Up-Regulation of Oxytocin Receptor Messenger Ribonucleic Acid and Protein by Estradiol in the Cervix of Ovariectomized Rat¹

^a Laboratory for Pregnancy and Newborn Research, Department of Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853–6401

	ABSTRACT

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Oxytocin receptor (OTR) regulation has been extensively studied in uterine myometrium and endometrium. However, studies in the cervix are limited. The present studies utilized in situ hybridization and immunocytochemistry to localize OTR mRNA and protein distribution in cervices of nonpregnant ovariectomized (OVX) rats and examined the effect of combined and independent treatments with estradiol and progesterone on cervical OTR. Thirteen nonpregnant rats were bilaterally OVX under general anesthesia. At least 7 days later, the rats were exposed to one of four different treatments 24 h prior to necropsy: 1) estradiol (50 µg, n = 4); 2) progesterone (10 mg, n = 3); 3) both estradiol (50 µg) and progesterone (10 mg) (n = 3); 4) corn oil vehicle (n = 3). After 24-h estradiol treatment, OTR mRNA increased significantly (p < 0.05) in smooth muscle cells of the rat cervix as a result of increased copy numbers of OTR mRNA per cell as well as an increased population of OTR mRNA-positive cells. Progesterone alone had no effect on OTR mRNA expression; however, progesterone combined with estradiol significantly inhibited the up-regulation of OTR mRNA by estradiol alone. The increase of OTR mRNA in cervical epithelial cells was minimal in all situations. Intensity of cervical OTR immunostaining in both the epithelial cells and cervical smooth muscle cells was also elevated after estradiol treatment. The anti-rat OTR antiserum used for immunocytochemistry was validated by Western blot analysis. In conclusion, OTR and OTR mRNA were localized in smooth muscle cells and in epithelial cells of rat cervix. Estradiol-dependent activation of OTR gene expression and active OTR synthesis in smooth muscle cells account for the increased OTR level in rat cervix in vivo, in which progesterone acted as an antagonist of estradiol on OTR gene expression.

	INTRODUCTION

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Oxytocin (OT), a nonapeptide hormone, is involved in processes associated with parturition in addition to stimulation of myometrial contraction, including the relaxation and softening of the cervix prior to birth [1]. The cervix is composed of several basic tissue types: epithelium lining the cervical canal, smooth muscle, and elastin and collagen connective tissues [2]. During late gestation, the cervix undergoes marked changes in preparation for labor. Most notably, the cervix increases in vascularization and water content, and a reorganization of connective tissue matrix takes place [3–8]. Numerous studies have shown that estrogen and prostaglandin (PGs) produced within the cervix play a major role in the complex process of cervical softening [9]. OT is known to stimulate the synthesis and release of PGs in uterine tissues in several species including rabbits, cattle, and humans [10–13]. OT may have a similar function in the tissues of the cervix. If such a mechanism exists, OT may indirectly contribute to the ripening of the cervix prior to labor through an induction of PG release.

In order for the cervix to demonstrate sensitivity to OT, the cervix must contain functional OT receptors (OTR) that bind the hormone. Although OTR has been demonstrated in uterine myometrial and endometrial glandular cells by both ligand-binding and mRNA measurements, studies investigating the presence and the regulation of OTR in the cervix are limited, and there are no reports on rat cervical OTR.

We had three objectives in the present study: 1) to establish the presence of OTR and its mRNA in the cervix of the rat; 2) to determine the specific cell types expressing OTR and its mRNA; 3) to determine the role of estradiol and progesterone in the regulation of OTR synthesis. Determining the effects of individual sex steroids on OTR regulation in vivo during late pregnancy is exceedingly difficult because of the numerous endocrine, paracrine, and autocrine changes that occur simultaneously at this time. To diminish the effects of such confounding influences, the combined and individual actions of estradiol and progesterone on the regulation of OTR were analyzed on the ovariectomized (OVX) rat model.

	MATERIALS AND METHODS

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Care of Animals

Virgin female CD rats (205–240 g) were obtained from Charles River Laboratories (St. Constant, PQ, Canada). They were fed rat chow ad libitum and kept in a temperature-controlled environment. All experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee. The Cornell facilities are approved by the American Association for the Accreditation of Laboratory Animal Care.

