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Endocrinology, doi:10.1210/en.2003-1007
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Endocrinology Vol. 145, No. 1 407-417
Copyright © 2004 by The Endocrine Society

Temporal and Tissue-Specific Expression of Prostaglandin Receptors EP2, EP3, EP4, FP, and Cyclooxygenases 1 and 2 in Uterus and Fetal Membranes during Bovine Pregnancy

J. A. Arosh, S. K. Banu, P. Chapdelaine and M. A. Fortier

Unité d’Ontogénie & Reproduction, Centre de Recherche du Centre Hospitalier de l’Université Laval, Ste-Foy, Québec, Canada G1V 4G2; and Centre de Recherche en Biologie de la Reproduction and Département d’Obstétrique et Gynécologie, Université Laval, Ste-Foy, Québec, Canada GIV 4G2

Address all correspondence and requests for reprints to: Dr. Michel A. Fortier, Ph.D., Unité D’Ontogénie & Reproduction, Bloc T1-49, Centre de Recherche du CHUL, 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: mafortier{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2{alpha} receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days < 50 to > 250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP > EP2 >EP3, intercaruncle EP2 > EP3 >= FP, and fetal membranes EP3 >= EP2 >> FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2{alpha} receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2{alpha} in uterine activities during pregnancy in bovine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) ARE central mediators involved in several female reproductive functions such as ovulation, fertilization, establishment and maintenance of pregnancy, and parturition (1, 2, 3, 4). Arachidonic acid (AA), an essential fatty acid stored in membrane phospholipids, is the primary precursor of PGs. AA is converted into PGH2 by the rate-limiting enzymes cyclooxygenase (COX) 1 and 2. PGH2 is then converted into different primary PGs, including PGE2, PGF2{alpha}, PGD2, PGI2, and TxA2, by cell-specific isomerases and synthases (5). PGs exert their effects primarily through G protein-coupled receptors designated EP, FP, DP, IP, and TP, respectively. EP receptor has four subtypes (EP1, EP2, EP3, and EP4). EP2, EP4, IP, and DP receptors are coupled to adenylate cyclase and generate cAMP that activates the PKA signaling pathway, and have been termed "relaxant" receptors. TP, FP, and EP1 receptors are coupled to phospholipase C, generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium and diacyl glycerol, an activator of protein kinase C, and constitute the "contractile" receptor group. Bovine EP3 receptors exist in four isoforms (A–D) having a wide range of action, from inhibition of cAMP production to increases in intracellular calcium, and IP (3) and are termed "inhibitory" receptors (6, 7, 8).

In most mammals, including ruminants, PGF2{alpha} is the luteolytic hormone (9) and a myometrial stimulant (3, 4). PGE2 has been proposed to have multiple roles as a temporary luteotrophic, luteostatic, or luteoprotective signal at the time of establishment of pregnancy (10, 11); as an immunomodulatory mediator at fetal-maternal interface (12); as a mitogenic, antiapoptotic, and angiogenic factor (13, 14); and either as a myometrial relaxant (15) or stimulant (16). Bovine EP3 and FP receptors have been cloned (17, 18) but not EP1. We recently cloned bovine EP2 and EP4 receptors and studied their regulation in the uterus during the estrous cycle and early pregnancy (19). Several studies documented the selective expression of COXs, and PG’s relaxant and contractile receptors in uterine and intrauterine tissues at the time of establishment of pregnancy (20, 21) and at term pregnancy and parturition (3, 4, 22, 23) in a variety of species. However, no detailed information is available in relation to maintenance of pregnancy. It has been suggested that changes in the expression of PG relaxant or contractile receptors could be involved in the maintenance of uterine quiescence for the majority of gestation and activate the uterus to contract at the time of parturition for expulsion of the fetus (24). Therefore, it is necessary to obtain information on tissue-specific and temporal expression and regulation of PG receptors in uteroplacental tissues to understand the mechanisms by which PGs regulate uterine activity during pregnancy.

