h before labor onset at 56.6 ± 0.8 h, whereas control, saline-treated ewes did not exhibit a change in EMG activity and did not enter labor [19]. Free estradiol levels in the maternal circulation started to increase about 20 h before delivery after labor induction by fetal dexamethasone infusion [18].

It is likely that the termination of pregnancy and the onset of parturition require a combination of complex messages in a timed order to allow precise control of delivery. Estrogen stimulates the expression of its receptor [22, 26, 27], the formation of gap junctions in the myometrium [28, 29], and dilatation of the cervix [30–32]. Therefore, the induction of aromatase, which is responsible for metabolism of androgen to estrogen during pregnancy, could have a role as a physiological regulator of the timing of these events.

A low but measurable concentration of aromatase mRNA was observed in the fetal villi in the control group (0 h), suggesting that aromatase activity in this tissue was established by Day 132. At this stage of pregnancy, estrone sulphate is the predominant estrogen produced [16]. The elevation of aromatase mRNA corresponds to its activity, which has previously been reported to increase progressively towards term in sheep [6]. This suggests that the regulation of aromatase expression is probably at the transcriptional level. Free estradiol, which is the major biologically active estrogen in the sheep, does not increase significantly until approximately 24 h before parturition [16,17, 18, 33]. This is about 18 h after we detected a significant increase in aromatase mRNA expression. It is also possible that the concentration of 17-hydroxysteroid dehydrogenase, which is required to convert estrone to estradiol after aromatization, may also be involved in regulating this increase, but this enzyme was not investigated in the present study. A significant increase in aromatase mRNA in the fetal villi was observed just 14 h after the administration of betamethasone, suggesting that the fetal tissues were highly sensitive to, and up-regulated by, the increased level of cortisol in the fetus.

The expression of estradiol receptor mRNA was partially consistent with the previous studies by Wu et al. [7, 8] who observed a significant increase in both estradiol receptor mRNA and protein in the endometrium and myometrium during parturition. In the present study, neither estradiol receptor mRNA nor its protein were detected at Day 132 of pregnancy, and a significant increase in estradiol receptor mRNA, but not its protein, was observed in the LE 28 h after the administration of betamethasone. The low concentration of estradiol receptor protein detected in the endometrial LE and myometrium in labor suggests that labor does not require a high level of estradiol receptor protein. It is also possible that the expression of estradiol receptor may be regulated at the translational level. The data obtained in this study support the latter hypothesis since the estradiol receptor mRNA and protein were not proportionately increased during the induction period. However, a different isoform of estradiol receptor (ß), which shares a low homology with the traditional type ({alpha}), has recently been reported in the mouse and human [34, 35]. It is possible that the probes and antiserum used in this study were unable to detect the ß isoform and that this new form of receptor may be important in parturition.

It is widely believed that estrogen up-regulates OTR expression in the uterus [36–40]. In this study, OTR mRNA was detectable at a basal level before the appearance of estradiol receptor mRNA, suggesting that estrogen may be responsible for only the up-regulation, but not the constitutive expression, of OTR. This conclusion is supported by studies in the ovariectomized ewe, in which OTR are present in the LE [41]. Furthermore, in tissue culture experiments using explants of ovine uterus collected at a similar stage of gestation, oxytocin binding increases spontaneously in the absence of any estradiol in the medium (unpublished observations). On the other hand, OTR mRNA in the myometrium remained basal when estradiol receptor protein was present in these tissues, indicating that a differential regulation from estradiol is operating in a tissue-specific manner. Previous evidence that estradiol can influence OTR translation as well as transcription was provided by a study of rat kidney which showed that estradiol stimulates the production of OTR transcripts of different sizes [40]. The expression of both estradiol receptor and OTR in the LE increased at 28 h after induction in the present study, so it is possible that the two events were related in this tissue.

