American Journal of Obstetrics and Gynecology (2006) 195, 275–87

www.ajog.org

Pregnancy and estradiol modulate myometrial G-proteinpathways in the guinea pig

Carl P. Weiner, MD,a,* Clifford Mason, BA,b Gentzon Hall, BS,c Usma Ahmad, MD,d

Peter Swaan, PhD,b Irina A. Buhimschi, MDd

Departments of Obstetrics and Gynecology, University of Kansas School of Medicine,a Kansas City, KS; Departmentof Pharmaceutical Sciences, University of Maryland School of Pharmacyb; Department of Obstetrics, Gynecology,and Reproductive Sciences, University of Maryland School of Medicine,c Baltimore, MD; Department of Obstetricsand Gynecology, Yale University School of Medicine, New Haven, CTd

Received for publication October 3, 2005; revised December 9, 2005; accepted December 22, 2005

KEY WORDSG-proteinsGTPaseUterus

MyometriumQuiescenceOxytocin

Objective: Coupled to hundreds of receptors, G-proteins modulate signal transduction path-ways and are important hormonal targets. The first objective was to determine the effect of preg-

nancy and estradiol on myometrial guanosine triphosphatase activity. The second objective was tobegin dissecting the molecular mechanism(s) underlying alterations in guanosine triphosphataseactivity.

Study design: Myometrial tissue was obtained from pregnant, nonpregnant, and ovariectomizeduntreated and estradiol-treated guinea pigs. Myometrial membranes were prepared by homoge-nization and differential centrifugation. Basal high-affinity specific guanosine triphosphatase

activity was quantitated by enzymatic assay and expressed in rmol 32Pi per milligram proteinper minute. Guanosine triphosphatase activity was stimulated using oxytocin, isoproterenol,and prostaglandin F2a. Specific G-protein subunits were quantitated using Western blots.G-protein associated gene expression was semiquantitated using HGU133A gene array chips from

Affymetrix.Results: Basal myometrial guanosine triphosphatase activity was increased in pregnant com-pared with nonpregnant animals. Estradiol increased basal myometrial guanosine triphosphatase

activity, compared with untreated controls. The effect of estradiol on stimulated activity was ag-onist dependent. Both Gas and Gai isoform 1 protein levels were increased in myometrium fromlate pregnant compared with nonpregnant animals. By late gestation, the messenger ribonucleic

acid levels of those genes were unaltered, compared with the nonpregnant animal. In general, theimpact of pregnancy on G-protein family member gene messenger ribonucleic acid expression wasmodest. Only the small guanosine triphosphatase Rap1b demonstrated altered expression more

than 2-fold during either myometrial quiescence (midpregnancy) or activation (term pregnancy)(up 3-fold during quiescence). Genomic network analyses revealed that the expression of anothersmall guanosine triphosphatase, Rab7, was exclusively up-regulated (80%) during quiescence.During late pregnancy, network analysis showed that only G-protein b was exclusively altered

Supported by Grants HL49041 (to C.P.W.), HD049185 (to C.P.W.), and U01-DP000187 (to C.P.W.) from the United States Public Health Service.

* Reprint requests: Dr. Carl P. Weiner, Department of Obstetrics and Gynecology, University of Kansas School of Medicine, 3901 Rainbow

Boulevard, MS 2028, Kansas City, KS 66160-7316.

E-mail: cweiner@kumc.edu

0002-9378/$ - see front matter � 2006 Mosby, Inc. All rights reserved.

doi:10.1016/j.ajog.2005.12.050

276 Weiner et al

(up-regulated). Estradiol mimicked the pregnancy effect on both transcription and translation ofG-protein family members for some but not all potentially relevant genes.Conclusion: The increase in functional myometrial guanosine triphosphatase activity duringpregnancy may reflect increased synthesis of 1 or more small guanosine triphosphatase.

� 2006 Mosby, Inc. All rights reserved.

Regulation of myometrial contractility is critical forboth the maintenance and timely end of pregnancy. Themyometrium spends 80% to 90% of pregnancy in astate of quiescence, a genomically distinct phase when itis relatively unresponsive to contractile agonists. Quies-cence is modulated at least in part by estrogen andprogesterone, although the cellular mechanisms respon-sible for their effects are poorly understood.1

Numerous cell membrane receptors are coupled to theireffector units via G-proteins (guanine-nucleotide-bindingregulatory proteins). G-protein-mediated signaling linksextracellular stimuli to their targeted intracellular re-sponse by effectors such as adenylate and guanylatecyclases, phospholipases A and C, and Ca2C activatedpotassium channels. G-proteins are known targets ofhormonal modulation. There are 2 main families: GsandGi. At least 14 different G-proteins have been cloned.Each is a heterodimeric complex consisting of 3 subunits:Gas-guanosine diphosphate (GDP), Gb, and Gg. In thepresence of a stimulatory agonist, the high energy mole-cule guanosine triphosphate (GTP) binds Gas, causingit to dissociate from the Gsabg complex. Both the Gas-GTP and the bg complex then activate their targetenzymes. Activation is halted by the cleavage of GTPfrom Gas by a high-affinity GTPase enzyme, allowingthe inactive Gsabg complex to reform. The interpositionof G-proteins between the triggering event (receptorbinding by an agonist) and effector protein allows forsubstantial amplification of the stimulus and an efficientoff switch.

