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Département d’Ontogénie et Reproduction, Centre de Recherche en Biologie de la Reproduction, Centre de Recherche du CHUL (J.A.A., S.K.B., P.C., V.E., J.J.K., M.A.F.), and Département d’Obstétrique et Gynécologie (M.A.F.), Université Laval, Ste-Foy, Québec, Canada GIV 4G2; and Departments of Plant and Animal Sciences, Nova Scotia Agricultural College (L.A.M.), Truro, Nova Scotia, Canada B2N 5E3
Address all correspondence and requests for reprints to: Dr. Michel A. Fortier, Département d’Ontogénie et Reproduction, Centre de Recherche en Biologie de la Reproduction, Centre de Recherche du CHUL, Université Laval, Ste-Foy, Québec, Canada GIV 4G2.
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Introduction |
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, PGD2, PGI2, and thromboxane A2, by cell-specific isomerases and synthases (2). These PGs exert their effects through G protein-coupled receptors, designated EP, FP, DP, IP, and TP, respectively (3, 4, 5). PGE2 has four receptor subtypes, EP1, EP2, EP3, and EP4. Each receptor is encoded by different genes. EP1 is coupled to phospholipase C, generating two second messengers, inositol triphosphate, which is involved in the liberation of intracellular calcium (Ca2+), and diacylglycerol, an activator of protein kinase C. EP2 and EP4 are coupled to adenylate cyclase and generate cAMP that activates the protein kinase A signaling pathway. The signaling of EP3 is more complex. EP3 receptor has four subtypes, A, B, C, and D, with a wide range of action from inhibition of cAMP production to increases in Ca2+ and inositol triphosphate. EP2 and EP4 are known as relaxant receptors, EP1 is considered as a contractile receptor, and EP3 is termed an inhibitory receptor (3, 4, 5).
PGE2 plays a vital role in the survival of cells acting as a mitogenic, antiapoptotic, and angiogenic factors (7, 8). PGE2 increases vascular endothelial growth factor, basic fibroblast growth factor, and COX-2 mRNA expression via the cAMP-protein kinase A signaling pathway (9, 10). PGE2 increases cAMP in many tissues, including kidney, intestine, and uterus, but the relative contribution of EP2 vs. EP4 receptors to these effects is incompletely defined (11). EP2 and EP4 receptors were cloned in rat (12), mouse (13, 14), rabbit (15, 16), dog (17, 18), and human (19, 20). EP2 and EP4 mRNAs are expressed in uterine tissues of different species, such as rat (21), mouse (22), sheep (23), baboon (24), and human (25). EP2 and EP4 receptors mediate relaxation of uterine smooth muscle during pregnancy in different species (21, 22, 23, 24, 25). The effect of PGE2 in endometrial angiogenesis and vasodilatation, uterine receptivity, and decidualization is mediated by cAMP-dependent mechanisms in rat (21), mouse (22), rabbit (26), and human (27). Knockout studies in mice indicated that EP2, but not EP1, EP3, or EP4, was associated with reproductive failures (5).
During the bovine estrous cycle, d 15–17 are critical for either the establishment of pregnancy or the return to a new estrous cycle (28). The bovine endometrium produces PGE2 throughout the estrous cycle, but its production is comparatively higher at mid and late luteal phases of the estrous cycle (29). The exact role of PGE2 in bovine endometrium is not fully elucidated. Recently, we have shown that two PGE2 biosynthetic enzymes, COX-2 and PGE synthase (PGES) are coexpressed in bovine endometrium in the mid and late luteal phases of the estrous cycle (30). High levels of PGE2 have been found in the uterine vein during early pregnancy in the sheep (31). We have shown previously that interferon-
, the pregnancy recognition signal in ruminants, increases the expression of COX-2 and PGES as well as PGE2 production in endometrial cells in vitro (32, 33). Very recent evidence suggests that pregnancy up-regulates COX-2 expression in the bovine and ovine endometrium (34, 35) in vivo. At the time of establishment of pregnancy, PGE2 is considered an important immunomodulatory (36) and a temporary luteostatic and/or luteoprotective factor in ruminants (37). Early findings from our laboratory have shown that treatment with PGE2 increases cAMP production in bovine endometrial epithelial and stromal cells in vitro (38).
