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Regular Article |
a Canadian Institutes for Health Research Groups in Fetal and Neonatal Health and Development, Departments of Physiology and Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
b Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cortisol, estradiol, placenta, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, with gestation and parturition was hypothesized to be the result of estrogen-stimulated PG synthesis by intrauterine tissues [5]. However, recent evidence in sheep has prompted investigators to suggest that the upregulation of PGE2 production is a direct result of cortisol stimulation of the expression and activity of prostaglandin H synthase type 2 (PGHS-2) in ovine placental trophoblast tissue [6, 7]. Increasing numbers of studies have focused on the regulation of PG production with the onset of labor and delivery, with particular attention paid to the expression and regulation of PGHS-2, the inducible form of PGHS. The substrate for PG synthesis, arachidonic acid, is first metabolized by PGHS (types 1 or 2) to an intermediate PG, PGH2, which is converted by a specific PG isomerase/synthase to one of a variety of bioactive PGs. Synthesis of PGE2 therefore is not only dependent on PGHS (type 1 or type 2) activity but is also dependent on the expression and activity of the specific PGE isomerase/synthase (PGES). PGES is a member of a protein superfamily consisting of membrane-associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family) [8]. The activity of the enzyme is dependent on glutathione, which may act as a coenzyme [8–10]. Although purification of sheep and bovine seminal vesicle PGES has been difficult, human PGES has recently been identified and characterized [8]. Expression of the PGES enzyme has been detected in various tissues, including the human placenta [8]; however, there is no published information on the localization, expression, or regulation of this enzyme in the placental tissue in relation to the onset of parturition.
In sheep, levels of PGE2 rise gradually in fetal plasma over the last 15–20 days of gestation [11]. To date, this rise has been correlated positively with a rise in placental PGHS-2 mRNA, protein, and activity [7, 11–15]. In addition, Whittle et al. [6] demonstrated that PGHS-2 mRNA and protein expression in the sheep placentome were stimulated by fetal cortisol infusion and were correlated with an increase in fetal plasma PGE2 concentration. Furthermore, the effects of cortisol were independent of a rise in estrogen [6]. However, in several cell-culture systems PGES and the inducible form of the PGHS enzyme (PGHS-2) couple functionally to increase PGE2 levels [9, 16]. Therefore we could not exclude the possibility that PGES and PGHS-2 act in concert to increase PGE2 output. We hypothesized that PGES expression in the ovine placenta rises through the end of gestation to the onset of labor, with a similar time course for the ontogenic expression of PGHS-2. In addition, we hypothesized that cortisol increases PGES expression, independent of a change in estrogen, as has been observed for PGHS-2 [6].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Placental tissues collected from three groups of pregnant ewes (ontogeny group, labor group, and treatment group) of mixed breed and known gestational age (day of natural mating = Day 0) were used in these studies. Studies were performed in accordance with the guidelines of the Canadian Council for Animal Care and the National Institutes of Health and according to protocols approved by the Animal Care Committees of the University of Toronto.
Ontogeny group The first group of animals (n = 28) consisted of both noninstrumented animals and animals catheterized as controls for vehicle infusion that were killed for tissue collection at specific gestational ages (term = 147–150 days). The first set of animals (n = 14) ranged in gestational age from Day 65 to Day 80 at the time they were killed and represented midgestation (MG). The second set of animals in this group (n = 14) represented late gestation (LG) and ranged in gestational age from Day 120 to Day 128 at the time they were killed. In each group, 10 tissues were processed for immunohistochemistry and four were processed for in situ hybridization (ISH).
Labor group
In this group, maternal and fetal catheters were implanted for blood sampling, and uterine activity was monitored via an electrode sewn into the myometrium (n = 17). The animals were subsequently killed at term before labor (not in labor [NIL] = Days 140–145; n = 5); ewes in early labor (EL = Days 143–149; n = 6); and ewes in active labor (L = Days 145–149; n = 6). Determination of progression of labor was assessed during the last 6 h before the animal was killed and was based on the criteria discussed by Lye et al. [17]. Animals NIL displayed long wave contractions lasting 5–8 min (contracture) at a frequency of three or four contractures in 2 h on their electromyogram traces with a parallel slow wave rise of the intrauterine pressure (IUP) of <5 mm Hg on average. Those going into EL had their contractures breaking up into contractions (duration of <1.0 min, frequency of 
30 contractures in 2 h) that were mostly coincident with small but precise rises in IUP (on average 
5 mm Hg) during the last 2 h before being killed and were approximately 0.5–1 min apart. The animals in L had traces containing mainly contractions (duration <1.0 min , frequency >30 contractures in 2 h) that were strongly paralleled by sharp rises (>5 mm Hg) in IUP 2 h prior to the time the ewes were killed. Details of the experimental protocol have been published elsewhere [7, 11].
