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a MRC Group in Fetal and Neonatal Health and Development,
b Departments of Physiology and Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
c MRC Group in Development and Fetal Health,
d Samuel Lunenfeld Research Institute, Mount Sinai Hospital Toronto, Toronto, Ontario, Canada M5G 1X5
e Department of Cellular and Molecular Medicine and Obstetrics and Gynecology, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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in maternal plasma increased significantly only in active labor. Messenger RNA encoding four EP receptor subtypes and FP receptor were present in myometrium, but levels did not change with labor. We suggest that spontaneous labor in sheep is associated with a progressive increase in PGHS-2 expression in a temporal and tissue-specific manner from trophoblast to maternal tissues, rather than alteration in PG receptor gene expression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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production increases at the time of labor in intrauterine tissues, leading to a rise in plasma as well as in amniotic fluid concentrations of PGs that precede the laborlike contractile activity of the myometrium [2, 3]. Primary PGs are produced from free arachidonic acid through the activity of prostaglandin H synthase (PGHS). Recent studies have demonstrated the existence of two isoforms of PGHS [4, 5]. PGHS-1 is a constitutively expressed housekeeping-type gene, while the expression of PGHS-2 can be induced rapidly by growth factors and cytokines [6]. It is the inducible PGHS-2 isoform that is specifically up-regulated in intrauterine tissues at the time of human and ovine labor [7–13].
In the sheep, PGE2 concentrations rise in fetal plasma progressively over the last 15–20 days of pregnancy, while PGF2 levels in maternal plasma rise sharply 12–24 h before delivery [14, 15]. The differential time course of appearance of these two primary PGs in fetal and maternal circulation might suggest that their site(s) of production are different. Several studies have confirmed the presence and induction of PGHS-2 in ovine placentomes at term [7–9, 16]. Recently Wu et al. [9] have shown using Western blot analysis that there is increased PGHS expression in fetal placenta and endometrium, as well as in myometrium, with spontaneous labor onset. Gibb et al. [16] localized PGHS-2 mRNA and protein by in situ hybridization and immunohistochemistry to the trophoblast cells in the fetal component (cotyledon) but not the maternal component (caruncle) of sheep placentomes at the time of parturition.
Primary PGs (PGE2 and PGF2) exert effects through specific receptors belonging to the rhodopsin-type family of receptors, part of the superfamily of G protein-coupled receptors. The action of PGF2 is mediated by the FP receptor, while four receptor subtypes (EP-1, EP-2, EP-3, and EP-4) differing in their structure, ligand-binding affinity, and signal transduction pathway subserve the action of PGE2 [17]. The EP-2 and EP-4 receptors cause relaxation of smooth muscle, while the EP-1, EP-3, and FP receptors cause smooth muscle contraction [18]. The presence of excitatory (EP-1, EP-3, FP) as well as of inhibitory (EP-2) receptors has been established in myometrium from nonpregnant and pregnant women at term [19–21]. Stimulatory but not inhibitory receptors have been detected in myometrium from nonpregnant and pregnant sheep [22, 23]. Even though PGs have been well established as stimulators of myometrial contractility at term, there is no information concerning changes in PG receptor expression in intrauterine tissues from the time of uterine activation until delivery.