Progesterone and Estradiol Treatment

Thirteen nonpregnant female rats were bilaterally OVX under general anesthesia. At least 7 days later, the rats were exposed to one of four different treatments 24 h prior to necropsy: 1) estradiol (50 µg, n = 4); 2) progesterone (10 mg, n = 3); 3) both estradiol (50 µg) and progesterone (10 mg) (n = 3); 4) corn oil (n = 3). These are referred to as E, P, EP, and C groups, respectively. The estradiol solution was prepared by dissolving 10 mg of 1,3,5[10]-estratriene-3,17ß-diol (#E-8875; Sigma Chemical Co., St. Louis, MO) in 0.5 ml of ethanol and 100% pure corn oil; 100% pure corn oil was the control vehicle in this study. The progesterone solution was purchased at 50 mg/ml from Schein Pharmaceutical, Inc., Florham Park, NJ (NDC 0364–6683–54).

The steroid concentrations used and the 24-h exposure protocol were based on the results of past studies from several laboratories, including our own, indicating that such concentrations were sufficient to produce effects on myometrial contraction [14]. Previous studies have indicated that the rat myometrium is most sensitive to OT at 24 h after estradiol exposure [15]. Thus we examined OTR in the rat cervix 24 h after estradiol administration. All treatments were accomplished by a single s.c. 0.2-cc injection, and the combined treatment of progesterone and estradiol was administered in two successive injections, one immediately after the other.

Collection and Fixation of Tissues

Animals were killed under CO₂ 24 h after treatment, and cervices were removed immediately. After rinsing with 0.01 M PBS to remove red blood cells, cervices were fixed in 4% paraformaldehyde in 0.01 M PBS at room temperature for 24 h before being processed and embedded in paraffin for evaluation by immunocytochemistry and in situ hybridization. Uterus collected from one pregnant rat at 19 days of gestation age and one nonpregnant rat was used to perform Western blot analysis to validate OTR antibody in this species.

In Situ Hybridization

Paraffin sections (4 µm) of OVX rat cervices were cut and thaw-mounted onto poly-L-lysine-coated slides. Tissue sections were dewaxed and pretreated with 1) 0.2 N HCl at room temperature for 20 min and 2) double-strength SSC (single-strength SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) at 70°C for 15 min; they were then washed in 0.01 M PBS (three times, 5 min each). Prehybridization was carried out by placing 100 µl prehybridization solution (4-strength SSC, 40% deionized formamide, 10% [w:v] dextran sulfate, 500 µg/ml denatured sonicated salmon sperm DNA, 250 µg/ml transfer RNA, 2.5-strength Denhardt's, 4 mM EDTA, 0.1% [w:v] pyrophosphate, and 50 nM -thio-dATP) on each section. Sections were coverslipped with parafilm, and prehybridization was performed at 37°C for 2 h in a humidified chamber. Prehybridization solution was then removed, and fresh prehybridization solution (100 µl) containing 10 mM dithiothreitol and 1 x 10⁶ cpm ³⁵S-radiolabeled OTR probe was placed over each section and coverslipped with parafilm; hybridization was performed at 37°C for 20 h. Oligonucleotides were labeled with terminal deoxynucleotidyl transferase, using [-³⁵S]dATP (1000–1500 Ci/mmol; Dupont NEN, Boston, MA) to a specific activity of 4200–4600 Ci/mmol DNA. After hybridization, sections were briefly washed twice in single-strength SSC at 23°C, twice (30 min each time) in single-strength SSC at 55°C, and once (30 min) in single-strength SSC containing 0.1% (v:v) Triton X-100 at 23°C; they were briefly rinsed in water, briefly immersed in 70% ethanol, and air dried. The sections were then coated with liquid photographic emulsion (NTB2; Eastman Kodak, Rochester, NY). After 3-wk exposure, the sections were developed and stained with hematoxylin. The oligo probe sequence for rat OTR is generated from rat OTR cDNA reported by Rozen: 5'-ACGACACAGCAGGTCGGGCCCATAGAAGCGGAAGGTGATGTC-3', which corresponds to nucleotides 2218–2260 of cDNA encoding rat OTR [16]. An OTR sense oligo probe was used as control.

Western Blot Analysis

To prepare solubilized cell membrane extracts, uterine tissues from one pregnant and one nonpregnant rat were ground into small pieces; they were homogenized with a Polytron (Brinkmann, Westbury, NY) in TED buffer (50 mM Tris [pH 7.4], 10 mM EDTA, and 1 mM diethyldithiocarbamic acid [DEDTC; Sigma]) containing 2 mM octyl glucoside (Sigma) and centrifuged at 30 000 x g for 30 min at 4°C. The crude pellets containing cell membranes were sonicated (8-sec cycle; three cycles; Branson Sonifier, Danbury, CT) in 500 µl TED sonication buffer (20 mM Tris [pH 7.4], 50 mM EDTA, and 0.1 mM DEDTC containing 45 mM octyl glucoside). The sonicates were centrifuged at 13 000 x g for 25 min at 4°C. The recovered supernatant was stored at -80°C until electrophoretic analysis. The protein concentration was determined by the method of Bradford (Bio-Rad Laboratories, Richmond, CA).