Overall, the available information converges to indicate that cAMP generated in response to PGE2 is an important mediator of diverse functions during the estrous cycle and at the time of establishment of pregnancy. EP2 and EP4 are the primary PGE2 receptors coupled with cAMP generation, but they have not been cloned in the bovine. Therefore, the objectives of the present investigation were 1) to clone and characterize bovine EP2 and EP4 receptors, and 2) to study the expression of EP2 and EP4 receptors in bovine endometrium and myometrium during the estrous cycle and early pregnancy.
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-32P]ATP and [-32P]deoxy-CTP from Perkin-Elmer, Markham, Canada); goat antirabbit biotinylated Ig from DAKO Corp. (Mississauga, Canada); goat antirabbit IgG conjugated with horseradish peroxidase from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA); human antirabbit EP2 polyclonal antibody from Cayman Chemicals (Ann Arbor, MI); frozen specimen embedding medium Cryomatrix from Shandon (Pittsburgh, PA); Vectastain Elite ABC kit from Vector Laboratories, Inc. (Burlingame, CA); rabbit preimmune serum from Solution Recherche, Inc. (Québec, Canada); Renaissance from NEN Life Science Products, Inc. (Boston, MA); tissue culture plates from BD Biosciences (Franklin Park, NJ); RPMI 1640 from ICN Biomedicals, Inc. (Aurora, OH); fetal bovine serum and fetal calf serum (Multicell) from Wisent, Inc. (Québec, Canada); BioMax film from Eastman Kodak Co. (Rochester, NY); plasmid and mRNA purification kits from QIAGEN (Mississauga, Canada); and PGE2, isobutylmethylxanthine, goat serum, collagenase type II, deoxyribonuclease I, and Mayer’s hematoxylin solution from Sigma-Aldrich Corp. (Oakville, Canada). All oligonucleotide primers were chemically synthesized using ABT 394 synthase (Perkin-Elmer). The other chemicals used were molecular biological grade available from Laboratoire Mat or Fisher Biotech (Québec, Canada).
Preparation of tissues
All of the bovine tissues used in the present study, endometrium, myometrium, caruncles, intercaruncles, fetal membranes, corpus luteum, testis, epididymis, seminal vesicle, and other nonreproductive tissues (brain, eye, lung, heart, liver, spleen, kidney, adrenal, rumen, and intestine) were collected at a local abattoir immediately after the slaughter. Tissues were placed on ice and transported to the laboratory within 1–1.5 h. Tissues were cut into small pieces, snap-frozen in liquid nitrogen, and stored at -80 C until used.
Total RNA extraction and preparation of polyadenylated (poly A+) RNA and genomic DNA
Total RNA was extracted from different bovine tissues and cell cultures using TRIzol according to the manufacturer’s protocols. Poly A+ RNA was isolated from 500 µg total RNA using Oligotex beads suspension (30 µl; QIAGEN kit) as described by the manufacturer. Bovine genomic DNA was isolated from bovine testis using a phenol-chloroform extraction method (39).
Cloning and sequencing of bovine EP2 receptor
Bovine EP2 receptor cDNA was cloned in three steps. The primers used are shown in Table 1A
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Step 2. For completing rapid amplification of 5' cDNA ends (5'RACE), bovine genomic DNA (2 µg) was used as a template, and primer (no. 3) was used to complete the first round asymmetric PCR. According to the known human and mouse EP2 gene structures (40, 41), no intron is present between ATG and the sixth transmembrane domain; primer 3 was selected near by the sixth transmembrane region. For the second round PCR, the reaction medium (2 µl) from the first round PCR was used as a template, and specific primers (no. 4 and 5) were used. The upstream primer was designed based on the known sequences of human and mouse 5' end. The downstream primer was deduced from the sequencing of the PCR product obtained in the first step. The PCR conditions were the same as in step 1, except for the annealing temperature, which was 40 C. The amplified PCR product (0.14 kb) was cloned into TOPO cloning pCR 2.1 and sequenced.
Step 3. For completing the 3'RACE, caruncular RNA from early pregnancy (d 20–25) was used as a template. Poly A+ RNA (2.5 µg) was reverse transcribed with Superscript II using adapter primer as anchor (no. 6). Primer 7 was used to complete the first round asymmetric PCR, deduced from the sequencing of PCR product obtained in the first step. For the second round PCR, reaction medium (2 µl) from the first round PCR was used as a template, and specific primers (no. 8 and 9) were used for PCR amplification. Primer 8 was deduced from the sequencing of the PCR product obtained in the first step, and primer 9 served as an adapter. The PCR conditions were the same as those in step 1. The RT-PCR product (1.63 kb) was cloned into pEF6/V5-His TOPO TA cloning vector and sequenced.