We previously demonstrated that uterine artery high-affinity GTPase activity is functionally decreased duringpregnancy.2 The net result of this pregnancy adaptationis an increased activated receptor life span and thus gen-eration of a greater number of secondary messengers peractivated receptor. We proposed that it is this event thatdirectly leads to the increased release of nitric oxide byreceptor-mediated stimuli, an adaptation characteristicof some organ beds and vessels during pregnancy.3,4,5

Because myometrial quiescence is characterized by de-creased responsiveness to contractile agents, we hypoth-esized that pregnancy modulates G-protein-mediatedresponses in a fashion that favors uterine relaxation asopposed to those favoring contraction. Involved regula-tory mechanisms could include an increase in GTPaseactivity or an increase in the concentration of a-subunitsof the heterotrimeric G-protein complex, given thatmost of the GTPase activity is associated with its ele-ments. Because uterine smooth muscle responsiveness

varies by gestational age, we hypothesize that any damp-ening of G-protein activity during myometrial quies-cence would be lost prior to the onset of labor duringthe phase of myometrial activation. The purpose of thecurrent study was to test aspects of this hypothesis bydetermining the effects of pregnancy and 17b-estradiolreplacement on myometrial GTPase activity and G pro-tein transcription and translation.

Material and methods

Animals and treatment

Spontaneously cycling nonpregnant [NP] and timedpregnant (midpregnancy [MP], 0.50-0.60; late pregnancy[LP], 0.8-0.9 gestation; and term pregnancy [TP] greaterthan 0.9 gestation) female Hartley albino guinea pigswere obtained from a commercial breeder (HarlanSprague Dawley, Indianapolis, IN). These gestationalages were selected to represent stages of myometrialquiescence, activation, and prelabor, respectively. Theanimals were of similar age (5 months) and weight(nonpregnant: 500-600 g; pregnant: 800-1100 g).

To determine the effect of estradiol on the studyparameters, some animals were ovariectomized throughflank incisions under sterile surgical conditions. Theanimals were allowed to recover for 100 days when acholesterol based, time-release pellet (21-day release)containing 0.0, 0.1, 0.25, 0.5, or 1 mg 17b-estradiol (17b-E2, Innovative Research of America, Sarasota, FL) wasimplanted subcutaneously. The release of estradiol fromthe pellet is reported by the manufacturer to be linear.We have previously demonstrated that this range ofestradiol ensures the circulating levels cover the fullphysiological range with the upper level achieving themaximum measured concentration during the normalguinea pig pregnancy.6 All experiments were approvedby the Committee on Animal Care at the University ofMaryland at Baltimore. Laparotomy was performedwhile the animal respired spontaneously under terminalanesthesia (ketamine hydrochloride 80 mg/kg intraperi-toneally, xylazine 3 mg/kg intramuscarly). Myometriumwas excised, carefully separated from the fetal mem-branes, and immediately frozen in liquid nitrogen.

All chemicals were purchased from Sigma ChemicalCo (St Louis, MO) unless specified.

GTPase activity was assayed as described by Casseland Selinger and modified by Sokolovsky7 and furtheroptimized as described below.

Weiner et al 277

Crude membrane fraction preparation

Frozen myometrium was pulverized in liquid nitrogenand homogenized in 10 mL 10 mM triethanolamine/HCland 140 mM NaCl (pH 7.4) and centrifuged at 3500 ! gfor 20 minutes at 4(C. The pellets were suspended in thesame buffer and centrifuged at 4300 ! g for 20 minutesat 4(C. The final pellet was resuspended in a buffercontaining 0.25 M sucrose, Tris/HCl (pH 7.5), 5 mMMgCl2, 1 mM of the high energy phosphate moleculeadenosine triphosphate (ATP), and protease inhibitors(100 mM phenylmethylsulphonyl fluoride, 10 mg/mL leu-peptin, 10 mg/mL soybean trypsin inhibitor, 2 mg/mLaprotinin, and 1 mMDL-dithiothreitol), filtered through3 layers of cheesecloth, and the resulting supernatantcentrifuged at 15,000 ! g for 20 minutes at 4(C. Themembrane pellet waswashed 3 times and uniformly resus-pended at 2 mg protein per milliliter by vigorous shakingin a Fastprep Bio101 homogenizer (Savant Instruments,Farmingdale, NY). The protein content was measuredwith a Bicinchoninic Acid kit (Pierce, Rockford, IL)against albumin standards.

Measurement of GTPase activity

Hydrolysis of [g-32P]-GTP (0.1 mCi/tube) was performedat 37(C (pH 7.30) in 120 mL reaction mixtures contain-ing 50 mM triethanolamine/HCl, 1 mM EDTA, 0.5 mMATP, an ATP regeneration system (5 mM creatine phos-phate, 0.4 mg/ml creatine kinase), 100 mg/tube bovineserum albumin 5 mM MgCl2, and either 1 mM (total ac-tivity reaction buffer) or 50 mM GTP (low-affinity reac-tion buffer). Duplicate reactions were initiated by theaddition of membranes (40 mg protein) to prewarmedreaction mixtures, allowed to proceed for 10 minutesat 37(C, and terminated by the addition of 0.7 mLice-cold sodium phosphate buffer (10 mM, pH 2.0) con-taining 5% (weight per volume) activated charcoal. Insome experiments reaction mixtures contained oxytocin,isoproterenol, or prostaglandin (PG)F2a (final concen-tration 10�8, 10�7, or 10�6 M). After incubation for10 minutes at 0(C, the reaction mixtures were clearedby low-speed centrifugation and radioactivity (32Pi)measured in 0.5 ml of supernatant.

