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Research Article |
Department of Obstetrics and Gynecology,3 University of Cincinnati, Medical College, Cincinnati, Ohio, 45267
Imperial College Parturition Research Group,4 Department of Maternal Fetal Medicine, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, SW10 9NH, United Kingdom
ABSTRACT
The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1–4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor.
All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes.
Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.
fetal membranes, mechanisms of hormone action, myometrium, parturition, prostaglandin receptors, steroid hormones, steroid hormone receptors
INTRODUCTION
Challis and Lye [1] first proposed that myometrial activation from a state of uterine quiescence resulted from the coordinated expression of a cassette of contraction-associated proteins (CAPS) that include ion channels, agonist receptors (i.e., oxytocin and prostaglandins) and gap junctions. This concept has lead to the suggestion of a functional partitioning of the myometrium during pregnancy, whereby the lower segment displays a contractile phenotype throughout gestation changing to a relaxatory phenotype at labor; allowing passage of the fetal head, whereas the upper uterine segment maintains a relaxatory phenotype throughout most of gestation to accommodate the growing fetus and adopts a contractile phenotype during labor. We are particularly interested in the mechanisms that regulate the expression of the prostaglandin (PG) receptors in the myometrium during pregnancy and the possible involvement of these receptors in myometrial activation. We have suggested that the change from uterine quiescence to an enhanced contractile phenotype may be due to the direct effect of differential expression of PG receptors within the myometrium, in a temporal and topographically distinct manner [2]. Evidence in support of this concept is now beginning to emerge in many species [3–6]. Furthermore, the expression of these receptors in the fetal membranes may suggest paracrine and autocrine roles for PGE2 in the signaling pathways associated with human parturition, with a possible indirect role contributing to the maintenance of uterine quiescence and contractility [7].
The effects of PGE2 and PGF2
on the myometrium are mediated through specific G-protein coupled receptors, each encoded by a separate gene, acting through different intracellular second messenger pathways—principally cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and intracellular calcium. PG receptors can produce a broad range of biological actions depending on which receptor subtype is expressed. Prostaglandin F2 action is mediated by PTGFR (also known as the FP receptor) and is coupled to signal transduction pathways leading to the mobilization of intracellular calcium. The PGE2 receptor family (PTGER) is composed of four distinct subtypes. PTGER1 and PTGER3 (also known as EP1 and EP3) increase phosphoinositol turnover and calcium mobilization, or decrease adenylate cyclase and cAMP levels, respectively [8]. PTGER2 and PTGER4 (also known as EP2 and EP4) both stimulate adenylate cyclase and cAMP production [9, 10]. Generally these receptors have been grouped into two categories; PTGER1, PTGER3, and PTGFR are stimulatory receptors resulting in smooth muscle contraction, whereas PTGER2 and PTGER4 are considered inhibitory receptors causing relaxation of smooth muscle.
High expression of PTGER2 mRNA in the myometrium before term is consistent with the influence of PTGER2 in activating intracellular adenylate cyclase, increasing cAMP and resulting in the maintenance of uterine quiescence [2, 11–14]. Conversely, low expression of the contractile PTGFR early in gestation with an increase at the onset of labor is consistent with its involvement in myometrial activation [2, 11–13, 15–17]. Furthermore, the contractile responses of the baboon myometrium vary; PGE2 contracts myometrial strips from the fundus but not the lower uterine segment [3]. Indeed, PTGER3 is more prominently expressed in the fundal part of the primate uterus, whereas the relaxatory PTGER2 and PTGER4 dominate the lower uterine segment [12].
A distinct localization and expression profile for the PGE2 receptor subtypes has recently been described [6], comparing upper and lower uterine segments from nonpregnant and pregnant women in the presence and absence of labor. The overall expression of PTGER2 was greatest in the lower segment compared with the upper segment and PTGER3 mRNA expression was significantly higher in the upper segment compared with the lower uterine segment. No topographical changes were observed for PTGER4, a finding consistent with previous observations in the primate model [3, 12]. Together these observations support the idea that varying PG receptor populations may be responsible for the difference in contractile response of the upper and lower uterine segments to PGs during pregnancy.
