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Endocrinology Vol. 147, No. 8 3719-3726
Copyright © 2006 by The Endocrine Society

Regulation of Membrane-Associated Prostaglandin E2 Synthase 1 in Pregnant Sheep Intrauterine Tissues by Glucocorticoid and Estradiol

Qi Zhang, Valta Collins, Kaushik Chakrabarty, Roman F. Wolf, Nobuya Unno, David Howe, James C. Rose and Wen Xuan Wu

Department of Obstetrics and Gynecology (Q.Z., J.C.R., W.X.W.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; The University of Oklahoma Health Sciences Center (V.C., K.C., R.F.W.), Oklahoma City, Oklahoma 73104; Department of Obstretrics/Gynecology (N.U.), Kitasato University, School of Medicine, Kanagawa 228-8555, Japan; and Department of Obstetrics and Gynecology (D.H.), The Queen Mother’s Hospital, Yorkhill, Glasgow G3 8SJ, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Dr. Wen Xuan Wu, Department of Obstetrics and Gynecology, Wake Forest University Baptist Medical Center, Winston Salem, North Carolina 27157. Email: wenwu@wfubmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was designed to determine the regulatory effect of glucocorticoid and estradiol on expression of ovine intrauterine membrane-associated prostaglandin E2 synthase 1 (mPTGES1) in late gestation and at labor. For gestational and labor groups, 16 pregnant ewes from 95–147 d gestational age (dGA) and four pregnant ewes at spontaneous term labor were used. The fetal glucocorticoid group, 14 pregnant ewes at 123–125 dGA with fetuses, was divided into the following groups: after sham adrenalectomy (n = 5), adrenalectomy (n = 4), and adrenalectomy with fetal cortisol replacement to late gestation levels (n = 5). For the maternal glucocorticoid group, nine pregnant ewes were treated with saline (n = 4) and three courses of maternal dexamethasone (n = 5). For the estradiol group, 10 pregnant ewes at 119–121 dGA were treated with sesame oil (n = 5) or estradiol (n = 5) to produce labor levels of estradiol in maternal plasma. Endometrial, myometrial, and placental mRNA and proteins were analyzed by Northern and Western blot and immunocytochemistry for mPTGES1. Data were analyzed by Student’s t test and ANOVA. There was a significant increase of placental mPTGES1 in late gestation. Glucocorticoids, given to the mother or fetus, significantly stimulated mPTGES1 in placenta. mPTGES1 was elevated only in the endometrium during spontaneous term labor and after estradiol treatment. The mPTGES1 was localized in the myometrial smooth muscle cells, endometrial stromal cells, and placental trophoblast cells. Our study suggested that increased expression of placental mPTGES1 throughout late gestation might result from the increased fetal and maternal circulating glucocorticoids, whereas elevated maternal plasma estradiol concentration might be responsible for the induced mPTGES1 expression in the endometrium during labor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDIN E2 (PGE2) HAS BEEN identified as one of the key prostanoids inducing fetal organ maturation (1), maternal cervical ripening (2, 3, 4), and myometrial contraction (5) in late gestation. Pregnant intrauterine tissues are believed to be the major PGE2 source (1, 6). In the PGE2 synthesis pathway, cyclooxygenase (COX) is one of key enzymes that metabolize arachidonic acid to PGG2 and PGH2. There are two isoforms of COX, which are designated COX1 and COX2 (7, 8, 9). The increase in prostaglandin synthesis at term and parturition is associated with increased expression of intrauterine COX2 (10, 11).

However, there are also other enzymes, such as PGE2 synthases (PTGES, also known as PGES) that are potential regulating steps in PGE2 synthesis in addition to COX. PTGES acts downstream of COX to catalyze the conversion of PGH2 into PGE2 (12, 13). At least three forms of human PTGES have been cloned and characterized, including membrane-associated PTGES1 (mPTGES1) (12), mPTGES2 (14), and cytosolic PTGES (15). Among theses, mPTGES1 shows coordinated induction with COX2 by inflammatory stimuli in various cells and tissues (12, 16). In contrast, mPTGES2 is constitutively expressed in various cells and tissues (17) and can be coupled with both COX1 and COX2 (14, 17), whereas cytosolic PTGES can be functionally coupled only with COX1 to promote immediate PGE2 production. Therefore, the expression and regulation of mPTGES1 is the focus of the current study.