Reactions without protein (blanks) and reactionswithout protein or charcoal (total counts) were run inparallel in the 2 incubation buffers. The counts obtainedfrom the blank reactions with the respective buffer weresubtracted from the average counts of the duplicatesample reactions. Specific GTPase activity was calcu-lated by subtracting the activity of the low-affinityreaction (activity in the presence of 50 mM GTP) fromthe total activity reaction (activity in the presence of0.1 mM GTP) and then normalized for total counts tocorrect for interassay variability. Because radioisotopedecay is the main determinant of interassay variability,samples were run simultaneously to eliminate interassay

variation. GTPase activity was expressed as rmol 32Pi

per milligram protein per minute. In preliminary exper-iments, we determined that high-affinity GTPase activityremained linear from 2 to 100 mg protein (r = 0.97) andfrom 2- to 30-minute incubation (r = 0.93).

Western blots

Crude membrane fractions were prepared from themyometrium of nonpregnant, MP (quiescence), or17b-E2 chronically treated guinea pigs. Membrane pro-tein (5 mg) diluted 1:2 volume/volume with electrophore-sis sample buffer (BioRad, La Jolla, CA) was solubilizedand then reduced by boiling for 5 minutes. One lane oneach gel was loaded with identical amounts of an immu-noblot standard mixture of G-protein a-subunits (Cal-biochem, EMD Biosciences, Darmstadt, Germany).

Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) was performed on a 10% sepa-rating gel and a 4% stacking gel using a BioRadMiniprotean II (Bio-Rad, Richmond, CA). Broad-rangeprestained SDS-PAGE standards (Bio-Rad) molecular-weight markers were used. After electrophoretic transferto polyvinyl difluoride membrane (Bio-Rad) at 100 Vfor 70 m and blocking with 5% milk, the blots wereincubated with polyclonal primary antibodies againstGa-subunits (Gsa, Gia-1, Gia-2, Gia-3, Goa, Gq/11a;Calbiochem) in dilutions of 1:1500 or 1:6000 for 1 hourat room temperature. Enhanced chemiluminescence wasapplied using a Western blotting detection system(Amersham, Arlington Heights, IL) with enzyme conju-gate anti-rabbit IgG/horseradish peroxidase servingas the secondary antibody. Autoradiography was per-formed and luminographs scanned densitometrically andthen analyzed using the National Institutes of HealthImage J software.8 The measurements were normalizedagainst the optical density of the positive control bandwithin the same gel in permit gel to gel comparisons.

Gene array

We previously applied these techniques to define someof the genomic effects of pregnancy on human andguinea pig myometrium.9,10

Preparation of ribonucleic acid (RNA)

Total RNA was isolated from frozen myometrium usinga homogenizer and TRIzol (Invitrogen, Carlsbad, CA)according to the manufacturer. Purity and integrity wereconfirmed by spectroscopy and formaldehyde-agarosegel electrophoresis.

Microarray hybridization

Gene profiling was performed in triplicate with 3 prep-arations from myometrium obtained from NP, MP(0.67 gestation), TP (0.96 gestation), castrate, and

278 Weiner et al

estradiol-treated castrate guinea pigs using the Gene-Chip human genome U133A array (Affymetrix, SantaClara, CA) containing 22,215 human gene probe sets.Each experiment was conducted on different days usingchips from different manufacturing lots. Prior to itsapplication to the GeneChip, RNA quality was assessedusing the Agilent 2100 bioanalyzer (Agilent Technolo-gies, Palo Alto, CA). Labeled complementary deoxyri-bonucleic acid (cDNA) was prepared from 20 mg totalRNA. Hybridization and washing were performed usingthe Affymetrix Fluidics Station 450 and HybridizationOven 640 under standard conditions. Image processingutilized the Affymetrix GeneArray 3000 scanner.

Statistical analysis

All data sets were tested for normality of distribution(Kolmogorov-Smirnov) before applying parametrictests. The results are presented as their mean G SEM.Statistical comparisons between 2 groups were madeusing the Student t test. Among multiple groups, a 1-wayor 2-way analysis of variance (ANOVA) followed bypost hoc testing with Student-Newman-Keuls tests wasapplied as appropriate. A 2-tailed P ! .05 was consid-ered indicative of statistical significance.

Microarray statistical analysis

Gene expression levels were normalized using Micro-array Suite 5.0 (Affymetrix). The average intensity forall probes on the array was scaled to 500. A detectionP value was used to make a reliable prediction of geneexpression (present, marginal, or absent). Only thosepresent were analyzed. Fold changes in transcript levelswere calculated from the mean measured signal values ofMP and TP versus the mean of their NP controls.The genes were further filtered to exclude those presentin only 1 of the three experiments.