Although previous studies have established the presence and induction of PGHS-2 at term in sheep intrauterine tissues, the pattern and site of expression of this enzyme with the progression of term spontaneous labor have not been elucidated. We hypothesized that labor would involve a progressive evolution of PGHS-2 expression from fetal to maternal tissues, and would involve both increased synthesis of agonist PGs (increased PGHS-2 expression) and potential for myometrial responsiveness, reflected in increased mRNA for stimulatory PG receptors or an altered ratio of stimulatory to inhibitory PG receptors in myometrium. Therefore, we examined the cellular distribution and level of expression of PGHS-2 mRNA and protein in the placenta and maternal uterine tissues with the onset of labor. We determined the levels of mRNA encoding the EP-1, EP-2, EP-3, EP-4, and FP receptors using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in sheep myometrium during this same time period. The changes in PGHS-2 expression were correlated with levels of PGE2 in fetal plasma and with 13,14-dihydro-15-keto-PGF2 (PGFM), a stable metabolite of the primary prostaglandin, PGF2, in the maternal circulation; they were then related temporally to the evolving pattern of myometrial activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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Pregnant ewes of mixed breed and of known gestational age (day of natural mating = Day 0) were used in these studies. Catheters were implanted into mothers and fetuses under general anesthesia between Days 121 and 126 of gestation as described previously [24], and pregnancies were allowed to progress to term. Fetal and maternal health was assessed by monitoring blood gases and pH each day (Blood Gas System AVL 995 and Co-oxylite AVL 92; AVL Scientific Corporation, Roswell, GA). Fetal (2.0 ml) and maternal (4.0 ml) blood samples were collected every 12 h and immediately prior to death into heparinized tubes containing indomethacin (200 µl of 1 mg/ml indomethacin-heparinized saline solution). Plasma was obtained by centrifugation at 1500 x g for 10 min at 4°C and stored at -20°C. Uterine activity (electromyographic [EMG] activity from myometrial electrodes; intrauterine pressure [IUP] from amniotic fluid catheter) was monitored continuously starting on Day 135 until the animals were killed. The uterine EMG signal was processed by a Grass wide-band AC preamplifier, model 7P511J; and the IUP was monitored by connecting the amniotic fluid catheter to a Statham P23 pressure transducer (Gould, Medical Product Division; Oxnard, CA) that was calibrated to give a 0.66-mm deflection on the polygraph paper for every 1 mm Hg rise in IUP, covering a range of 0–60 mm Hg. The signal from the pressure transducer was processed through a Grass low-level DC preamplifier (7P122D). Both signals (EMG and IUP) were recorded using a Grass 78 D EEG & Polygraph data recording system (Grass Instruments, Quincy, MA). Animals were killed with an overdose of Euthanyl (sodium pentobarbital; MTC Pharmaceuticals, Cambridge, ON, Canada).
Timing of Tissue Collection
Tissues were collected at three distinct stages during the progression of labor: from ewes not in labor (NIL, 140–145 days gestation, n = 5), from ewes in early labor (EL, 143–149 days, n = 6), and from animals in active labor (L, 145–149 days, n = 6). Progression of labor was assessed during the last 6 h before death based on the criteria discussed by Lye et al. [25]. NIL animals displayed long-wave myometrial contractions lasting 5–8 min (contracture) at a frequency of ~3–4 per 2 h on their EMG traces with a parallel slow-wave rise of the IUP of < 5 mm Hg on average [26]. Those in EL had uterine contractures breaking up into contractions (duration < 1.0 min, frequency of 
30 per 2 h) that were mostly coincident with small but precise rises in IUP (on average 
5 mm Hg), spread approximately 0.5–1 min apart, during the last 2 h before the animal was killed. L animals had uterine activity traces containing mainly contractions (duration < 1.0 min, frequency > 30 per 2 h) that were associated with sharp rises (> 5 mm Hg) in IUP for at least 2 h prior to death.
After animals were killed, some tissues were fast frozen in liquid nitrogen for RT-PCR and Western blot analysis. Samples of myometrium were separated from endometrium by blunt dissection, or were rolled together to retain tissue integrity. Other tissues were slow frozen on dry ice (solid CO2) for in situ hybridization (placentomes, uterus, and fetal membranes as one unit) or fixed in 4% paraformaldehyde and 0.2% glutaraldehyde for immunohistochemistry (placentomes, uterus, and fetal membranes as one unit). Not all tissues were collected or available from all animals, hence the variations in the number of animals indicated in the text and figure legends.