Finally, full-length EP2 cDNA was amplified using the specific primers sense 5'-ATGAATACTTCCAACGACTCC-3' and antisense 5'-TCAAAGGTCAATCTGTTTAC TGGC-3'. These primers were deduced from the sequencing of the PCR products obtained in the three steps. Comparisons were made between the sequences of full-length cDNA and partial amplified products generated in the three steps to establish the complete sequence of bovine EP2 receptor.
Cloning and sequencing of bovine EP4 receptor
Bovine EP4 receptor was cloned using the strategies described under EP2 using different sets of primers (Table 1B
). Primers 10, 11, and 13 were deduced from the known EP4 sequences of human and mouse. Primers 12, 14, 16, and 17 were deduced from the sequencing of bovine EP4 cDNA obtained in the first step. Primers 15 and 18 served as adapters. In step 1, 1.28-kb bovine EP4 partial cDNA was generated by RT-PCR with primers 10 and 11. In step 2 for the 5' end, a 0.10-kb EP4 receptor fragment was obtained with primer 12 in an asymmetric reaction (annealing at 43 C) on genomic DNA, followed by PCR with primers 13 and 14. In step 3 for the 3' end, a 1.46-kb EP4 receptor fragment was obtained with primer 15 in an asymmetric reaction (annealing at 50 C) on cDNA transcribed with Superscript II, followed by PCR with primers 17 and 18. At each step the PCR product was cloned into TOPO cloning pCR 2.1 and sequenced. Repeated sequencing of the overlapping PCR products from both sides of the amplified products was performed to confirm the accuracy and reproducibility of the results and to establish the complete sequence of bovine EP4 receptor.
DNA sequencing and sequence analyses
The plasmid DNA was isolated using the QIAGEN plasmid purification system. After using the appropriate restriction enzymes, the clones were sequenced using a T7 DNA polymerase kit and automated analyzer. Multiple sequence alignments were obtained using the CLUSTALX program. Hydropathy analysis (42), Shaw-Kaman sequence consensus (43), and consensus sequences for phosphorylation and glycosylation (44) were performed.
Northern blot analysis
Northern blotting and hybridization were performed as described in our recent publication (30). Briefly, total RNA (
20 µg) or poly (A) RNA (10 µg) was loaded in each lane and electrophoresed on 1.2% formaldehyde-agarose gel. RNA was transferred overnight onto a nylon membrane in 10x standard saline citrate (SSC). Blots were prepared separately for EP2 and EP4 and stored at -20 C until used. The cDNA probe for EP2 or EP4 was labeled with [-32P]deoxy-CTP (3000 Ci/mmol) using 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. Full-length EP2 cDNA (1.05 kb) and partial-length EP4 cDNA (1.28 kb) served as probes.
Southern blot analysis
Bovine genomic DNA was prepared as described above and digested (10 µg/reaction) with EcoRI, BamHI, and HindIII overnight at 37 C and precipitated in ethanol (45). After electrophoresis on 0.8% agarose in 1x TBE, the gel was denatured in 0.5 M NaOH containing 1.5 M NaCl (twice, 15 min each time) and neutralized in 1 M Tris-HCl, pH 8.0, containing 1.5 M NaCl (twice, 30 min each time). The DNA was transferred overnight onto a nylon membrane by capillary blotting in 10x SSC. The membrane was hybridized as described above for Northern blot using EP2 (1.05 kb) or EP4 (1.28 kb) 32P-labeled cDNA probe.