In most mammalian species, there is an increase in glucocorticoid concentration in maternal and fetal circulations toward the end of gestation (18, 19). In parallel with the increase of glucocorticoids, there is a gradual increase of PGE2 in maternal and fetal circulations throughout late gestation (1). At the end of gestation, there is an additional increase of fetal cortisol, which results in an increase in estrogen and a decrease in progesterone in maternal plasma through induction of the P450c17{alpha} hydroxylase enzyme (20). Estrogen then recruits many positive systems, including additional enhanced prostaglandin production in the intrauterine tissues to initiate onset of labor (21). One of the induced prostaglandins associated with onset of labor is PGE2. Therefore, there are two phases of increased PGE2 production during pregnancy: gestation-associated gradual increase and labor-associated additional increase (1).

The labor-associated increase of PGE2 might result from estrogen-stimulated prostaglandin synthesis by intrauterine tissues. The mechanisms that regulate the gestation-associated increase of PGE2 production are less clear. Recent studies have suggested that fetal cortisol may directly stimulate the intrauterine prostaglandin synthesis (22, 23). For example, in vitro studies have demonstrated that glucocorticoid stimulates prostaglandin production directly in cultured human fetal membranes (24). Furthermore, evidence has also been presented for the existence of a cortisol-dependent/estradiol-independent mechanism for COX2 induction within trophoblast tissue leading to the elevation of fetal plasma PGE2 (23) in pregnant sheep. Recently, we have shown that cortisol administration to adrenalectomized fetuses to clamp fetal cortisol at levels present early in the late gestation rise stimulates intrauterine COX2 expression in the absence of increased estradiol action in maternal plasma (22). Taken together, these findings support a direct action of glucocorticoids on prostaglandin production in intrauterine tissues, which may play a critical role in induction of the gestation-associated increase of the intrauterine prostaglandin system. In light of the findings that mPTGES1 is coupled with COX2 and COX2 rises progressively in placenta in late gestation (10, 11), we hypothesized that there would be a gestation-associated increase of mPTGES1 in intrauterine tissues of pregnant sheep, which might result from the increased fetal and maternal glucocorticoids in late gestation. We further hypothesized that estradiol might play an important role in regulation of mPTGES1 in intrauterine tissues during labor. Therefore, this study was designed to examine regulatory roles of estradiol and glucocorticoids on mPTGES1 expression in the myometrium, endometrium, and placenta of pregnant sheep. Immunocytochemical staining of mPTGES1 was applied to sheep myometrium, endometrium, and placenta to determine the cell types that contained mPTGES1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue collection
Pregnant Rambouillet-Dorset ewes bred on a single occasion and carrying fetuses of known gestational age were studied. Experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee and conducted in facilities approved by the American Association for the Accreditation of Laboratory Animal Care. Pregnant ewes from which tissues were obtained were instrumented with fetal and maternal carotid arterial and jugular venous catheters. Maternal and fetal arterial blood samples were taken daily for the determination of pH and blood gases to evaluate maternal and fetal well-being.

Gestational and spontaneous term labor (STL) groups
Myometrium, endometrium, and placenta were removed under halothane general anesthesia from ewes at 95–110 d gestational age (dGA) (n = 5), 115–134 dGA (n = 5), and 135–147 dGA (n = 6). Tissues were also collected from ewes (n = 4) in STL (143–147 dGA) as judged from a well established myometrial electromyogram contraction pattern.