In ruminants, endometrial caruncles (CARs) take part in the formation of placentomes with fetal cotyledons and are involved in fetal-maternal communication and maintenance of pregnancy. The intercaruncular regions have been primarily associated with maintenance of uterine quiescence and also involved in other essential fetal-maternal interactions (25, 26). No information is available on selective expression and regulation of PGE2 and PGF2{alpha} receptors in placentome CAR, intercaruncle (ICAR), and fetal membrane (FM) tissues throughout the pregnancy in cattle. Furthermore, expression of COX enzymes is poorly studied in these uteroplacental tissues during the same period. Therefore, the objectives of the present investigation were: 1) to study the expression of PGE2 receptors (EP2, EP3, EP4) and PGF2{alpha} receptor (FP) in CAR, ICAR, and FM throughout the bovine pregnancy; 2) to study the coexpression of COX-1 and COX-2 with PGE2 and PGF2{alpha} receptors in these uteroplacental tissues at different stages of pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Reagents used for this study were purchased from the following suppliers: Superscript II RT, DNA ladder, RNA ladder, dithiothreitol, T4 kinase, 5 x forward reaction buffer, 5 x first-strand buffer, TRIzol and Topo cloning kits (Invitrogen Life Technologies Inc., Burlington, Ontario, Canada); Random primer-pd(N)6, deoxynucleotide triphosphates, RNA guard, rTaq DNA polymerase, PCR 10 x buffer and Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech Montreal, Quebec, Canada); EcoRI, EcoRV, BamHI, HindIII, and prestained protein markers (New England Biolabs Inc., Mississauga, Ontario, Canada); enzyme cutting buffers and trypsin (Boehringer Mannheim Corp., Montreal, Quebec, Canada); T7 sequencing kit (USB Corp. Inc., Cleveland, OH); Bright Star-plus nylon membrane and UltraHyb (Ambion Inc., Austin TX); Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA); [{gamma}32P]ATP and [{alpha}32P]deoxy-CTP (Perkin-Elmer life Sciences, Markham, Ontario, Canada); COX-1 and COX-2 antibodies (Merk-Frost, Montreal, Canada); goat antirabbit biotinylated Ig (Dako Diagnostics of Canada Inc., Mississauga, Ontario, Canada); goat antirabbit or mouse IgG conjugated with horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA); monoclonal antimouse ß-actin antibody and antihuman rabbit EP2 polyclonal antibody (Cayman Chemicals, Ann Arbor, MI); Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, CA); rabbit preimmune serum (Solution Recherché Inc., Quebec, Canada); Renaissance (Life Science Products Inc., Boston, MA); BioMax film (Eastman Kodak Corp., New York, NY); plasmid and mRNA purification kits (QIAGEN Inc., Mississauga, Ontario, Canada); and Mayer’s hematoxylin solution (Sigma-Aldrich Canada Ltd., Oakville, Ontario); LightCycler FasterStart DNA Master SYBR Green I mix and MgCl2 (Roche Diagnostics, Laval, Quebec, Canada). All oligonucleotide primers were chemically synthesized using ABT 394 synthase (Perkin-Elmer, Foster City, CA). The other chemicals used were molecular biological grade available from Laboratoire Mat or Fisher Biotech (Quebec, Canada).

Tissue collection
Bovine pregnant uteri were collected at local abattoir immediately after slaughter. Care was taken to eliminate pathological conditions during processing as described previously (27). Uteri were opened longitudinally along the greater curvature. Day of pregnancy was determined by measurement of the crown-rump length of fetuses present in the uterus (28). CAR, ICAR, and FM tissues were collected. CAR was composed of endometrial CARs and fetal cotyledons (chorion and chorioallantois); ICAR tissues consisted of endometrium and myometrium (full thickness of uterus); and FM was composed of intercotyledonary portions of amnion, chorion, and chorioallantois. Cross-sections of tissues were prepared and processed for immunohistochemistry as described below. Tissues were cut into small pieces and snap-frozen in liquid nitrogen and stored at -80 C until used.

Experimental design
Based on the days of pregnancy, the CAR, ICAR, and FM tissues were classified into six groups as days less than 50 (n = 3), 51–100 (n = 6), 101–150 (n = 4), 151–200 (n = 5), 201–250 (n = 4), and more than 250 (n = 3). Total RNA was isolated using TRIzol according to the manufacturer’s protocol. Total proteins were extracted and quantified (29). Expression of EP2, EP3, EP4, and FP mRNAs was studied using RT-PCR and real time quantitative RT-PCR (LightCycler). Expression of COX-1 and COX-2 mRNA was studied using Northern blot. EP2, COX-1, and COX-2 proteins were analyzed by Western blot. Cellular localization of EP2 and COX-2 proteins were performed by immunohistochemistry.

