Estradiol-17β, Prostaglandin E 2 (PGE 2 ), and the PGE 2 Receptor Are Involved in PGE 2 Positive Feedback Loop in the Porcine Endometrium

Estradiol-17 , Prostaglandin E2 (PGE2), and the PGE2

Receptor Are Involved in PGE2 Positive Feedback Loopin the Porcine Endometrium

A gnieszka W acla w ik, Henry N. Jabbour, A gnieszka Blitek, and A dam J. Ziecik

Institute of A nimal Reproduction and Food Research of Polish Academy of Sciences (A . W ., A .B., A .J.Z.), Tu w ima 10,10-747 O lsztyn, Poland; and M edical Research C ouncil Human Reproductive Science Unit (H.N.J.), The Q ueens M edicalResearch Institute, Edinburgh EH16 4TJ, United Kingdom

Before implantation, the porcine endometrium and trophoblast synthesize elevated amounts ofluteoprotective prostaglandin estradiol-17! (E2) (PGE2). We hypothesized that embryo signal, E2,and PGE2 modulate expression of key enzymes in PG synthesis: PG-endoperoxide synthase-2(PTGS2), microsomal PGE synthase (mPGES-1), PGF synthase (PGFS), and PG 9-ketoreductase (CBR1)as well as PGE2 receptor (PTGER2 and -4) expression and signaling within the endometrium. Wedetermined the site of action of PGE2 in endometrium during the estrous cycle and pregnancy.Endometrial tissue explants obtained from gilts (n ! 6) on d 11–12 of the estrous cycle were treatedwith vehicle (control), PGE2 (100 nM), E2 (1–100 nM), or phorbol 12-myristate 13-acetate (100 nM,positive control). E2 increased PGE2 secretion through elevating expression of mPGES-1 mRNA andPTGS2 and mPGES-1 protein in endometrial explants. By contrast, E2 decreased PGFS and CBR1protein expression. E2 also stimulated PTGER2 but not PTGER4 protein content. PGE2 enhancedmPGES-1 and PTGER2 mRNA as well as PTGS2, mPGES-1, and PTGER2 protein expression. PGE2 hadno effect on PGFS, CBR1, and PTGER4 expression and PGF2" release. Treatment of endometrialtissue with PGE2 increased cAMP production. Cotreatment with PTGER2 antagonist (AH6809) butnot PTGER4 antagonist (GW 627368X) inhibited significantly PGE2-mediated cAMP production.PTGER2 protein was localized in luminal and glandular epithelium and blood vessels of endome-trium and was significantly up-regulated on d 11–12 of pregnancy. Our results suggest that E2

prevents luteolysis through enzymatic modification of PG synthesis and that E2, PGE2, and endo-metrial PTGER2 are involved in a PGE2 positive feedback loop in porcine endometrium.(Endocrinology 150: 3823–3832, 2009)

Porcine embryos secrete enhanced levels of estrogens, mainlyestradiol-17! (E2) between d 10 and 13 after fertilization,

just before implantation in the process of the maternal recogni-tion of pregnancy (1–4). It has been demonstrated that systemicestrogen administration between d 11 and 13 of the estrous cycleprevents corpus luteum regression and extends this gland’s lifes-pan in the pig (5). The action of estrogens may be luteoprotectiveand antiluteolytic (3, 4, 6).

However, estrogen alone does not fully exert the effect of theembryo on growth and development of corpus luteum (7). It hasbeen suggested that besides estrogens, prostaglandin (PG) E2

(PGE2) could be involved in the maternal recognition of preg-nancy as a luteoprotective/antiluteolytic factor (8, 9). Endome-

trial PGE2, and PGF2", which has an opposite luteolytic effect,are involved in reproduction processes in many species (10,11). It was demonstrated that inhibition of PG synthesis be-fore implantation causes pregnancy failure in different spe-cies, including the pig (11, 12).

PGs are synthesized by PG-endoperoxide synthase (PTGS)and specific terminal PG synthases: PGE synthase (PGES) andPGF synthase (PGFS) (13, 14). Moreover, PGE2 can be con-verted into PGF2" by PG 9-ketoreductase/carbonyl reductase(CBR1) (15–17). It was found that highly inducible forms ofPTGS and PGES in the porcine endometrium are PTGS2,(known also as PGHS-2 or COX-2) (18, 19) and microsomalPGES-1 (mPGES-1), respectively (14).

ISSN Print 0013-7227 ISSN O nline 1945-7170Printed in U.S. A .C opyright © 2009 by The Endocrine Societydoi: 10.1210/en.2008-1499 Received October 24, 2008. Accepted M arch 30, 2009.First Published O nline A pril 9, 2009

A bbreviations: CBR1, PG 9-ketoreductase/carbonyl reductase; E2, estradiol-17!; EIA , en-zyme immunoassay; mPGES-1, microsomal PGES-1; PG , prostaglandin; PGE2, prostaglan-din E2; PGES, PGE synthase; PGFS, PGF synthase; PM A , phorbol 12-myristate 13-acetate;PTGER, PGE2 receptor; PTGS, PG-endoperoxide synthase.

