Molecular Cloning and Spatiotemporal Expression of Prostaglandin F Synthase and Microsomal Prostaglandin E Synthase-1 in Porcine E
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《内分泌学杂志》
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences (A.W., A.B., M.M.K., A.J.Z.), 10-747 Olsztyn, Poland
Department of Physiology (A.R.-M., L.J.S.B., N.A.R.), University of Turku, 20520 Turku, Finland
Graduate School of Integrated Science and Art (K.W.), University of East Asia, Yamaguchi 750-8503, Japan
Abstract
Endometrial prostaglandins (PGs) and the PGE2/PGF2 ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2 ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10–13, intermediate on d 14–23, and high on d 24–25). In pregnancy, expression of mPGES-1 was intermediate on d 10–11 and low on d 14–17 and then increased after d 22, reaching the maximum on d 24–25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10–13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2 ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.
Introduction
UTERINE SYNTHESIS OF prostaglandin (PG) E2 and F2 plays an important role in regulation of the estrous cycle and establishment of pregnancy through autocrine, paracrine, and endocrine actions in many domestic species (1, 2, 3). Because PGE2 and PGF2 differ in many of their actions, the PGE2/PGF2 ratio may integrate information from different sources. The PGE2/PGF2 ratio affects corpus luteum (CL) function, endometrial cell growth and differentiation, blood flow, vascular permeability, embryo migration, and implantation (4).
The estrous cycle of the pig is dependent on the uterus as the source of luteolysin, PGF2 (1), which together with PGE2 reaches the CL by local and/or systemic mechanisms (5). In pigs, the CL is caused to regress on d 15–16 of the estrous cycle by an increase in pulsatile endometrial secretion of PGF2 that occurs after d 13. During this period the PGE2/PGF2 ratio in the utero-ovarian vein reaches 1:3 (6). Moreover, mean concentrations, peak frequency, and peak amplitude of PGF2 in utero-ovarian vein plasma are higher in cyclic than in pregnant gilts on d 12–17 (7, 8, 9). On the other hand, uterine flushings of pregnant gilts contain significantly higher amounts of PGF2 than those from cyclic gilts (10). Bazer and Thatcher (7) proposed that the maternal recognition of pregnancy in the pig involves redirection of the PGF2 secretion from the uterine venous drainage (endocrine) to the uterine lumen (exocrine) by conceptus estrogen secretion. A part of the putative mechanism of CL protection during early pregnancy could also be the retrograde transfer of PGF2 from venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF2 (11). However, the above-mentioned hypotheses do not satisfactorily explain the mechanisms involved in maintaining CL function during early pregnancy in the pig, as discussed below.
Another potential mechanism by which a conceptus could inhibit luteolysis is by changing the PGE2/PGF2 ratio, i.e. in favor of the luteoprotective/antiluteolytic PGE2 (6, 12, 13). On d 11–13 of pregnancy, at approximately the time of maternal recognition of pregnancy, the PGE2/PGF2 ratio in the uterus and uterine vein increases (>1:1) (4, 6), suggesting that PGE2 can overcome the luteolytic effect of PGF2, thus preventing CL regression (6). It is possible that one of the important mechanisms of the maternal recognition of pregnancy could occur by the conceptus alerting the endometrial expression of enzymes involved in PG synthesis. PGs are produced from arachidonic acid, which is further metabolized to an unstable PGH2 by the cyclooxygenase (COX) enzymes COX-1 and COX-2. However, it has been reported that no differences in staining intensity of COX between cyclic and pregnant gilts on d 10, 12, and 15 were observed (14). Thus, it seems unlikely that the porcine conceptus targets COX expression as a means to modulate PGF2 and PGE2 release.
PGH2 is rapidly converted into different prostanoids (PGE2, PGF2, PGD2, PGI2, and TxA2) by specific terminal PG synthases or reductases (15). Additionally, PGE2 could be converted into PGF2 by PG-9-ketoreductase (PG-9-KR). PGF synthase (PGFS) has been primarily studied in the bovine species, and three characterized isoforms (16, 17, 18, 19) belong to the AKR1C subclass of the aldo-keto reductase family. Lung-type PGFS (20, 21) or 20-hydroxysteroid dehydrogenase (AKR1B5), a distinct enzyme with potent PGFS activity recently identified in bovine endometrium (3, 22), is down-regulated in bovine endometrial cells by interferon-. It suggests that the conceptus could target expression of enzymes involved in PGF2 synthesis to decrease PGF2 levels. However, in the available literature, no information could be found on the expression and regulation of PGFS in the pig endometrium.
