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Regular Article |
a Departments of Obstetrics and Gynecology,
b Molecular and Cellular Physiology, and
c Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0526
d Samuel Lunenfield Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1X5
ABSTRACT
A polyclonal antibody was raised against amino acids 7–18 in the first extracellular loop of rat prostaglandin F (FP) receptor to monitor expression and localization in pregnant rat myometrium at Gestational Days 16, 18, 20, 21, 21.5, 22 (delivery), and 23 (1-day postpartum; n = 5 per group). The antibody recognized a protein of approximately 43 kDa on Western blot analysis in both membrane (soluble and nonsoluble) and cytosolic fractions of myometrium on each day of gestation. Expression of FP protein increased significantly (P < 0.05) during late gestation in both soluble membrane and cytosolic fractions, being significantly greater at Day 21.5 than at Day 20 of gestation in the soluble membrane fraction and in the cytosolic fraction of tissues collected during labor compared with those obtained before labor. The total concentration of FP receptor in the membrane (soluble plus nonsoluble) remained high throughout late gestation and fell significantly (P < 0.05) in the postpartum period. The FP receptor in the soluble membrane fraction (compared to the total membrane FP receptor) was significantly (P < 0.05) higher in late gestation than earlier, whereas the ratio of FP protein in cytosolic to that in the total membrane was significantly (P < 0.05) higher on Day 23 than earlier in gestation, suggesting a dynamic movement of FP with advancing gestational age. Immunoreactive FP receptor localized to circular and longitudinal smooth muscle at all gestational ages, but changes in intracellular localization were observed in late gestation with a staining pattern similar to
-actin, suggesting an association with myofibrils. Our study suggests an increase in FP-receptor protein in myometrium with advancing gestation and a marked elevation at term. This supports a role for uterine FP receptors in mediation of uterine contractility at term.
parturition, pregnancy, uterus
INTRODUCTION
Synthesis and metabolism of uterine prostaglandins (PGs), especially PGF2 and PGE2, are important in regulation of uterine activity and participate in the process of the estrous cycle, implantation of embryos, progression of pregnancy, and in parturition [1, 2]. The principal site of action of prostaglandins during labor is the myometrium, where they act via specific receptors to modulate contractility [3]. Prostaglandin receptors were first classified on the basis of pharmacological specificity [4] as TP, DP, IP, FP, and EP receptor isoforms specific for thromboxane A2, PGD2, PGI2, PGF2, and PGE2, respectively [4–6]. The EP receptors are further subdivided into EP1 to EP4 [7] subtypes encoded by separate genes [8].
Cloning studies of PG receptors show that they are members of the regulatory G protein with seven transmembrane domain receptor superfamily [9]. These membranous receptors are characterized by seven hydrophobic stretches of amino acids that are predicted to form transmembrane helices [10] linked by extra- and intracellular peptide loops [11, 12]. The PGF receptor has been cloned from rodent (mouse [13] and rat [14, 15]) and ruminant (bovine [16] and ovine [17]) ovarian and astrocyte complementary DNA libraries and from human uterine and placental tissues [14, 18].
Prostaglandin F2 is believed to play an important role in regulation of myometrial contractility and initiation of parturition. Measurements of PGF2 in the uterine vein of pregnant rats showed that the concentrations increased gradually from Day 15, with the highest concentrations around delivery (Day 22) [19, 20]. Exogenous PGF2 has been shown to induce labor [21], and administration of the cyclooxygenase inhibitor indomethacin inhibits uterine contractions and labor [22]. Interestingly, in FP knockout mice, spontaneous labor did not commence even 1 wk after term due to failure of PGF2-induced corpus luteum regression [23]. Previously, we have demonstrated changes in expression of mRNA for the contractile FP and relaxatory EP2 receptors in rat [24] and human [25] myometrium in late gestation, labor, and postpartum. However, it remains unclear how FP-receptor protein expression changes during pregnancy and at parturition.
The objectives of this study were to monitor changes in the expression of FP-receptor protein and in the distribution and localization of FP receptor in uterus obtained from rat during late gestation, labor, and postpartum using Western blot analysis and immunohistochemistry techniques, respectively, with specific antibody raised against the FP receptor.