RT-PCR
Total RNA isolated from bovine endometrium and corpus luteum was used as templates for EP2, EP3, EP4, and FP, respectively. The PCR-cloning strategies were as described previously (19). Briefly, total RNA (1 µg) was reverse transcribed using random primer and Superscript II RT. Sets of specific primers were deduced from the known sequences of bovine EP2, EP3, EP4, and FP (Fig. 1Go). Based on gene structure analysis of PG receptors, the forward primer was selected within exon 1, and the reverse primer was selected within exon 2 to eliminate nonspecific amplification of genomic DNA (Fig. 1Go) (6, 7, 8). The RT-PCR products were cloned into pCR 2.1 Vector. First, the expression of EP2, EP3, EP4, and FP mRNAs in CAR, ICAR, and FM was studied by standard RT-PCR. As an internal standard, bovine ß-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using specific primers. The PCR conditions were: 94 C/1 min, 60 C/30 sec, and 72 C/1 min for 35 cycles for each gene. The results demonstrated that EP2, EP3, and FP were detectable, but EP4 was undetectable, by RT-PCR in all tissue studied (data not shown).



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  		FIG. 1. A, PCR strategies. The gene structure analysis of PG receptors revealed no intron between ATG and the VIth transmembrane domain. Transmembrane domains I–VI are encoded by exon 1, and transmembrane domain VII and the remaining part of the coding sequence by exon 2. B, Based on that, the forward primer was selected within exon 1, and the reverse primer was selected within exon 2, to eliminate nonspecific amplification of genomic DNA. C, Details of the primers used and cDNA size and position.

 
Quantitative RT-PCR (LightCycler)
Standard RT-PCR was used to confirm the specificity of the sets of primers and amplification of single PCR product at the expected size as described above. Then, the same sets of primers (Fig. 1Go) were used for LightCycler using SYBR Green I. The reaction was performed in a total vol of 20 µl in microcapillary tubes according to the manufacturer’s instruction. Standard curve derivation was as follows. Recombinant plasmid containing specific inserts of EP2, EP3, and FP, and the purified PCR product for GAPDH were used as templates. The plasmid DNA or PCR product was quantified and serially diluted from 100 pg to 0.01 pg/2 µl. Each reaction mixture contained 2 µl of cDNAs, 2 µl FasterStart DNA Master SYBR Green I mix, 2 µl of sense and antisense primers each (0.5 µM), 1.6 µl of 25 mM MgCl2 and 10.4 µl of PCR-grade H2O. The LightCycler programs for each gene were as follows: denaturation (95 C/10 min); PCR amplification and quantification (95 C/10 sec, 60 C/5 sec, 72 C/20 sec) with single fluorescence measurement at specific temperature (acquisition) for 5 sec, repeated for 30–50 cycles respective to the gene studied; a melting program (70–95 C at the rate of 0.1 C/sec with continuous fluorescence measurement); and finally, a cooling step to 40 C (Fig. 2Go). At all steps, the transition temperature was 20 C/sec. Quantification was performed using LightCycler analysis software. Second-derivative maximum analysis, arithmetic base line adjustment, and polynomial calculation methods were used. Base line curve, melting curve, melting point, crossing point, slope, error (0.1–0.5), and correlation (r-1) were monitored (Fig. 2Go). For each gene, standard curves were repeated three to four times to obtain reproducible results. Earlier, different concentrations of cDNA, MgCl2, and various steps in LightCycler (such as denaturation, PCR, melting and cooling programs) were tested and standardized. The amplified products were analyzed in 1.2% agarose gel.



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  		FIG. 2. Real-time RT-PCR (LightCycler) quantification using SYBR Green I. Amplification quality was validated by analysis of (A) amplification curves, (B) melting curves, and agarose gels for, respectively, EP2, EP3, FP, and GAPDH transcripts. Recombinant plasmid DNA, containing specific inserts of EP2 or EP3 or FP receptors and purified PCR products for GAPDH, were serially diluted from 100 to 0.01 pg and used for derivation of the standard curve. PCR-grade water was used as negative control. Placentome CAR and/or intercaruncular tissues were used as unknown samples. Both melting curves and gel analysis show a single peak and band at the expected size, respectively. The acquisition temperatures for EP2, EP3, FP, and GAPDH are 87, 88, 80, and 88 C, respectively (indicated by vertical arrows). The numbers of PCR cycles were 40 for EP2, 50 for EP3, 36 for FP, and 30 for GAPDH. More details are given in Materials and Methods.

 
Quantification of unknown.
Based on analysis, a suitable PCR program was selected for each gene, and one appropriate standard curve was exported and used as an external standard for quantification of the unknown. PCR, quantification and validation methods were as described above. For each reaction, 2 µl cDNA was used. The level of mRNA was expressed as a ratio between EP2 or EP3 or FP and GAPDH.