In many species, endometrium also secretes PGE2, which in contrast to PGF2 exerts a luteoprotective action. Recent evidence suggests the existence of three forms of PGE synthase (PGES); among them, microsomal PGES-1 (mPGES-1) is highly inducible along with COX-2 (23, 24) and was found to be the main enzyme in the bovine endometrium associated with increased PGE2 production in vitro (25). We hypothesize that mPGES-1 may have a supportive role in maintenance of pregnancy by modulating the PGE2/PGF2 ratio on d 11–13 of pregnancy and that it may be involved in increasing uterine PGE2 after initiation of implantation in pigs.
Achieving an optimal PGE2/PGF2 ratio is essential for luteolysis or maintenance of the CL, which are the critical events in domestic animal female reproduction. We further hypothesized that the enzymes downstream of COX-2 may determine the ratio of PGE2/PGF2 in porcine uterus and have an influence on both PG concentrations in utero-ovarian circulation. Therefore, the objectives of the present study were 1) to clone and characterize porcine PGFS and mPGES-1 cDNA sequences; 2) to determine the temporal expression profiles of PGFS and mPGES-1 in the endometrium during the estrous cycle and early pregnancy in the pig; and 3) to determine the spatial distribution of PGFS and mPGES-1 proteins in the porcine uterus.
Materials and Methods
Tissue collection
Uteri were collected from cyclic crossbred gilts from a local abattoir. Days of the estrous cycle were determined by utero-ovarian morphology (26, 27). Uteri were classified into seven groups corresponding to d 1–4 (n = 7), 5–8 (n = 8), 9–12 (n = 8), 13–15 (n = 4), 16–17 (n = 5), and 18–21 (n = 6) or every 2 d starting from d 10 (n = 3–7) for comparison of enzyme expression in endometrium from cyclic vs. pregnant animals. Gilts randomly assigned to a pregnant group, after exhibiting two estrous cycles of normal length, were bred at the onset of estrus (d 0) and then 12 and 24 h later. Pregnant gilts were slaughtered at a local abattoir on d 10–11 (n = 8), 12–13 (n = 3), 14–15 (n = 7), 16–17 (n = 5), 18–19 (n = 4), 20–21 (n = 4), 22–23 (n = 3), and 24–25 (n = 4) of pregnancy, and uteri were collected. Pregnancy was confirmed by the presence of conceptuses. Endometrium dissected from myometrium was collected from the middle portion of the uterine horn. During the implantation stage, endometrium was also separated from trophoblasts and was collected from implantation sites.
Endometrial and other tissue samples (liver, kidney, lung, CL, 20-d embryo, oviduct, brain, heart, and myometrium) were snap-frozen in liquid nitrogen and stored at –80 C until further use. Cross sections of uterus samples were also fixed for immunohistochemical analyses. All procedures involving animals were approved by the Local Research Ethics Committee and were conducted in accordance with the national guidelines for agricultural animal care.
Total RNA isolation
Total RNA was extracted from endometrial and other tissue samples using the acid guanidinium thiocyanate-phenol-chloroform method (28) and treated with DNase I (Invitrogen Life Technologies Inc., Carlsbad, CA) as described by the supplier’s protocol.
Cloning and sequencing of the porcine PGFS and mPGES-1 cDNAs
The porcine PGFS and mPGES-1 cDNAs were isolated in fragments using a multistep cloning strategy. The 978-bp PGFS and 510-bp mPGES-1 RT-PCR products were initially cloned from porcine endometrial total RNA obtained from a gilt on d 14 of the estrous cycle. Briefly, total RNA (2 μg) was reversed transcribed using an oligo(dT) primer and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Amplification of obtained cDNA was performed with oligonucleotide primers designed according to homologous sequences (Table 1; primers 1/2 for PGFS and primers 5/6 for mPGES-1). The PCR conditions for PGFS and mPGES-1 were 95 C for 35 sec, 55 C for 35 sec, and 72 C for 1 min for 33 cycles and 94 C for 30 sec, 56 C for 30 sec, and 72 C for 45 sec for 30 cycles, respectively. The partial cDNAs were cloned into a pCR 4-TOPO cloning vector using TOPO TA cloning kit, version N (Invitrogen) according to the manufacturer’s protocol. Proper recombinant plasmids were identified and isolated from transformed bacterial colonies using standard techniques (29). Sequencing of the insert was performed by the Institute of Biochemistry and Biophysics of Polish Academy of Sciences (Warsaw, Poland) using vector-based T3 and T7 oligonucleotide primers. The nucleotide sequence of the clone was determined on both strands to verify the clone’s identity.