Cluster and pattern analysis

Because of the highly redundant nature of the oligonu-cleotide arrays used, probe sets corresponding to thesame gene were identified using Netaffx (Affymetrix).Cluster analysis was performed by first separating thegenes into those that were up- or down-regulated duringeach gestational time period using MetaCore (GeneGo,St. Joseph, MO).11 Each category was subdivided intogenes specific for quiescence and activation using logicaloperations. Gene ontology (GO) annotations were usedas indicators of biological function. Associations withGO biological process, molecular function, and cellularcomponent groups were obtained through MetaCore.

Network visualization and analysis

MetaCore is a web-based computational platform de-signed primarily for the analysis of regulatory networks

and pathways.11 It includes a database of protein interac-tions, metabolism, and bioactive compounds. Whereas amap represents the preset expert curated representationoffunctional blocks and pathways, a network displays theinteractions and reactions compiled from different exper-iments and experimental conditions. Identification andvisual analysis of the resulting gene networks were per-formed using the MetaCore Analytical Suite (version2.0,GeneGo). For a network of a given size, statistical sig-nificance is calculated based on the probability of the net-work’s assembly from a random set of genes the same sizeas the input list (based on their P value). The network’srelevance to GO processes is then ranked according totheir P value as determined by preassessed algorithms.

Results

Effects of pregnancy and estradiol on basalGTPase activity

High-affinity specific GTPase activity was some 80%higher in myometrium from LP, compared with NPguinea pig animals (LP: 23.4 G 1.6 versus NP: 13.0 G1.4 rmol 32Pi per milligram protein per minute, t test,P ! .001) (Figure 1A).

17b-E2 replacement (0.25 mg) increased myometrialGTPase activity, compared with untreated ovariecto-mized controls (17b-E2: 15.8 G 2.1 versus OVX: 4.0 G0.6 rmol 32Pi per milligram protein per minute, 1-wayANOVA, P ! .001) (Figure 1B), but achieved absolutelevels no different from the intact, NP guinea pig(t test, P = .278). We previously demonstrated that the17b-E2 serum levels in ovariectomized, estradiol-replaced, and intact pregnant guinea pigs were increased,compared with nonpregnant control. The estradiol levelsassociated with the 0.25- and 0.5-mg pellets most closelyduplicated the levels achieved during pregnancy (23.9 G3.3, 28.3 G 6.0, and 16.0 G 2.4 pmol/mL, respectively).6

Although these results demonstrate an effect ofcastration and presumably hypoestrogenism on GTPaseactivity, estrogen replacement alone failed to generateGTPase activity levels seen during pregnancy.

Effects of pregnancy and estradiol onagonist-modulated GTPase activity

Isoproterenol (a Gas agonist) decreased GTPase activityin pregnant and stimulated it in nonpregnant animals.For 2 of the doses tested (10�8 M and 10�7 M), GTPaseactivity in response to isoproterenol was decreased inmyometrium from LP, compared with NP, controls(2-way, repeated-measures ANOVA, P = .002 for theeffect of pregnancy, P = .641 for isoproterenol doses)(Figure 2). This finding suggests that G-protein medi-ated signal transduction in response to b-mimetic agentsis more efficient in pregnant, compared with the NP,

Weiner et al 279

Figure 1 Basal myometrial high-affinity GTPase activity. A, NP versus LP. B, Castrate (ovariectomized [OVX]) versus chronically

17b-E2 treated. OVX guinea pigs were chronically implanted with pellets containing 0.1, 0.25, 0.5 mg 17b-E2. The 0.25 mg 17b-E2

induces serum estradiol levels similar to pregnancy.

Figure 2 Myometrial high-affinity GTPase activity in response to isoproterenol. A, NP versus LP. B, Castrate (ovariectomized[OVX]) versus guinea pigs chronically 17b-E2 treated (0.25-mg pellet).

state (ie, the activated effector-Gas complex persistslonger). Interestingly, long-term castration (100 days) re-duced isoproterenol-stimulated GTPase activity, whereasestradiol replacement increased it (2-way, repeated-measures ANOVA, P = .036 for the effect of estradiol;P= 0.643 for isoproterenol concentration). This suggeststhe effect of pregnancy on isoproterenol-stimulatedGTPase activity is not estrogen dependent.

LP was associated with increased oxytocin-stimulated(10�8 M) GTPase activity, compared with the NP state,suggesting a reduced half-life for the activated oxytocinreceptor (Figure 3). In contrast to the LP findings, estra-diol replacement decreased oxytocin-stimulated GTPaseactivity. Prior study reveals that myometrial sensitivityto oxytocin begins to increase during activation buthas its greatest increase just prior to and during labor.

280 Weiner et al

Figure 3 Myometrial high-affinity GTPase activity in response to oxytocin. A, NP versus LP. B, Castrate (ovariectomized [OVX])versus guinea pigs chronically 17b-E2 treated (0.25-mg pellet).

Figure 4 Myometrial high-affinity GTPase activity in response to prostaglandin F2a oxytocin. A, NP versus LP. B, Castrate(ovariectomized [OVX]) versus guinea pigs chronically 17b-E2 treated (0.25-mg pellet).

An increase in stimulated GTPase activity would func-tion as an efficient on-off switch for contractions.