In Situ Hybridization (ISH) for PGHS-2
Sections (10 µm) of slow-frozen tissues (placentomes and full membranes) were used for ISH analysis. The 50-mer antisense nucleotide probe (GGG ACA GCC CTT CAC GTT ATT GCA GAT GAG AGA CTG AAT TGA GGC AGT GT) used for ISH of PGHS-2 was complementary to bases 1734–1783 of the human PGHS-2 gene [4]. The probe was synthesized in the molecular biology facility at the University of Ottawa using an Oligo 1000 DNA synthesizer (Beckman Instruments, Fullerton, CA) and was purified by HPLC. The sequence of the DNA had been verified previously also at the University of Ottawa [27]. Hybridization with the corresponding sense probe (prepared in a similar fashion) served as a negative control.
ISH was performed as previously described [16, 28]. Briefly, probes were labeled using terminal deoxynucleotide transferase and -33P-labeled deoxyadenosine 5'-triphosphate, tetra(triethylammonium) salt (10 mCi/ml; NEN DuPont Canada, Mississauga, ON, Canada). The labeled probe was purified using a Nensorb 20 column (NEN DuPont Canada) and used at a concentration of 10 000 cpm/µl. Slides were incubated overnight at 45°C in a moist chamber with the radiolabeled oligonucleotide probe in hybridization buffer. Positive and negative control slides were also processed with the antisense or sense probes, respectively. After the overnight incubation, slides were washed in single-strength SSC (0.15 M sodium chloride and 0.015 M sodium citrate) containing 10 mM dithiothreitol (DTT) at room temperature for 20 min and in single-strength SSC (containing 10 mM DTT) at 55°C for 45 min, and rinsed in single-strength SSC, 0.1-strength SSC, and distilled (d)H2O at room temperature for 10 sec each. Slides were finally dehydrated in a graded series of ethanols, air dried, and exposed to x-ray film (Biomax; Eastman Kodak, Rochester, NY) in conjunction with 14C standards (RPA504; Amersham Life Science, Buckinghamshire, England) to allow computer-assisted quantification of autoradiograms within the linear range of the film (Image Research, St. Catherine, ON, Canada). Silver emulsion autoradiography was carried out according to standard procedures using Ilford K5 (Mobberley, UK) liquid emulsion. Emulsion sections were developed according to standard procedures and counterstained with Carrazi's hematoxylin.
Immunohistochemistry (IHC)
Placentomes were paraffin embedded, sectioned at 5 µm, and processed for IHC as described previously [29] using primary antiserum generated against the C-terminus of human PGHS-2 (used at 1:250 dilution; Oxford Biomedical Research, Oxford, MI; product #PG27). The avidin-biotin-peroxidase technique for immunostaining was utilized with diaminobenzidine as substrate [30]. Slides were counterstained with Carrazi's hematoxylin followed by dehydration in a graded series of ethanols, cleared in xylene, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). For negative controls, the primary antibody was either substituted with antibody dilution buffer or preimmune rabbit serum or was preabsorbed with 5 µg of native ovine PGHS-2 standard (Oxford Biomedical Research; product #NP 04) overnight at 4°C (shaking) before being used for the IHC.
Western Analysis
Protein extraction Frozen placental and myometrial samples were pulverized with mortar and pestle under liquid nitrogen and homogenized (Ultra Turrax T-25; Janke & Henkel, IKA-Labortechnik, Germany) for 1 min on ice in RIPA lysis buffer, which comprised 50 mM Tris-HCl, pH 7.5 (Trizma hydrochloride; Sigma Chemical Co., St. Louis, MO), 150 mM NaCl (Fisher), 1% v:v Triton X-100 (Fisher), 1% w:v sodium deoxycholate (deoxycholic acid; Sigma), 0.1% w:v SDS (Fisher), and 100 µM sodium ortho-vanadate and complete, mini EDTA-free protease inhibitors (Boehringer Mannheim Biochemicals, Mannheim, Germany). Fetal liver was processed as negative control. Homogenates were transferred into Eppendorf tubes (1.5 ml; Diamed, Mississauga, ON, Canada) and centrifuged at 15 000 x g at 4°C for 15 min. Supernatants were collected and transferred to new Eppendorf tubes, and protein concentrations were determined by the Bradford assay [31] using BSA as standard (Bio-Rad, Richmond, CA) and absorbance at 595 nm.