Sequences obtained from initial RT-PCR products served to design specific oligonucleotides for 5' and 3' rapid amplification of cDNA ends procedures. The 5' and 3' rapid amplification of cDNA ends for PGFS and mPGES-1 was performed using GeneRacer Kit (Invitrogen) as directed by the manufacturer. The primers used for the cloning are shown in Table 1. Endometrial total RNA (2 μg) obtained from a gilt on d 14 of the estrous cycle was reverse transcribed using Superscript III reverse transcriptase and a poly-dT oligonucleotide with anchor sequences at its 5' end. Amplification of the 5'-terminal sequences was performed with primer 2 (for PGFS) or primer 6 (for mPGES-1) and adapter primer 9. The obtained products were used in a second nested PCR with primer 3 (for PGFS) or primer 7 (for mPGES-1) and adapter primer 10. To obtain 3' ends of the porcine sequences, amplification was performed with primer 1 (for PGFS) or primer 5 (for mPGES-1) and adapter primer 11. The first PCR rounds were followed by the second-round PCR with specific primer sets (primers 4/12 for PGFS and primers 8/12 for mPGES-1). At each step, the PCR product was cloned into a pCR 4-TOPO cloning vector (TOPO TA cloning kit, version N; Invitrogen) and sequenced.
As additional confirmation, clones containing the entire coding region were isolated by RT-PCR (not shown) and found to correspond with the deduced primary PGFS and mPGES-1 transcripts reported herein. Nucleotide and amino acid sequence comparisons were performed online by standard BLAST analyses at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov) and by ClustalW multiple sequence alignment (30).
RT-PCR
RT was used to generate cDNA for real-time PCR. RT-PCR was used to determine PGFS and mPGES-1 mRNA expression in porcine tissues. Briefly, 2 μg of total RNA sample was reverse transcribed in a total reaction volume of 25 μl containing 5.5 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP mix, 1 U/μl ribonuclease inhibitor, 1 μg oligo(dT) primer, and 15 U/μl avian myeloblastosis virus reverse transcriptase (all from Promega). RNA was first denatured at 70 C for 10 min, followed by 42 C for 60 min for RT, and then 94 C for 5 min to terminate the reaction and chilled on ice. Resulting cDNA was amplified in PCR with 200 μM dNTP mix, 0.5 μM of the appropriate pair of primers, and 0.04 U/μl Taq DNA Polymerase (Sigma-Aldrich Co., St. Louis, MO).
The amplification for PGFS was performed with oligonucleotide primers designed according to the cloned porcine sequence: sense primer, 5'-GGACTTGGCACTCTCGTCTC-3', and antisense primer, 5'-AAACCCTCTTCACAGCCCTA-3'. The PCR conditions for PGFS were 95 C for 35 sec, 55 C for 35 sec, and 72 C for 1 min for 29 cycles. For mPGES-1 amplification, 24 cycles (94 C for 30 sec, 56 C for 30 sec, and 72 C for 45 sec) were performed with primers designed according to the cloned porcine cDNA: sense primer 13, 5'-ATCAAGATGTACGTGGTGGC-3', and antisense primer 14, 5'-GAGCTGGGCCAGGGTGTAGG-3'. -Actin amplification was performed with sense primer 15 (5'-ACATCAAGGAGAAGCTCTGCTACG-3') and antisense primer 16 (5'-AGGGGCGATGATCTTGATCTTCA-3') in the same conditions as for mPGES-1.
PCR products were run on 1.2% agarose gels containing 0.5 μg/ml ethidium bromide and photographed under UV light. To check for the specificity of RT-PCR products, three controls were set: 1) RNA samples were directly amplified without RT; 2) RT was done without adding reverse transcriptase followed by PCR amplification; and 3) RNA samples were replaced by nuclease-free water in RT-PCR.
Real-time PCR quantitation
Real-time PCR was performed with a DNA Engine Opticon continuous fluorescence detection system (MJ Research, Inc., San Francisco, CA) using QuantiTect SYBR Green PCR master mix (QIAGEN GmbH, Hilden, Germany), following the manufacturer’s instructions. Briefly, total RNA was reverse transcribed as described above. Real-time PCR (50 μl) included 25 μl QuantiTect SYBR Green PCR master mix, 0.5 μM sense and antisense primers each, and reverse-transcribed cDNA (3 μl of diluted RT product). To evaluate mRNA levels of both terminal synthases, specific primers were used: sense 5'-ACGCTGCTGGTCATCAAGA-3' and antisense 5'-GAACAGCTCCTCCCTCTTCA-3' for PGFS, primers 13/14 for mPGES-1, and primers 15/16 for -actin, respectively. For quantification, standard curves consisting of serial dilutions of the appropriate purified cDNA were included. Before amplification, an initial denaturation (15 min at 95 C) step was used. The PCR programs for each gene were performed as follows: 38 cycles of denaturation (15 sec at 95 C), annealing (30 sec at 52.5 C for PGFS and at 55 C for mPGES-1 and -actin), and elongation (60 sec at 72 C). After PCR, melting curves were acquired by stepwise increases in the temperature from 50–95 C to ensure that a single product was amplified in the reaction. Data obtained from the real-time PCR for PGFS and mPGES-1 were normalized against -actin. Intraassay coefficients of variations for single PGFS and mPGES-1 assays were 8.7 and 6.3%, respectively. Sensitivity was at least 0.5 ng/ml (PGFS) and 0.1 ng/ml (mPGES-1). Control reactions in the absence of reverse transcriptase were performed to test for genomic DNA contamination. Furthermore, specificity of RT-PCR products was confirmed by gel electrophoresis and sequencing.