Fetal glucocorticoid treatment groups
Endometrium, myometrium, and placenta were also collected from pregnant ewes with bilateral fetal adrenalectomy (ADX) with (n = 5) or without (n = 4) cortisol replacement or sham adrenalectomy (n = 5) at 110–115 dGA. ADX was performed as we described previously (25, 26). Cortisol was infused continuously at a rate of 4 µg/min to five ADX fetuses via the fetal venous catheter in physiological saline on d 5–7 after surgery until necropsy. This cortisol infusion rate produced fetal plasma cortisol levels present early in the late gestation rise but that were inadequate to produce labor (15–25 ng/ml in fetal plasma after 3 d infusion until the end of experiments) (25). Saline vehicle was infused at the same rate to the other nine fetuses (sham and ADX groups). In sham and ADX fetuses, fetal plasma cortisol concentration remained below the assay sensitivity level for the volume of plasma extracted (4.9 ng/ml) throughout the observation period (25). At 123–125 dGA, the fetuses were delivered by cesarean section and euthanized by exsanguinations under halothane general anesthesia. This experiment was designed to examine the effect of fetal glucocorticoid on regulation of intrauterine mPTGES1.

Maternal glucocorticoid treatment
To determine the effect of maternal glucocorticoid, dexamethasone (2 mg) or vehicle was administered im for 2 d to pregnant ewes as a course of four injections at 12-h intervals for three courses. Treatment was given at 103–104, 110–111, and 117–118 dGA. The myometrium, endometrium, and placenta were collected at the end of 48 h after the third course. The dose of dexamethasone was chosen after extensive trials in which 2, 4, and 6 mg were administered without progesterone coverage. Delivery within 48 h of receiving the first four injection courses occurred in 84 and 100% of animals in the 4- and 6-mg groups, respectively (n = 4–5 per group). All animals in the 2-mg group delivered at term. Dexamethasone, not cortisol, was chosen for two reasons: 1) the pilot study provided a dose that would not induce labor, and 2) dexamethasone specifically bound to glucocorticoid receptor but not the mineralocorticoid receptor. This experiment was designed to examine the effect of maternal glucocorticoid on regulation of intrauterine mPTGES1.

Estradiol treatment groups
Ten ewes at 119 dGA were treated with sesame oil (n = 5) or estradiol (n = 5), 5 mg twice a day, administered im for 2 d to produce labor levels of estradiol in maternal plasma (350 pg/ml) (26, 27). At 121 dGA, necropsies were performed under halothane anesthesia after which the animals were euthanized. The myometrium, endometrium, and placenta were collected for later RNA and protein analysis. This experiment was designed to examine estrogen’s effect on regulation of intrauterine mPTGES1.

Northern blot analysis
Total RNA was extracted from placenta, and polyadenylated RNA was extracted from endometrium by oligo dT cellulose affinity chromatography using a commercial kit (Invitrogen, San Diego, CA). Samples of total RNA (30 µg) or polyadenylated RNA (2 µg) were separated by electrophoresis on a 1.4% (wt/vol) agarose/0.66 M formaldehyde gel and transferred onto a nylon membrane (NEN Life Science Products, Wilmington, DE) and then subjected to Northern blot analysis for mPTGES1. ß-Actin or 18S were used to control RNA loading.

Synthesis of probes
Our cloned ovine mPTGES1 cDNA in pCR II vector (Invitrogen), which includes promoters for phage polymerases SP-6 to produce antisense probe and T-7 to produce sense probe, was linearized by an appropriate restriction enzyme. Antisense and sense riboprobes were synthesized using a commercial kit (MAXIscript; Ambion, Austin, TX) labeled with [{alpha}-32P]UTP (800 Ci/mmol) (NEN Life Science Products). The cloned cDNA of mPTGES1 was sequenced from at least two different clones and from both the coding and noncoding strands at the core sequencing facility of the University of Oklahoma. The DNASTAR program (DNASTAR Inc., Madison, WI) was used for alignments and sequence analysis of mPTGES1 cDNA clones. The plasmid containing ß-actin cDNA with RNA polymerase promoters was purchased from Ambion (catalog no. 7424).