We have demonstrated the presence of the four PGE2 receptor subtypes in human fetal membranes and placenta, suggesting paracrine and autocrine roles for PGE2 in the signaling pathways associated with parturition [7]. This may also suggest an indirect role of PGE2, acting through specific receptors within the fetal membranes on uterine quiescence and enhanced uterine activity required for labor. Similarly, tissue specific expression of the PG receptors has been demonstrated in the baboon, where PTGER2, PTGER3, and PTGER4 were present in the cervix, decidua, and fetal membranes (including the chorion) and the PTGFR receptor was predominately found in the myometrium and the cervix [3, 18]. This suggests that myometrial activation (from quiescence to contractile activity) may be directly mediated by a combination or balance of the PG receptor subtypes within maternal tissue and indirectly mediated by receptors within the fetal tissues. The combination or balance of receptor expression between the maternal and fetal tissues may vary differently over the course of pregnancy and with labor.
Consequently, to achieve a more comprehensive understanding of the role of the PG receptors in uterine activation, we characterized the temporal and tissue specific localization and expression of the PGE2 receptor subtypes (PTGER1–4) and the PGF2 receptor (PTGFR) in matched human upper and lower segment myometrium, amnion, and choriodecidual samples during pregnancy. We included samples obtained at various gestational ages in the presence and absence of labor. The distribution of PGE2 and PGF2 receptors between the maternal and fetal tissues during pregnancy was also examined to determine whether the expression of these receptors (presence or absence) in the fetal membranes is coordinated with that seen in the myometrium.
MATERIALS AND METHODS
Tissue Collection and Preparation
All procedures involving human myometrium and fetal membranes were conducted in compliance with the Institution Review Board of the University of Cincinnati (Cincinnati, OH). Informed consent was obtained from all women prior to any tissue collection.
Paired upper and lower segment human myometrial samples were obtained from four groups of women (n = 6, in each group), at preterm no labor (PTNL; 30.4 ± 1.5 wk), preterm with labor (PTL; 33.9 ± 1.5 wk), term no labor (TNL; 38.4 ± 0.4 wk), or term with labor (TL; 39.2 ± 0.5 wk). Labor was defined as the presence of regular uterine contractions (every 3–4 minutes) resulting in cervical effacement and dilation. A lower transverse cesarean section was performed where the uterus was exteriorized after delivery of the fetus and placenta. Myometrial samples were removed from the upper margin of the incision made in the lower uterine segment (LS). For the upper segment (US), an Allis clamp was used to grasp a small segment (1.0 x 0.5 cm) of myometrium below the fundus, which was then excised using Mayo scissors (this included the serosal surface but not the endometrium). Hemostasis was obtained using a single figure of 8 suture.
The indications for cesarean section included: fetal distress (n = 3), previous cesarean section (n = 13), failure to progress (n = 2), PPROM with abruption and/or breech presentation (n = 4), multiple fetuses (twins, triplets; n = 4) severe PE with IUGR (n = 1), HSV+ with lesions and active labor (n = 1). Separated amnion and choriodecidua were also collected from the reflected portion of membranes and individually flash frozen in liquid N2 prior to storage at –80°C. For some patients, fetal membranes were unable to be collected; myometrial samples from those patients (n = 7) were age matched with amnion and choriodecidua samples that had previously been collected. Indications for cesarean section for those patients (n = 4) are shown, while the 3 remaining patients delivered vaginally.
Immunohistochemistry
Polyclonal antibodies against human PGE2 and PGF2 receptor subtypes (PTGER1–4, PTGFR) and respective blocking peptides were purchased from Cayman Chemical (Ann Arbor, MI). Vectastain Elite ABC and aminoethyl carbazole (AEC) were purchased from Vector Labs (Burlingame, CA). Cryosections of upper and lower segment myometrium (n = 6 patients, in each group) were sectioned at 8 µm and immunostained using the Vectastain Elite ABC method (Vector Labs, Burlingame, CA), as per manufacturer's instructions and as previously described [7]. Polyclonal antibodies PTGER1–4 and PTGFR were used at a final concentration of 10 µg/ml, while alpha smooth muscle actin (ACTA2; Gift from Dr. J. Lessard, Cincinnati Children's Hospital, Cincinnati, OH), was used at a final dilution 20 µg/ml. Tissue sections that were not treated with the anti-human PTGER1–4 antibodies and those which were treated with the primary antibody preabsorbed with its respective blocking peptide were included as negative controls. Assessment of receptor localization in each smooth muscle cell layer (i.e., presence or absence) and localization within cells was performed on all patient samples (n = 6, in each group).