Preparation of cytosol and membrane fractions for Western blot
Protein fractions for immunoblotting were obtained using the following procedure. Briefly, endometrial and other tissues were homogenized on ice in homogenization buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 μg/ml aprotinin, 52 μM leupeptin, 1 mM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). Homogenates were then centrifuged for 10 min at 1000 x g at 4 C. The supernatant was centrifuged for 1 h at 105,000 x g at 8 C, and the resulting supernatant and precipitate were used as the cytosol and membrane fraction, respectively. The fractions were stored at –70 C for further analysis. The protein concentration was determined by the Bradford (31) method.
Western blot analysis
Equal amounts (30 μg) of cytosol (for PGFS) and membrane (for mPGES-1) fractions were dissolved in SDS gel-loading buffer [50 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 2% -mercaptoethanol], heated to 95 C for 4 min, and separated on 12% (for PGFS) and 15% (for mPGES-1) SDS-PAGE. Separated proteins were electroblotted onto 0.2-μm nitrocellulose membrane in transfer buffer [20 mM Tris-HCl buffer (pH 8.2), 150 mM glycine, and 20% methanol). After blocking in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1.5 h at 25.6 C, the membranes were incubated overnight with 1:2000 anti-lung-type PGFS antiserum (17) , 1:1500 anti-liver-type PGFS antiserum (16), or 1:1000 polyclonal anti-mPGES-1 antibodies (Cayman Chemical, Ann Arbor, MI) at 4 C. Subsequently, PGFS and mPGES-1 were detected by incubating the membrane with 1:20,000 dilution of secondary polyclonal antirabbit alkaline phosphatase-conjugated antibodies (Sigma-Aldrich) for 1.5 h at 25.6 C. Immune complexes were visualized using a standard alkaline phosphatase visualization procedure (29). Western blots were quantitated using Kodak 1D program (Eastman Kodak, Rochester, NY). Interassay coefficients of variations for PGFS and mPGES-1 Western blots assays were 8.1 and 13.2%, respectively. Intraassay coefficients of variations of PGFS and mPGES-1 assays were 5.6 and 9.4%, respectively.
Immunohistochemistry
Immunohistochemical localization of PGFS and mPGES-1 was performed using Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, uterine tissues were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 4 h at room temperature and then rinsed three times for 10 min with PBS. Fixed tissues were stored in 18% sucrose in PBS containing 0.01% sodium azide and then frozen at –80 C. Cryostat cross-sections (6 μm in thickness) were dehydrated in ethanol ascending concentrations series (50, 70, and 96% absolute ethanol). After blocking of endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 30 min, the sections were treated in 0.75% glycine in TBS for 30 min and incubated in 10% normal goat serum. Incubations with the anti-lung type PGFS antiserum (1:500), anti-liver type PGFS antiserum (1:250), or polyclonal anti-mPGES-1 antibodies (1:50; Cayman Chemical) were performed overnight at 4 C. For the negative control, normal goat serum (1:10) was used instead of primary antibodies. The sections were further incubated with secondary antibodies (biotinylated goat antirabbit IgG, 1:200) for 30 min at room temperature and then with AB complex (Vectastain ABC kit; Vector) for 30 min. Between each step, sections were washed in TBS. The immunoreaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as a substrate and then examined under a light microscope (Olympus BX 60; Olympus Optical Co. Ltd., Tokyo, Japan) and photographed with an Olympus digital camera.
Statistical analysis
Statistical analyses were performed using ANOVA, followed by Tukey multiple comparison test (Graphpad Prism 4.0; Graphpad Software, Inc., San Diego, CA). All numerical data are presented as the mean ± SEM, and differences were considered as statistically significant at the 95% confidence level (P < 0.05).