MATERIALS AND METHODS
Sprague-Dawley rats (Zivic Miller, Zelienople, PA) were maintained on a 12L:12D schedule. Animals received rat chow and water ad libitum. Animals were killed at 0900 h either on Day 16, 18, 20, or 21 of gestation; Day 21.5; Day 22 (at labor); or 1-day postpartum (n = 5 animals/time point). The day of mating was designated as Day 1 of pregnancy, with delivery occurring between 2100 h on Day 21 and 0900 h on Day 22. All procedures on the care and use of animals were approved by the University of Cincinnati Institutional Animal Care and Use Committee.
Preparation and Solubilization of Rat Tissues
Rat uteri were excised and opened, and pups and placentas were discarded. Decidua were removed by wiping with the back of a scalpel blade. Uteri were then rinsed in ice-cold saline, blotted dry, cut in small pieces, and then frozen in liquid nitrogen and stored at -80°C until processed. Homogenization buffers contained protease inhibitors (1 mM benzamidine, 0.1 mg/ml of chicken egg-white trypsin inhibitor, and 60 kallikrein inhibitor units/ml of aprotinin). The tissue was pulverized to powder under liquid nitrogen and homogenized using a polytron homogenizer (Polytron PT 300; Kinematica AG, Littou, Switzerland) three times for 15 sec each time in three volumes of ice-cold TED buffer containing 50 mM Tris (pH 7.4), 10 mM EDTA, 1 mM diethyldithiocarbamic acid (DEDTC), and 2 mM octyl glucoside as previously described for oxytocin receptor in sheep uterus [26]. The homogenate was centrifuged at 30 000 x g for 30 min at 4°C, and the 30 000 x g supernatant was collected and stored at -80°C (cytosolic fraction). The resulting crude pellets containing cell membranes were sonicated three times for 8 sec each time in 500 µl of ice cold TED sonication buffer containing 20 mM Tris (pH 7.4), 50 mM EDTA, 0.1 mM DEDTC, and 45 mM octyl glucoside for solubilization. The sonicates were centrifuged at 13 000 x g for 25 min at 4°C, and the supernatant containing solubilized protein from nuclei and cell membranes was collected and stored at -80°C (soluble membrane fraction). The residual pellets (insoluble membrane fraction) were further homogenized in 0.5 ml of sample buffer (250 mM Tris-HCl [pH 6.8], 2% [w/v] SDS, 20% [v/v] glycerol, 5% [v/v] 2-ß-mercaptoethanol, and 0.02% [w/v] bromophenol blue) using a polytron followed by a glass homogenizer. The mixture was then sonicated and stored at -80°C (nonsoluble fraction). Protein concentration was determined by Pierce BCA kit (Pierce, Rockford, IL) for all fractions and for purified concentrated antibody using bovine serum albumin as protein standard. The rat ovary, which was homogenized under the same conditions, served as a source of positive-control FP receptor due to its high FP-receptor concentration as reported previously [27].
Because immunohistochemical studies revealed FP receptor to be present in various cellular fractions, we prepared, in a separate experiment, specific cellular fractions (cytosolic, microsomes, nuclei, and plasma membranes) from myometrium at Day 18 of gestation by differential and density gradient centrifugation as reported previously [28]. Briefly, the myometrial homogenate was centrifuged at 960 x g for 10 min at 4°C.
The pellet was resuspended in 1.6 M SHM (1.6 M sucrose, 10 mM Hepes, 1 mM MgCl2; overall pH 7.5), then overlayered with 0.25 M SHM and centrifuged at 70 000 x g. The pellet (nuclei) was resuspended with 2.2 M SHM and centrifuged at 70 000 x g for 60 min at 4°C. The supernatant was discarded, the sides of the tubes wiped, and the nuclei gently suspended in 2.2 M sucrose containing 1 mM MgCl2. The 70 000 x g (0.25 M/1.6 M) interface was collected (plasma membrane and mitochondria), diluted with 0.25 M SHE (0.25 M sucrose, 10 mM Hepes, 1 mM EDTA; overall pH 7.5), and centrifuged at 1200 x g for 10 min at 4°C. The pellet was resuspended in 0.25 M SHM and centrifuged at 1200 x g for 10 min at 4°C, then resuspended in 1.45 M SHE, overlayered with 0.25 M SH (0.25 M sucrose, 10 mM Hepes), and centrifuged at 68 000 x g for 60 min at 4°C. The plasma membrane that appeared at the sucrose interface was collected, diluted with 0.25 M SHE, and centrifuged at 17 600 x g for 10 min at 4°C. The pellet was resuspended with 1.35 M SHE, overlayered with 0.25 M SH, and centrifuged at 231 000 x g for 30 min at 4°C. The plasma membrane collected from the interface was diluted with two volumes of cold H2O, then with 0.25 M sucrose to fill the tube, and centrifuged at 14 500 x g for 10 min at 4°C. The whitish pellet (pure plasma membrane) was resuspended in 0.25 M sucrose (pH 7.0).