Treatment group In this group (n = 20) maternal and fetal catheters were implanted for blood sampling and intrafetal infusion. Uterine activity was monitored via an electrode sewn into the myometrium. Details of the experimental protocol have been published elsewhere [6]. Fetuses at 125–128 days gestation received a continuous infusion of saline (3 ml/h; n = 10) or cortisol (1.35 mg/h; n = 10) (Steraloids, Wilton, NH). After 24 h of infusion, a subset (n = 5) of each group received an additional intrafetal infusion of 1.44 mg/h 4-hydroxyandrostendione (4-OHA) (Lentaron; CIBA-Geigy, Basel, Switzerland), a competitive suicide inhibitor of the P450aromatase enzyme, that completely attenuated the cortisol-induced rise in maternal and fetal estrogen [6]. The infusions continued until 80 h (a time sufficient to induce a uterine contraction pattern consistent with labor in the animals infused with cortisol alone), and the animals were then killed.
Tissue Collection
Ewes were killed with an overdose (25–30 ml i.v.) of sodium pentobarbital (Euthanyl, 240 mg/ml; MTC Pharmaceuticals, Cambridge, ON, Canada). The uterus was exposed and opened, and the fetus(es) was removed and killed with 5 ml sodium pentobarbital via cardiac puncture. Placentomes were collected from the pregnant horn of the uterus and were 1) fast frozen in liquid nitrogen and placed into vials for Western blot analysis, 2) slowly frozen on dry ice (solid CO2) for ISH, or 3) fixed in 4% paraformaldehyde (Sigma, St. Louis, MO) and 0.2% glutaraldehyde (8% EM grade; Polyscience, Warrington, PA) for immunohistochemistry (IHC).
ISH for PGES
The 50-mer antisense nucleotide probe 5'-CTTCTTCCGCAGCCTCACTTGGCCCGTGATGATGGCCACCACGTACATCT-3' used for ISH of PGES was complementary to bases 95–144 of the human PGES gene [18]. 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 cartridge purification. A corresponding sense probe was prepared in a similar fashion to serve as a negative control.
Probes were labeled using terminal deoxynucleotide transferase (Gibco BRL, Burlington, ON, Canada) and [-35S]dATP (12.5 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 5000 cpm/µl.
The method used for ISH has been described previously by Matthews et al. [19]. Tissue sections (10 µm) were mounted on Fisher Superfrost glass slides (Fisher Scientific, Nepean, ON, Canada), fixed with 4% paraformaldehyde, dehydrated through graded ethanol, and stored in 95% ethanol at 4°C. Slides were removed from ethanol and allowed to air dry at room temperature. Tissues were incubated overnight at 45°C in a moist chamber with the radiolabeled oligonucleotide PGES probe diluted in hybridization buffer. Hybridization buffer was composed of 4x saline sodium citrate (SSC; 1x SSC is 150 mM sodium chloride, 15 mM sodium citrate) (Sigma), 50% deionized formamide (Gibco BRL), 10% dextran sulfate (Pharmacia LKB, Baie d'Urfe, PQ, Canada), 25 mM sodium phosphate (pH 7.0), 1 mM sodium pyrophosphate, 200 µg/ml acid-alkali hydrolyzed salmon sperm DNA, 100 µg/ml polyadenylic acid, 120 µg/ml heparin, 40 mM dithiothreitol (DTT; Sigma), and 5x Denhardt solution. Negative control slides were processed with sense probes. After overnight incubation, slides were washed in 1x SSC (containing 10 mM DTT) at room temperature for 20 min and then again in 1x SSC (containing 10 mM DTT) at 55°C for 45 min. Slides were rinsed in graded SSC, dehydrated in graded ethanol, air dried, and exposed to x-ray film for 2 days (Biomax; Eastman Kodak, Rochester, NY) in conjunction with a 14C standard (RPA504; Amersham Life Science, Baie d'Urfe, PQ, Canada) to establish linearity. The autoradiograms were analyzed using computerized analysis software (Image Research, St. Catharines, ON, Canada). Relative optical density (ROD) values were obtained using a minimum of four sections per animal. Silver emulsion radiography was carried out by dipping the slides into Ilford K5 (Mobberley, UK) liquid emulsion and exposing them for 3 wk. Emulsion sections were developed in Developer D-19 (Eastman Kodak), counterstained with Carazzi hematoxylin, dehydrated, and mounted with Permount (Fisher).