RT-PCR/Southern blot analysis
Total RNA (1 µg) was reverse transcribed using random primer and Superscript II reverse transcriptase. The gene structure analysis of human and mouse EP2 (40, 41) and EP4 (46, 47) receptors revealed that transmembrane domains I–VI are encoded by exon 1, and trans-membrane domain VII and the remaining part of the coding sequence are encoded by exon 2. Based on the gene structures, the forward primer was selected within exon 1, and the reverse primer was selected within exon 2 to eliminate nonspecific amplification of genomic DNA. Specific primers were used for EP2 [sense, 5'-GTGCTGCCCACTATCTACATAGTC-3' (positions 466–489); antisense, 5'-TCTA AGAGAGCTTGGAGGTCCCACTT-3' (positions 888–865)] and for EP4 [sense, 5'-TTCAGTTCCTTCCTCATCCTCGCC-3' (positions 571–594); antisense, 5'-CTGTCTTCCG CAGGAGGATGTATA-3' (positions 1024–1001)]. Oligoprobes were selected between these two primers to identify the mature amplified fragment. For EP2, primer 5'-GCCGTCTGC TCCTTGCCTTTCACG-3' (positions 802–825), and for EP4, primer 5'-TGGCTCCAGTTGTGGCCGAT ATAA-3' (positions 936–913) were used. The PCR conditions were 94 C for 1 min, 60 C for 30 sec, and 72 C for 1.30 min for 35 cycles. The expected size of the amplified EP2 cDNA was approximately 422 bp, and that of EP4 cDNA was approximately 454 bp. The RT-PCR products were confirmed by sequencing. As an internal standard, bovine ß-actin cDNA (443 bp) was amplified using specific sense (5'-CAACTGGGACGACATGGAGAAGATCTGGCA-3') and antisense (5'-GAGGATCTTCATG AGGTAGTCTGTCAGGTC-3') primers. The PCR conditions were similar to those described above. After RT-PCR, 10 µl PCR product were run on a 1.2% agarose gel. Southern blotting was performed as described above. Classical hybridization solution (6x SSC, 5x Denhardt’s solution, 50 µg /ml salmon sperm DNA, and 0.2% sodium dodecyl sulfate) was used. Prehybridization was carried out for 1 h at 60 C. The EP2 or EP4 oligoprobes were labeled with [-32 P]ATP (3000 Ci/mmol) using T4 kinase. Hybridization was carried out for 3–4 h at 60 C. The blots were washed and exposed to BioMax film. Densitometry of autoradiograms was performed using an Imager ( Innotec Corp., Montréal, Canada).
Nested PCR
Total RNA (1 µg) was reverse transcribed as described above. First, a PCR was performed using specific sense (5'-AAATCGCGCAAGGAGCAGAAGGAGAC-3'; positions 130–156) and antisense (5'-GACTTCCAGGGAGCTCCCCTTAGG-3'; positions 1434–1410) primers for EP4. Then, a 2-µl aliquot from the first PCR was amplified using a different set of primers. The sense (5'-TTCAGTTCCTTCCTCATCCTCGCC-3'; positions 571–594) and antisense (5'-CTGTC TTCCGCAGGAGGATGTATA-3'; positions 1024–1001) primers were chosen to amplify a fragment within the first PCR product. The amplified PCR product (454 bp) was cloned and sequenced as described above to confirm the identity of the EP4 messenger. The PCR conditions for each PCR program were 94 C for 1 min, 60 C for 30 sec, and 72 C for 1.30 min for 35 cycles. Bovine ß-actin was used as an internal standard as described above. The primers were deduced from the bovine EP4 sequence determined earlier.
Western blot analysis
Western blot analysis was performed as described in our recent paper (30). In brief, total proteins (20 µg) extracted from tissues (48) were loaded in each lane and electrophoresed on 10% SDS-PAGE, followed by electrotransfer onto nitrocellulose membrane. Antihuman polyclonal EP2, which possessed 83% homology with bovine (human, SLRTQDA TQTSCSTQSDASKQADL; bovine, SLRTQEATQTSCSTQSNASKQIDL) was used as the primary antibody (1:500). Amino acids different from human sequences are underlined. 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 of autoradiograms was performed using an Imager.
Immunohistochemistry
Tissues were embedded using embedding medium (Cryomatrix), and cryosections (6 µm) were made using a cryotome (Shandon, Pittsburgh, PA). Immunohistochemical localization of EP2 protein was performed using Vectastain Elite ABC kit according to the manufacturer’s protocols. Briefly, tissue sections were fixed in 4% paraformaldehyde for 30 min at room temperature. 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 (antihuman polyclonal EP2, 1:500) was performed overnight at 4 C. The sections were further incubated with the second antibody (goat antirabbit IgG biotinylated, 1:200) for 30 min at room temperature. For the negative control, preimmune rabbit serum (1:5000) was used instead of EP2 antibody. Between each step, tissues were washed in PBS. Finally, tissues were stained with Mayer’s hematoxylin. Photos were captured using the Spot program (Carsen Group, Inc., Markham, Canada), and quantification was performed using Image-Pro-Plus (Media Cybernetics, Silver Spring, MD).