Solubilized cell membrane extraction and Western blot analysis
Solubilized cell membrane extracts from myometrium, endometrium, and placenta were prepared as described previously (11). The protein concentration was determined by the method of Bradford (Bio-Rad Laboratories, Hercules, CA). The solubilized proteins (25 µg/lane) were then separated on 10% SDS-PAGE and electrophoretically transferred to a nylon membrane (Immobilon; Millipore Corp., Bedford, MA), using a Bio-Rad transfer blot cell. Western blot analysis was performed as described previously (11). The protein bands were visualized using an enhanced chemiluminescence Western blotting detection kit (ECL; Amersham Life Sciences, Arlington Heights, IL). The molecular sizes of the proteins were determined by running standard molecular weight marker proteins (Bio-Rad) in an adjacent lane. G protein ß-subunit (Gß) was detected on each blot to control protein loading. Chemiluminescence signals were analyzed and quantified with the scanner, and data were analyzed with a densitometry program-scan analysis and quantified against an arbitrary scale in the plot.

Immunocytochemistry
Frozen sections (4 µm) of the pregnant sheep myometrium, endometrium, and placenta were immunostained for mPTGES1 using the avidin-biotin immunoperoxidase method as described previously (11) to localize the cellular distribution of mPTGES1 protein in intrauterine tissues. Specificity of immunostaining for the mPTGES1 was confirmed by three approaches: 1) omission of the primary antibody, 2) incubation of the slides with the normal rabbit serum instead of the primary antibody, and 3) Western blot analysis.

Antibodies for Western blot analysis and immunocytochemistry
A rabbit polyclonal antibody for human mPTGES1 (Cayman Chemical Co., Ann Arbor, MI) was used at 1:500 dilutions and incubated at 4 C for 20 h. Specificity of Western blot analysis for mPTGES1 antibody was confirmed by preabsorption of mPTGES1 antibody with synthetic polypeptides that were used for generating the antibody (Cayman; data not shown). The primary antibody used for immunocytochemistry was the same antibody used for Western analysis of mPTGES1. A rabbit polyclonal antibody for bovine Gß was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A second antibody, horseradish-peroxidase-conjugated donkey antirabbit IgG, was used for Western blotting (Amersham), and a biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) was used for immunocytochemistry.

Statistical analysis
Comparison of two means was made with the Student’s t test. Comparison of three or more means was made by ANOVA and multiple post hoc comparisons with Tukey’s method for 95% confidence interval of pairwise differences. Statistical significance was assumed at the 5% level. Data are presented throughout as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of gestation and labor
mPTGES1 significantly increased in the placenta after 115 dGA to term (Fig. 1Go). There were no gestational-associated changes of mPTGES1 in the endometrium and myometrium (data not shown). There was a significant increase of mPTGES1 during STL in endometrium (Fig. 2Go) but not in placenta or myometrium (data not shown).


Figure 1
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  		FIG. 1. A, Gestational-associated changes in the abundance of mPTGES1 protein in the placenta: lanes 1–5, 95–110 dGA (n = 5); lanes 6–10, 115–134 dGA (n = 5); lanes 11–14, 135–145 dGA (n = 4); B, Gß in each corresponding lane; C, densitometric analysis of placental mPTGES1 protein as a ratio to Gß. mPTGES1 protein in placenta significantly increased at 115–134 dGA, and there was no additional increase at term. *, P < 0.05 compared with 115–134 dGA and >135 dGA. Values are presented as mean ± SEM.

 

Figure 2
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  		FIG. 2. A, Western blot analysis of mPTGES1 protein in the endometrium collected from term controls not in labor (TCNL, lanes 1–4) and STL (lanes 5–8); B, Gß in each corresponding lane; C, densitometric analysis of endometrial mPTGES1 in TCNL and STL groups (n = 4 in each group). mPTGES1 increased significantly in the STL group. *, P < 0.05 compared with TCNL. Values are presented as mean ± SEM.

 
Effect of fetal and maternal glucocorticoid
Fetal cortisol at late gestation level (15–20 ng/ml in fetal plasma) was able to stimulate mPTGES1 mRNA (Fig. 3Go) and protein (Fig. 4Go) expression in placenta but not in myometrium or endometrium (data not shown). In addition, maternal dexamethasone also up-regulated placental (Fig. 5Go) but not endometrial or myometrial mPTGES1 expression (data not shown).