Results
Cloning and sequencing of the porcine PGFS and mPGES-1 cDNAs
We have cloned and characterized the primary structures of porcine PGFS and mPGES-1 (GenBank accession nos. AY863054 and AY857634, respectively). The 1129-bp PGFS and 1175-bp mPGES-1 cDNAs contained 966-bp and 459-bp open reading frames that encoded proteins of 322 and 153 amino acids, respectively (Fig. 1, A and B). Calculated molecular mass for porcine PGFS was approximately 36.8 kDa, and for porcine mPGES-1, it was approximately 17.3 kDa. The deduced amino acid sequence of porcine PGFS possessed 73% identity with all the bovine isoforms, lung-type PGFS (18), liver-type PGFS (16), and PGFS II (19), and 75, 72, 71, and 67% with ovine (32), human (33), horse (34), and canine (Kowalewski, M. P., and B. Hoffman, unpublished; GenBank no. AAW69917) PGFS, respectively. PGFS consisted of a single conserved aldo-keto reductase domain (Fig. 1A), which was supposed to have an (/)8-barrel three-dimensional structure (35). Furthermore, critical amino acid residues that are required for catalytic activity, NADP+ cofactor binding, and the substrate pocket (35) were found to be conserved in porcine PGFS (Fig. 1A). The predicted amino acid sequence of porcine mPGES-1 possessed 91% identity with the homologs of cow (36) and horse (Sirois, J., N. Bouchard, and F. Filion, unpublished; GenBank no. AY057096), 86% with human (23), and 77% with mouse (37) and rat homologs (37). Porcine mPGES-1 showed about 40% similarity to microsomal glutathione S-transferase 1, another membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily member. Arg110, the residue strictly conserved in all MAPEG superfamily members and essential for catalytic function (37, 38), and the putative MK-886-binding motif were present in porcine mPGES-1 (Fig. 1B). The motif ERXXXAXXNXXD/E has been proposed to represent a consensus sequence for interaction with arachidonic acid and/or several of its oxygenation products (39).
Expression of PGFS and mPGES-1 in different porcine tissues
RT-PCR analysis demonstrated high abundance of PGFS mRNA in liver, kidney, CL, lung, oviduct, and embryo; an intermediate abundance in endometrium and heart; very low expression in myometrium; and no detectable expression in brain (Fig. 2A). Western blot analyses showed a similar tissue distribution pattern of PGFS protein using both anti-liver-type PGFS antiserum and anti-lung-type PGFS antiserum (Fig. 2B). The highest levels of PGFS protein were detected in liver. An intermediate amount of PGFS protein was found in CL, kidney, brain, and heart, and the lowest levels were found in lung, endometrium, myometrium, embryo, and oviduct (Fig. 2B).
The abundance of mPGES-1 mRNA and protein was the highest in CL and intermediate in lung, kidney, myometrium, and embryo (Fig. 2, A and B). Low mPGES-1 mRNA and high mPGES-1 protein expression was observed in endometrium. Although an intermediate abundance of mPGES-1 protein was detected in oviduct, mPGES-1 mRNA could not be detected in this tissue. Neither mPGES-1 mRNA nor protein was detected in liver, brain, and heart.
Endometrial PGFS expression during the estrous cycle and during early pregnancy
To evaluate PGFS expression levels during the estrous cycle and early pregnancy, real-time RT-PCR and Western blotting were performed (Fig. 3). The PGFS protein expression profiles were identical for both anti-liver-type and anti-lung-type antisera in Western blotting analyses. Quantification of PGFS expression (Fig. 3, A and C) revealed significant up-regulation on d 13–15 in both mRNA (vs. metestrus and the follicular phase; P < 0.05) and protein levels (vs. all other days of the estrous cycle; P < 0.001). However, induction of PGFS mRNA expression occurred earlier, on d 5–8 of the estrous cycle (Fig. 3A). The increase of PGFS mRNA (d 5–15) and protein levels (d 13–15) was 8.5-fold and 3.7-fold, respectively, in comparison with the low mRNA and protein levels on d 18–21 of the estrous cycle.
In early pregnancy, no significant variation in PGFS mRNA levels was observed (Fig. 3B). However, PGFS protein levels were significantly lower on d 10–11 and d 12–13 when compared with d 14–25 and d 14–17 and 22–25, respectively (Fig. 3D). Protein expression was intermediate on d 14–23 and the highest on d 24–25 of pregnancy (P < 0.001).
Comparison of PGFS and mPGES-1 expression in the endometrium at the corresponding stages of the estrous cycle and early pregnancy required regrouping cyclic gilts every 2 d starting from d 10 of the estrous cycle (Table 2). Interestingly, the levels of PGFS mRNA on d 10–15 of the estrous cycle and the corresponding days of pregnancy were comparable; however, PGFS protein tended to decrease (P = 0.057) on d 10–13 of pregnancy when compared with respective days of the estrous cycle (Table 2). Furthermore, in pregnancy, up-regulation of PGFS at both the mRNA and protein levels was observed on d 16–21 when compared with respective days of the estrous cycle (P < 0.05).
Endometrial mPGES-1 expression during the estrous cycle and during early pregnancy
Quantification of mPGES-1 mRNA revealed no significant variation throughout the estrous cycle (Fig. 4A); however, an increase in protein expression on d 13–15, when compared with d 18–21, was observed (P < 0.05; Fig. 4C).