Northern blot analysis
Northern blotting and hybridization were performed as described previously (17, 27). Briefly, total RNA (~20 µg) was loaded in each lane and electrophoresed on 1.2% formaldehyde-agarose gel. RNA was transferred overnight onto a nylon membrane in 10 x saline sodium citrate. Blots were prepared separately for COX-1 and COX-2 and stored at -20 C until used. The cDNA probes for COX-1 and -2 were labeled with [{alpha}32P]deoxy-CTP (3000 Ci/mmol) using the Ready-To-Go DNA labeling kit. Prehybridization was carried out for 1 h at 45 C, and hybridization was carried out overnight at 45 C using UltraHyb. The blots were stripped of COX-1 and COX-2 probes by boiling in 1% SDS for 30 min and rehybridized with {gamma}-32P[ATP]-labeled oligoprobe specific to 18S ribosomal RNA to normalize each level of COX-1 and COX-2 mRNA. The blots were exposed to BioMax film, and densitometry of autoradiograms was performed using an Alpha Imager (Alpha Innotech Corp., Montreal, Canada). Bovine COX-1 (777 bp) and COX-2 (449 bp) cDNA were obtained and used as probes as described previously (27, 30).

Western blot analysis
Western blot analysis was performed as described previously (19, 27). Briefly, total proteins (~20 µg) were loaded in each lane and electrophoresed on 10% SDS-PAGE followed by electrotransfer onto nitrocellulose membrane. Rabbit antihuman polyclonal EP2 antibody (1:500) and rabbit antisheep COX-1 and COX-2 (1:3,000) were used as the primary antibodies. Goat antirabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (1:20,000). Chemiluminescent substrate was applied according to the manufacturer’s instructions. The blots were exposed to BioMax film, and densitometry was done using an {alpha} Imager. As an internal standard, ß-actin (1:5,000) was measured.

Immunohistochemistry
CAR and ICAR cross-sections (1 cm3) were taken. Tissues were fixed in 4% paraformaldehyde-buffered saline for 4 h at 4 C and processed using standard procedures. Paraffin sections (3 µm) were made. Immunohistolocalization of EP2 and COX-2 protein was performed using Vectastain Elite ABC kit according to the manufacturer’s protocols and as described previously (19). Endogenous peroxidase activity was removed by fixing sections in 0.3% hydrogen peroxide in methanol. Tissue sections were blocked in 10% goat serum for 1 h at room temperature. Incubation with the primary antibody EP2 (1:500) or COX-2 antiserum (1:4,000) was done overnight at 4 C. The sections were further incubated with the second antibody (goat antirabbit IgG biotinilated, 1:200) for 30 min at room temperature. For the negative control, preimmune rabbit serum was used instead of EP2 (1:20,000) antibody or COX-2 (1:4,000) antiserum. Between each step, tissues were washed in PBS. Finally, tissues were stained with Mayer’s hematoxylin. Photos were captured using Spot program (Carsen Group Inc. Corp., Markham, Ontario, Canada).

Statistical analysis
All numerical data were presented as the mean ± SEM. Data were analyzed using two-way ANOVA followed by Fischer’s protected LSD and Duncan new multiple-range comparison and Scheffé’s tests (SUPER ANOVA, ABACUS Concepts, Inc., Berkeley, CA). The data were also analyzed to study the effect of stage of gestation on tissue and gestation-tissue interaction. Differences were considered as statistically significant at 95% confidence level (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of EP2 receptor
EP2 mRNA and protein (Fig. 3Go) were expressed and modulated in CAR, ICAR, and FM tissues throughout pregnancy. In CAR, the level of expression of EP2 mRNA and protein was higher (P < 0.05) during the first trimester than at other stages of pregnancy. However, expression of EP2 in ICAR was highest during midgestation but not affected by stage of gestation in FM. The level of expression of EP2 mRNA and protein was higher (P < 0.05) (2- to 3-fold) in CAR and ICAR than in FM. Immunohistochemistry (see Fig. 5Go) showed that in CAR, on the maternal side, EP2 protein was expressed in epithelial cells of caruncular crypts and stromal cells of caruncular septa. On the fetal side, EP2 protein was expressed in secondary branches of chorioallantoic villus; by contrast, it was absent in its primary branches. EP2 protein was selectively expressed in mononuclear, binuclear, and giant cells of trophectoderm. In ICAR, EP2 protein expression was high in endometrial luminal epithelium and myometrial smooth muscle, moderate in glandular epithelium, and low in endometrial stroma.