Specificity of EP2 antibody
A transient transfection study was performed to confirm the specificity of the EP2 antibody. Embryonal human kidney cells (transformed cell line, Invitrogen) 293 FT were cultured in 24-well plates in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin. Full-length bovine EP2 cDNA was cloned in PEF6V5His-TOPO TA cloning vector downstream of the T7 promoter. At 70–80% confluence, cells were transfected with bovine EP2 cDNA (0.8 µg/well) premixed with Lipofectamine 2000 (1.0 µl/well). During transfection cells were grown in the absence of antibiotics. As a control, 293FT cells were transfected with PCR 3.1 vector containing enhanced green fluorescence protein (EGFP) (49). After 24–48 h, transfected cells were lysed, and total protein was collected as previously described (48). Total protein (20 µg) was electrophoresed in 10% SDS-PAGE, and Western blot was performed as described above.
Study during the estrous cycle
Cyclic uteri were collected from abattoir. Days of the estrous cycle were determined by utero-ovarian morphology (30). Uteri were classified into seven groups as d 1–3 (n = 4), 4–6 (n = 3), 7–9 (n = 3), 10–12 (n = 3), 13–15 (n = 6), 16–18 (n = 8), and 19–21 (n = 7). The endometrium and myometrium were isolated. Total RNA and proteins were extracted, and the expression of EP2 and EP4 receptors was studied.
Study during early pregnancy
Beef cattle were used in this study. All heifers (cycle length, 19 ± 1 d) were maintained at the Novo Scotia Agricultural College Research Farm. All procedures performed were in accordance with the guidelines of the Canadian Council on Animal Care, 1993. Estrus was synchronized using a standard double PGF2 regimen. In the pregnant group (n = 3), heifers were inseminated with proven semen at estrus. In the control group (n = 3), heifers were not inseminated. On d 18 of the estrous cycle heifers were slaughtered at a local abattoir, and uteri were collected. Ipsi- and contralateral uterine horns were identified. Pregnancy was confirmed by the presence of a conceptus. One-cubic centimeter cross-sectioned blocks were dissected in the middle portion of the uterine horn ipsilateral to the corpus luteum in both cyclic and pregnant uteri. Tissues were immediately snap-frozen in liquid nitrogen and stored at -80 until further use. The expression of EP2 protein was studied by immunohistochemistry.
Functional characterization of EP2 in bovine endometrium
Cyclic uteri (d 1–3) were collected from the abattoir. Endometrial epithelial and stromal primary cells were cultured as described previously (38). Medium (RPMI 1640 and 10% fetal bovine serum depleted of steroids by dextran-charcoal extraction) was changed every 2 d until the cells were used. Cells reached confluence within 7–10 d. Cell cultures were incubated at 37 C in an atmosphere of 95% air and 5% CO2. At confluence the medium was replaced with serum-free RPMI 1640 and incubated for 24 h. Total RNA and proteins were extracted to study the expression of EP2 and EP4 mRNAs and proteins. The functionality of EP2 receptor was studied in epithelial and stromal cells by measurement of cAMP generation. Cells were treated with PGE2 at different concentrations from 10-9–10-5 M. At confluence, the cells were incubated for 15 min at 37 C in the presence of Krebs solution containing 0.5 mM isobutylmethylxanthine. The reaction was initiated by adding PGE2 for 10 min at 37 C. The reaction was stopped by the addition of an equal volume of ethanol/0.02 N HCl. All samples were lyophilized and resuspended in 50 mM sodium acetate buffer, pH 6.2. cAMP was measured by RIA as described previously (50). The experiment was repeated three times using different uterine tissues.
Statistical analysis
All numerical data are presented as the mean ± SEM. Where appropriate, data were subjected to statistical analysis with ANOVA, followed by Fischer’s protected least significant difference and Duncan’s new multiple range comparison tests (SUPER ANOVA, Abacus Concepts, Inc., Berkeley, CA).