The solubilized proteins (50 µg/ml) were then separated on 10% SDS-PAGE and electrophoretically transferred to nylon membrane (Immobilon; Millipore Corp., Bedford, MA), using a Bio-Rad transfer blot cell. The filters for immunostaining were blocked with 2% BSA in 10 mM Tris-Cl buffer containing 0.1% Tween 20. After blocking, the blots were washed three times with wash buffer (5 min each) containing 10 mM Tris-Cl and 0.1% Tween 20; they were then incubated with the polyclonal rabbit anti-OTR receptor antiserum generated by a synthetic dodecapeptide (WQNLRLKTAAAA) corresponding to the third intracellular loop of the rat OTR sequence, which tends to be the area of least homology between receptors of the arginine vasopressin/OT family [17] (#3579, 1:1000 dilution), or the preabsorbed OTR antiserum at 4°C overnight. The blots were then washed and were incubated with peroxide-conjugated goat anti-rabbit IgG at room temperature for 1 h. After each antibody incubation, the blots were washed three times (15 min each) in wash buffer. The protein bands were visualized by using an enhanced chemiluminescence Western blotting detection kit (Amersham Life Sciences, Arlington Heights, IL). The molecular size of the proteins was determined by running standard molecular weight marker proteins (Bio-Rad) in an adjacent lane.

Immunocytochemistry

Paraffin sections (4 µm) of rat cervices were dewaxed in two changes of xylene (5 min each) and rehydrated in a series of alcohol. Unless otherwise specified, all the slides were sequentially incubated at room temperature with each of the following reagents for the time indicated: 1) 3% (v:v) H₂O₂ in PBS for 30 min; 2) 10% (v:v) normal goat serum with 5% (w:v) BSA in 0.05 M Tris-Cl with 0.15 M NaCl (TBS) for 1 h; 3) polyclonal rabbit anti-rat oxytocin receptor antiserum (#3579, 1:1000 dilution) at room temperature for 1 h; 4) biotinylated goat anti-rabbit IgG (Vector Labs., Burlingame, CA) for 1 h; 5) avidin-biotin complex (Vector) for 1 h; 6) 3,3-diaminobenzidine tetrahydrochloride (Sigma), 4 mg/10 ml 0.05 M Tris buffer (pH 7.6), and 10 ml 3% H₂O₂ for 20 min. After each incubation except that of step 2, the slides were washed with TBS for 15 min. The slides were then counterstained with hematoxylin and mounted. The specificity of the anti-OTR antibody in the current study was controlled by 1) omission of the primary antibody, 2) incubation of the slides with the normal rabbit serum instead of the primary antibody, 3) Western blot analysis using the rabbit anti-rat OTR antiserum or the preabsorbed anti-rat OTR antiserum.

Quantitative and Statistical Analysis of In Situ Hybridization

Six sections from a block of rat cervix from each rat were cut and thawed onto the polylysine-coated slides. Thus a total of 24 (E group) or 18 (C, P, or EP groups) sections of cervix from each group were assessed. Five areas were selected at random from each section, and the number of in situ positive cells as a percentage of total cells was determined in each area. The mean of the six sections was then calculated to give an n = 1. The percentage of OTR in situ positive cells or the grain density of OTR per cell was determined in a total of 120 (E group) or 90 (C, P, or EP groups) areas of cervix in each group. Differences in OTR mRNA concentration among different groups for in situ hybridization were examined by ANOVA. Data throughout are presented as the mean ± SEM.

	RESULTS

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Western Blot Analysis

As shown in Figure 1, after Western blot analysis, anti-OTR antiserum mainly stained a protein with an approximate molecular mass of 66 kDa in the rat uterine tissue. This staining was abolished by incubation with the preabsorbed OTR antiserum. Only one major protein species reacted with this OTR antiserum in the pregnant rat uterine tissue (Fig. 1). However, in the nonpregnant rat uterine tissue, an additional band at lower molecular mass (approximate 35 kDa) was observed. The nature of the additional band in nonpregnant rat uterine tissues is not clear.