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  		FIG. 3. Expression and regulation of EP2, EP3, and FP receptors in placentome CAR, ICAR, and FM tissues throughout bovine pregnancy. A, Level of expression of EP2, EP3, and FP mRNAs based on real time RT-PCR (LightCycler) quantification. As an internal standard, GAPDH mRNA was measured. Values are expressed as mean ± SEM of ratios between EP2 or EP3 or FP and GAPDH mRNAs. B, Analysis of PCR-amplified products of EP2, EP3, FP, and GAPDH, after the stipulated number of cycles using LightCycler in 1.2% agarose gel. C, Western analysis of EP2 protein. As an internal standard, ß-actin was measured. D, Densitometry of EP2 protein is expressed as the mean ± SEM of the ratio between EP2 and ß-actin protein. Each group consisted of three to six samples; representative samples are shown. a and e, EP2—days < 50–100 vs. others; c and f, EP2—days 51–250 vs. others; b, EP3—days 201–>250 vs. others; d, EP3—days < 50 vs. others; P < 0.05. More details are given in Materials and Methods. Note: The scales used for relative expression are not the same for placentome CAR, ICAR, and FM.

 


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  		FIG. 5. Cellular localization of EP2 and COX-2 proteins in placentome CAR and intercaruncular tissues during bovine pregnancy (50–150 d). A, Normal structure of placentome CAR. B, Immunohistochemistry. EP2 and COX-2 proteins are selectively expressed in epithelial cells of caruncular crypts, stromal cells of caruncular septum, and uninucleated, binucleated, and giant cells of trophoblast luminal epithelial, glandular epithelial, and stromal cells of endometrium and myometrial smooth muscle cells. EP2 and COX-2 proteins are colocalized in most cell types. BV, Blood vessels; CEP, caruncular epithelial cells; CC, caruncular crypts; CS, caruncular stalk; CSE, caruncular septum; CST, caruncular stromal cells; GLE, glandular epithelial cells; LE, luminal epithelium; LU, lumen; PCAV, primary chorioallantoic villus; SCAV, secondary chorioallantoic villus; SMC, smooth muscle cells; ST, stromal cells; TRV, trophoblastic-villous; UW, uterine wall. 1, Mononuclear; 2, binuclear; and 3, giant cells of trophectoderm. Immunohistochemistry was performed using Vectastain Elite ABC kit as described in Materials and Methods.

 
Expression of EP3 receptor
Changes in EP3 mRNA (Fig. 3Go) expression in CAR, ICAR, and FM were tissue specific and vary with stage of gestation. In CAR, EP3 mRNA expression increased (P < 0.05) with advancing stage of gestation. In ICAR and FM, EP3 mRNA levels were affected by stage of gestation, but changes in EP3 mRNA in CAR and FM were inversely related during pregnancy. Expression of EP3 mRNA was higher (P < 0.05) in CAR than in ICAR and FM at gestation days greater than 201.

Expression of FP receptor
Expression of FP mRNA differed (P < 0.05) among tissues (Fig. 3Go); high in CAR, moderate in ICAR, and low in FM. Expression pattern of FP mRNA did not change in any of the tissues studied during pregnancy.

Temporal and tissue-specific expression of EP2, EP3, and FP receptors
In CAR, the expression levels of PG receptor mRNAs were FP > EP2 > EP3 (Fig. 3Go). The level of FP expression was higher (P < 0.05) than EP2 from days > 100 and EP3 from days < 50–200 of pregnancy. Near-term (days > 250), the expression level of EP3 was similar to that of FP. The expression patterns of EP2 and EP3 mRNAs were inversely related during pregnancy and differed (P < 0.05) between early (days < 50–100) and late (days > 200) stages of gestation. In ICAR, the expression levels indicated EP2 > EP3 >= FP throughout pregnancy. The level of expression of EP2 mRNA was higher (P < 0.05) than FP and EP3, but no differences were detected between EP2 and EP3 at any stage of gestation. In FM, the levels of expression indicated EP3 >= EP2 >> FP throughout pregnancy. Expression of EP2 and EP3 mRNAs were higher (P < 0.05) than for FP throughout pregnancy, but no differences between EP2 and EP3 were detected.