IHC for PGES
Immunoreactive PGES was localized in paraffin-embedded ovine placentome tissue sections (10 µm) using a polyclonal antibody raised in rabbits against a peptide corresponding to amino acids 59–75 (CRSDPDVERSLRAHRND) of human PGES (Cayman Chemical, Ann Arbor, MI) at a dilution of 1:100 in antibody dilution buffer (1% BSA, 0.02% sodium azide in 0.01 M PBS [150 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.5 mM monosodium dihydrogen phophate, pH 7.4]). The PGES antibody reacts with human, murine, and ovine PGES [8].
Sections were deparaffinized with xylene substitute (EM Diagnostic Systems, Gibbstown, NJ) and rehydrated in graded ethanol washes. Endogenous peroxidase activity was inhibited by treatment with 0.3% hydrogen peroxide in PBS for 30 min. The sections were then washed in PBS and incubated for 20 min with immune serum to block nonspecific binding. Tissue sections were incubated overnight at 4°C with primary antibody. Primary antibody binding was visualized using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). A 2-h incubation with biotinylated secondary antibody was followed by a PBS wash and a 2-h incubation with ABC (avidin-biotin-peroxidase complex). Diaminobenzidine was used for color development. The sections were counterstained with Carazzi hematoxylin, dehydrated, and mounted with Permount (Fisher). For negative controls, the primary antibody was either replaced with antibody dilution buffer or was preabsorbed with the PGES antigen (Cayman Chemical) overnight at 4°C before being used for IHC.
Western Analysis
Frozen placental samples were homogenized on ice for 1 min in RIPA lysis buffer: 50 mM Tris-HCl, pH 7.5, 1% (w/v) sodium deoxycholate, 100 µM sodium orthovanadate, 150 mM sodium chloride, 1% (v/v) Triton X-100, 0.1% SDS, and Complete Mini EDTA-free Protease Inhibitors (Boehringer Mannheim Biochemicals, Mannheim, Germany). Homogenates were centrifuged at 15 000 rpm at 4°C for 15 min, and supernatants were collected. Protein concentrations were determined by the Bradford Assay [20] using BSA as a standard (Bio-Rad, Richmond, CA) and absorbance at 595 nm.
Protein samples (50 µg) were separated by PAGE (4%-14% gel gradient) as described by Laemmli [21]. Proteins were electrophoretically transferred to a 0.45-µm pure nitrocellulose membrane (Bio-Rad), transfer was confirmed by protein visualization with Ponceau S solution (Sigma), and Ponceau S staining was scanned and analyzed to correct for any differences in loading. Nitrocellulose blots were incubated with blocking solution (5% nonfat milk in PBS with 0.1% [v/v] Tween-20 [PBS-T]) at 4°C for 18–24 h with constant agitation. Blots were incubated with primary antibody for PGES (1:1000 dilution in blocking solution) for 1 h at room temperature. Blots were then rinsed six times for 5 min each with PBS-T and incubated with rabbit secondary antiserum conjugated with horseradish peroxidase for 1 h (1:2000 dilution in blocking solution; Amersham Life Science). Blots were washed six times for 5 min each, and the antibody was detected after 1-min incubation with enhanced chemiluminescence detection reagents (Amersham Life Sciences, Buckinghamshire, U.K.) followed by exposure to X-OMAT blue film (Kodak). The intensities of immunoreactive bands were measured by scanning the image (6200C scanner; Hewlett Packard Canada Ltd., Mississauga, ON, Canada) and analyzing the image on a desktop computer with Scion Image (v. 4.0.2; Scion Corporation, Frederick, MD). Protein bands were digitized, and the mean pixel density for each band was analyzed to obtain ROD units for each protein. To standardize for sample loading, the ROD units of PGES for each band were standardized to the ROD units of the total protein in the band as analyzed by Ponceau S. To compare measurements between blots prepared at different times, a reference sample from sheep placenta was included in each gel.