R E P R O D U C T I O N - D E V E L O P M E N T

Endocrinology, A ugust 2009, 150(8):3823–3832 endo.endojournals.org 3823

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Before implantation, the endometrium and trophoblastsynthesize elevated amounts of PGE2, which results in a highcontent of this prostanoid in the uterine lumen and/or utero-ovarian circulation in the pig (9, 14, 17, 20). Our recent studyhas demonstrated that expression of PG synthesis pathwayenzymes is altered in the porcine conceptus and endometriumto favor luteoprotective PGE2 synthesis between d 10 and 13of pregnancy (14, 17).

PGE2 mediates its effect via binding to either of four subtypesof G protein-coupled receptors (PTGER1-4 also known as EP1-4), which are encoded by four separate genes (21). The effect ofPGE2 in endometrial angiogenesis, uterine receptivity, and de-cidualization is mediated by cAMP-dependent mechanisms inrats (22), rabbits (23), and humans (24). Only two subtypes ofPGE2 receptors (PTGERs), PTGER2 and PTGER4, are coupledto adenylate cyclase and generate cAMP, which activates theprotein kinase A signaling pathway (21). Knockout studies inmice indicated that PTGER2, but not PTGER1, PTGER3, orPTGER4, is associated with reproductive dysfunction (25).PTGER2-deficient mice demonstrate impaired ovulation anddramatic reduction in litter size (25). In pigs, the PGE2-bind-ing sites were detected in both cycling and pregnant endome-trium, but their location within endometrium was not deter-mined (26). Moreover, no mRNA or protein expression of anyPGE2 receptor subtype has been identified in the porcine endo-metrium yet. To date, regulation of expression of PGE2 receptorsin porcine endometrium remains unknown. Therefore, the ob-jective of the present study was to elucidate whether the well es-tablished porcine embryo signal, E2, and the putative embryo sig-nal, PGE2, regulate expression of PG synthesis pathway enzymesPTGS2, mPGES-1, PGFS, and CBR1 as well as PGE2 receptors(PTGER2 and PTGER4) in the porcine endometrium. Moreover,the aim of study was to determine the site of action of PGE2 in theporcine endometrium during the estrous cycle and early pregnancy.

Materials and Methods

Tissue collectionPeripubertal crossbred gilts of similar age (approximately 5–5.5

months) were observed daily for onset of estrus. After exhibiting twonatural estrous cycle gilts were assigned into two groups: pregnant andcycling. Gilts assigned to the pregnant groups were artificially insemi-nated at 12 h after onset of estrus (d 0) and 24 h later. Gilts were slaugh-tered at a local abattoir on d 9, 11, 12, or 15 of pregnancy or the estrouscycle (n ! 4–6 per group). The stage of the estrous cycle was verified byuteroovarian morphology (14), and pregnancy was confirmed by thepresence of morphologically normal conceptuses. Uterine tissues from allgroups were fixed in 4% neutral buffered formaldehyde overnight at 4C, stored in 70% ethanol, and wax embedded (for immunohistochemicalanalyses). The endometrium was snap-frozen and stored until further use(for RNA and protein extraction). Additionally, uteri collected from giltson d 11–12 of the estrous cycle were immediately placed in ice-cold PBSand transported to the laboratory within 1–1.5 h for in vitro explanttissue and cell culture.

To determine whether there are differences in expression of mPGES-1protein between regions near the conceptus and without proximity tothe conceptus, endometrial samples were collected from the implan-tation sites (n ! 4) and interimplantation sites (n ! 4) in d-25 preg-nant gilts (n ! 4).

All procedures involving animals were approved by the Local Re-search Ethics Committee and were conducted in accordance with thenational guidelines for agricultural animal care.

Endometrial explant cultureUteri collected from gilts on d 11–12 of the estrous cycle (n ! 6) were

washed three times in sterile PBS. The endometrial tissue from the middleof uterine horn was dissected and cut into small pieces (2 " 3 mm) andthen washed three times in medium M199 (Sigma-Aldrich Co., St. Louis,MO). A total of 100 mg tissue was placed into each glass vial with 2 mlM199 medium containing 0.1% BSA (ICN Biomedicals, Inc., CostaMesa, CA), 5% dextran/charcoal-stripped newborn calf serum (Sigma-Aldrich), penicillin (100 IU/ml), and streptomycin (100 #g/ml). The en-dometrial tissue explants were preincubated in a shaking water bath at37 C in a humidified atmosphere of 5% CO2 in air for 2 h.

After this period, explants were treated with vehicle only (control),PGE2 (100 nM; ICN), E2 (1, 10, or 100 nM; Sigma-Aldrich), or phorbol12-myristate 13-acetate (PMA; 100 nM; Sigma-Aldrich) and incubatedfor an additional 24-h period. The structure of PMA is analogous todiacylglycerol and serves as an agonist for activation of protein kinase C.Because it is known that PMA increases PG secretion, it was used as apositive control (27, 28).

All treatments were performed in duplicate in six independent ex-periments. After culture, the medium was collected and stored at #20 Cuntil PG concentrations were measured by enzyme immunoassay (EIA).Furthermore, endometrial tissue explants were snap-frozen in liquid ni-trogen (for RNA and protein extraction) and stored at #80 C untilfurther use.