Expression of COX-1 and COX-2
COX-2 mRNA and protein were highly regulated in CAR, ICAR, and FM throughout pregnancy, whereas COX-1 mRNA and protein were constantly expressed at low levels (Fig. 4Go). In CAR, the levels of expression of COX-2 mRNA and protein were significantly (P < 0.05) higher in early (days < 50) and late (days > 251) than in midpregnancy (days 51–250). In ICAR, COX-2 mRNA and protein were expressed at low levels without change during gestation. In FM, the levels of expression of COX-2 mRNA and protein were high at late (days > 201), moderate at early (days < 50–100), and low at mid (days 101–200) stages of pregnancy. The level of expression of COX-2 mRNA and protein were higher (P < 0.05) in CAR than in ICAR. Immunohistochemistry (Fig. 5Go) revealed that COX-2 protein was expressed more intensely on the fetal than on the maternal compartment. In CAR, COX-2 protein was moderately and diffusely expressed in epithelial cells of caruncular crypts and stromal cells of caruncular septum. On the fetal side, COX-2 protein was selectively expressed in mononuclear, binuclear, and giant cells of trophectoderm. Moreover, COX-2 protein was expressed in secondary branches of chorioallantoic villus; by contrast, it was absent in its primary branches. In ICAR, COX-2 expression was high in endometrial luminal epithelial cells, moderate in myometrium and glandular epithelium, and low in endometrial stroma.



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  		FIG. 4. Expression of COX-1 and COX-2 in placentome CAR, ICAR, and FM tissues throughout bovine pregnancy.A, Northern analysis of COX-1 and COX-2 mRNA expression. As an internal standard, 18S RNA was measured. B, Western analysis of COX-1 and COX-2 proteins. As an internal standard, ß-actin was measured. C, Densitometric values for COX-1 and COX-2 mRNAs are expressed as the mean ± SEM of ratios between COX-1 or COX-2 mRNA and 18S RNA. D, Densitometric values for COX-1 and COX-2 proteins are expressed as the mean ± SEM of ratios between COX-1 or COX-2 and ß-actin proteins. Each group consisted of three to six samples, representative samples are shown. a, b, and c, COX2 mRNA; d, e, and f, COX-2 protein; a and d, days 51–250 vs. others; b and e, days 101–200 vs. others; c and f, days > 201 vs. others; P < 0.05. More details are given in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In cattle, the duration of the pregnancy is 270 ± 5 d. The placenta is epitheliochorial, cotyledonary, and noninvasive. In bovine species, 75–120 endometrial CARs take part in the formation of the placentomes with fetal cotyledons for exchange of nutrients, wastage, and gas. Intercaruncular regions are associated with maintenance of uterine quiescence and physiological hypertrophy of the uterus to accommodate the growing fetus. The uterine glands present in intercaruncular regions secrete histotroph that is absorbed by placental areolae by fluid phase pinocytosis and release these secretions into the fetal circulation (25, 26, 31, 32, 33). Thus, caruncular and intercaruncular tissues of the pregnant uterus play selective and distinctive roles in the maintenance of successful pregnancy (31, 32, 33). Uteroplacental PGE2 and PGF2{alpha} are considered important mediators involved in recognition and maintenance of pregnancy (4, 31, 32, 33). In the present study, we have characterized the temporal and tissue-specific expression of PGE2 and PGF2{alpha} receptors and COX 1 and 2 in uteroplacental tissues during bovine pregnancy.

In CAR, EP2 mRNA and protein are more highly expressed during early than at mid and late stages of pregnancy. EP2 protein is localized in maternal caruncular epithelial and stromal cells and in fetal trophoblast cells. EP2 expression is absent in primary chorioallantoic villi (superficial part of cotyledon) but is highly expressed in its secondary branches (deeper part of cotyledon interdigited with caruncular tissue). Activation of EP2 results in generation of cAMP, which, in turn, activates several signaling cascades (6, 7, 8). The effects of PGE2 mediated by cAMP-dependent mechanisms are mitogenic, angiogenic, antiapoptotic, and immunomodulatory in different cell types (34, 35). Moreover, PGE2 has long been proposed as a temporary luteostatic and/or luteoprotective factor in ruminants (10, 11). PGE2 regulates its own production in uteroplacental tissues, depending on stage of bovine gestation (36). PGE2 is involved in placental functions and maintenance of pregnancy in ewes (37, 38). Expression of EP2 in CAR supports a role for PGE2 as paracrine and an autocrine factor involved in the regulation of growth, differentiation, and function of the placentome CAR. Furthermore, the presence of EP2 receptors in maternal caruncular epithelial, stromal, and fetal trophoblast cells indicates that PGE2 could act through cAMP to effect the maternal-fetal cross-talk between the different cell types to maintain successful pregnancy.