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, A and B) consist of an open reading frame of 1056 and 1476 bp and a 3'-untranslated region of 1441 and 1348 bp nucleic acids, respectively. The open reading frames of EP2 and EP4 encode 352 (39 kDa) and 492 (55 kDa) amino acids, respectively. The deduced amino acid sequences of bovine EP2 and EP4 receptors (Fig. 2, A and B) indicate the presence of seven transmembrane domains typical of rhodopsin type G protein-coupled receptors. Bovine EP2 amino acid sequences possess 84, 82, 80, and 79% identity with the homologs of human, mouse, dog, and rat, respectively. Bovine EP4 possesses 89, 89, 88, 87, and 87% identity with the homologs of human, rabbit, dog, rat, and mouse, respectively. Most of the sequence divergence is found in the extracellular and cytoplasmic loops, whereas a high degree of sequence conservation (90%) is observed in the putative transmembrane domains. Arginine (R) is located at positions 296 and 322 in the seventh transmembrane domain of bovine EP2 and EP4 receptors, respectively. Putative N-glycosylation and serine/threonine phosphorylation sites are predicted (44) in the amino acid sequences of bovine EP2 and EP4, as was found for other species.
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, A and B) using total RNA (20 µg) or poly A+ RNA (10 µg). Both results indicate that EP2 mRNA is present in endometrium, but not in lung or kidney. The results (Fig. 3A) using poly A+ RNA further demonstrate that the EP2 transcript is present in two forms, primarily at about 4.4 kb and, to a lesser extent, at about 2.7 kb. EP4 mRNA is undetectable in any of the tissues using either total RNA or poly A+ RNA. The cDNA probes used for Northern analysis of EP2 and EP4 were also used for Southern analysis of bovine genomic DNA digested with different restriction enzymes. The results (Fig. 3C) show that a hybridization signal is present in at least two lanes for each probe. This confirms that the bovine EP2 and EP4 receptors are encoded by single copy genes.
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, A–C). The results indicate that EP2 mRNA is expressed in every tissue studied, and EP4 mRNA is expressed only in intestine and testis. The absence of expression of EP4 mRNA as determined by Northern and RT-PCR/Southern blotting was surprising given the pattern of expression in other species. A much more sensitive method, nested PCR, was used to validate our results. With this more sensitive method, EP4 mRNA could be detected in kidney, adrenals, lung, and endometrium (Fig. 4D). The results suggest that the limited number of copies of EP4 expressed in bovine tissues does not permit detection by classical RT-PCR/Southern blotting methods.
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). HEK298 FT cells were transiently transfected with full-length bovine EP2 cDNA. The immunoreactivity was tested by Western analysis that highlighted a strong signal appearing at about 54 kDa in transfected cells. EP2 is also expressed at a low level in control cells. This expression could be expected, because the cells were derived from embryonal human kidney, a tissue in which EP4 expression has been reported. Our results thus confirm the specificity and validity of the commercial EP2 antibody in bovine tissues.
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). RT-PCR/Southern blot analysis show that EP2 mRNA is expressed in endometrium and myometrium with little modulation, whereas EP4 mRNA is undetectable in either tissue throughout the estrous cycle. RNA from intestine was used as a positive control. Western blot analysis confirms the presence of EP2 protein and its modulation in endometrium and myometrium. EP2 protein appears as one form (54 kDa) in endometrium and as two forms (54 and 60 kDa) in myometrium. Based on densitometric analysis, the expression of EP2 protein in endometrium is significantly (P < 0.05) higher between d 10 and 18 of the estrous cycle. In the myometrium, EP2 protein expression begins to increase on day 7, is maximal (P < 0.05) between d 10 and 18, and decreases on d 19 of the estrous cycle.
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). Quantitative analysis indicates that pregnancy significantly (P < 0.05) increases EP2 protein expression in both endometrium and myometrium. The level of increase is 1.5-fold in luminal epithelium, 3-fold in stroma, 4-fold in circular, and 5-fold in longitudinal smooth muscle cells. No change is observed in glandular epithelial cells. The effect of pregnancy on EP2 expression is mainly observed in endometrial stroma and myometrial smooth muscle cells.
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). EP2 mRNA and protein are expressed in both epithelial and stromal cells. The level of expression does not differ significantly between the two cell types. Treatment with PGE2 increases cAMP production in endometrial epithelial and stromal cells in a dose-dependent manner from 10-9–10-5 M (P < 0.05). The maximal cAMP production is observed at 10-5 M, and the levels of increase are 13- and 27-fold in epithelial and stromal cells, respectively. Overall, cAMP production is significantly higher (P < 0.05) in stromal than in epithelial cells.