Data Analysis
Results are expressed as the mean ± SEM. The changes in the levels of PGES mRNA or protein with gestation, labor onset, and/or treatment were determined using a one-way ANOVA followed by a Tukey test ( = 0.05), as required (SigmaStat; Jandel Scientific Software, San Rafael, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of PGES mRNA in the ovine placentome The expression of PGES mRNA in placental tissue as assessed by autoradiography rose with gestation and did not change with the onset or progression of labor. PGES mRNA expression increased significantly in LG (n = 4) compared with MG (n = 4, P < 0.05) but did not increase further to term (where "term" includes all labor tissues: NIL, EL, L; n = 12). There was also no significant difference between MG and term levels of mRNA expression (Fig. 1). PGES mRNA was distributed throughout the placentome from MG (Days 65–80) to term (Fig. 1) without clear demarcation between trophoblast and maternal tissue (data not shown). There was no signal above background with the sense probe (Fig. 1).
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Immunoreactive PGES protein expression Immunoreactive (ir) PGES protein was detected in sheep placentomes by IHC at all gestational ages. Immunoreactive staining was localized to the uninucleate trophoblast cells, to the maternal stroma, and to the cytoplasm of the endothelial cells of maternal blood vessels (Fig. 2). Preabsorption of the primary antibody with purified PGES antigen completely eliminated the staining pattern. The ir-PGES protein was present as a single band with the expected molecular mass of 16 kDa, and the ROD increased significantly from MG (n = 6) to term (n = 17, P < 0.05) (Fig. 3).
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Effect of Cortisol Treatment on the Pattern of PGES Expression
Cortisol infusion in these animals results in increased estrogen production, and this increase is inhibited by coinfusion of 4-OHA [6]. In the present study, cortisol infusion increased placental expression of PGHS-2 whether or not estrogen levels were increased.
Cortisol treatment in either the presence (-4-OHA) or absence (+4-OHA) of increased placental estradiol production did not alter the ir-PGES expression as detected by IHC (data not shown) and Western blot analysis (Fig. 4) nor did these treatments alter levels of PGES mRNA when compared with the control animals (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PGES converts PGH2, the cyclic endoperoxide intermediate of PG synthesis, to PGE2. PGH2 is formed by the sequential cyclooxygenase and peroxidase activities of PGHS, which exists as two isoforms (PGHS-1 and PGHS-2). In human embryonic kidney cell lines, PGES, known to be a membrane-associated protein, is colocalized with both PGHS enzymes in the perinuclear envelope [9]. In the present study, ir-PGES was localized to the endothelial cells of the maternal vessels in the placentome and to the fetal uninucleate trophoblast cells. The intense endothelial cell staining from MG through term supports strongly the hypothesis that PGE2 has a local influence on blood flow within the ovine placenta [23, 24]. In the uninucleate trophoblast cells, ir-PGES was localized to both perinuclear and peripheral regions of the cell, corresponding to areas of cellular membrane. In the ovine placenta, ir-PGHS-2 is also expressed primarily in the perinuclear and cytosolic regions of the fetal uninucleate trophoblast tissue [7]. Colocalization of PGES and PGHS-2 to similar regions of the trophoblast cell may allow for efficient conversion of arachidonic acid to PGE2 and would be in accord with coregulation of these enzymes.