Western blotting Protein samples (5 µg/lane for placentomes, 100 µg lane for myometrium and liver) were separated by PAGE as previously described [32] using a 4–12% gradient premade polyacrylamide gel (Tris-Glycine Gel, 1.0 x 10 well, Novex; Helixx Technologies, Scarborough, ON, Canada) and electrophoretically transferred to 0.45-µm polyvinylidene diflouride membrane (Millipore, Bedford, MA). Membranes were air dried overnight after electroblotting, rinsed in methanol (10 sec), and rehydrated in dH2O for 3 min. All incubations took place at room temperature with constant agitation. Blots were blocked in 5% skim milk powder in TBS-T (20 mM Tris-base [Boehringer Mannheim], 137 mM NaCl [Fisher], and 0.1% Tween 20 [Sigma]) for 1 h; they were then rinsed in TBS-T (once for 15 min and twice for 5 min). Primary antibody (PG 27; Oxford Biomedical Research) was diluted (1:1000) in blocking solution and incubated with the blots for 1 h. Blots were rinsed in TBS-T once for 15 min and twice for 5 min. Rabbit secondary antiserum conjugated to horseradish peroxidase was also diluted in blocking solution (1:1000, anti-rabbit immunoglobulin, horseradish peroxidase; Amersham Life Science) and incubated with membranes for 1 h, followed by one 15-min and four 5-min washes in TBS-T. Detection of proteins on Western blots was accomplished using the Amersham ECL Detection System (Amersham International, Buckinghamshire, England). Blots were placed in a 1:1 mixture of detection reagents #1 and #2 for 1 min, drained slightly, placed in a hybridization bag, and exposed to x-ray film (X-Omat Blue XB-1; Eastman Kodak). The relative intensity of protein signals was quantified using computerized image analysis (Image Research; laser scanner and ImageQuant software from Molecular Dynamics, Sunnyvale, CA).
Isolation of Total RNA
Frozen tissue samples (200–300 mg) were pulverized in liquid nitrogen, transferred into polypropylene tubes (12 ml; Becton Dickinson, Rutherford, NJ), and homogenized in 2.5 ml Trizol reagent (Gibco/BRL, Life Technologies, Grand Island, NY) at room temperature. Total RNA was extracted from tissues using a method based on principles described by Chomczynski and Sacchi [33].
Design of Primers
Primers for the various PGE2 receptor subtypes (EP1, EP2, EP3, and EP4), as well as those for ß-actin, were initially designed by Tai et al. [34], targeting highly conserved regions among species for each gene (Table 1). Sequence alignment for cDNAs for a specific gene from different species was performed using the GCG software package (Ver. 7.0; Genetic Computer Group, Madison, WI). Primers for the PGF2 receptor (FP) were designed using the published sequence of the ovine FP receptor [35] and were analyzed and confirmed using an Oligo Software Package (Primer Analysis Software, Ver. 5.0; National Biosciences, Plymouth, MN). Attempts were made to design the primer sets to span an intron; thus any contamination of the original RNA extract with DNA (genomic or otherwise) would yield multiple bands instead of one specific band when visualized on an agarose gel. All primer sets spanned an intron except for primers targeting EP-4.
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RT-PCR |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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PCR products were all sequence verified by the Samuel Lunenfeld Sequencing Facility using an ABI Prism, 377 DNA Sequencer and provided software (Perkin-Elmer, Irvine, CA). The sequences were then compared and verified against the database in GenBank on the World Wide Web using a BLAST search. All sequences showed high homology (95–100%) to the published ovine PG receptor sequences, thus confirming their identity.