Quantitative RT-PCR
Total RNA was extracted and purified from the upper and lower segment myometrial samples and separated amnion and choriodecidua, using the Tri-reagent method (Trizol, Sigma Chemical Company). After quantification, 2.0µg RNA was pretreated with DNase I (Amp Grade, Invitrogen) and then reverse transcribed with Oligo dT random primers using SuperScript II reverse transcriptase (Invitrogen). Paired oligonucleotide primers for amplification of human PGE2 receptors were designed using Primer Designer (Scientific and Educational Software, Durham, NC) against the sequence downloaded from GenBank (Table 1). The primer sets used produced amplicons of the expected size and flanked intron/exon junctions. Assays were validated for all primer sets by confirming that single amplicons of appropriate size and sequence were generated. Quantitative real-time PCR was performed in the presence of SYBR Green (QiagenLtd., Crawley, West Sussex, UK), and amplicon yield was monitored during cycling in a RotorGene Sequence Detector (Corbett Research Ltd., Mortlake, Sydney, Australia). Pre-PCR cycle was 7 min at 95°C followed by 35 cycles of 95°C for 10 sec, 58–60°C for 10 sec, and 72°C for 10 sec, followed by final extension 72°C for 1 min. The cycle threshold in each assay was set at a level where the exponential increase in amplicon abundance was approximately parallel between all samples. All target gene mRNA abundance was calculated from a known standard curve and expressed relative to an internal control sample included in every real-time PCR run. Subsequently target gene mRNA was normalized to the housekeeping gene beta actin (ACTB).
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Statistical Analysis
All results are presented as mean ± SEM. Data were first tested for normality using a Kolmogorov-Smirnov test. Data were normalized using a logarithmic transformation. The effects of gestational age (group) and tissue type were tested with multifactorial analysis of variance for repeated measures (ANOVA; Statistical Packages for Social Sciences, SPSS-X, Data Analysis Systems; SPSS Inc, Chicago, IL). Where significant group or group-tissue interactions were found, the upper and lower myometrium were compared across gestational groups individually using a 1-way ANOVA. Within each gestational group, the upper and lower segment myometrial samples were compared using a Tukey HSD posthoc test. The amnion and choriodecidua were compared in a similar manner; across and within gestational groups as described for myometrial analysis. To assess the differences between the maternal and fetal relative target gene expression, myometrial samples (all gestational groups) were combined together and compared with the combined fetal membranes (all gestational groups) as described. Statistical significance was reported at a level of 0.05.
RESULTS
Immunohistochemistry
Serial tissue sections stained without the primary antibody or with the preabsorbed primary antibody showed no positive staining or markedly reduced immunostaining for all four PGE2 receptor subtypes and the PTGFR receptor in the upper and lower segment myometrium; this confirmed the specificity of the antibodies used (Fig. 1).
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Contractile/stimulatory receptors (PTGER1, PTGER3, and PTGFR) Diffuse staining within the cytosol of the myocytes together with some intense punctate striated bundles were observed throughout the circular and longitudinal muscle layers in the upper and lower uterine segments for the contractile PG receptor subtypes, PTGER1, PTGER3, and PTGFR (Fig. 2). Staining in the upper and lower segments was similar in each of the gestational groups and no dramatic changes in overall staining intensity or localization were observed with gestational age or with labor, except at TL, where PTGER1 was predominately associated with the nucleus (Fig. 2C).
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Relaxatory/inhibitory receptors (PTGER2 and PTGER4) Similar to the contractile receptor subtypes, PTGER2 and PTGER4 showed diffuse immunostaining within the cytosol of the myocytes throughout the muscle layers in both myometrial segments in all gestational groups (Fig. 3). There were no observable differences in overall staining intensity with gestational age, labor, or between the upper and lower segments. At higher magnification PTGER2 clearly showed intense punctate staining in striated bundles throughout the myometrium similar to the contractile receptor subtypes (Fig. 3C), while PTGER4 was predominately associated with the nucleus in all gestational groups (Fig. 3F), though more so at TL, and was similar to that seen for PTGER1 at TL (Fig. 2C).
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The striated staining pattern observed in the cytosol for the PGE2 receptor subtypes and PTGFR suggested an association with myofibrils, therefore immunostaining for these receptors was compared with that of alpha-smooth muscle actin in serial tissue sections. Immunoreactive alpha actin was observed throughout the circular and longitudinal muscle layers in the upper and lower uterine segments in all gestational groups (data not shown). The striated intracellular staining pattern of alpha-smooth muscle actin was similar to that observed for the PGE2 receptors subtypes and PTGFR (data not shown), indicating an association with myofibrils.