Figure 3
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  		FIG. 3. A, Northern blot analysis of mPTGES1 mRNA in placenta collected from sham (lanes 1–5), ADX fetuses (lanes 6–9), and ADX plus cortisol (ADX+F; lanes 10–14); B, 18S in each corresponding lane; C, densitometric analysis of placental mPTGES1 mRNA in sham (n = 5), ADX (n = 4), and ADX+F groups (n = 5). mPTGES1 mRNA increased significantly in the ADX+F group. *, P < 0.05 compared with sham and ADX. Values are presented as mean ± SEM.

 

Figure 4
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  		FIG. 4. A, Western blot analysis of mPTGES1 protein in placenta collected from sham (lanes 1–3), ADX fetuses (lanes 4–6), and ADX+cortisol (ADX+F; lanes 7–10); B, Gß in each corresponding lane; C, densitometric analysis of placental mPTGES1 protein in sham (n = 3), ADX (n = 3), and ADX+F groups (n = 4). mPTGES1 protein increased significantly in the ADX+F group. *, P < 0.05 compared with sham.

 

Figure 5
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  		FIG. 5. A, Western blot analysis of mPTGES1 protein in the placenta collected from controls (Cont; lanes 1–4) and dexamethasone-treated ewes (DEX; lanes 5–9); B, Gß in each corresponding lane; C, densitometric analysis of placental mPTGES1 protein in control (n = 4) and dexamethasone-treated (n = 5) groups. Placental mPTGES1 protein increased significantly after dexamethasone treatment compared with the control. *, P < 0.05. Data are presented as mean ± SEM.

 
Effect of estradiol
Estradiol at labor level (350 pg/ml in maternal plasma) stimulated mPTGES1 mRNA (Fig. 6Go) and protein (Fig. 7Go) expression in the endometrium at the gestation studied. There was no significant effect of estradiol on mPTGES1 expression in the myometrium or placenta (Fig. 8Go).


Figure 6
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  		FIG. 6. A, Northern blot analysis of mPTGES1 mRNA in endometrium from controls (Cont; lanes 1–5) and estradiol-treated animals (E; lanes 6–10); B, ß-actin in each corresponding lane; C, densitometry analysis of mPTGES1 mRNA in endometrium from control and estradiol-treated animals (n = 5 for each group). There was a significant increase of endometrial mPTGES1 mRNA (*, P < 0.05) in the estradiol-treated group compared with the control group. Values are presented as mean ± SEM.

 

Figure 7
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  		FIG. 7. A, Western blot analysis of mPTGES1 protein in endometrium from controls (Cont; lanes 1–5) and estradiol-treated animals (E; lanes 6–10); B, Gß in each corresponding lane; C, densitometry analysis of mPTGES1 protein in endometrium from control and estradiol-treated animals (n = 5 for each group). There was a significant increase of endometrial mPTGES1 (*, P < 0.05) in the estradiol-treated group compared with the control group. Values are presented as mean ± SEM.

 

Figure 8
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  		FIG. 8. Densitometry analysis of mPTGES1 protein in myometrium (Myo) and placenta (Plac) from control (Cont) and estradiol-treated (E) animals (n = 5 for each group). There was no change of myometrium and placenta mPTGES1 (*, P > 0.05) in the estradiol-treated group compared with the control group. Values are presented as mean ± SEM.

 
Localization of mPTGES1 in the endometrium, myometrium, and placenta
The mPTGES1 immunostaining was mainly localized in the myometrial smooth muscle cells, endometrial stromal cells, and placental trophoblast cells. Intensive staining for mPTGES1 was also observed on smooth muscle cells and endothelial cells along the blood vessels in all three intrauterine tissues (Fig. 9Go).