Mitochondria, lysosomes, peroxisomes, and the heavy microsomal fraction enriched in Golgi complex were removed from the 960 x g supernatant by centrifugation at 25 000 x g for 10 min and at 34 000 x g for 30 min, respectively. The purified supernatant (light microsomes and cell supernatant) was centrifuged at 124 000 x g for 100 min at 4°C, and the supernatant was collected (cytosol). The pellet (microsomes) was resuspended in 0.25 M sucrose. All fractions were stored at -80°C.
FP Antibody Production
The antibody was raised against a synthetic peptide corresponding to amino acids 7–18 (KQPASSAAGLIA) in the first extracellular loop of the rat FP-receptor sequence [29]. Peptide conjugated to BSA was mixed with sterile PBS and injected into a vial of RIBI adjuvant system (RAS; Sigma Chemical Co., St. Louis, MO). A combination of s.c. and i.m. injections were given to 6-mo-old New Zealand White rabbits 3 wk after collection of preimmune sera. The blood was obtained 7 days after each boost, and sera were collected, divided into aliquots, and stored at -80°C. The antibody was affinity purified using the peptide against which the antibody was raised attached to a HiTrap affinity column, and the IgG was dialyzed, then concentrated, and used for Western blot analysis. For immunohistochemistry, the antiserum was purified using a protein A sepharose column (ImmunoPure IgG purification kit; Pierce), then concentrated. Preimmune serum collected before immunization was treated similarly for each technique.
Western Blot Analysis
The SDS gel (12.5%; Owl Separation Systems, Portsmouth, NH) electrophoresis was conducted under denaturing conditions. The solubilized proteins from rat myometrium and ovary (15 µg/lane) were boiled for 2.5 min in sample buffer (1:1 [v/v]) before electrophoresis. The separated proteins were transferred onto a polyvinylidene fluoride (PVDF) microporous membrane (Millipore Corporation, Bedford, MA). The PVDF membrane was blocked with 3% goat serum in TTBS (0.05% Tween-20 in Tris-buffered saline containing 0.5 M sodium chloride and 0.025 M Tris [pH 7.5]) at room temperature for 1.5 h. Following three washings of 5 min each with TTBS, either primary polyclonal FP-receptor antibody (5 µg/ml), preimmune sera, or preabsorbed FP antiserum in blocking buffer was applied for 2 h at room temperature. After four washings of 15 min each with TTBS, secondary antibodies, donkey anti-rabbit Ig horse radish peroxidase (HRP; 0.08 µg/ml; Amersham Pharmacia Biotech, Piscataway, NJ), and goat anti-mouse IgG-HRP (1:10 000 [v/v]; Santa Cruz Biotechnology, Santa Cruz, CA, for Cruz markers) in blocking buffer were applied for 1.5 h at room temperature. The blot was washed three times for 15 min each in TTBS and then given a final 15-min wash with TBS. The molecular sizes of the proteins were determined by comparison with standard proteins (Cruz Marker MW Standards) in an adjacent lane. The protein bands were visualized by using an enhanced chemiluminescence reagent (ECL kit; NEN Life Science Products, Inc., Boston, MA) and then exposed to autoradiographic double-emulsion film (Eastman Kodak, Rochester, NY). Scanning densitometry of immunoreactive bands was used to quantify changes of FP-receptor expression at different time points. Each gel contained the positive-control FP protein (rat ovary) plus one sample from each day of gestation. Expression in each sample was then normalized against expression of the positive-control protein on that gel to control variation between gels. In the experiments using purified subcellular fractions, qualitative Western blot analysis was performed as described above.
Immunohistochemistry
Frozen rat uterus was cross-sectioned at 7 µm. Serial sections were then air-dried and fixed in buffered formaldehyde with picrate (2% para-formaldehyde and 15% picric acid in sodium phosphate buffer [pH 7.3]) for 10 min at room temperature. The sections were washed once for 5 min in PBS/Triton (0.2% Triton X-100 in PBS), then twice for 5 min each in PBS alone. Slides were then incubated for 30 min in ice-cold ethanolamine (0.15 M in PBS), followed by three 5-min washes in PBS.