In vitro studies using human macrophage cells, osteoblast cells, and lung adenocarcinoma-derived cell lines have demonstrated functional coupling of PGES with either of the PGHS isoforms for efficient biosynthesis of PGE2 [9, 16, 25]. Expression of PGHS-2, but not PGHS-1, mRNA and protein in the sheep placenta increases in LG and continues to rise until term, with no further change through labor [7, 11, 15, 26]. Furthermore, the rise in PGHS-2 expression was correlated positively with the rising levels of PGE2 in the fetal plasma in LG [11]. We hypothesized that both the timing of expression during gestation and the regulation of PGES and PGHS-2 is similar. However, placental PGHS-2 expression increases in LG around Days 120–125 and continues to rise to term [7, 15]. Increases in both PGES mRNA and protein levels occurred earlier in pregnancy than did the changes in PGHS-2 expression, although this difference in timing could reflect different sensitivities in the methods used to detect these enzymes. In LG, the concurrent rise in both proteins is consistent with the temporal rise in fetal plasma levels of PGE2 [11].
What regulates the increase in ovine placentome PGES expression is still unclear. Although PGHS-2 mRNA and protein levels in the ovine trophoblast rose with fetal glucocorticoid injection/infusion [6, 25], there was no change in PGES protein or mRNA expression in placentae collected from ewes whose fetuses were infused with cortisol in the presence or absence of 4-OHA. This finding suggests that the gestational rise in ovine placental PGES expression is independent of the LG rise in cortisol and/or estrogen and is separate from the cortisol-stimulated rise in PGHS-2 expression. Alternatively, in several cell systems PGES expression is induced by cytokines, such as interleukin ß and tumor necrosis factor [9, 16, 18, 27, 28]. Intrauterine cytokine levels may therefore play a role in the regulation of PGES expression during gestation. Cortisol also may have an effect on PGES earlier in gestation. In LG PGES expression is at a maximum, and the final surge in PG production may be a result of the direct stimulation of PGHS-2 in the fetal trophoblast cell by cortisol. Further investigation of possible mediators of PGES expression in the overall regulation of PG production with the onset of labor is required.
PGES is expressed in increasing amounts in the uninucleate trophoblast cells of the ovine placenta in late pregnancy. This cell type also expresses the glucocorticoid receptor and shows increased levels of PGHS-2 in response to glucocorticoid infusion(s). The present findings suggest that at least during Days 125–130 of ovine pregnancy cortisol has differential effects on PGHS-2 and PGES and does not coregulate these enzymes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 We gratefully acknowledge the support of the Canadian Institutes for Health Research (CIHR, operating grant MOP 14097) and the CIHR Group in Development and Fetal Health. 
2 Correspondence: J.R.G. Challis, Department of Physiology, Medical Sciences Building, University of Toronto, 1 King's College Circle, Toronto, ON, Canada M5S 1A8. FAX: 416 978 4940; j.challis{at}utoronto.ca
Accepted: April 10, 2002.
Received: December 11, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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during parturition in sheep. J Reprod Fertil 1976 48:190 inversely regulates prostaglandin D2 and prostaglandin E2 production in murine macrophages (synergistic action of cyclic AMP on cyclooxygenase-2 expression and prostaglandin E2 synthesis). J Biol Chem 1997 272::31065-31072This article has been cited by other articles:
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  Q. Zhang, V. Collins, K. Chakrabarty, R. F. Wolf, N. Unno, D. Howe, J. C. Rose, and W. X. Wu Regulation of Membrane-Associated Prostaglandin E2 Synthase 1 in Pregnant Sheep Intrauterine Tissues by Glucocorticoid and Estradiol Endocrinology, August 1, 2006; 147(8): 3719 - 3726. [Abstract] [Full Text] [PDF]
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 K. Sun, R. Ma, X. Cui, B. Campos, R. Webster, D. Brockman, and L. Myatt Glucocorticoids Induce Cytosolic Phospholipase A2 and Prostaglandin H Synthase Type 2 But Not Microsomal Prostaglandin E Synthase (PGES) and Cytosolic PGES Expression in Cultured Primary Human Amnion Cells J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5564 - 5571. [Abstract] [Full Text] [PDF]
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D. Giannoulias, N. Alfaidy, A. C. Holloway, W. Gibb, M. Sun, S. J. Lye, and J. R. G. Challis Expression of Prostaglandin I2 Synthase, but Not Prostaglandin E Synthase, Changes in Myometrium of Women at Term Pregnancy J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5274 - 5282. [Abstract] [Full Text] [PDF]
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