Figure 9
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  		FIG. 9. A–C, Immunocytochemical analysis of mPTGES1 in endometrium (A), myometrium (B), and placenta (C). The brown staining represents mPTGES1 antibody hybridized with mPTGES1 protein. Note the brown staining was associated with endometrial stromal cells (ESC) (A), myometrial smooth muscle cells (SMC) (B), and placental trophoblast cells (PTC) (C). D–F, mPTGES1-positive staining was also localized in endothelial cells (EC) along the blood vessels in endometrium (D), smooth muscle cells (SMC) of myometrial small artery (E), and endothelial cells (EC) of placental vascular beds (F) of pregnant sheep. G–I, The specific staining for mPTGES1 was abolished in endometrium (G), myometrium (H), and placenta (I) when the primary antibody of mPTGES1 was replaced with normal rabbit serum. Magnification, x200 (A and B) and x400 (C–I); scale bar, 40 µm (A and B) and 20 µm (C–I).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression of COX2 in pregnant ovine placenta and endometrium increases in late gestation (10, 11), and the rise in intrauterine COX2 is correlated positively with the rising levels of PGE2 in fetal plasma (1, 28). Because COX2 is functionally coupled with mPTGES1 as shown in several cell culture systems (16, 17), we propose that mPTGES1 and COX2 are simultaneously regulated in intrauterine tissues to increase PGE2 level in maternal and fetal plasma in late gestation and at labor. To test this possibility, we examined the development of the intrauterine mPTGES1 in pregnant sheep in late gestation and at labor and correlated the changes of intrauterine mPTGES1 with the classic hormonal changes in fetal and maternal plasma associated with late gestation and onset of labor, which have been very well presented in the literature. As has been seen for COX2 (10, 11, 28), there was a significant increase of mPTGES1 in placenta from 95 dGA to term, with no additional change during labor, which is consistent with report by Martin et al. (29). In contrast to the gestational change of placental mPTGES1, endometrial and myometrial mPTGES1 concentration remained the same throughout the late gestation. Endometrial, but not myometrial, mPTGES1 increased during labor. Our data suggested that ontogenic expression of intrauterine mPTGES1 is similar to that of COX2, although differences also exist. For example, endometrial COX2 rose progressively in the late gestation, with another rise at the time of labor (11), in contrast with the steady level of endometrial mPTGES1 throughout late gestation with an increase during labor. Taken together, our past and present observations have consistently shown the tissue-specific ontogenic regulation of the prostaglandin synthesis pathway in intrauterine tissues of pregnant sheep, suggesting that specific prostaglandin production from different intrauterine tissue origin may play differential functions in preparation, activation, and completion of labor. In addition, our data suggest that PGE2 synthesis is controlled at multiple enzymatic levels. COX2 is not the only key enzyme to differentiate gestation- and labor-associated changes in PGE2 production by intrauterine tissues.

In sheep, fetal cortisol starts to increase from 125 dGA and reaches its maximal level at term (19), whereas estrogen level is constantly low. Increased glucocorticoid in maternal and fetal plasma in late gestation is a common feature shared across species (18, 30, 31). The ontogenic change of placental mPTGES1 in late gestation is parallel with the increase of fetal plasma cortisol concentration. This temporal association between fetal cortisol and placental mPTGES1 in late gestation and at labor suggests that fetal cortisol might be an important candidate in regulating intrauterine mPTGES1 expression. To test this hypothesis, we first administered cortisol at concentrations seen in late gestation (15–20 ng/ml) to adrenalectomized fetuses to clamp fetal cortisol at levels present early in the late gestation rise but higher than the cortisol level present in sham fetuses at the gestation studied. Fetal cortisol administration at the late gestation level was able to stimulate placental mPTGES1 expression. We then determined the effect of maternal dexamethasone administration in amounts inadequate to produce labor on intrauterine mPTGES1 expression. Maternal dexamethasone administration also induced placental mPTGES1 expression. Results from those two parallel experiments suggested that increased glucocorticoids might be the cause of increased placental mPTGES1 expression in late gestation in pregnant sheep.

In contrast to our results, Martin et al. (29) and Palliser et al. (32) found that placental mPTGES1 did not respond to fetal cortisol infused at labor level (29) or decreased in fetal-dexamethasone-induced labor (32). The cause for the different response of placental mPTGES1 to infused fetal cortisol and dexamethasone is not well understood. However, in the studies of Martin et al. (29) and Palliser et al. (32) and the study by us, there was no significant rise of placental mPTGES1 mRNA/protein associated with onset of labor, which suggested that induction of placental mPTGES1 could not be precipitated by the full range of changes associated with onset of labor. By conducting fetal adrenalectomy in all animals investigated in the present studies, we removed endogenous fetal cortisol as well as other steroids of fetal adrenal origin with concurrent replacement of a known amount of cortisol to produce a cortisol level at late gestation but not at labor level (15–20 ng/ml in fetal plasma). Removal of endogenous fetal cortisol production provided the ability to identify the specific function of carefully controlled levels of cortisol in distinction from other steroids of fetal adrenal. Infused fetal cortisol at late gestation level induced placental mPTGES1 at both mRNA and protein levels. Furthermore, placental mPTGES1 was also induced by maternal dexamethasone administration at a dose inadequate to produce labor, which again supported our hypothesis for glucocorticoids functioning as a potential inducer of placental mPTGES1. Thus, we present the first evidence to show that fetal and maternal glucocorticoid administration at a dose inadequate to induce labor is able to induce placental mPTGES1 expression.