Myometrial GTPase activity in response to PGF2awas increased in the NP state, whereas there was nochange in activity in pregnant animals (Figure 4) (2-way,repeated-measures ANOVA, P = .018 for the effect ofpregnancy, P = .015 for PGF2a concentration). Thissuggests the activated G-protein receptor will remainfunctional longer during LP, compared with NP.Thus, the effect of PGF2a will be greater during preg-nancy. This was the sole agonist-mediated effect mim-icked by estradiol but only for the lowest PGF2aconcentration (10�8 M). This was not sufficient for astatistically significant impact of estradiol replacementon PGF2a-mediated GTPase, compared with the

ovariectomized state (2-way, repeated-measures AN-OVA, P = .154 for the effect of estradiol, P = .033for PGF2a concentration).

In summary, pregnancy induces changes in basal andagonist-stimulated GTPase activity of guinea pig myo-metrium that cannot be explained solely by the hormo-nal effect of estradiol.

Effects of pregnancy and estradiol on G-proteina-subunit component content

Total Gas protein (both the 59- and 48-kDa bands) wasincreased in myometrium obtained from animals at LP,compared with NP control (Figure 5A). Estradiol (0.25-mg pellet) increased only the 59-kDa band of Gas.

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Figure 5 Myometrial G-protein levels in NP, LP, castrate (ovariectomized [OVX]), and 17b-E2-treated (0.25-mg pellet) guineapigs. A, Gas. B, Gai isoform 1.

Figure 6 Myometrial G-protein levels in NP-, LP-, castrate (ovariectomized [OVX]), and 17b-E2-treated (0.25-mg pellet) guineapigs. A, Gao. B, Gaq/11.

Both LP and estradiol (0.25-mg pellet) increased theprotein level of myometrial Gai isoform 1 (Figure 5B)but not either Ga isoform 2 or 3 (not shown).

There was no detectable effect of LP, castration, orestradiol treatment on either the Go subunit (Figure 6A)or Ga q/11 (Figure 6B) protein levels. The latter is theGa-subunit that couples the oxytocin receptor.

Effects of pregnancy and estradiol on G-proteinassociated gene expression

The impact of pregnancy and estradiol on the expressionof G-protein-signaling pathway components, comparedwith control, were modest. Many of the genes expressedon the arrays were unaltered by pregnancy (Table I, A

282 Weiner et al

Table I Potential changes in G-protein related genes duringpregnancy

A. Quiescence (MP)

Gene symbol Affinity identification Fold change

Up-regulationRAP1B 200833_s_at 3.0GNAS1 200780_x_at 1.9RAB7 211960_s_at 1.8RAP2C 218668_s_at 1.7GNAS1 212273_x_at 1.7GNAS1 200981_x_at 1.7GNAS1 214548_x_at 1.5GNAS1 217673_x_at 1.5RAB6A 201047_x_at 1.3GNAS1 211858_x_at 1.2RANBP9 202583_s_at 1.2RANBP9 202582_s_at 1.1RAB1A 208724_s_at 1.1RAB5A 206113_s_at 1.0

Down-regulationGPRK7 204559_s_at �1.7RAB1A 207791_s_at �1.6GPRK7 218290_at �1.2RANBP16 208459_s_at �1.2ROCK1 213044_at �1.2GNAO1 204762_s_at �1.1GPR27 221306_at �1.1RAB14 211503_s_at �1.1RAB11B 34478_at �1.0

B. Activation (TP)

Gene Affinity identification Fold change

Up-regulationRAB5A 209089_at 1.7RAP1B 200833_s_at 1.5RAP2C 218668_s_at 1.4GNB2 200852_x_at 1.4RANBP9 202583_s_at 1.2ROCK1 213044_at 1.1GNAS1 214548_x_at 1.1GNAS1 200981_x_at 1.1GNAS1 217673_x_at 1.1

Down-regulationGNAO1 204762_s_at �1.3RAB1A 207791_s_at �1.3RAB1A 208724_s_at �1.3RAB14 211503_s_at �1.3RAB11B 34478_at �1.3GPRK7 218290_at �1.2GNAS1 211858_x_at �1.2GNAS1 200780_x_at �1.1GNAS1 212273_x_at �1.1RAB6A 201047_x_at �1.1RANBP9 202582_s_at �1.1RANBP16 208459_s_at �1.1GPR27 221306_at �1.0

Table I (Continued)

C. 0.25 mg 17b-E2 replacement

Gene Affinity identification Fold change

Up-regulationRAB1A 207791_s_at 1.6RANBP9 202582_s_at 1.6RAB1A 208724_s_at 1.5RAP1B 200833_s_at 1.5RANBP7 200993_at 1.4RAC1 208640_at 1.3PDZGEF1 203097_s_at 1.2RAB6A 201047_x_at 1.2GNAS1 217673_x_at 1.2GNAS1 212273_x_at 1.2GNAS1 200981_x_at 1.1GNAS1 211858_x_at 1.1RAB5A 206113_s_at 1.1RAB14 211503_s_at 1.1RANBP9 202583_s_at 1.0

Down-regulationRANBP7 200994_at �1.6GPRK7 204559_s_at �1.5GPRK7 218290_at �1.4RAB9A 221808_at �1.3GNAS1 200780_x_at �1.2RAB11B 34478_at �1.1GNAS1 214548_x_at �1.0