Real-Time PCR
Contractile/stimulatory receptors (PTGER1, PTGER3, and PTGFR)
To assess the differences in overall PGE2 and PGF2 receptor mRNA expression between the maternal and fetal tissues, myometrial samples (i.e., preterm labor and nonlabor plus term labor and nonlabor) were initially grouped and compared with grouped fetal membrane samples. PTGER1 mRNA expression was significantly greater in the fetal tissues when compared with the maternal tissue, with a significant intra-tissue difference between the amnion and choriodecidua (Fig. 4A). In contrast, while the relative expression of PTGER3 mRNA between maternal and fetal tissues was similar, there was a significant intra-tissue difference within both the maternal and fetal samples (Fig. 4B), as opposed to PTGER1, which showed a significant variation between the maternal and fetal side, but with a significant intra-tissue difference only within the fetal membranes (Fig. 4A). Additionally, the differences in the fetal membranes were reversed between the two contractile PGE2 receptors subtypes; PTGER1 expression was greatest in the amnion and least in the choriodecidua, while PTGER3 expression was greatest in choriodecidua and least in the amnion.
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The significant difference in PTGER1 expression between the maternal and fetal tissues was evident across all gestational groups (Fig. 4C), with an increase in PTGER1 expression in both the fetal and maternal tissues with increasing gestational age (Fig. 4C; PTNL vs. TL). On the other hand, PTGER3 expression decreased in maternal tissue with advancing gestation (Fig. 4D; PTNL vs. TL).
When studying the maternal tissues separately (upper and lower uterine segments), there was a trend of increased PTGER1 expression in the upper and lower uterine segments with advancing gestation, achieving significance only in the lower uterine segment (Fig. 4E; PTNL and TNL vs. TL). However, PTGER3 expression decreased in the lower uterine segments with advancing gestation (Fig. 4F). Interestingly, at term in both TNL and TL samples, the upper segment myometrium expressed significantly more PTGER3 when compared with the lower segment, in part due to the significant reduction in PTGER3 expression in the lower segment at this time (Fig. 4F). Analysis of the separated fetal membranes also showed contrasting patterns for PTGER1 and PTGER3 expression in the amnion to that of the choriodecidua. The amnion expressed predominately more PTGER1 in each gestational group when compared with the choriodecidua, despite a significant increase in PTGER1 expression in the choriodecidua with increasing gestational age (Fig. 4G; PTNL vs. PTL, TNL, and TL). Conversely, PTGER3 expression was greater in the choriodecidua compared with the amnion, and was statistically significant in both nonlabor groups, PTNL and TNL (Fig. 4H).
Relative expression of PTGFR between maternal and fetal tissues was equivalent (Fig. 5A), and there was consistent expression in all gestational groups except at PTL, where a significant reduction in the maternal tissue was observed (Fig. 5B). In the both uterine segments there was a trend for decreased PTGFR expression at PTL, followed by a modest increase at TL (Fig. 5C). The difference in the expression of PTGFR between the upper and lower segments was significant at PTNL (Fig. 5C). There was a trend for decreased PTGFR expression in the amnion with increasing gestational age, resulting in a reduced difference between the amnion and choriodecidua at term (Fig. 5D).
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Relaxatory/inhibitory receptors (PTGER2 and PTGER4) PTGER2 mRNA expression was significantly greater in the maternal tissues when compared with the fetal membranes, with a significant intra-tissue difference between the amnion and choriodecidua (Fig. 6A). Conversely, PTGER4 mRNA expression was greatest in the fetal membranes compared with the maternal tissues, with an intra-tissue difference between the upper and lower segment myometrium (Fig. 6B). The significant difference in PTGER2 expression between the fetal and maternal tissues was observed in all gestational groups, except at PTL (Fig. 6C). In addition, there was a significant increase in PTGER2 expression in the maternal tissues with advancing gestation (Fig. 6C; PTNL vs. TL). The significantly increased PTGER4 expression in the fetal membranes compared with maternal tissue was also observed in all gestational groups (Fig. 6D). Both the upper and lower myometrial segments showed a significant increase in PTGER2 expression with gestational age (Fig. 6E; PTL vs. TL). Although there was a trend for increased PTGER4 expression in the upper uterine segment at TL, the difference failed to reach significance (Fig. 6F; PTNL vs. TL). A significant difference in PTGER2 expression between the amnion and choriodecidua was seen at PTNL and TL, where the expression of the amnion was higher than that of the choriodecidua (Fig. 6G). The only difference seen in the expression of PTGER4 between the amnion and choriodecidua was at TNL, where PTGER4 expression was greatest in the choriodecidua (Fig. 6H).