Real-time PCR quantitationTotal RNA was extracted from endometrial samples and tissue ex-

plants collected after in vitro experiment using the Total RNA Prep Pluskit (A&A Biotechnology, Gdansk, Poland) and treated with deoxyribo-nuclease I (Invitrogen Life Technologies, Carlsbad, CA) according to themanufacturer’s protocol. Real-time PCR was performed with the Ap-plied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Fos-ter City, CA) using QuantiTect SYBR Green PCR master mix (QIAGENGmbH, Hilden, Germany), as described previously (14, 17). Briefly, totalRNA was reverse transcribed using oligo(dT) primer and avian myelo-blastosis virus reverse transcriptase (Promega, Madison, WI). Real-timePCR (25 #l) included 12.5 #l QuantiTect SYBR Green PCR master mix,0.5 #M sense and antisense primers each, and reverse-transcribed cDNA(3.5 #l diluted RT product). The specific primers were used to evaluatethe mRNA levels (Fig. 1G). For quantification, standard curves consist-ing of serial dilutions of the appropriate purified cDNA were included.Before amplification, an initial denaturation (15 min at 95 C) step wasused. The PCR programs for each gene were performed as follows: 36cycles of denaturation (15 sec at 95 C), annealing (30 sec at 52.5 C forPGFS or at 55 C for PTGS2, mPGES-1, CBR1, and !-actin), and elon-gation (60 sec at 72 C). The PCR programs for PTGER2 and PTGER4genes were performed as follows: 36 cycles of denaturation (15 sec at 95C) and annealing (60 sec at 60 C). After PCR, melting curves were ac-quired by stepwise increases in the temperature from 60–95 C to ensurethat a single product was amplified in the reaction. Data obtained fromthe real-time PCR for PTGS2, mPGES-1, PGFS, CBR1, PTGER2, andPTGER4 were normalized against !-actin. Control reactions in the ab-sence of reverse transcriptase were performed to test for genomic DNAcontamination.

Protein extractionEndometrial samples and tissue explants were homogenized on ice in

buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM

EDTA and supplemented with protease inhibitor cocktail (Sigma-Aldrich).Homogenates were then centrifuged for 15 min at 800" g at 4 C and storedat #70 C for further analysis. The protein concentration was determined bythe Bradford (29) method.

3824 W acla w ik et al. E2 and PGE2 and PG Synthesis Enzymes and PTGER Endocrinology, A ugust 2009, 150(8):3823–3832



Western blot analysisWestern blot analysis was performed as we previously described

(14, 17). Protein extracts (30 #g) were dissolved in SDS gel-loadingbuffer [50 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 2%!-mercaptoethanol], heated to 95 C for 4 min, and separated on 15%(for mPGES-1), 12% (for PGFS and CBR1), and 10% (for PTGS2,PTGER2, and PTGER4) SDS-PAGE. Separated proteins were electro-blotted onto 0.2-#m nitrocellulose membrane in transfer buffer [20 mM

Tris-HCl buffer (pH 8.2), 150 mM glycine, and 20% methanol]. Afterblocking in 5% nonfat dry milk in Tris-buffered saline containing 0.1%Tween 20 for 1.5 h at 25.6 C, the membranes were incubated overnightwith 1:750 anti-COX-2 antibody (anti-PTGS2 antibody; CaymanChemical, Ann Arbor, MI), 1:1000 polyclonal anti-mPGES-1 antibody

(Cayman Chemical), 1:2000 anti-lung-type PGFS anti-serum (kindly donated from Prof. Kikuko Watanabe, Uni-versity of East Asia, Yamaguchi, Japan), 1:2000 poly-clonal antihuman carbonyl reductase 1 antibody (Abcam,Cambridge, UK), 1:200 rabbit polyclonal antibodiesagainst human EP2 (anti-PTGER2 antibody; CaymanChemical), or 1:50 rabbit polyclonal antibodies againsthuman EP4 (anti-PTGER4 antibody; Cayman Chemical)at 4 C. Subsequently, the studied proteins were detected byincubating the membrane with a 1:20,000 dilution of sec-ondary polyclonal antirabbit alkaline phosphatase-conju-gated antibodies (for PTGS2, mPGES-1, PGFS, PTGER2,and PTGER4; Sigma-Aldrich) and a 1:2000 dilution ofantigoat alkaline phosphatase-conjugated antibodies (forCBR1; Abcam) for 1.5 h at 25.6 C. Immunecomplexeswerevisualized using an alkaline phosphatase visualization pro-cedure. Western blots were quantitated using the Kodak 1Dprogram (Eastman Kodak, Rochester, NY). Sample loadingwas standardized to expression of !-actin using specific an-tibodies (1:3000; Abcam). Control experiments were per-formed for PTGER receptors by incubating the membraneswith anti-PTGER2 or anti-PTGER4 antibodies preabsorbedwith thecorresponding immunogenicblockingpeptide (Cay-man Chemical). Western blot analyses for PTGERs by incu-bating the membranes with the primary antibodies preab-sorbed with the related blocking peptides did not give anysignal. Specificity of other antibodies used was confirmedpreviously (14, 17, 30).