D. 1.0 mg 17b-E2 Replacement

Gene Affinity identification Fold change

Up-regulationRAB1A 207791_s_at 1.9RAP1B 200833_s_at 1.9RAB1A 208724_s_at 1.7RAB5A 206113_s_at 1.5RANBP9 202583_s_at 1.4ROCK1 213044_at 1.4RAB14 211503_s_at 1.3RANBP9 202582_s_at 1.2RAP2C 218668_s_at 1.2PDZGEF1 203097_s_at 1.1RAP2C 218669_at 1.0RAB6A 201047_x_at 1.0GNAS1 211858_x_at 1.0

Down-regulationGNAS1 212273_x_at �1.6GNAS1 200780_x_at �1.6GNAS1 214548_x_at �1.3GNAS1 217673_x_at �1.3GPRK7 218290_at �1.3GNAS1 200981_x_at �1.2RAB11B 34478_at �1.1ARFGAP1 217888_s_at �1.0GPR27 221306_at �1.0RAB5A 209089_at �1.0

Weiner et al 283

Table II Differential gene expression changes throughout gestation

Gene Affinity identification MP TP 0.25 mg estrogen 1.0 mg estrogen

RAB-6A 201047_x_at 1.3 �1.1 1.2 1.0RAB-1A 207791_s_at �1.6 �1.3 1.6 1.9

208724_s_at 1.1 �1.3 1.5 1.7RAB-5A 206113_s_at 1.0 d 1.1 1.5

209089_at d 1.7 d �1.0ROCK1 213044_at �1.2 1.1 d 1.4Gas 200780_x_at 1.2 �1.2 1.1 1.0

200981_x_at 1.7 1.1 1.1 �1.221185_x_at 1.2 �1.2 1.1 1.0212273_x_at 1.7 �1.1 1.2 �1.6214548_x_at 1.5 1.1 �1.0 �1.3217673_x_at 1.5 1.1 1.2 �1.3

PDZ-GEF1 203097_s_at �1.3 �1.1 1.2 1.1

d, Signal was not detected with significant probability on array.

and B), estradiol (Table I, C and D), or castration (notshown). In contrast to the Western blots performed onmyometrium from LP animals in which Gai proteinlevels were increased, myometrial GNA01 (an isoformof Gai) expression was reduced at TP.

There was an apparent increase in myometrial Gasexpression during MP, compared with NP (Table II)that was lost by TP. Estradiol (0.25mg) had no effecton Gas expression, whereas 1 mg estradiol was associ-ated with a decrease (Table II).

Pregnancy and estradiol on G-proteinpathway maps

Network analyses proved helpful identifying candidategenes whose change in expression might explain theincrease in GTPase activity during pregnancy and afterestradiol. Figure 7 illustrates the expression of thoseG-protein-associated genes consistently altered duringMP and TP, compared with their NP control. Two specificpathways emerged. One was gestationally regulated andconsisted of r-associated kinase (ROCK) and ROCK1.As noted in Table II, these changes are modest andphysiologic importance should not be assumed.

The second pathway identified revealed down-regula-tion of Gai. Although these changes were fundamental topregnancy, they were not unique to either quiescence oractivation. The multiple identification tags representingthe same gene (ie, GNAS1) may designate sequences ofsplice variants that may or may not have the samephysiological activity. We observed several minor differ-ences among the expression patterns of these potentialvariants.

We next applied network analyses to identify G-protein-related genes whose expression was specificallyaltered during either quiescence or activation: the so-called signature networks. We subtracted those genesexpressed during TP from those expressed during MP to

produce the MP signature network (Figure 8). Thisanalysis revealed that only Rab-7, a small GTPase,was exclusively altered during quiescence when it wasup-regulated by 80%, compared with NP. We thenrevised the equation to subtract those genes altered dur-ing MP from those altered during TP to generate the TPsignature network (Figure 9) and found that the onlyG-protein-related genes altered only during myometrialactivation was Gb, which was up-regulated by 40%,compared with NP.

Pregnancy and gene ontology

Lastly, we grouped myometrial gene array messengerRNA (mRNA) expression by gene ontology: the per-centage of genes expressed on the array within majorthemes or functional processes describing molecularfunction, biological process, and cellular componentsof the gene network (Table III A and B). During quies-cence, the gene categories with the greatest percentage ofexpression tended to fall within those categories impor-tant for cell growth and division. In contrast, those pro-cesses uniquely expressed during myometrial activationtended to be involved with cell-cell communicationand energy provision.

Comment

The conversion of quiescent myometrium into an activeand reactive state ready for labor, a condition domi-nated by rhythmic, synchronous, and forceful contrac-tion, results from a series of complex interactions amongmultiple systems and events. Normal as well as patho-logic labor is thought to be preceded by a series ofhormonal changes that alter the synthesis and respon-siveness of the myometrium to prostanoids and cyto-kines. These events trigger a number of smooth muscle

284 Weiner et al

cell processes that shift the myometrium to a higherplane of excitability and conductivity. Simultaneously,the mechanisms responsible for myometrial quiescencemust be down-regulated.