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DISCUSSION
The model of a functional partitioning of the myometrium during pregnancy is logical given the profound physiological and biochemical changes occurring over the course of pregnancy. This concept suggests that the lower uterine segment displays a contractile phenotype throughout most of gestation that changes to a relaxatory phenotype at labor, whereas the upper uterine segment maintains a relaxatory phenotype throughout most of gestation and then adopts a contractile phenotype during labor [1]. To our knowledge no other study has simultaneously compared the PGE2 and PGF2 receptor subtypes in the upper and lower segment myometrium as well as the corresponding fetal membranes (amnion and choriodecidua) during human pregnancy.
We hypothesized that the differential expression of contractile and relaxatory PG receptors between the upper and lower uterine segments and fetal membranes would occur in a temporal and topographically distinct manner, and may account for changes in uterine contractility throughout gestation and labor. However, we found no dramatic changes in the PGE2 receptor subtypes or the PGF2 receptor in the myometrium or fetal membranes with advancing gestation or labor, or any topographical differences between the upper and lower uterine segments. This suggests that the coordination of changes, if they occur, is perhaps more subtle and/or complex. Furthermore, there were no clear coordinated increases or decreases in the relative expression of these receptor subtypes between the maternal and fetal tissues, except for PTGER1 subtype, which showed a significant increase with gestational age in both the maternal and fetal tissues. Those changes that did occur in the maternal tissue with gestational age were independent of the fetal membranes.
Interestingly, the relative expression of the PG receptor subtypes differed significantly between the maternal and fetal tissues; PTGER1 and PTGER4 were predominately expressed in the fetal membranes and PTGER2 expression was greatest in the myometrium, while overall, PTGER3 and PTGFR were consistently expressed in both the myometrium and fetal membranes. The presence of one or several of the PG receptor subtypes predominately in the myometrium and/or fetal membranes and the lack of coordinated expression between these tissues may indicate different regulatory mechanisms for these receptors between the myometrium and fetal membranes, in addition to an independent function of these receptors in the amnion and choriodecidua from that seen in the myometrium. Tissue-specific expression of the PG receptors has been demonstrated in the baboon, where PTGER2, PTGER3, and PTGER4 were present in the cervix, deciduas, and fetal membranes, including the chorion, whereas PTGFR was predominately found in the myometrium and the cervix [3, 18]. Thus myometrial activation (from quiescence to contractile activity) may not be mediated by a single specific receptor subtype, but rather a combination or balance of several or all of the major PG receptor subtypes between maternal and fetal tissues. This balance may be different between intrauterine tissues during pregnancy.
There were no topographical differences between the upper and lower uterine segments for the contractile PTGER1; however, a significant increase in expression was seen in the lower uterine segment with advancing gestation and at labor, which is consistent with previous published observations [6]. While this pattern of expression may be explained by preparing the uterus for postpartum contraction [6], the present observations showing significantly elevated levels of PTGER1 in the fetal membranes compared with the myometrium suggest PTGER1 may be more involved in intracellular signaling pathways associated with parturition within the fetal membranes rather than contraction of the myometrium during labor. Conversely, the expression profile for PTGER3, another contractile receptor subtype, decreased in the upper and lower segment myometrium with advancing gestation; however, in agreement with our hypothesis, at term the upper segment myometrium expressed significantly more PTGER3 when compared with the lower segment. Surprisingly, the upper segment myometrium expressed significantly more PTGFR early in gestation at PTNL as opposed to term. We have previously shown a reduction in PTGFR expression in the lower uterine segment with increasing gestational age, followed by a modest increase at term labor [2]; our present observations are consistent with this and both myometrial segments follow this expression pattern.
The opposite expression pattern was also seen in the relaxatory receptors, where PTGER2 mRNA expression was significantly greater in the maternal tissues when compared with the fetal membranes, and PTGER4 mRNA expression was greater in the fetal membranes compared with the maternal tissues. In contrast to other published data, there was a significant increase in PTGER2 expression in both the upper and lower myometrial segments with increasing gestational age, and a modest increase was seen in PTGER4 expression in the upper uterine segment. While this increased expression in the lower segment may help facilitate relaxation of the myometrium for delivery of the fetus, the reason for increased expression in the upper segment remains unclear. It is likely due to the upregulation seen in the lower segment, suggesting that the uterine segments may not work independently of each other as our hypothesis might indicate. These observations have lead us to question whether PTGER2 and PTGER4 subtypes are regulated in a similar manner, even though these subtypes share a common signaling pathway that is coupled with cAMP production.