Sections were immunostained using the Vectastain Elite ABC anti-mouse or anti-rabbit kit as appropriate (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer's instructions, with some modifications. In brief, following a 90-min incubation of sections at 37°C in FP antibody (7 µg/ml) or -smooth muscle actin antibody (3 µg/ml; Sigma), sections were washed once in PBS/Triton X-100, followed by two 5-min washes in PBS/BSA (0.1% BSA in PBS). Following a 30-min incubation at 37°C in secondary antibody, slides were incubated for 10 min in hydrogen peroxide (3% in water). Remaining steps followed the manufacturer's protocol. Slides were incubated according to the supplied protocol for 15 min at room temperature in peroxidase substrate, 3-amino-9-ethylcarbazole (Vector Laboratories), which forms a red precipitate.
Specificity of the Antibody
Although the FP antibody was raised against a unique peptide sequence from FP receptor, we utilized several approaches to verify the specificity of FP-receptor antibody used for Western blot analysis or immunohistochemistry. We substituted the primary antibody with the purified preimmune serum from the same rabbit in which the immune serum was raised or with purified anti-FP receptor preabsorbed by incubation with a 10-fold excess (by weight; compared to FP antibody concentration) of synthetic FP peptide for 2 h at room temperature and then overnight at 4°C. In addition, the purified antibody was utilized to isolate FP protein from a rat ovarian homogenate using IgG plus orientation kit (Pierce). This isolated FP protein was electrophoresed on SDS gel and then silver stained using Gelcode color silver stain (Pierce) for estimation of molecular weight.
Statistics
The band intensity for each sample was calculated and normalized to that of the standard rat ovarian sample loaded on the same gel. Expression at each time point was determined relative to that at Day 16 (100%). One-way ANOVA followed by post-hoc test with Student-Newman-Keuls was used to evaluate the significance of changes in expression of FP receptor at different time points. Values are shown as the mean ± SEM and are considered to be statistically significant if P < 0.05.
RESULTS
Western Blot Analysis
In the rat uterus, a single FP protein band with an apparent mass of approximately 43 kDa was expressed in the soluble and nonsoluble membrane and cytosolic fractions of myometrium on each day of gestation. No immunoreactive bands were observed if the preimmune serum was used in place of primary antibody for Western blot analysis (Fig. 1A), and immunoreactivity was almost entirely abolished when the primary antibody was substituted by antibody preabsorbed with the peptide against which the antibody was raised (Fig. 1B). This confirmed the antibody specificity. The protein that was affinity purified from rat ovary using the FP antibody also gave a band of approximately 43 kDa on a silver-stained gel, agreeing with the predicted size of FP (Fig. 1C). When we employed an established cell fractionation protocol that gave semipurification of cell fractions, expression of FP protein was detected in myometrial cytosol, microsome, nuclei, and plasma membrane fractions by Western blot analysis (Fig. 1D).
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The expression of FP protein in rat myometrium changed significantly (P < 0.05) throughout late gestation in both the soluble membrane and cytosolic cellular fractions. The expression of FP protein in the soluble membrane fraction was significantly greater in tissues collected at Day 21.5 compared with those obtained at Day 20 of gestation (Fig. 2A) and then declined in the postpartum period. The expression of FP protein in the cytosolic fraction was significantly (P < 0.05) greater in tissues collected during labor compared with those obtained at Days 16, 18, 20, and 21 of gestation (Fig. 2B).
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However, our investigations revealed that octyl glucoside did not totally solubilize receptor from the membrane fraction. Indeed, receptor could be found in the nonsoluble membrane fraction. The total concentrations of FP receptor measured by Western blot analysis (densitometric units/mg protein; Fig. 2C) in the membrane (soluble plus nonsoluble membrane fraction) was high throughout late gestation and then fell significantly (P < 0.05) in the postpartum period. The percentage of FP receptor in the membrane that was solubilized by octyl glucoside (soluble) compared to the total FP receptor in the membrane (soluble and nonsoluble) was significantly (P < 0.05) higher during late gestation (Days 21.5 and 22) than at Days 16, 18, and 20 (Fig. 2D), which suggests that membrane structure or anchoring of receptor in the membrane changes at this time, hence altering the ability of octyl glucoside to extract receptor.