We previously identified a paracrine substance fromhuman and guinea pig chorion that inhibits myometrialcontractility by opening large conductance calcium-dependent KC channels. The amount of this quiescentfactor released declines with advancing gestation.12,13

One line of evidence suggests that the modulation andphosphorylation of these KC channels by Pyk2 and aSrc-family kinase may be a general cellular mechanismby which G-protein-coupled receptor activation leadsto the regulation of membrane ion channels.14,15,16,17

The working hypothesis for the present investigation isrelated: We suggest that a gestational-dependent changein myometrial G-proteins/G-protein-mediated signaltransduction favors either myometrial quiescence (repre-sented by MP) or myometrial activation (LP). Thepresent findings, functional, transcriptional, and transla-tional in nature, each support the working hypothesis.

Late pregnancy and physiological 17b-E2 replacement(for pregnancy) increases myometrial basal high-affinityGTPase activity. This suggests an increase in eitherG-protein expression and/or the absence of enzymaticreceptor activation. It also indicates that myometrial

Figure 7 Logical operation intersection. Network illustratesgene and gene interactions common among both MP and LPwhen compared with NP. Colored symbols (nodes) represent

genes and red and blue circles correspond to genes with signif-icant up- and down-regulation, respectively. The small coloredhexagons on vectors between nodes describe positive (green),

negative (red), unspecified (black) interactions, or logical rela-tionships (blue).

G-proteins may be targets for hormonal modulation.However, levels of estradiol higher than that typicallyobserved during a normal guinea pig pregnancy(0.5-mg pellet) have no significant effect on high-affinityGTPase activity, compared with untreated controls, sug-gesting a bell shaped dose-response curve to estradiol.We observed a similar estradiol effect on endothelium-derived nitric oxide synthase.18 Furthermore, the in-crease generated by estradiol returned the basal GTPaseactivity level to only that of the nonpregnant animal.This suggests that estradiol does not account for theincrease in myometrial basal high-affinity GTPaseactivity during pregnancy.

In contrast to the myometrial basal GTPase activity,the effects of pregnancy and estradiol replacement onreceptor-stimulated GTPase activity vary with the ago-nist. High-affinity GTPase activity in response to b-adrenergic receptor activation (isoproterenol) is reducedin the myometrium from LP, compared with NP,controls. This finding suggests that G-protein-mediated

Figure 8 Logical operation subtraction: the MP signature

network. Genes specific for MP when compared with NP(MP/NP) were determined by subtracting from genes meetingthe required criteria in LP when compared with NP (LP/NP)

using a logical operations algorithm within the software. Theresulting data are built into a network of genes that have achange in expression (compared with NP) only during MP.Just one gene remains. Colored symbols (nodes) represent

genes. Red solid circle corresponds to genes with microarraydata significantly up-regulation.

Weiner et al 285

signal transduction in response to b-adrenergic receptoractivation is more efficient during gestation (ie, theactivated effector-Gas complex has a longer half-life).

One suggested mechanism of myometrial desensitiz-ation to b-mimetic agents used for tocolysis is uncou-pling of this G-protein.19 On the other hand, PGF2a,whose receptor is coupled to Gaq, failed during LP toalter receptor-stimulated high-affinity GTPase activity.Rather, it was increased in myometrium from NP con-trols. This finding also suggests more efficient PGF2a re-ceptor action during LP, compared with the NP state.Oxytocin increased stimulated high-affinity GTPaseactivity in LP myometrium. Because this action wouldserve to decrease the half-life of the activated receptor,the finding may seem counterintuitive. However, al-though myometrial sensitivity to oxytocin is increasedduring LP, compared with MP, the greatest increase inoxytocin effectiveness occurs immediately prior to andduring labor.20 It is also consistent with the observationthat Atosiban, an oxytocin receptor antagonist, is mosteffective in inhibiting contractions at gestational agesgreater than 34 weeks’ gestation in humans and is con-sidered ineffective early in pregnancy.21

A series of Western blots and microarray cDNAstudies were performed to seek potential mechanismsunderlying the pregnancy-mediated increase in myome-trial high-affinity GTPase activity. There is a clearincrease in myometrial Gas protein content in samplesfrom LP animals, compared with their NP controls.Gas is coupled to multiple receptors including theb2-adrenoceptor, prostaglandin E2 receptor EP2 sub-type (prostanoid EP2) prostacyclin receptor (IP), andsome 5-hydroxytryptamine (5HT) receptors. Activationtriggers an increase in myometrial cAMP. In contrast,Gsell et al22 found no change in myometrial Gas frompregnant women. Unfortunately, the samples were ob-tained across a broad gestational age range from women,most of whom were in labor (many premature) and by

Figure 9 Logical operation subtraction: the LP signature net-

work. Similar to Figure 8, genes specific for LP, compared withNP (LP/NP) were determined by subtracting from genes meet-ing the required criteria in MP when compared with NP (MP/

NP) using a logical operations algorithm within the software.The resulting genes are incorporated into a direct interactionsnetwork associated with LP. Colored symbols (nodes) represent

genes. Red and blue solid circles correspond to genes withmicroarray data showing significant up- and down-regulation,respectively.

definition had already undergone myometrial activation.It is likely heterogeneity of their sample explains the dif-ferences with the current study.

Isoform 1 of Gai protein level was also increasedduring TP and after estradiol, compared with controls.Gai is coupled to a2-adrenoceptors, muscarinic, and5HT receptors.23 Their activation tends to promote con-traction, probably by inhibiting cAMP production. Wewill test the effect of muscarinic and 5HT receptor acti-vation on GTPase activity in the future.