Semiquantitative RT-PCR
Using a protocol modified from that previously described by Kinoshita et al. [36] and Choi-Lungdberg and Bohn [37], semiquantitative RT-PCR was performed in order to compare the level of expression of the PG receptors and ß-actin (internal control) mRNA in myometrial and placental samples of the three groups of animals (NIL, EL, and L). For semiquantitative analysis of gene expression, RT-PCR reactions were set up as described above. PCR was conducted at three cycles in the linear range for each message, still allowing its detection (Table 1). Electrophoresis of PCR products took place as described above, followed by photography of agarose gels under UV light using Polaroid Positive/Negative Film (Type 665; Polaroid Corp., Cambridge, MA). The relative intensity of cDNA signals for the PG receptors was quantified using computerized image analysis performed on the negative films (Image Research; Laser Scanner by Molecular Dynamics; software by ImageQuant). In order to control for possible differences in the initial amount of total RNA template used in each sample for RT-PCR, all values were normalized against ß-actin. This was achieved by dividing the optical density values obtained for each of the three cycles of the receptors by those obtained for each of the three cycles of ß-actin.
RIA
Extraction of plasma samples for fetal PGE2 and maternal PGFM RIAs was performed as previously described [38].
RIAs for fetal PGE2 and maternal PGFM were performed and measurements quantified as reported previously [38, 39]. The intraassay coefficients of variation for PGE2 and PGFM were 13.9% and 1.5%, respectively.
Statistical Analysis
Results were examined by one-way ANOVA followed by Student-Newman-Keuls test or the Student's t-test as appropriate, with significance set at P < 0.05. For some measurements, not all tissues were available, and the number of different tissues analyzed is indicated in the text and/or figure legend. The data were assessed for normality and homogeneity of variance, and those that did not satisfy either underwent mathematical manipulation according to Zivin and Bartko [40]. Data are presented as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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EMG traces of NIL animals were characterized by long-wave contractions lasting 5–8 min (contracture), with rises in IUP of < 5 mm Hg, at a frequency of ~3–4 per 2 h (Fig. 1A). Animals in the EL group had contractures breaking up into contractions (lasting ~0.5–1.0 min) that in most cases were coincident with small rises in IUP (Fig. 1B). L animals had traces containing mainly contractions that were strongly associated with sharp rises in IUP (Fig. 1C).
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There was a significant (P < 0.01) increase in the average number of myometrial contractions with the onset of labor, measured during the last 6 h before animals were killed, from 1.11 ± 0.5 contractions/2 h (NIL) to 18.8 ± 2.0 (EL) and further (P < 0.01) to 49.6 ± 2.9 contractions/2 h (L) (Fig. 1D). Similarly, the values for IUP rose significantly (P < 0.01) from an average of 3.6 ± 0.9 mm Hg (NIL) to 5.5 ± 0.5 mm Hg (EL) and 11.6 ± 2.7 mm Hg (L) in the last 2 h before animals were killed (Fig. 1E).
Pattern of Expression of PGHS-2 mRNA in Intrauterine Tissues with Labor Onset
The mean value of PGHS-2 mRNA in placental tissue was higher in EL and L compared to NIL animals, as analyzed by film autoradiography of ISH of placental sections, and approached statistical significance (P = 0.066) between groups (Fig. 2A). Analysis of mRNA expression levels in endometrium indicated that levels of expression did not change with EL but that mean values tended to increase with active labor, lagging slightly behind the changes in placental PGHS-2 mRNA expression (Fig. 2B).
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Pattern of Expression of PGHS-2 Protein in Intrauterine Tissues with Labor Onset
The pattern of change in immunoreactive (ir)-PGHS-2 was similar to the pattern of expression for PGHS-2 mRNA. Ir-PGHS-2 localized to distinct regions of placental and full-membrane tissue sections (Fig. 3). In placental tissue, ir-PGHS-2 was confined to the trophoblast, was already evident in tissues from NIL animals, and increased with onset of labor (Fig. 3, A–C). The intensity of ir-PGHS-2 staining in endometrial epithelium was greater in EL and L than in NIL animals (Fig. 3, D–F). Staining was also detected in myometrium, but remained relatively constant between the groups (Fig. 3, D–F). There was no staining for PGHS-2 in control incubations performed using antiserum preabsorbed with purified ovine PGHS-2 or with preimmune rabbit serum (data not shown).