To test whether increased estradiol concentration associated with onset of labor may be responsible for regulation of mPTGES1 expression in maternal intrauterine tissues in pregnant sheep at labor, we first examined the labor effect on mPTGES1 expression in the endometrium and myometrium. To our surprise, it was only the endometrium, but not the myometrium, with higher mPTGES1 concentration during labor. Because the labor-associated rise of endometrial mPTGES1 correlated with the time of sharp increase of maternal plasma estradiol (33), we administered estradiol to pregnant sheep at 120 dGA at a dose that produces estradiol concentrations in maternal plasma observed during labor, 350 pg/ml, a value within the range observed in dexamethasone-induced premature labor (34) and STL (33). It was only endometrial mPTGES1 that was induced by administered exogenous estradiol. Our data indicate that induction of endometrial mPTGES1 is strongly dependent on the stimulation by estrogen.

It is of interest that glucocorticoid and estradiol regulation of mPTGES1 expression is strictly tissue specific. Similar tissue specificity has been observed for glucocorticoids and estradiol in regulating COX2. Glucocorticoids stimulate COX2 expression in cultured human amnion cells (24) but inhibit COX2 expression in numerous other cell types (35). In addition, estradiol stimulates COX2 expression in pregnant sheep endometrium and myometrium but not in placenta (27). These results indicate that cell-type-specific subcellular signals are important to direct responses to steroid hormone regulation in some cell types while leaving the prostaglandin synthetic activity of other cells unaltered. This form of differential regulation is of fundamental importance for prostaglandin synthesis, which appears ubiquitous, if not universal, in cell function.

The precise cellular localization of mPTGES1 in the sheep uterus was determined by immunocytochemistry. mPTGES1 was immunolocalized in the smooth muscle cells of the myometrium and stromal cells of endometrium. In placenta, mPTGES1 was localized primarily to the fetal trophoblast villi. We have previously demonstrated that glucocorticoid and estrogen receptors were localized in ovine intrauterine tissues by immunocytochemistry in a similar distribution of mPTGES1 (36). These findings support a direct control of glucocorticoid and estrogen in regulating mPTGES1 synthesis in the ovine intrauterine tissues. One of the important features of mPTGES1 is its localization in endothelial and smooth muscle cells of blood vessels in all three intrauterine tissues studied, which is consistent with the report by Martin et al. (29). Localization of mPTGES1 in blood vessels further supports the critical function of PGE2 in regulating utero-placental blood flow.

Conclusions
Our results suggest that regulation of mPTGES1 by glucocorticoid and estrogen is similar to that of COX2 in sheep endometrium and placenta. Cortisol-dependent activation of the COX2/mPTGES1 system may account for the increased PGE2 synthesis in late gestation, whereas estradiol may be responsible for the labor-associated induction of the COX2/mPTGES1/PGE2 system in maternal uterine tissues of pregnant sheep.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD 39247 and HD 21350.

Disclosure statement: D.H. is currently employed by Pfizer and has equity interests in Pfizer. All the other authors have nothing to disclose.

First Published Online May 11, 2006

Abbreviations: COX, Cyclooxygenase; dGA, days gestational age; Gß, G protein ß-subunit; mPTGES1, membrane-associated prostaglandin E2 synthase 1; PGE2, prostaglandin E2; PTGES, PGE2 synthase; STL, spontaneous term labor.

Received March 7, 2006.

Accepted for publication May 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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