EIA of PGE2 and PGF2

Concentrations of PGE2 in medium were determinedby an EIA as described previously (31). Cross-reactivitiesof the anti-PGE2 antiserum (donated by Dr. Seiji Ito, Kan-sai Medical University, Osaka, Japan) were as follows:PGE1 18%, PGA1 10%, PGA2 4.6%, PGB2 6.7%, PGD2

0.13%, PGF2" 2.8%, PGJ2 14%, and 15-keto-PGE2

0.05%. Assay sensitivity was 0.19 ng/ml, and the intra-and interassay coefficients of variation were 4.9 and 8.5%,respectively. Concentrations of PGF2" were determined byEIA test as described previously (31). Cross-reactivities ofthe anti-PGF2" antiserum (Sigma-Aldrich) were as follows:PGF1" 60%, PGE1 and PGE2 less than 0.1%, and PGA1,PGA2, PGB1, and PGB2 less than 0.01%. Assay sensitivitywas 0.23 ng/ml, and the intra- and interassay coefficientsof variation were 8.9 and 11.9%, respectively.

cAMP assayEndometrial tissue (100 mg) collected from gilts on d

11–12 of the estrous cycle (n ! 6) were minced finely intosmall pieces (2 " 3 mm). The tissue explants were incubatedovernight at 37 C in a humidified 5% CO2 incubator in 2 mlM199 medium (Sigma-Aldrich) containing 100 IU penicillin,100 #g streptomycin, and 3 #g/ml indomethacin (Sigma-Al-drich). After overnight treatment, the tissue was incubated inM199 medium, containing 100 IU/ml penicillin, 100 #g/ml

streptomycin, 3 #g/ml indomethacin, and 1 mM 1-methyl-3-iso-butylxan-thine (Sigma-Aldrich) in the absence or presence of the appropriate receptorantagonist as shown in Fig. 5C legend at 37 C for 30 min. It was then treatedwith vehicle, 100 nM PGE2 in the presence or absence of 10 #M AH6809(PTGER2 antagonist; Sigma-Aldrich), or 5 #M GW 627368X (PTGER4antagonist; Cayman Chemical) (32) for 10 min at 37 C and washed inice-cold PBS and snap-frozen. Tissue explants were homogenized in lysisbuffer (100 mg tissue/ml buffer; R&D Systems, Abingdon, UK), and cAMPconcentration was quantified in the homogenates by ELISA using a cAMPkit (R&D Systems) according to the manufacturer’s protocol. The resultswere normalized to the protein concentration, which was determined usinga protein assay kit (Bio-Rad, Hemel Hempstead, Herts, UK).

FIG. 1. A–F, Effect of E2 (1, 10, or 100 nM) on mRNA expression of PTGS2 (A), PGFS (B), mPGES-1(C), PG 9-ketoreductase (D), PTGER2 (E), and PTGER4 (F) in endometrial tissue explants in vitro.Asterisks indicate significant differences between control (C) and experimental groups: *, P $ 0.05;**, P $ 0.01. G, Primers used for real-time PCR.




ImmunohistochemistryImmunohistochemical localization of PTGER2 in the endometrium

ond9,11,12, and15of the estrous cycle andpregnancy (n !3–4 ineverygroup) was performed using Bond-X automated immunostaining ma-chine (Vision Biosystems, Newcastle, UK). The method on the Bond-Xautomated machine uses a specific polymer high-contrast program.Briefly, cross-sections were made in the middle portion of the uterinehorn. Five-micrometer paraffin sections of uterine tissue were cut, de-waxed in xylene, and dehydrated using decreasing grades of ethanol.Antigen retrieval was performed by pressure cooking in 0.01 M sodiumcitrate (pH 6) before being placed on the Bond-X machine. Then, en-dogenous peroxidase activity was removed by fixing sections in 3%hydrogen peroxide for 5 min in methanol. Afterward, slides were incu-bated with rabbit polyclonal anti-PTGER2 (1:1000; Cayman Chemical)antibody for 2 h and treated with the polymer reagent (Vision Biosys-tems) for 15 min. The detection step was performed using 3,3%-diami-nobenzidine tetrahydrochloride for 10 min, and sections were counter-stained in hematoxylin for 5 min. Slides were then removed from themachine and dehydrated and mounted using Pertex. To confirm antibodyspecificity, negative controls were performed by incubating sections with acontrol rabbit IgG at the matched concentrations to the primary antibodiesand with the primary antibody preabsorbed with the peptide with which itwas raised (Cayman Chemical). Immunoreactivity was negligible with bothpreabsorbed antibody and with a control rabbit IgG.

Endometrial cell culture in vitroEndometrial epithelial and stromal cells were isolated from uteri on

d 11–12 of the estrous cycle (n ! 5) and pregnancy (n ! 3), as describedpreviously (31), and seeded in six-well plates. Cell cultures were incu-bated in M199 medium containing 2% BSA (ICN Biomedicals), 10%newborn calf serum (Sigma-Aldrich), penicillin (100 IU/ml), and strep-tomycin (100 #g/ml) at 37 C in an atmosphere of 95% air and 5% CO2.At 80–90% cell confluence, medium was replaced with serum-freeM199. After 24 h, cells were harvested and lysed in RIPA buffer [50mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 mM EDTA, and protease inhibitor cock-tail], and the obtained protein extracts were used to study the expressionof PTGER2 and PTGER4 by Western blot analyses.