Presently we hybridized labeled cDNA generatedfrom guinea pig myometrium to human oligonucleo-tides. An animal model was important to assure accessto myometrium at the appropriate time points. Theguinea pig has a high degree of similarity to the humanin both its cardiovascular responses and sex hormoneprofile during pregnancy, and its placenta is most similarto the human among the nonprimates. The completeguinea pig genome has yet to be sequenced, and specificDNA microarrays are unavailable from commercial ven-dors. However, genes previously cited as significant forthe myometrium and sequenced completely (mitochon-dria) or partially (ie, uroguanylin, aquaporin 2, insulin-like growth factor-I) share high-percentage homology(data not shown) when aligned with their human

Table III Differentially expressed genes from the corre-sponding MP and LP signature (subtraction) networks

# Process P value

A. Quiescence (MP)1. Dephosphorylation 4.73e-072. Cell growth 1.70e-063. T-cell differentiation 1.85e-064. Muscle contraction 2.22e-065. Viral assembly, maturation, egress,

and release4.32e-06

6. Negative regulation of mitosis 4.32e-067. Regulation of cell cycle 1.15e-058. Intermediate filament-based process 1.30e-059. Regulation of protein-nucleus import 1.30e-05

10. Peripheral nervous system development 2.14e-05

B. Activation (LP)1. Protein biosynthesis 1.92e-122. Copper ion homeostasis 2.59e-103. Microtubule polymerization 1.51e-074. Establishment and/or maintenance

of chromatin architecture2.14e-06

5. Ribosome biogenesis 2.20e-066. Cell death 2.63e-067. Microtubule-based movement 1.52e-058. Nucleosome mobilization 2.23e-059. N-linked glycosylation via aparagine 6.68e-05

10. Regulation of viral genome replication 6.68e-05

The genes are grouped into associated processes and scored according

to the P value.

286 Weiner et al

orthologs. We recognized that mismatched sequencesmay have led to an underestimate of the expression ofsome critical genes. With that possible limitation, thedirectional array results are consistent with both theWestern blot data and, in part, the reports of priorinvestigators.

We observed an increase myometrial Gas mRNA insamples from MP, compared with NP control. How-ever, the myometrial expression of Gas near term wasessentially unchanged from the nonpregnant state. It ispossible that high-affinity GTPase activity either imme-diately before or during labor would have been reduced,compared with MP. In support of this possibility,Europe-Finner et al24 report that total levels of humanmyometrial Gas mRNA remain similar in nonpregnantand pregnant women until labor when they decline sub-stantially. The investigators also provided evidence thatalternative splicing of Gas precursor mRNA had a rolein regulating expression of Gas protein isoforms duringpregnancy and labor, which is in accordance with ourcurrent Gas findings.

Oxytocin is considered by some to play an active rolein the onset of labor, whereas others suggest its pre-dominant role is the maintenance of labor once initiatedand hemostasis postpartum.23 The oxytocin receptor iscoupled to Gaq/11.25 Other contractile agonists coupledto Gaq include the prostagladin F2 alpha receptor (pros-tanoid FP) and thromboxane AZ receptor (prostanoidTP) and the endothelin receptors.23 It is, therefore, of in-terest that the myometrial protein level of Gaq/11 fromeither TP- or estradiol-treated animals appears un-changed, compared with controls. This is consistentwith the findings of Harris et al26 in rat, which differedfrom Lajat et al,27 who observed a gestation-dependentincrease in myometrial Gaq protein levels in the rat.

The findings of the present study suggest anothersource of GTPase activity other than the components ofthe heterotrimeric complex is required to explain therelative resistance of myometrium to the contractileeffects of oxytocin in midpregnancy. Park et al28 investi-gated regulators of G-protein signaling (RGS proteins)that may interact with Gaq and Gai to accelerateGTPase activity and found in human myometrial cellculture that oxytocin increased RGS2 mRNA in adose-dependent fashion. We too sought the expressionof a variety of genes that may modulate G-protein-cou-pled receptor cycling including the RGS and SRC familyof proteins. What is most intriguing is that we foundthat of the many potential GTPases increased expres-sion of Rab7 unique to a time in gestation consistencewith myometrial quiescence. We also observed thatRap1 is increased during quiescence and most of thatincrease is lost by the time of myometrial activationat term.

Finally, we examined the cellular processes (ie, notjust those genes associated with G-proteins) in the

myometrium that had had an impact by pregnancy.Those affected most during quiescence were more oftenassociated with cell division and growth, whereas thoseduring activation were associated with cell communica-tion and energy transfer.

In conclusion, myometrial quiescence results from aseries of events, all designed to safeguard the growingconceptus from preterm birth. Pregnancy alters thekinetics of G-protein coupling in the myometrium in agestation-dependent fashion, facilitating transduction ofproquiescent signals (such as b-adrenergic receptor me-diated). This is opposite to the effect of pregnancy andestradiol on GTPase activity in the uterine artery. Itappears that much of the increase in GTPase activitycan be explained by the enhanced synthesis of smallGTPases such as Rab7 and Rap1 by a mechanismindependent of estradiol.

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