During pregnancy, higher levels of mPGES-1 mRNA occurred on d 10–11 when compared with d 12–17 (P < 0.05). Intermediate levels of mPGES-1 protein were observed on d 10–13. Afterward, mPGES-1 mRNA and protein expression decreased, followed by an increase from d 18–19 or 22–23 of pregnancy, respectively (Fig. 4, B and D). The highest mPGES-1 mRNA and protein levels were reached on d 24–25 of pregnancy (vs. d 10–21, P < 0.001; and vs. d 22–23, P < 0.05).
Comparison of the expression patterns between pregnancy and the estrous cycle (Table 2) revealed no significant differences on d 10–13 in expression of mPGES-1 in pregnancy vs. the corresponding stage of the estrous cycle. When comparing the expression patterns in pregnancy with the estrous cycle, the protein and mRNA levels in pregnancy were lower on d 14–15 and on d 16–17 (P < 0.05 vs. the corresponding days of the estrous cycle), respectively, but the protein levels were significantly higher on d 18–19 (P < 0.05 vs. the corresponding days of the estrous cycle).
The mPGES-1:PGFS ratio
The paired expression data (mRNA and protein) analyzed as the mPGES-1:PGFS ratio are presented in Fig. 5. Except for the periovulatory stages (d 1–4 and 18–21), patterns of both mRNA and protein ratios during the estrous cycle were parallel (Fig. 5, A and C). The mRNA and protein ratio of mPGES-1 to PGFS were decreased on d 13–15 of the estrous cycle when compared with d 16–21 and d 5–12 and 16–17 of the cycle, respectively. The most dynamic changes occurred in the ratio of protein expression between d 13–15 (low) and 16–17 of the estrous cycle (high) (P < 0.001). The pattern of mPGES-1:PGFS mRNA ratio during early pregnancy generally corresponded to the pattern of mPGES-1:PGFS protein ratio, except d 24–25 (Fig. 5, B and D). Moreover, significantly higher levels of mPGES-1:PGFS protein ratio (4–7.5 fold higher than on d 14–21 and 24–25; P < 0.05) were observed on d 10–13 of pregnancy.
Immunohistochemical localization of endometrial PGFS and mPGES-1 expression
Cyclic and pregnant uteri were used to study PGFS and mPGES-1 protein expression immunohistochemically. The strongest immunostaining of both terminal PG synthases was observed in the luminal and glandular epithelium (Fig. 6). PGFS and mPGES-1 staining was also detected in myometrium, vascular endothelium, and stroma.
Discussion
In the present study, we have cloned and characterized the porcine PGFS and mPGES-1 cDNAs. There is no existing report or data on porcine PGFS and mPGES-1 cDNA cloning or their characterization. Comparative analysis of the amino acid sequences of the porcine terminal PG synthases revealed a high percentage (67–75% for PGFS and 77–91% for mPGFS-1) of identity with corresponding known mammalian homologs. By contrast, porcine mPGES-1 exhibited only about 40% identity to microsomal glutathione S-transferase 1, another member of the MAPEG family. These results are in line with what has been reported for mPGES-1 of other species (23, 37). Porcine mPGES-1 possessed the conserved residue for the MAPEG family members, Arg110, which is critical for catalytic function (38) and the putative MK-886-binding motif (Fig. 1B). The motif ERXXXAXXNXXD/E in mPGES-1 has been proposed to represent a consensus sequence for interaction with arachidonic acid and/or several of its oxygenation products (39). Porcine PGFS (Fig. 1A) contained the aldo-keto reductase domain with highly conserved, critical amino acid residues that were required for catalytic activity, NADP+ cofactor binding, and the substrate pocket (35).
We found that PGFS and mPGES-1 represented distinct tissue distribution patterns and were widely expressed in various tissues in the pig. PGFS was abundant in liver, kidney, lung, and CL, whereas expression of mPGES-1 was high in CL and intermediate in lung, kidney, embryo, and myometrium. The fact that we found no detectable mRNA and protein levels of mPGES-1 in liver, brain, or heart is consistent with very low mRNA expression in the same tissues in the rat (40).
The immunohistochemistry data demonstrated the localization of PGFS and mPGES-1 mostly in the epithelial cells of endometrium, indicating that this type of cell is the main source of PG synthesis. This finding is in agreement with in vitro studies showing high PGF2 release from porcine epithelial cells (41, 42, 43). The present results are consistent with immunohistochemical localization of terminal PG synthases in uterus in other species (3, 25, 34, 44).
This study provides the first demonstration that endometrial PGFS is up-regulated around the time of luteolysis in the pig. High expression of PGFS in the endometrium corresponds to high levels of luteolytic uterine secretion of PGF2 (6, 45) and significant up-regulation of PGF2 receptors in the CL (46, 47). Interestingly, the temporal increase of PGFS protein expression also correlates with the period of luteolytic capacity of porcine CL, which occurs after d 13 (9, 48). During this period, PGF2, reaching the CL, acts through luteal PGF2 receptors and causes a decrease of expression of many genes involved in the progesterone biosynthesis process (49).