Statistical analysisStatistical analyses were performed using repeated-measures one-

way ANOVA or paired t test (GraphPad Prism version 4.0; GraphpadSoftware, San Diego, CA). Two-way ANOVA was used to evaluatechanges in abundance of PTGER2 and PTGER4 mRNA and protein dueto day of the estrous cycle and pregnancy as well as an effect of repro-ductive status. All numerical data are presented as the mean & SEM, anddifferences were considered as statistically significant when P $ 0.05.

Results

Effect of E2 on PTGS2, mPGES-1, PGFS, CBR1, PTGER2,and PTGER4 expression and PGE2 and PGF2 secretion inendometrial tissue explants

E2 elevated expression of mPGES-1 mRNA (10 and 100 nM,P $ 0.05 and P $ 0.01, respectively) but had no effect on PTGS2,PGFS, CBR1, PTGER2 and PTGER4 mRNA content in endo-metrial tissue explants (Fig. 1). E2 stimulated expression ofPTGS2 (10 and 100 nM, P $ 0.05 and P $ 0.01, respectively),mPGES-1 (1 and 10 nM, P $ 0.05) and PTGER2 protein (10 and100 nM) but decreased (P $ 0.05) expression of PGFS (10 and100 nM) and CBR1 protein (100 nM) and had no effect onPTGER4 protein content (Fig. 2, A–F). E2 treatment increasedPGE2 secretion in a dose-dependent manner (Fig. 2G). However,

there was no effect of E2 treatment on PGF2" secretion by en-dometrial tissue explants (Fig. 2H).

Effect of PGE2 on PTGS2, mPGES-1, PGFS, CBR1, PTGER2,and PTGER4 expression and PGE2 and PGF2 secretion inendometrial tissue explants

PGE2 stimulated mPGES-1 (P $ 0.01, Fig. 3C) and PTGER2mRNA expression (P $ 0.05; Fig. 5A) and had no effect onPTGS2, PGFS, and CBR1 mRNA content in endometrial ex-plants (Fig. 3, A, B, and D). PGE2 enhanced PTGS2 (Fig. 4A),mPGES-1 (P $ 0.05, Fig. 4C), and PTGER2 protein expression(P $ 0.5, Fig. 5B) as well as PGE2 secretion by endometrial tissueexplants (P $ 0.05, Fig. 4E). By contrast, PGE2 had no effect onPGFS and CBR1 expression and PGF2" release (Fig. 4, B, D, andF) and PTGER4 mRNA and protein expression (Fig. 5, C and D).PMA used as a positive control stimulated both PTGS2 andmPGES-1 mRNA and protein expression (P $ 0.05, Fig. 3, A andC, and Fig. 4, A and C) and PGFS protein levels (P $ 0.05, Fig.4B) but had no effect on CBR1 expression (Figs. 3D and 4D).Moreover, PMA increased secretion of PGE2 (P $ 0.01, Fig. 4E)and PGF2" (P $ 0.05, Fig. 4F).

PGE2 and E2 elevated about 2-fold the mPGES-1/PGFS pro-tein ratios in endometrial tissue explants (Fig. 3F).

Endometrial expression of mPGES-1 protein inimplantation and interimplantation sites

Expression of mPGES-1 protein was up-regulated in endo-metrium collected from the implantation sites when comparedwith interimplantaion sites (P $ 0.01, Fig. 3G).

Effect of PGE2 on cAMP accumulation in endometrialexplants

Treatment with PGE2 increased cAMP production in endo-metrial tissue explants in absence of any PTGER antagonists(P $ 0.001, Fig. 5J). Cotreatment with PTGER2 antagonist(AH6809) inhibited significantly the PGE2-mediated cAMP pro-duction. However, the response to PGE2 was not suppressed bycotreatment with the PTGER4 antagonist (GW 627368X). Nosignificant alteration in basal levels of cAMP was observed inendometrial tissue treated with the antagonists alone.

Expression of PTGER2 and PTGER4 in the endometriumduring the estrous cycle and early pregnancy

No effect of day or reproductive status was detected forPTGER4 mRNA expression (Fig. 5F). PTGER4 protein andPTGER2 mRNA expression was affected by day but not reproduc-tive status (Fig. 5, H and E). PTGER2 mRNA content was signif-icantly higher on d 12 and 15 in cyclic and pregnant gilts whencompared with d 9 and 11 of the estrous cycle and pregnancy (Fig.5E). Expression of PTGER2 protein was affected by reproductivestatus but not day. PTGER2 protein expression was up-regulatedon d 11 and 12 of pregnancy when compared with correspondingdays of the estrous cycle (P $ 0.05, Fig. 5G).

PTGER2 protein was highly expressed in luminal and glan-dular epithelium of endometrium and in blood vessels and waspresent in myometrium (Fig. 5I). Protein expression of PTGER2was detected on d 9–15 of the estrous cycle and pregnancy.




Moreover, protein expression of PTGER2 and PTGER4 wasconfirmed in both luminal epithelial and stromal cells isolatedfrom the uterus on d 11–12 of the estrous cycle and d 11–12 ofpregnancy (Fig. 5K).