Because FP receptor is thought primarily to be a plasma membrane receptor, the FP receptor that we detected in the cytosolic fraction may be an artifact of homogenization. However, the ratio of FP protein measured in the cytosolic fraction compared to the combined soluble and nonsoluble membrane fractions was significantly (P < 0.05) higher on Day 23 than on Days 16, 18, 20, and 21 (Fig. 2E). Therefore, in late gestation, more receptor is on the membrane, but labor causes loss or movement of some receptor to the cytosol.
Immunohistochemistry
The FP receptor could be detected by immunohistochemistry in the vascular smooth muscle of myometrial vessels throughout the uterus. In addition, expression of FP receptor was predominantly localized to myocytes in both circular and longitudinal smooth muscle at all gestational ages (Fig. 3). Although a drastic change in overall staining intensity was not observed with gestational age or with labor, changes in intracellular localization of immunoreactive FP receptor were observed in myocytes (Fig. 4). At Day 16, FP receptor appeared to be associated with the nucleus as well as evenly dispersed throughout the cytosol. At Day 18, nuclear staining intensified, and cytosolic receptors formed a striated pattern that became more marked at Days 20–22. During the postpartum period (Day 23), however, the nuclear staining was decreased, and FP receptor protein again became finely dispersed throughout the cytosol. Observation of the stained sections under the light microscope revealed that FP immunostaining was greatly reduced in all tissues incubated in either preabsorbed FP antibody or preimmune IgG (Fig. 5).
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Because the striated staining pattern observed in the cytosol suggested an association between FP receptor and myofibrils, FP-receptor staining was compared with that of -smooth muscle actin in serial sections. Immunoreactive -actin appeared in circular and longitudinal smooth muscle in myometrium and in vascular smooth muscle of vessels throughout the uterus. In serial sections, a striated, intracellular staining pattern of -smooth muscle actin similar to that for the FP receptor was seen in circular and longitudinal smooth muscle (Fig. 6).
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DISCUSSION
In the present study, using Western immunoblotting with a specific polyclonal antibody, we demonstrated the presence of an approximately 43-kDa FP protein in rat myometrium during late gestation, labor, and postpartum. The specificity of the anti-FP receptor was confirmed by Western blot analysis, showing that the antiserum specifically hybridized to a protein and that binding was abolished by substituting preabsorbed FP antibody or preimmune serum in place of primary FP antibody. Cross-reactivity with other prostanoid receptors seems unlikely, because the peptide sequence used to generate the FP antibody receptor is unique to FP receptor, with no more than 2 of 12 bases of the deduced amino acids from this sequence in the first extracellular loop being homologous to other rat prostanoid receptors. In addition, the apparent molecular size of the FP receptor that we observed was in close agreement with that estimated by Kitanaka et al. [15] from sequence data and was different from those reported for other isoforms. Orlicky and Williams-Skipp [30], using a polyclonal antiserum produced against purified FP receptor from bovine corpora lutea, reported an apparent molecular weight of FP receptor in rat corpus luteum of 135 kDa, which is much larger than that predicted from sequence data and from that estimated in both our and previous studies.
Pierce et al. [31] have identified a distinct isoform for FP receptor, termed FPB receptor, with an mRNA of approximately 3.2 kilobases (kb) in the ovine corpus luteum. The FPB isoform is virtually identical to the original isoform, FPA receptor (
0.3 kb), and arises from alternative mRNA splicing. The two isoforms share nine amino acids in the carboxy terminus. However, in FPA receptor, the carboxyl terminus continues for 46 amino acids behind the 9 shared amino acids, whereas the FPB receptor terminates after only 1 amino acid. Furthermore, Ristimäki et al. [32] reported that human granulosa cells express FP-receptor mRNA with a major transcript isoform (5.2 kb) and two minor transcript isoforms (4.5 and 1.6 kb), and other investigators have shown minor FP-receptor mRNAs of various sizes [14, 17]. The biological significance of these transcript isoforms is presently unknown. However, the presence of these splice isoforms was not apparent at the protein level in this study, in which we only found a 43-kDa isoform.
Our study demonstrates a significant increase in FP-receptor protein in the myometrium with advancing gestational age, with maximal levels occurring at the time of delivery (Day 22). The changes in FP-receptor protein demonstrated here parallel changes we have previously reported for FP-receptor mRNA expression in rat [24] and human [25], and this suggests that expression may be primarily regulated at the transcriptional level. Increases in FP-receptor concentration would increase sensitivity of the myometrium to PGF2 action at term, even in the absence of increases in PGF2 concentration. However, PGF2 production also increases at term [20].