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Examination of placental and full-membrane silver emulsions and immunohistochemical sections confirmed localization of PGHS-2 mRNA and protein to the trophoblast compartment in placentomes, to the chorionic epithelium, and to the maternal endometrial epithelium (Fig. 3, C and F, and G and H).
Further analysis by Western blotting revealed no significant change in ir-PGHS-2 (72-kDa protein) in placentomes with labor, but a significant (P < 0.01, Student's t-test) increase in the level of PGHS-2 protein in myometrium between the NIL and L groups (Fig. 4, A and B). The level of PGHS-2 protein in the placentomes was much greater than that in the myometrium because only 5 µg of placental protein was loaded onto the gel in contrast to 100 µg of myometrial protein. While Western blot analysis revealed only one band for PGHS-2 in placentomes (72 kDa), several preabsorbable protein bands were present in myometrium, ranging from ~50 to 120 kDa in addition to the expected 72- and 74-kDa bands (data not shown). This may account for the lack of change in PGHS-2 protein observed in myometrium with progression of parturition with the use of IHC.
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PG Levels in Fetal (PGE2) and Maternal (PGFM) Plasma with Labor
Levels of PGE2 in fetal plasma closely followed the pattern of increase in placental PGHS-2 mRNA and protein, whereas PGFM in maternal peripheral plasma closely followed the endometrial and myometrial pattern of PGHS-2 mRNA and protein expression. Thus, fetal PGE2 levels progressively increased with the onset of labor from 370 ± 43 pg/ml (NIL) to 831 ± 64 pg/ml in the L group (P < 0.01, ANOVA; L compared to NIL and EL groups; Fig. 5A). Changes in maternal plasma PGFM concentration followed the pattern of PGHS-2 mRNA and protein in endometrium. PGFM levels remained low within the NIL and EL groups but then increased at the time of active labor (L; P < 0.01, ANOVA; Fig. 5B). Thus the general profile of PGFM increase prepartum was similar to that of uterine IUP, both rising significantly in the L group.
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PG Receptor mRNA Expression with Labor Onset in Myometrium
Products for all four PGE2 receptor subtypes (EP-1, -2, -3, -4), as well as that for the PGF2 receptor (FP), were identified in the myometrium with the onset of term spontaneous labor (Fig. 6). However, there was no significant change in the level of mRNA for any PG receptor subtype with onset of spontaneous labor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRT-PCRRESULTSDISCUSSIONREFERENCES |
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output) in maternal plasma, thus corresponding to the observed temporal pattern of PGHS-2 message and protein expression between fetal and maternal tissues. We demonstrated the presence of all four subtypes of the PGE2 receptor (EP-1, -2, -3, and -4) as well as the presence of the PGF2 receptor (FP) in ovine myometrium with the onset of term spontaneous labor in sheep, but using semiquantitative RT-PCR we were unable to show significant changes in the expression of any PG receptor mRNA in this tissue with the progression of parturition. Thus, changes in PG synthesis appear to be of greater importance than alterations in expression of PG receptor subtypes in ovine intrauterine tissues with progression of labor. Complete resolution of this issue will require measurement of PG receptor protein and determination of PG binding kinetics.
The changes in maternal PGFM concentrations were better related temporally than fetal PGE2 levels to the changes in contractile activity (EMG) of the uterus, as well as with changes in uterine IUP. This relationship is consistent with PGF2 of maternal origin as provider of the major drive to myometrial contractility, while the earlier rise in fetal PGE2 may be of greater importance to the endocrine and biophysical responses of the fetus and the induction of placental P450C17 expression. In this sequence of changes, the factors regulating PGHS-2 expression in placental trophoblast and maternal uterus may be different, and up-regulation of maternal PGHS-2 may be analogous to that of other contraction-associated protein genes at the time of labor [1].