Discussion

Present hypotheses about the antiluteolytic mechanism and rec-ognition of pregnancy in the pig are mainly focused on PGF2"

secretion (2, 4, 33). Although PGE2 has been reported to haveluteoprotective effects (8, 9), currently, there are no studies de-scribing the molecular mechanisms regulating PGE2 synthesis inthe endometrium during early pregnancy. To our knowledge,this is the first study to address the effect of PGE2 and embryosignal, E2 on the PG biosynthesis pathway and PGE2 signaling in

the porcine endometrium. PTGS2 is an en-zyme involved in nonselective production ofPGs, whereas PG synthases (mPGES-1 andPGFS) and CBR1 control the selective pro-duction and associated specific functions ofPGE2 and PGF2". We have found that PGE2

biosynthetic pathways are selectively acti-vated in the porcine endometrium (14). Ourhypothesis was that selective changes in ex-pression of enzymes involved in PG synthe-sis and in the porcine endometrium are aresult of embryo signals.

Even spherical d 10–11 blastocysts sized5–7 mm in diameter are capable of enhancedestrogen synthesis (34, 35). Synthesis and se-cretion of estrogens by conceptuses dramati-cally increase during trophoblast elongationon d 11–12 of pregnancy, decline rapidly on d13, and then initiate a second more sustainedincrease on d 16–25 of pregnancy (4, 36). Inthe present studies, we demonstrated that E2

stimulated PGE2 synthesis through increaseof PTGS2 protein and mPGES-1 mRNAand protein expression and decreased theprotein content of enzymes involved inPGF2" production: PGFS and CBR1 in theporcine endometrium on d 11–12 after es-trus. Stimulation of PTGS2 protein expres-sion in endometrial explants by E2 in vitroobserved by us is in agreement with the ob-servation that administration of estrogen invivo affects the expression pattern ofPTGS2 (18). The data presented herein arealso consistent with our previous findingsthat mPGES-1 expression is higher andPGFS and CBR1 lower in the porcine endo-metrium between d 10 and 13 of pregnancywhen compared with the following days ofgestation (14, 17). The observations in theexplant cultures after treatment with con-ceptus factors corresponds also with com-

parison of CBR1 and PGFS protein expression in pregnant vs.cyclic animals on d 10–11 and 10–13, respectively (14, 17). Incontrast, our previous data showed no differences in mRNA andprotein expression of mPGES-1 in the endometrium on d 11–12of pregnancy and d 11–12 of the estrous cycle (14). This dis-crepancy between stimulation of mPGES-1 expression in ex-plants by conceptus factors (E2 and PGE2) and lack of effect ofpregnancy on mPGES-1 mRNA and protein can be a result oflocal changes induced in the endometrium by the conceptus. It isnot possible to distinguish which endometrial samples collectedon d 11–12 of pregnancy had direct contact with the embryobecause implantation begins on d 14. It is tempting to speculatethat the effect of conceptus factors on endometrial explants canmimic local changes in the endometrium that is near the con-ceptus. This hypothesis, although it needs further study, is sup-ported by results concerning the later stage of pregnancy and

FIG. 2. A–F, Effect of E2 (1, 10 or 100 nM) on protein expression of PTGS2 (A), PGFS (B), PGES (C), PG9-ketoreductase (D), PTGER2 (E), and PTGER4 (F) in endometrial tissue explants in vitro. The representativesamples of W estern blots are sho w n in the upper panels. G and H, Effect of E2 (1, 10, or 100 nM) on PGE2

(G) and PGF2" secretion (H) in endometrial tissue explants in vitro. Asterisks indicate significant differencesbetw een control (C) and experimental groups: *, P $ 0.05; **, P $ 0.01.




indicating 2-fold up-regulation of mPGES-1 protein expressionin the endometrium in the implantation sites when comparedwith interimplantation sites (Fig. 3G). It is likely that effects ofestrogen on the endometrium are restricted to regions in close prox-imity to the conceptus due to metabolic activity of trophectoderm.During pregnancy, pig endometrium rapidly converts E2 to the bi-ologically inactive estrone sulfate that is present in high concentra-tions within the uterine lumen of pregnant pigs (37). The trophec-toderm has sulfatase enzyme activity that restores the biologicalactivity of estrogen, allowing for a localized effect of estrogen toup-regulate genes such as SPP1 in luminal epithelium (38).

The profile of estrogen secretion by the conceptus (36) highlycorresponds with selective and dramatic changes in PG synthesisenzymes during early pregnancy, reported previously by us (14,

17). Endometrial mPGES-1 expression exhibits a biphasic pro-file, similar to estrogen synthesized and secreted by conceptuses.By contrast, rapid increase of PGFS and CBR1 after initiation ofimplantation corresponds with the decline in conceptus estrogensynthesis. Our findings are in agreement with in vivo observa-tions that demonstrate that estrogens increase PGE2 content inthe porcine uterus (5) and therefore may be an important factorin increase of the PGE2 to PGF2" ratio during early pregnancy.

CBR1 is the enzyme that can modulate both PG concentra-tions because it converts PGE2 into PGF2" (15, 16). In the presentstudy, E2 inhibited CBR1 protein expression. This is consistentwith reports demonstrating inhibition of PG 9-ketoreductase ac-tivity in the sheep endometrium during the maternal recognitionof pregnancy (39). Similarly, interferon-$, the conceptus signal inruminants, down-regulates expression of the PG 9-ketoreduc-tase gene and increases PGE2 secretion in epithelial cells of bo-vine endometrium (40). Moreover, E2 inhibits PG 9-ketoreduc-tase activity in human placenta (41).