View larger version (57K): [in this window] [in a new window]   FIG. 1. Western immunoblot analysis of rat uterine solubilized cell membranes with OTR antibody. A) Solubilized cell membranes from the nonpregnant rat uterus (lane 1, 50 µg/lane) and the pregnant rat uterus (lane 2, 50 µg/lane) were electrophoresed on 10% SDS-polyacrylamide gels, transferred to Immobilon membrane, and immunoblotted with OTR antibody as described in the Materials and Methods. A major protein band with a molecular mass at approximately 66 kDa reacted with the anti-rat OTR antiserum. B) The same samples were subjected to the anti-OTR antibody preabsorbed with the synthetic dodecapeptide specifically synthesized to raise the OTR antibody. Note: The major protein band identified by the anti-rat OTR antiserum was abolished.

Localization of OTR and OTR mRNA in Rat Cervix

OTR was localized in the cytoplasm of a variety of cell types. OTR was present in the epithelium of glands (Fig. 2) as well as in the luminal epithelium (data not shown). Staining was also observed in the muscle cells of the cervix (Fig. 3). Moreover, OTR was localized in the epithelium and the muscle cells of the veins and arteries that supply the cervical tissues (Fig. 4). The distribution of OTR mRNA was largely similar to that for the OTR protein in the rat cervix (Fig. 5).

View larger version (135K): [in this window] [in a new window]   FIG. 2. OTR immunostaining in the cervical glandular epithelium of the nonpregnant OVX rat. The brown stain represents localized OTR. A) 24-h control; B) after 24-h estradiol treatment; C) after 24-h progesterone treatment; D) after 24-h estradiol and progesterone treatment. FIG. 3. OTR immunostaining in the cervical muscle cells of the nonpregnant OVX rat. The brown stain represents localized OTR. A) 24-h control (longitudinal muscle cells); B) after 24-h estradiol treatment; C) after 24-h progesterone treatment; D) after 24-h estradiol and progesterone treatment.

View larger version (134K): [in this window] [in a new window]   FIG. 4. OTR immunostaining in the smooth muscle and epithelium of blood vessels supplying the nonpregnant rat cervix. A) OTR was localized in the epithelium and the muscle cells of the cervical blood vessels of controls. B) Animals treated with estradiol for 24 h demonstrated intense OTR staining in the cervical blood vessels as compared with controls. C) OTR staining in the cervical blood vessels after 24-h progesterone treatment. D) OTR staining in the cervical blood vessels after 24-h estradiol and progesterone treatment. The intensity of OTR staining was reduced after combined estradiol and progesterone treatment.

View larger version (109K): [in this window] [in a new window]   FIG. 5. In situ hybridization of OTR mRNA of the nonpregnant OVX rat cervix shown in brightfield (left panels) and darkfield (right panels). A, B) 24-h control; C, D) after 24-h estradiol treatment; E, D) after 24-h progesterone treatment; G, H) after 24-h estradiol and progesterone treatment. Note: There was a significant increase in OTR mRNA level in the cervix of the rat that received estradiol treatment. Progesterone antagonized the effect of estradiol on OTR mRNA expression. x40 (reproduced at 70%).

Effect of Estradiol and Progesterone on OTR and OTR mRNA in OVX Rat Cervix

After 24-h estradiol treatment, the level of OTR mRNA increased significantly (p < 0.05) in smooth muscle cells of the rat cervix (Figs. 5 and 6). This increase was due to an increase in copy number of OTR mRNA per cell as well as an increased population of OTR mRNA-positive cells (Fig. 6). Progesterone alone had no effect on OTR mRNA expression; however, the combination of progesterone and estradiol significantly inhibited the up-regulation of OTR mRNA by estradiol (Figs. 5 and 6). The changes in OTR mRNA in cervical epithelial cells in all situations were small compared with the changes in OTR protein. Intensity of OTR immunostaining was elevated in both smooth muscle cells and epithelial cells of the cervix after estradiol treatment; staining in these locations was inhibited by combined treatment with progesterone. In addition, estradiol administration affected the morphology of the cervical epithelium, increasing the thickness of the glandular epithelium of the cervix as compared with that in controls (Fig. 2).

View larger version (15K): [in this window] [in a new window]   FIG. 6. Densitometric analysis of cervical OTR mRNA in control (C, n = 3), estradiol-treated (E, n = 4), progesterone-treated (P, n = 3), and estradiol plus progesterone-treated (EP, n = 3) nonpregnant OVX rats. Significant increase of OTR mRNA was observed in smooth muscle cells of the rat cervix after 24-h estradiol treatment. *p < 0.05 compared with C, P, and EP. Progesterone antagonized the stimulatory effect of estradiol on OTR mRNA when EP were compared with E; **p < 0.05.