In ICAR, EP2 is expressed highly during midpregnancy, and its level of expression is higher than that of EP3 and FP at all stages. EP2 protein is localized in luminal epithelium, stroma, and glandular epithelium of endometrium and myometrial smooth muscle cells. Other studies have demonstrated the expression of EP2 in ovine endometrium at near-term pregnancy (22). Recently, we have reported increased expression of EP2 in bovine endometrium and myometrium during early pregnancy (19). The role of endometrial PGs during pregnancy is not completely understood and may include regulation of endometrial receptivity (39). The EP2 has been considered as a relaxant receptor in the myometrium of different species (4, 15, 22, 23, 40, 41, 42). Butaprost, an agonist of EP2, increases cAMP production and reduces myometrial contraction (41) and abolishes oxytocin-induced myometrial activity (15). The action of butaprost is significantly greater in pregnant myometrium than in nonpregnant tissues, suggesting that there are more EP2 receptors in the pregnant myometrium (15). Our results, showing increased expression of EP2 receptor in myometrium, support the concept that PGE2 of endometrial and/or myometrial origin is involved in maintenance of uterine quiescence during pregnancy.

The EP3 receptor has different isoforms encoded by a single gene and generated by alternative C-terminal splicing (6, 7). Species variation does exist in the number of EP3 subtypes: three in mouse, four in rat, four in bovine, five in rabbit, and seven in human (43). Pharmacological and molecular characterization of bovine EP3 isoforms indicated that EP3B and EP3C increased, whereas EP3A inhibited cAMP production, and EP3D decreased cAMP and increased IP3 (6, 7, 43). Although some EP3 subtypes were pharmacologically associated with cAMP formation by activation of adenylate cyclase, there was no report of EP3-induced cAMP production in uterine tissues (44). In this study, we designed specific primers that did not distinguish the four isoforms of the bovine EP3. In CAR, EP3 mRNA is more highly expressed during late than at mid and early stages of pregnancy. The expression pattern of EP3 mRNA is inversely related to that of EP2 receptor. In ICAR, EP3 mRNA expression is low compared with EP2, and it is not modulated. In FM, EP3 is expressed at a level comparable with ICAR, and changes during gestation are those of EP2. Abundant expression of EP3 in human uterus is associated with increased myometrial contraction at late stages of gestation (16). During human pregnancy, EP3 receptor mRNA expression in the myometrium is reduced, suggesting that loss of EP3 may be an important regulatory mechanism to maintain quiescence of myometrium (45). In this study, there are contrasting expression patterns for EP3 and EP2. Distinct functions of EP2 (relaxation and vasodilation effects on smooth muscle cells) vs. effect of EP3 (contraction and vasoconstriction of smooth muscle) suggest that high levels of expression of EP3 mRNA near the end of pregnancy contribute to mechanisms associated with the initiation of parturition. However, the existence of different isoforms with distinct functions does not permit one to define the exact role of EP3. Indeed, it remains to be determined whether different EP3 isoforms are expressed in a cell-specific manner under different physiological conditions in bovine uteroplacental tissues during pregnancy. Taken together, these results suggest a significant role for PGE2 in uterine receptivity and quiescence, two prerequisite mechanisms for successful maintenance of pregnancy.

FP is generally considered as a contractile receptor with two isoforms (FPA and FPB), and generated by alternative splicing of C terminal of a single gene identified in ovine and bovine (46, 47). Bovine FPB acts as a negative regulator to attenuate the normal FPA-mediated protein kinase C function (47). Information on FPB is at a preliminary stage. Therefore, in this study, we evaluated only the bovine FPA mRNA. In CAR, expression of FP mRNA is high and not affected by stage of gestation. In ICAR and FM, FP is expressed at very low levels. Other studies have documented the expression of FP mRNA in endometrium and myometrium near-term in ovine (22) and in myometrium in human (24) and rat (24, 42). In human, FP protein is expressed at a low level in smooth muscle cells of myometrium at all stages of pregnancy but increases toward term (48). Increased placental apoptosis and parturition failure were identified in FP-deficient mice (49, 50). Normally, FP expression increases just before labor in most mammalian species (4, 20, 22, 42). In this study, high expression of FP in CAR is interesting but physiological intriguing. The physiological significance of this selective tissue-specific expression during the course of pregnancy is not known. Whether the FPB isoform, the negative regulator of FPA, is expressed in uteroplacental tissues remains to be elucidated.

In the present study, EP4 is undetectable in any of the uteroplacental tissue during pregnancy. In rat, mouse, and human uterus, expression of EP4 along with EP2 was observed during the implantation window and establishment of pregnancy (20, 21, 51). Recently, we found that EP2 was highly expressed in endometrium and myometrium at the time of establishment of pregnancy, whereas expression of EP4 was very low or undetectable (19). Thus, changes in EP4 expression are species specific. Although EP4 is expressed in human uterus, it has either no or minimal effect on myometrial relaxation (43). Moreover, EP4 null mice reproduces normally (8, 52), whereas EP2-deficient mice suffer from multiple reproductive failures (8, 53). The present findings show that EP2 is the primary cAMP generating PGE2 receptor expressed and modulated in uterine and intrauterine tissues during pregnancy in cattle.