Our results are consistent with reports showing that during adrenocorticotropic hormone-induced preterm labor the increase in the production and plasma levels of PGs precedes the occurrence of increased myometrial contractile activity, and that levels of PGE2 in fetal plasma increase significantly within 24 h of spontaneous or induced parturition whereas PGF2 levels in maternal plasma increase with the contractions of labor [14, 38, 41]. Thus we suggest that there is a temporal sequence in PG production in intrauterine tissues that starts in fetal tissues and progresses into maternal tissues with the progression of ovine parturition.
Our results are consistent with previously reported changes in PGHS-2 mRNA and protein in placentomes of sheep in late gestation and at term. Wimsatt et al. [7] reported that PGHS-2 protein increased in placental tissue at term in association with increased PGHS-2 activity. In addition, Wu et al. [9] reported a further rise in PGHS-2 protein expression with the onset of labor in the fetal placenta as well as the endometrium and myometrium. Rice and coworkers [8] also showed that PGHS-2 mRNA increased in placental tissue at term and, as in the present study, did not demonstrate further increases in PGHS-2 mRNA expression in placentomes at the time of active labor. Clearly, PGHS-2 is not the only enzyme determining PG output. We anticipate changes in phospholipases, specific PG isomerases, and 15-hydroxyprostaglandin dehydrogenase that could all contribute to net PG synthesis and output. Changes in PG concentrations in plasma were significant whereas those of PGHS-2, one enzyme in the cascade, only approached statistically significant differences. Thus, our results may also reflect the importance and up-regulation of other members of the enzyme cascade responsible for the production of PGs.
We have localized PGHS-2 mRNA and protein, using ISH and IHC to the trophoblast cells of the placenta, confirming the report of Gibb et al. [16]. In addition, we found discrete localization of PGHS-2 mRNA and protein to the chorionic and endometrial epithelium in association with labor. There was also a dramatic (over 30-fold) increase in ir-PGHS-2 protein in the myometrium with the onset of labor. The pattern of change in ir-PGHS-2 in myometrium differed between IHC and Western blotting, the latter indicating the presence of other immunoreactive bands that were absent in preabsorbed (negative) controls. Similar observations have been made in human fetal membranes [42–44], and it appears that these different bands reflect inactive as well as active PGHS-2 protein.
The present results cast doubt on the suggestion that the maternal compartment of the ovine placenta is a major site of PG production [45, 46], a discrepancy that may be explained by the difficulty in separating the maternal and fetal compartment of individual placentomes.
Previous researchers had inferred the presence of excitatory PG receptors (EP-1, EP-3, and FP) in myometrium from nonpregnant and pregnant sheep, through pharmacological characterization, but had not detected the presence of the EP-2 and EP-4 receptors [22, 23]. Those studies focused only on samples of myometrium collected at term, whereas here we report the presence of PG receptor gene expression at term and with the onset of parturition in myometrium. Molnar and Hertelendy [47] reported that whereas PGE2 and PGF2 binding increased in rat myometrium in late gestation, there were also no significant changes with labor. In vitro studies with sheep myometrial strips showed that although there is an increase in receptor-mediated myometrial activity in response to PGE2 and PGF2 during gestation, this reaches maximal values by Days 126–135 of gestation and does not increase further with labor [48]. Thus a body of evidence is emerging that indicates that increased PG synthesis and uterotonic drive to the myometrium, rather than altered PG receptor expression or binding, is of greater importance in late pregnancy. However, Brodt-Eppley and Myatt [49] have also reported that expression of stimulatory PG receptors increases and inhibitory PG receptors decrease in rat myometrium at the time of labor; thus this issue requires further examination.