We have previously shown the localization of the PGE2 receptor subtypes in the fetal membranes and placenta during gestation, with and without labor [7]. While we found no significant differences in PTGER1, PTGER2, and PTGER4 protein expression between the amnion and choriodecidua in any of the gestational groups or with advancing gestation, PTGER1, PTGER2, and PTGER4 all showed a different cellular location (basal membrane to diffuse cytoplasmic) in the amnion with labor, suggestive of different cellular functions with labor. The present observations are consistent with this idea of a different function of these receptors, particularly PTGER1 and PTGER4, in the fetal membranes (not directly related to contraction) as opposed to the myometrium. Indeed, PGF2 and PTGFR have been reported to play a role in the regulation of 11ß-HSD activity in fetal membranes, creating a positive feed-forward loop by which PGF2 acts to promote the production of cortisol and thus further PG output at the onset of labor [19]. The expression of these receptors in utero-placental tissues during bovine pregnancy also shows contrasting patterns across gestation for PTGER2, PTGER3, and PTGFR between the maternal and fetal tissues [20]. The presence of PTGER2 in the maternal caruncular epithelial, stromal, and fetal trophoblast cells suggests PGE2, acting via cAMP may effect maternal–fetal communication between the different cell types, thus supporting a role for PGE2 as an autocrine and paracrine factor in the regulation of placental function and possibly myometrial quiescence [20].
Smooth muscle contractions result from myosin and actin filament interactions, initiated by calcium and calmodulin-dependent myosin phosphorylation. The striated intracellular staining pattern seen for the PGE2 receptor subtypes and PTGFR was similar to that observed for ACTA2, suggesting an association with myofilaments and close coupling of PG receptors as part of the intracellular signaling pathways associated with muscle relaxation and contraction. Our preliminary data examining the localization of the oxytocin receptor in these tissues also shows a similar association with myofilaments (unpublished observations). Additionally, PTGER1 and PTGER4 showed an association with the nucleus in the myometrium at term, suggesting these receptors may mediate different cellular effects. This is in accordance with previously published work on the nuclear localization of the PGE2 receptor subtypes (PTGER1, PTGER3, and PTGER4) in a variety of cells types including the myometrium [21, 22]. The functionality of these receptors has been demonstrated by PTGER1-agonist-induced increase in intranuclear calcium concentrations and expression of c-fos transcripts by PTGER2 and PTGER4 stimulated nitric oxide formation. While there is abundant literature describing the effect of PGs on stimulating myometrial contractions via specific receptors on the cell membrane, these reports provide evidence for the localization of functional G protein-coupled receptors in the nuclear envelope that may regulate gene expression. Indeed, cytosolic phospholipase A2 and both cyclooxygenase enzymes are co-localized to the nuclear/perinuclear region, which suggests local generation of PGs can activate nuclear receptors that can then modulate effects other than direct contraction/relaxation (i.e., gene transcription) [23, 24]. This demonstrates the complexity of PG receptor function in the myometrium during pregnancy.
Myometrial activation via the PG receptors is not as clearly defined as we had hypothesized and may be mediated by a balance between one or several of the PG receptor subtypes, together with other known contraction-associated proteins. The specific reason for the regional variations in receptor populations within the uterus is yet to be fully elucidated, but would appear to explain the differential control of regions of the uterus before and after delivery of the fetus. The lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, as well as an independent function for these receptors in the amnion and choriodecidua compared with the myometrium. Further research is now required to define the functional role and involvement of the PG receptors in the signaling pathways that may or may not influence myometrial activation.
ACKNOWLEDGMENTS
We thank Dr. Rose Maxwell for her assistance in the statistical analysis of this data, and we thank Katherine Recht and Gail Kushner for their assistance in consenting patients and tissue collection.
FOOTNOTES
1 Supported by NIH Grant number HD 40285–01A1. 
2 Correspondence: Peta Grigsby, Department of Obstetrics and Gynecology, MSB 5456, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio, 45267. FAX: 513 558 6103; grigsbpl{at}ucmail.uc.edu
Received: 23 February 2006.
First decision: 20 March 2006.
Accepted: 13 May 2006.
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