During bovine pregnancy, COX-1 mRNA and protein are constitutively expressed, whereas COX-2 is highly modulated in uteroplacental tissues. In CAR, COX-2 is highly expressed at the beginning and at the end of the pregnancy. COX-2 protein is localized in maternal caruncular epithelial and stromal cells and fetal trophoblast. COX-2 is not expressed in primary chorioallantoic villi but is highly expressed in its secondary branches. Our findings support those reported for bovine (54), sheep (55, 56), and humans (57). In ICAR tissues, COX-2 is more highly expressed in luminal epithelium than in stroma, glandular epithelium, and myometrial smooth muscle cells. Increased expression of COX-2 was reported in endometrium during the implantation window and early pregnancy in bovine and ovine (25, 58, 59, 60). Other results indicated that COX-2 expression was decreased in myometrium during early and mid, compared with late and near term, pregnancy in several species (4, 54, 61, 62, 63, 64). In FM, COX-2 expression is moderate during early, low at mid, and high at late stages of pregnancy. Placental expression of COX-2 is gradually increased during the second half of pregnancy and at the time of labor in a variety of species, including ruminants (4, 56, 62, 65). In a given tissue, the eventual production of PGE2 and PGF2{alpha} is determined not only by COX isoenzymes but also by other enzymes, such as cPLA2, PG synthases, and PGDH. The expression level of cPLA2 mRNA is not modulated, but PGFS mRNA is highly regulated throughout pregnancy in mice (66). PGES mRNA is expressed in ovine intrauterine tissues during pregnancy (67). PGDH is more highly expressed in uteroplacental tissues during early and mid, than during late pregnancy, in ewe (68) and mice (66). Our recent studies demonstrate the expression of COX-1, COX-2, PGES, PGFS, and PGDH in bovine endometrium during the implantation window (17, 25, 69, 70). However, such coexpression of PG biosynthetic and catabolic enzymes in bovine uteroplacental tissues during pregnancy remains to be determined. Furthermore, among the three tissues studied, COX-2 first increases in fetal and then in maternal tissues at the late stage of pregnancy, supporting the notion that labor is initiated on the fetal side and then maternal tissues become involved (4, 64). Together, expression of COX-1 and COX-2, along with PG receptors, suggests that PGs biosynthetic and signaling machinery contribute to fetal-maternal communication and regulate uterine activities to maintain pregnancy.

In cattle, the biological contribution of uteroplacental PGs during maintenance of pregnancy is largely unknown. The physiological significance of an increase or decrease in the overall expression of a given PG receptor in the different uteroplacental tissues depends on the cell types involved, the second messenger generated, and the associated signaling cascades. Little information is available on the regulation of PG receptor in uterine tissues. Nevertheless, progesterone, estradiol, oxytocin, and corticosteroids are potential regulators of EP and FP receptors in uterine tissues during the estrous cycle, establishment of pregnancy, and at parturition (3, 4, 20, 21, 40). In this study, PGE2 and PGF2{alpha} receptors and COX-1 and -2 are expressed in temporal and tissue-specific patterns in CAR, ICAR, and FM during pregnancy in cattle. Further studies are required to unravel the molecular and cellular mechanisms involved in the dynamic expression and regulation of PG receptors and biosynthetic enzymes in uteroplacental tissues during maintenance of pregnancy. To our knowledge, this is the first study to characterize PG receptors in uterine and intrauterine tissues in association with maintenance of pregnancy in ruminants as well as in other species.


   Acknowledgments
 
We acknowledge Ms. Christine Legare and Dr. Fabrice Saez for technical advice in standardization of LightCycler, and Ms. Mariane Parent for assistance in collection of tissues.


   Footnotes
 
This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada (to M.A.F.).

J.A.A. and S.K.B. contributed equally to this work

Abbreviations: AA, Arachidonic acid; CAR, caruncle; COX, cyclooxygenase; FM, fetal membrane; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAR, intercaruncle; PG, prostaglandin.

Received August 5, 2003.

Accepted for publication September 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Kang, P. Chapdelaine, J. Parent, E. Madore, P. Y. Laberge, and M. A. Fortier
Expression of Human Prostaglandin Transporter in the Human Endometrium across the Menstrual Cycle
J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2308 - 2313.
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