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Southern analysis of bovine genomic DNA reveals that the EP2 receptor is encoded by a single gene in bovine, as was found in human (40), mouse (41), and dog (17). EP4 receptor is also encoded by a single gene in bovine. This is similar to the mouse (47) and dog (18), but differs from the human genome, where two pseudogenes are present in addition to the functional receptor gene (46). We have used cDNA probes to study the expression of EP2 and EP4 receptors in the bovine by Northern and Southern analyses. We were able to detect EP2 and EP4 genes in genomic DNA by Southern blot. Using the same method, it was difficult to detect EP2 mRNA in tissues other than the endometrium, and EP4 mRNA remained undetectable in all tissues tested. This indicates that the probing and hybridization procedures were functioning, but the number of copies of EP2 and EP4 mRNAs is low in the tissues studied. Using RT-PCR followed by Southern blotting, a much more sensitive method, it was possible to detect EP2 mRNA in every tissue studied, but EP4 mRNA was found only in intestine and testis. These results confirm the very low expression of EP2 and EP4 mRNAs in bovine tissues. When we compare the relative expression of bovine EP2 and EP4 mRNAs, we find that the number of EP4 mRNA copies is much lower than that of EP2. Indeed, optimal exposure time for autoradiograms was 3 h for EP2 mRNA and 3 d for EP4 mRNA. Low abundance of EP2 and EP4 mRNA was reported in human (19, 20), mouse (13, 14), rat (12), and dog (17, 18) tissues. Despite the low expression of EP2 mRNA, we were able to detect the EP2 protein by Western analysis and immunohistochemistry. We used a nested PCR procedure to confirm whether EP4 is expressed at an undetectable level or is not expressed in tissues such as lung, kidney, adrenals, and endometrium where EP4 expression was reported in other species (12, 14, 18, 20). We were able to detect EP4 in each tissue studied. The results confirm that EP4 is expressed at extremely low or undetectable levels, below the limit of sensitivity of Northern blot and RT-PCR/Southern blotting. This result demonstrates the high sensitivity of nested PCR, but does not support its use to quantitate mRNA expression. Therefore, we prefer to use the term undetectable instead of not expressed to describe the expression of EP4. In support of our results, a recent study also indicated that EP2 was detectable and EP4 was undetectable by RT-PCR in bovine granulosa cells (56).
In endometrium, EP2 mRNA and protein are expressed throughout the estrous cycle. EP2 protein (54 kDa) is maximally expressed between d 10 and 18 of the estrous cycle. It has been proposed that the function of the EP2 receptor is regulated by posttranslational modification of the protein (18, 21, 40). Immunohistochemical localization of the EP2 protein reveals its presence in epithelial, stromal, and glandular epithelial cells of the cyclic endometrium. Our in vitro studies show that EP2 protein is expressed at comparable levels in epithelial and stromal cells. The EP2 receptor is functional in both cell types, and PGE2 induces a dose-dependent increase in cAMP. The levels of cAMP generated are higher in stromal than in epithelial cells, suggesting that the coupling between the receptor and adenylate cyclase is better in stromal cells. In human endometrium, both EP2 and EP4 mRNAs are expressed throughout the menstrual cycle without modulation (25). In mouse and rat, EP2 mRNA is expressed between d 4 and 5 of the estrous cycle, corresponding to the implantation window (21, 22). We have previously found that in the rabbit maximal sensitivity of the endometrium to generate cAMP in response to PGE2 occurs on d 6.5, corresponding to the uterine receptivity (26).
In the myometrium we observed that EP2 mRNA is expressed uniformly throughout the estrous cycle, whereas EP2 protein is expressed maximally between d 10 and 18 of the estrous cycle. We observed two forms of EP2 protein at approximately 54 and 60 kDa in myometrium. By contrast only one form (54 kDa) was found in the endometrium. This may result from different processing of the receptor protein in endometrium and myometrium. Immunohistochemistry shows the presence of EP2 protein in circular and longitudinal smooth muscle cells of the myometrium. No information is available on the production PGE2 in the myometrium during the estrous cycle in the bovine.
During early pregnancy the expression of EP2 protein is up-regulated 1.5-fold in endometrial luminal epithelium and 3-fold in stroma. Interestingly, EP2 protein is also present in embryonic trophoblastic cells (Fig. 7C). It has been proposed for several years that PGE2 of embryonic origin could play a role in the establishment of pregnancy in ruminants (31). The presence of EP2 receptors on embryonic cells suggests that PGE2 may play a role in embryonic development as well. Recently, COX-2 expression was found in trophoblastic cells of the ovine embryo between d 10 and 17 of pregnancy (57). Roles for PGE2 in embryogenesis (58) and fetal development (59) have been proposed, and its immunomodulatory role (36) at the time of establishment of pregnancy is well documented in ruminants. Moreover, PGE2 has long been proposed as a temporary luteostatic and/or luteoprotective factor during the establishment of pregnancy in ruminants (37). Together, these data and our observation of the presence of EP2 receptors in epithelial, stromal, glandular epithelial, and embryonic trophoblastic cells indicate that PGE2 could act through cAMP to effect the cross-talk between the different cell types at the time of establishment of pregnancy.