The increase in OTR mRNA expression in the myometrium was small and failed to reach statistical significance, although the sheep were in the process of giving birth during the final collection time point. This result followed a trend similar to the findings in natural and cortisol-induced labor reported in previous studies [11, 12], but the increase in the myometrium was less pronounced in the present study. The result reported here indicates, therefore, that a low concentration of OTR in myometrium may be sufficient to facilitate the expulsion of the fetus. This view is supported by studies of mutant mice deficient in oxytocin that are able to give birth apparently normally [42, 43]. These mice still had a parturient rise in OTR, but no agonist was present to activate them. This suggests that alternative mechanisms involving, for example, PGF2{alpha} may be able to achieve parturition.

The up-regulation of OTR mRNA in the endometrial LE, however, was more pronounced than that found in the myometrium and had increased significantly within 28 h of induction and approximately 28 h before birth. In addition to its stimulating effect on muscle contraction as found in myometrium [10, 14, 44, 45], oxytocin, mediated by OTR, may have a regulatory role in inducing the production of PGF2{alpha} as found in the nonpregnant sheep during luteolysis [13]. This view is supported by our previous study using an identical induction protocol in which levels of PGH synthase-2 immunoreactivity in ovine cotyledons and concentrations of PGF2{alpha} in both fetal and maternal circulations increased within 56 h of labor induction with betamethasone [19]. Therefore, the development of OTR in the endometrium may enhance secretion of PGF2{alpha} and thus have a role in the onset of parturition. In contrast, there was no significant variation in OTR mRNA concentration in the cervix throughout the induction period, which is consistent with previous findings [11]. This suggests that oxytocin may not play an important role in cervical dilatation during parturition in this species. The results also indicate that OTR expression in the endometrium is differentially regulated from that in the myometrium and cervix, and is relatively more sensitive to the changes in the steroidal environment.

Previous studies have demonstrated that OTR mRNA concentration changes in parallel to its binding activity [11,23], leading to the belief that the regulation of the expression of OTR is mainly at the transcriptional level. The 125I-OTA binding results show that the binding activity in the myometrium, but not in the endometrial LE, corresponded to its mRNA expression. A significant drop in 125I-OTA binding was observed in the endometrial LE at 14 h. This suggests that the translation of OTR mRNA was disrupted after the administration of betamethasone into the fetus and that such disruption was reversed within 14 h since a significant increase in OTR binding was observed at 28 h. A similar biphasic action of betamethasone on PGH synthase activity on ovine uterine tissues in late gestation has also been observed by us (I.R. Young; personal communication). In the previous studies, OTR mRNA and its binding activity were monitored in days rather than hours so that such a brief translational disruption might not have been noticed.

Taken together with previously published data on changes in estrogen and progesterone concentrations that occur at cortisol-induced parturition [18], these data suggest that the major sequence of events that occurs during the induction of parturition may be 1) the elevation of fetal cortisol which 2) stimulated the expression of cytochrome P450 aromatase in the fetal tissues. The increase in aromatase activity raised the rate of estrogen synthesis and 3) subsequently increased the estrogen to progesterone ratio. In the presence of estrogen and the withdrawal of progesterone, 4) estradiol receptor and 5) OTR expression were up-regulated in the endometrial LE, whereas the rise observed in both estrogen receptors and OTR in the myometrium failed to achieve significance.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. S. Meier for her assistance with the induction of labor and collection of tissue.


   FOOTNOTES
 
1 This research was supported in part by an Australian Research Council Grant to Professor G. Jenkin. Back

2 Correspondence: D.C. Wathes, Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Road, Potters Bar, Herts., EN6 1NB, UK. FAX: 01707 647085; dcwathes{at}rvc.ac.uk Back

Accepted: November 3, 1998.

Received: December 12, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Thorbun GD, Liggins GC. Role of the fetal pituitary-adrenal axis and placenta in the initiation of parturition. In: Lamming GE (ed.), Marshall's Physiology of Reproduction, 4th Ed. Vol 3. London: Chapman and Hall; 1994: 1003–1028.
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