E2 had a significant effect mainly on protein levels but did notinfluence the mRNA content of most factors investigated (exceptmPGES-1). This can be explained by the fact that steroids affectstability of many mRNAs that are important in reproductive pro-

FIG. 3. Panels A–D, Effect of PGE2 (100 nM) and PM A (100 nM) on mRN Aexpression of PTGS2 (panel A), PGFS (panel B), mPGES-1 (panel C), and PG 9-ketoreductase (panel D) in endometrial tissue explants in vitro; panels E and F,effect of PGE2 and E2 on the ratios of mPGES-1 to PGFS mRN A (panel E) andprotein (panel F). Panel G , Expression of mPGES-1 protein in implantation andinterimplantation sites. Asterisks indicate significant differences bet w een control(C) and experimental groups: *, P $ 0.05; **, P $ 0.01.

FIG. 4. Effect of PGE2 (100 nM) and PM A (100 nM) on protein expression ofPTGS2 (panel A), PGFS (panel B), mPGES-1 (panel C), and PG 9-ketoreductase(panel D) in endometrial tissue explants in vitro. The representative samples ofWestern blots are shown in the upper panels. Panels E and F, Effect of PGE2 (100 nM)and PM A (100 nM) on PGE2 (panel E) and PGF2" (panel F) secretion in endometrialtissue explants in vitro. Asterisks indicate significant differences between control (C)and experimental groups: *, P $ 0.05; **, P $ 0.01; ***, P $ 0.001.




FIG. 5. Regulation of PTGER2 and PTGER4 expression, cA MP signaling, and localization of PTGER2 expression in the porcine endometrium. Panels A–D, Effect of PGE2

(100 nM) on mRN A and protein expression of PTGER2 (panels A and B) and PTGER4 (panels C and D) in endometrial tissue explants in vitro. Asterisks indicate significantdifferences bet w een control (C) and experimental group: *, P $ 0.05. Panels E and F, Expression of PTGER2 (panel E) and PTGER4 (panel F) mRN A during the estrouscycle (white bar) and early pregnancy (black bar). Panels G and H, Expression of PTGER2 (panel G) and PTGER4 (panel H) protein during d 9 –15 of the estrous cycle(white bar) and early pregnancy (black bar). Days w ith different letters for expression of PTGER2 mRN A and PTGER4 protein are statistically different. PTGER4 mRN A isnot affected by day or reproductive status (P ' 0.05). The asterisk represents differences (*, P $ 0.05; **, P $ 0.01) for PTGER2 protein abundance betw een cyclic andpregnant gilts on d 11 and 12 of the estrous cycle and pregnancy. Panel I, Immunolocalization of PTGER2 protein in luminal (LE) and glandular epithelium (G) ofendometrium, blood vessels (*), and myometrium (M) in the uterus of pregnant and cycling gilts. Representative immunohistochemical analyses are presented: 12c, d12 of the estrous cycle; 12p, d 12 of pregnancy. Insets, Negative control w ith preabsorbed antibodies. Scale bar, 50 #m. Panel J, cA MP accumulation in endometrialtissue explants in response to treatment w ith 100 nM PGE2 in the presence or absence of PTGER2 antagonist (A H6809) or PTGER4 antagonist (G W 627368X).A dditionally, endometrial explants w ere treated w ith PTGER2 or PTGER4 antagonists alone. M eans w ith different letters indicate significant differences (P $ 0.05).Panel K, Protein expression of PTGER2 and PTGER4 in luminal epithelial (LE) and stromal cells (ST) isolated from uterus on d 11–12 of the estrous cycle and pregnancy.




cesses and in this way may influence gene expression levels andfunction (42).

In vivo studies demonstrated that timing of endometrial ex-posure to estrogen is critical to establishment of pregnancy (18).Early administration of estrogen on d 9 and 10 of pregnancyprobably desynchronizes the uterine environment for conceptusimplantation, resulting in later embryonic loss, as well as altersthe pattern of many gene expression, including endometrialPTGS2 mRNA and protein expression (18, 43). Therefore, in ourexperiment, we used gilts on d 11–12 of the estrous cycle, whichcorresponds to the normal period of conceptus E2 secretion. Wedid not choose the 11- to 12-d pregnant gilts because their en-dometrium would have been exposed already to embryo signaland hence resulted in altered expression of target genes ofinterest.

These studies indicate for the first time the existence of a PGE2

autoamplification loop in the porcine endometrium that can beinvolved in the increase of the PGE2 to PGF2" ratio during thematernal recognition of pregnancy. Porcine endometrial tissue ex-plants produced cAMP in response to PGE2 treatment. Increase ofPGE2-mediated cAMP production occurred via PTGER2 becausecAMP production in response to PGE2 was inhibited after cotreat-mentwith thePTGER2-specificantagonist.Ourstudysuggests thatPGE2 acts through endometrial PTGER2 receptor, activates thecAMP signaling pathway, and elevates the mRNA and proteinexpression of enzymes involved in PGE2 synthesis (namelyPTGS2 and mPGES-1) and secretion of this prostanoid by theendometrium (Fig. 6). It is consistent with the reports that PGE2

itself may induce its own secretion in other cell types (44–46).Both PGE2 and E2 increased the mPGES-1 to PGFS protein ra-tios, which are indicative of the relative PGE2 to PGF2" produc-tion in the endometrium. Moreover, E2 and PGE2 stimulated

endometrial PTGER2 expression that corresponds to up-regu-lation of PTGER2 protein content in the endometrium on d11–12 of pregnancy when compared with corresponding days ofthe estrous cycle. These data provide a novel mechanism of thepositive feedback loop that contributes to increased PGE2 syn-thesis in the porcine uterus during the periimplantation window(Fig. 6). Similar observations have been reported for the bovineembryo recognition signal interferon-$ (47).