Our detection of immunoreactive FP-receptor protein in the membrane fraction of rat myometrium from Western blot analysis supports previous reports that the PGF2 receptor is a plasma membrane receptor [33, 34]. Thus, the FP receptor is apparently responsible for transducing the signal across the plasma membrane for a number of physiological responses, including cell proliferation [35], cell death [36], smooth muscle contraction [37], and inhibition of steroid synthesis [38].
However, our finding of FP receptor in the 30 000 x g supernatant (cytosolic) is intriguing. This could be either receptor present in vivo in the soluble cytosolic fraction or membrane receptor solubilized in the homogenization process and recovered in the soluble fraction. Our data reveal that the proportion of FP receptor found in the 30 000 x g fraction was significantly higher on Day 22 (labor) and Day 23 compared to earlier time points. This suggests that we are not simply solubilizing receptor in the homogenization process. Using an established subcellular fractionation technique, we find receptors in fractions other than the plasma membrane, which also agrees with our immunohistochemical data showing FP receptor in the cytosol. Collectively, these data suggest that FP receptor is found in vivo in the soluble fraction.
The observation of FP immunoreactivity in a nuclear compartment may reflect that the outer membrane of the nuclear envelope is contiguous with the endoplasmic reticulum (microsomes), where nascent FP protein is formed before trafficking to membrane or other cellular sites. To confirm the presence of FP-receptor protein in intracellular and nuclear compartments, subcellular fractionation of myometrium was performed, followed by Western blot analysis of cytosol, microsome, nuclear, and plasma membrane fractions. The presence of FP protein in each fraction strongly suggests that the immunohistochemistry data are real. These results are consistent with earlier data obtained by other investigators [39, 40], in which autoradiographic studies revealed radiolabeled prostaglandins over the nuclei. Recently, Bhattacharya et al. [41] observed EP3 and EP4 in the nuclear envelope of endothelial cells and demonstrated functionality as revealed by an increase of inducible nitric oxide synthase transcription and intracellular calcium on addition of EP3 agonist. The data obtained from the present study and others, therefore, raise the possibility of both intracellular and nuclear prostanoid receptors, possibly with a distinct mode of action from the plasma membrane receptors. Further studies are needed to clarify the mechanism of action of prostaglandins via nuclear FP receptor.
Determination of the precise distribution of FP receptor in rat uterus is essential for a complete understanding of the action of PGF2 in its different target tissues. In the present study, specific immunostaining for FP receptor was mainly localized to myocytes at all gestational ages, which provides histological evidence supporting a physiological function of PGF2 in rat uterus. In contrast to a previous study by Yang et al. [42], who demonstrated the expression of FP primarily in the circular muscle of the myometrium on Days 3–5 of mouse pregnancy, our results revealed that the expression of FP receptor was localized equally in circular and longitudinal smooth muscle of rat myometrium. We found that FP receptor gave a striated pattern of immunostaining similar to that seen for -actin, suggesting an association with myofibrils, the cells' contractile mechanism, thus reinforcing its role in uterine contractility at term.
Peripheral progesterone concentrations are high during gestation and decline before parturition in the rat; however, estrogen concentrations are low throughout gestation and rise just before parturition [21]. These changes in steroid hormones are reflected by changes in concentrations of rat myometrial nuclear estrogen and progesterone receptors [43]. Al-Matubsi et al. [44], using an ovarian autotransplanted ewe model, demonstrated that estradiol stimulates secretion of ovarian oxytocin and uterine PGF2 during the late estrous cycle. Therefore, alteration of FP-receptor protein expression in uterus during gestation and labor may be hormonally regulated.
Our findings on FP-receptor protein expression point to the potential use of FP-receptor antagonists as tocolytics. However, the possibility that other mediator(s) influence the uterine muscle contractions cannot be excluded.
FOOTNOTES
First decision: 25 September 2000.
1 Supported by NIH 1R03HD 36738. 
2 Correspondence: Leslie Myatt, Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Room 4459, 231 Albert B. Sabin Way, P.O. Box 670526, Cincinnati, OH 45267-0526. FAX: 513 558 6138; leslie.myatt{at}uc.edu
Accepted: May 15, 2001.
Received: August 17, 2000.
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