Our findings are in contrast with similar studies in the horse, which revealed no increase of PGFS expression at the time around luteolysis (34). However, similar to our research, an increase of PGFS protein levels in diestrus has been reported in mice (50). Accordingly, in bovine species, there is up-regulation of the enzyme AKR1B5 possessing potent PGFS activity in diestrus but also on later days of the estrous cycle (22).
In pigs, just before implantation, the conceptuses undergo rapid elongation (51) and signal their presence to the maternal system by estrogen synthesis and secretion, which is a prerequisite for maintaining the CL function (7, 52, 53). Until now, there has been no clear explanation for the mechanism protecting CL from the luteolytic action of PGF2 in pigs. Interestingly, during the time of maternal recognition of pregnancy (about d 10–13) and at a period corresponding to the time around luteolysis (d 14–15) in cyclic gilts, endometrial PGFS mRNA expression was not affected by pregnancy; however, PGFS protein levels tended to decrease on d 10–13 of pregnancy when compared with the corresponding days of the estrous cycle. Pregnancy does not appear to have an effect on the overall amount of PGF2 released by the uterus at a period corresponding to the time around luteolysis in cyclic gilts; rather, it may affect the pulsatility of its secretion (8, 9), as has been suggested in ruminants (1, 54). On the other hand, the lack of effect of pregnancy on porcine PGFS expression in the endometrium on d 14–15 post estrus is consistent with results of a study of PGFS expression carried out in the mare (34). However, unlike the pig, uterine PGF2 production in the mare is significantly reduced in pregnant compared with nonpregnant mares (55).
Continued synthesis of PGs is required for implantation and maintenance of pregnancy (56, 57). As anticipated, in pregnant gilts, induction of PGFS mRNA and protein expression occurred on d 16–21 when compared with the corresponding stage of the estrous cycle. Furthermore, PGFS protein expression increased markedly after d 22 of pregnancy. These findings indicate a significant contribution of endometrial PGFS to the increase of PGF2 in uterine lumen during the progression of implantation (4).
Our results revealed that mRNA levels of mPGES-1, the second studied enzyme, did not vary significantly throughout the estrous cycle. Only a slight increase of mPGES-1 protein expression during the late luteal phase was observed. These findings can be supported by previous reports showing that the secretion of PGE2 also increases from d 13–16 of the estrous cycle; however, it remains 3-fold lower than those of PGF2 (6). In bovine and equine endometrium, mPGES-1 expression was not modulated significantly during the estrous cycle (25, 34), showing a similar pattern of mPGES-1 expression as that described in this report. However, in humans, abundant levels of mPGES-1 protein were shown to be expressed in endometrium during the proliferative phase, whereas a very low expression was observed during the late secretory phase of the menstrual cycle (44).
The uterine PGE2/PGF2 ratio plays an important role on d 11–15 post estrus, the critical period either for luteolysis initiating a new estrous cycle or for the establishment of pregnancy in pigs. Our findings show low mRNA and protein ratios of mPGES-1 to PGFS on d 13–15 of the estrous cycle (Fig. 5, A and C) that correlate with decreased PGE2/PGF2 ratio observed in uterine vein just before luteolysis (6). During maternal recognition of pregnancy, the protein ratio of mPGES-1 to PGFS was significantly higher (4- to 7.5-fold) when compared with d 14–21 and 24–25 of pregnancy (Fig. 5D). In pregnancy, mPGES-1 mRNA expression was also relatively high on d 10–11, and the protein levels were intermediate on d 10–13. These findings correspond to the peak of PGE2 in endometrium, which occurs earlier in pregnancy than in the estrous cycle (6). Interestingly, two periods of intermediate/high endometrial mPGES-1 expression correlate with reported previously biphasic estrogen secretion synthesis by the conceptus (53, 58). Indeed, estrogen secreted by the conceptus may stimulate mPGES-1 expression in the endometrium as it increases endometrial PGE2 production (59); for this reason, this steroid may be a key factor in increasing the PGE2/PGF2 ratio (43, 60, 61). Estradiol may also be responsible for increased mPGES-1:PGFS mRNA ratio we found around the follicular and periovulatory stage of the estrous cycle. The luteoprotective action of elevated levels of uterine PGE2 reaching the CL is additionally supported by elevated levels of luteal binding sites for PGE2 on d 14 of pregnancy, in contrast to d 14 of the estrous cycle (62), coupled with reduced luteal PGF2 receptors vs. cyclic pigs at the corresponding stage (46). However, we expected a much higher increase of mPGES-1 expression around the time of maternal recognition of pregnancy and significantly higher expression levels of mPGES-1 on d 10–13 when compared with the corresponding stage of the estrous cycle. Therefore, the question remains as to whether the minimal changes seen in mPGES-1 and PGFS expression in the endometrium on d 10–13 provide a sufficient stimulus to modulate the PGE2/PGF2 ratio in the uterus and systemic circulation during the maternal recognition of pregnancy.