Even though uterine receptivity is primarily related with the endometrium, the regulation of uterine quiescence involves myometrial activity. During pregnancy EP2 expression increases 4-fold in circular and 5-fold in longitudinal smooth muscle cells. The level of up-regulation of EP2 protein is comparatively higher in myometrium than in endometrium, but its contribution to the mechanisms leading to the establishment of pregnancy has not been addressed in the bovine. The EP2 receptor has been considered a relaxant receptor in the myometrium of different species (23, 24, 60). Butaprost, an agonist of EP2, increases cAMP production and reduces myometrial contraction (61). Myometrial cells isolated from human pregnancy are capable of producing PGE2 in addition to PGI2 (60). No information is available on myometrial production of PGE2 in early pregnancy in the bovine. Our results suggest that increased expression of EP2 receptors in myometrium may respond to PGE2 of myometrial and/or endometrial origin to effect uterine quiescence at the time of establishment of pregnancy.
Uterine receptivity and quiescence are both time and hormone dependent (21). Not much information is available on the regulation of EP2 receptor in uterine tissues. Ovarian steroids have been documented as potential regulators of EP2 and other PG receptors in rat and mouse uterus during the estrous cycle and early pregnancy (21, 22). The precise hormonal regulation of PGE2 and the other PG receptors is tissue specific and likely to be different between the endometrium and the myometrium (23). Studies of the promoter regions of the human and mouse EP2 receptors indicate the presence of response elements for progesterone, cAMP, a few cytokines, and the CAAT box (40, 41). The EP4 receptor has been described as a housekeeping gene in most tissues (47) and as an inducible gene under specific conditions (62, 63).
PGE2 plays a vital role in the survival of cells as a mitogenic, antiapoptotic, and angiogenic (7, 8) factor. PGE2 increases vascular endothelial growth factor, basic fibroblast growth factor, COX-2, and EP2 (9, 10) expressions. EP2 (64) and COX-2 (1) knockout mice both suffer from ovulation, fertilization, and periimplantation defects. By contrast, no reproductive abnormalities were reported in EP4-deficient mice (5). The exact role of PGE2 in bovine endometrium is not fully elucidated. Bovine endometrium produces PGE2 throughout the estrous cycle, but its production is comparatively higher in the mid and late luteal phases, between d 13 and 17 of the estrous cycle (29). Recently, we found that COX-2 and PGES are coexpressed in bovine endometrium between d 15 and 18 of the estrous cycle (30). We have already shown that interferon- increases COX-2 and PGES expression and PGE2 production in endometrial cells (32, 33) in vitro. Increased expression of COX-2 at the time of establishment of pregnancy was recently demonstrated in bovine (34) and ovine (35) endometrium in vivo. Interestingly, in bovine endometrium the high expression of EP2 receptor protein between d 10 and 18 of the estrous cycle closely matches the expression of COX-2, PGES, and PGE2 production. Taken together, these results suggest a significant role for PGE2 in uterine receptivity and quiescence, which are the prerequisites for successful establishment of pregnancy.
In conclusion, we have cloned and characterized two subtypes of PGE2 receptors, EP2 and EP4, for the bovine. Our results indicate that EP2 is the major cAMP-generating PGE2 receptor expressed and regulated in uterine tissues during the estrous cycle and early pregnancy. Our study is the first to report the expression and regulation EP2 and EP4 receptors in uterine tissues during the estrous cycle and early pregnancy in the bovine as well as in ruminants.
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Present address for J.J.K.: Department of Obstetrics and Gynecology, University of Illinois, Chicago, Illinois 60612-7313.
Abbreviations: COX, Cyclooxygenase; EGFP, enhanced green fluorescence protein; PG, prostaglandin; PGES, PGE synthase; poly A+, polyadenylated; RACE, rapid amplification of cDNA ends; SSC, standard saline citrate.
Received November 27, 2002.
Accepted for publication March 26, 2003.
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), key residue (
), and phosphorylation sites (
) are shown across the species.