It is likely that PGE2 produced in the uterus acts in autocrine/paracrine manner via PGE2 receptors, resulting in the local in-crease of endometrial vascular permeability and preparation forangiogenesis and implantation (11, 48). We have identified themRNA and protein expression of two subtypes of PGE2 recep-tors coupled with cAMP signaling pathway, in the porcine en-dometrium, for the first time. PTGER2 protein was highly ex-pressed in the luminal and glandular epithelium of endometriumand in uterine blood vessels and moderately in myometrium inboth pregnant and cycling gilts. Both epithelial and stromal cellsisolated from the uterus of cycling and pregnant gilts expressedPTGER2 and PTGER4 protein. On the basis of our present andprevious results (14), it may be concluded that the site of PGE2

action in the porcine endometrium corresponds highly to local-ization of PGE2 synthesis. The present results are also consistentwith localization of PTGER2 in bovine and human endometrium(49, 50). It is suggested that in the rat and mouse, PTGER2 isinvolved in implantation, whereas PTGER4 is important inthe process of decidualization (11). The role of PTGER4 couldvary in different species. This hypothesis is supported by thefact that in contrast to rodents and human, in the bovineendometrium PTGER4 expression is very low (50). The spe-cific roles of these receptors in the porcine endometrium re-main to be elucidated. However, the present results indicatethat expression of PTGER4 is not affected by reproductivestatus or by factors secreted by the conceptus during the ma-ternal recognition of pregnancy. On the other hand, our stud-ies suggest an important role for PTGER2 in the porcine ma-ternal-embryo communication.

In contrast to PTGER2 and mPGES-1, expression of the en-zymes involved in PGF2" production (PGFS and CBR1) was notaffected by PGE2 treatment. As predicted, expression of PTGS2,mPGES-1, and PGFS as well as PGE2 and PGF2" secretion fromendometrial explants was stimulated by PMA, used in thepresent study as a positive control. PMA mimics diacylglycerolactivation of protein kinase C, and it was demonstrated that thisphorbol ester stimulates both PG secretion and expression of PGsynthesis enzymes (27, 28).

In summary, the present study provides the first direct evi-dence that the primary conceptus signal, E2, and another con-ceptus factor, PGE2, regulate the protein expression of endome-trial PG synthesis pathway enzymes to favor production ofluteoprotective PGE2 in pigs. Our novel findings indicate thatone potential mechanism of inhibition of luteolysis in the pig isthe up-regulation of PTGS2 and PGES protein expression andthe down-regulation of endometrial PGFS and CBR1 proteinconcentrations by E2. Our results suggest that E2 produced by theporcine trophoblast prevents luteolysis through enzymatic mod-ification of PG synthesis in endometrial cells.

FIG. 6. Potential mechanism involved in increasing the luteoprotective PGE2 leveland the PGE2 to PGF2" ratio during maternal recognition of pregnancy in the pig:involvement of E2, PGE2, and endometrial PTGERs (PTGER2) in a PGE2 positivefeedback loop in the porcine endometrium. The primary conceptus signal, E2, andanother conceptus factor, PGE2, regulate protein expression of the endometrialPG synthesis path w ay enzymes to favor production of luteoprotective PGE2. E2

up-regulates PTGS2 and mPGES-1 protein expression and do w n-regulatesendometrial PGFS and CBR1 protein concentrations, increasing PGE2 secretionand the PGE2 to PGF2" ratio. PGE2 acts through endometrial PTGER2, activatesthe cA MP signaling path w ay, and elevates the mRN A and protein expression ofenzymes involved in PGE2 synthesis (PTGS2 and mPGES-1) and its o w n secretionin the endometrium.




PGE2 could exert a luteoprotective effect, and it may act lo-cally through endometrial PGE2 receptors, especially PTGER2.Moreover, endometrial PTGER2 may be involved in a positivefeedback loop during increased PGE2 synthesis in the porcineuterus in the periimplantation window.

Acknowledgments

We thank Jan Klos, Katarzyna Gromadzka-Hliwa, Sheila Wright, andNancy Evans for technical assistance. We thank Michal Blitek for help incare and handling of animals. Moreover, the authors acknowledge Prof.Kikuko Watanabe (University of East Asia, Yamaguchi, Japan) for provid-ing anti-PGFS antibody.

Address all correspondence and requests for reprints to: AgnieszkaWaclawik, Institute of Animal Reproduction and Food Research of Pol-ish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland. E-mail:[email protected].

This research was supported by Grant N N311 319135 from the StateCommittee for Scientific Research in Poland and by COST (GEMINI)-STSM-FA0702-03685 Grant (A.W.). A.W. was awarded the DomesticGrant for Young Scientists from the Foundation for Polish Sciences.

Disclosure Summary: The authors have nothing to disclose.

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