	DISCUSSION

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OTR has been demonstrated in uterine myometrial and endometrial glandular cells by both ligand-binding [18] and mRNA measurements [19]. Studies in the cervix, however, are limited. Earlier experiments have characterized OTR in homogenized cervix using receptor-binding techniques [20, 21]. OTR in the pregnant [22] and nonpregnant [1] bovine cervix, demonstrated by immunohistochemistry, is present in epithelial cells at the luminal surface of the mucosa as well as smooth muscle cells. Epithelial cells at the luminal surface of the mucosa were the principal site of OTR. However, the precise localization and histological distribution of OTR protein and its mRNA in the intact cervix of the rat have not been examined.

Understanding the cellular and tissue distribution of OTR provides valuable information regarding the underlying mechanism of both the regulation of OTR and its action within the cervix. Specific localization of OTR and its mRNA was associated with smooth muscle cells as well as the glandular and luminal epithelium of the rat cervix. These results provide histological evidence supporting a biological function of OT in the nonpregnant and probably the pregnant rat cervix.

The present study is the first to investigate the regulation of OTR in the intact cervix by estradiol and progesterone. The results of the present study demonstrate that both OTR protein and OTR mRNA in cervical smooth muscle cells are dramatically up-regulated by estrogen. This action of estrogen is antagonized by progesterone. There is a limited increase in OTR mRNA in the cervical glandular epithelium as compared with OTR protein, which may indicate differential regulation of OTR expression in the different subtypes of cervical cells.

Previous studies have indicated that estradiol plays a major role in cervical softening [9]. We have demonstrated that androgen administration to pregnant rhesus monkeys at 80% gestation duration leads to a physiological rise in maternal plasma estrogen accompanied by normal cervical dilation and delivery of live young [23]. Aromatase inhibitors prevent the maternal estrogen rise and inhibit androgen-induced delivery and cervical dilation [24]. Cervical estrogen receptor (ER) mRNA increases in ewes during labor, providing further support for a function of estrogen in the softening and dilation of the cervix in labor [25]. In the present study, estradiol administered 24 h prior to necropsy markedly up-regulated OTR in all OTR-containing cervical tissues. This result matched the results of similar studies investigating effects of estradiol on myometrial OTR [18, 26, 27] and agrees with autoradiographic studies in OVX rats demonstrating increased OT binding to both myometrium and endometrium after estradiol treatment [28].

Simultaneous administration of estradiol and progesterone for 24 h down-regulated OTR to levels below that in tissues treated with estradiol. We combined estradiol and progesterone to study interactions that resemble the natural hormonal state in the rat, in which there is a balance between estradiol and progesterone. It is important to realize that OTR stimulation is not merely due to increase in stimulatory factors but is rather the result of the balance of stimulatory and inhibitory agents. It is likely that the increased maternal estradiol concentration and decreased progesterone concentration that occur immediately before parturition may together be responsible for an increased expression of cervical OTR during labor in the rat [29]. Previous studies show that labor increases the expression of OTR mRNA in rat myometrium, endometrium, and mammary glands [30]. The mechanism through which progesterone down-regulated OTR may involve ER. Several studies have demonstrated that progesterone down-regulates ER gene expression [31, 32]. Thus progesterone's down-regulatory effect on cervical OTR may be due, in part, to down-regulation of cervical ER preventing estradiol from increasing OTR.

The OTR antibody used for immunocytochemistry was validated in rat uterine tissues by Western blot analysis using OTR antiserum and preabsorbed OTR antiserum. The approximate molecular size of OTR in pregnant and nonpregnant rat tissues was about 66 kDa, in close agreement with the size estimate of OTR in sheep [19], rabbit [33], and rat mammary gland [34]. In addition, the anti-rat OTR antibody we used has been successfully applied to localization of OTR in several rat tissues including the uterus [17]. The novel observation of OTR immunostaining in smooth muscle of cervical blood vessels is of considerable interest. OTR in cervical blood vessels was most intense in rats treated with estradiol. OTR in the smooth muscles of cervical blood vessels may act to prevent postpartum hemorrhage. We have previously described significant amounts of OTR in the smooth muscle of blood vessels supplying the myometrium of pregnant sheep [19].

In summary, these results identify, for the first time, the precise location of OTR and its mRNA within the nonpregnant rat cervix. Estradiol-dependent activation of OTR gene expression and active OTR synthesis in smooth muscle cells account for the increased OTR level in the rat cervix in vivo, in which progesterone acted as an antagonist of estradiol on OTR gene expression.

	FOOTNOTES

 
¹ Supported by NIH HD 21350 and NS 28477.

² Correspondence: Peter W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Dept. of Physiology, Box 16, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853–6401. FAX: 607 253 3455; pwn1{at}cornell.edu

Accepted: June 18, 1998.

Received: February 17, 1998.

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