In summary, the present study suggests that there is a temporal and tissue-specific regulation and pattern of synthesis of PGs from fetal to maternal tissues, reflected in changes in PGHS-2 mRNA, PGHS-2 protein, fetal PGE2, and maternal PGFM concentrations during the course of ovine parturition. Furthermore, increased PGHS-2 expression and increased PG output at term are of greater importance than changes in PG receptor gene expression in myometrium. We speculate that a further understanding of these temporal associations may be informative with respect to similar patterns of change at the time of human labor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Medical Research Council of Canada (MT-14097). 
2 Correspondence: Sandor Gyomorey, Department of Physiology, University of Toronto, 1 King's College Circle, Medical Sciences Bldg., Room 3344, Toronto, ON, Canada M5S 1A8. FAX: 416 978 4940; j.challis{at}utoronto.ca
Accepted: October 22, 1999.
Received: June 11, 1999.
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P. L. Grigsby, J. J. Hirst, J.-P. Scheerlinck, D. J. Phillips, and G. Jenkin Fetal Responses to Maternal and Intra-Amniotic Lipopolysaccharide Administration in Sheep Biol Reprod, May 1, 2003; 68(5): 1695 - 1702. [Abstract] [Full Text] [PDF]
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S. Gupta, S. Gyomorey, S. J. Lye, W. Gibb, and J. R. G. Challis Effect of Labor on Glucocorticoid Receptor (GRTotal, GR{alpha}, and GR{beta}) Proteins in Ovine Intrauterine Tissues Reproductive Sciences, April 1, 2003; 10(3): 136 - 144. [Abstract] [PDF]
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R.L. Martin, W.L. Whittle, A.C. Holloway, S. Gyomorey, W. Gibb, S. Lye, and J.R.G. Challis Ontogeny and Regulation of Ovine Placental Prostaglandin E2 Synthase Biol Reprod, September 1, 2002; 67(3): 868 - 873. [Abstract] [Full Text] [PDF]
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 W. X. Wu, X. H. Ma, N. Unno, and P. W. Nathanielsz In Vivo Evidence for Stimulation of Placental, Myometrial, and Endometrial Prostaglandin G/H Synthase 2 by Fetal Cortisol Replacement after Fetal Adrenalectomy Endocrinology, September 1, 2001; 142(9): 3857 - 3864. [Abstract] [Full Text] [PDF]
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 C. B. Chan, D. De Leo, J. W. Joseph, T. S. McQuaid, X. F. Ha, F. Xu, R. G. Tsushima, P. S. Pennefather, A. M. F. Salapatek, and M. B. Wheeler Increased Uncoupling Protein-2 Levels in {beta}-cells Are Associated With Impaired Glucose-Stimulated Insulin Secretion: Mechanism of Action Diabetes, June 1, 2001; 50(6): 1302 - 1310. [Abstract] [Full Text]
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W.L. Whittle, F.A. Patel, N. Alfaidy, A.C. Holloway, M. Fraser, S. Gyomorey, S.J. Lye, W. Gibb, and J.R.G. Challis Glucocorticoid Regulation of Human and Ovine Parturition: The Relationship Between Fetal Hypothalamic-Pituitary-Adrenal Axis Activation and Intrauterine Prostaglandin Production Biol Reprod, April 1, 2001; 64(4): 1019 - 1032. [Abstract] [Full Text]
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 R. Agarwal, A. Loganath, A.C. Roy, Y.C. Wong, and S.C. Ng Expression profiles of interleukin-15 in early and late gestational human placenta and in pre-eclamptic placenta Mol. Hum. Reprod., January 1, 2001; 7(1): 97 - 101. [Abstract] [Full Text] [PDF]
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K.J. McKeown, J.R.G. Challis, C. Small, L. Adamson, A.D. Bocking, M. Fraser, D. Rurak, K.W. Riggs, and S.J. Lye Altered Fetal Pituitary-Adrenal Function in the Ovine Fetus Treated with RU486 and Meloxicam, an Inhibitor of Prostaglandin Synthase-II Biol Reprod, December 1, 2000; 63(6): 1899 - 1904. [Abstract] [Full Text]
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