An increase of the uterine PGE2 levels and increase of the PGE2/PGF2 ratio on d 11–13 of pregnancy could also be a result of the direct contribution of the conceptus to PGE2 secretion (63), correlating with the increased expression of PG biosynthetic enzymes in the conceptus around the time of elongation (64, 65). Another potential mechanism that may be responsible for the increase of the PGE2/PGF2 ratio during the establishment of pregnancy could be the inhibition of the activity of endometrial PG-9-KR (66). Although PG-9-KR has been identified in endometrium in other species (66, 67), the presence of this enzyme has not been reported in the porcine uterus yet.
It has been observed that products secreted by the porcine conceptus stimulate uterine production of PGF2 and PGE2 in vivo (68) and in vitro (69). In agreement, we found induction of mPGES-1 protein on d 18–19 in pregnant gilts when compared with the respective days of the estrous cycle. Furthermore, similar to PGFS, significant up-regulation of mPGES-1 protein expression was detected after d 22, with the maximum on d 24–25 of pregnancy. Up-regulation of both PG synthases affected by pregnancy could be a possible reason for the increase of PG secretion in uterus observed in early pregnancy (4). High levels of both terminal PG synthases after initiation of implantation may indicate a potential role of these enzymes in placentation and the establishment of pregnancy. The high endometrial expression of mPGES-1 that we found is in agreement with other reports on the significant role of mPGES-1 in implantation in other species (70, 71). Although PGE2 receptors (EP) have not been cloned in the pig, the PGE2-binding sites were reported in porcine endometrium (72). On the basis of knowledge about other species, we can speculate that PGE2 produced in uterus could act in an autocrine/paracrine role via EP2 and/or EP4, resulting in a local increase of endometrial vascular permeability and preparing for angiogenesis and placentation (73, 74). Endometrial up-regulation of mPGES-1 after initiation of implantation may be involved also in fetal allograft survival by suppressing maternal immune responses in an immunoregulatory role of PGE2 (75, 76).
To our knowledge, this is the first report of the cloning and the characterization of the two key PG synthases, PGFS and mPGES-1, in the pig. We have also shown simultaneous functional changes of expression of both PGFS and mPGES-1 in the porcine endometrium during the estrous cycle and early pregnancy. The spatiotemporal expression of PGFS throughout the estrous cycle indicates a significant role of PGFS in the regulation of luteolysis in this species. The comparison of endometrial PGFS and mPGES-1 expression on d 10–13 of the estrous cycle and pregnancy suggests a supportive rather than a major role of these enzymes in the increase of the uterine PGE2/PGF2 ratio during the period of maternal recognition of pregnancy. However, high endometrial expression of both terminal PG synthases after initiation of implantation may indicate their involvement in placentation and could be a result of local changes that occur in the uterus during the establishment of pregnancy.
Acknowledgments
We are grateful to Jan Klos, Katarzyna Gromadzka-Hliwa, and Michal Blitek for technical assistance. We also thank Dr. Wioletta Blaszczak for advice during the making of microscopy pictures.
Footnotes
This work was supported by Grant 2 P06D 041 26 from the State Committee for Scientific Research in Poland and by the Academy of Finland. M.M.K. was awarded the Domestic Grant for Young Scientists from the Foundation for Polish Sciences.
The PGFS and mPGES-1 sequences reported in this paper have been submitted to the GenBank database under accession numbers AY863054 and AY857634, respectively.
First Published Online October 13, 2005
Abbreviations: CL, Corpus luteum; COX, cyclooxygenase; EP, PGE2 receptors; MAPEG, membrane-associated proteins involved in eicosanoid and glutathione metabolism; mPGES-1, microsomal prostaglandin E synthase-1; PG, prostaglandin; PG-9-KR, PG-9-ketoreductase; PGES, PGE synthase; PGFS, PGF synthase; TBS, Tris-buffered saline.
Accepted for publication October 3, 2005.
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Department of Physiology (A.R.-M., L.J.S.B., N.A.R.), University of Turku, 20520 Turku, Finland
Graduate School of Integrated Science and Art (K.W.), University of East Asia, Yamaguchi 750-8503, Japan
Abstract
Endometrial prostaglandins (PGs) and the PGE2/PGF2 ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2 ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10–13, intermediate on d 14–23, and high on d 24–25). In pregnancy, expression of mPGES-1 was intermediate on d 10–11 and low on d 14–17 and then increased after d 22, reaching the maximum on d 24–25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10–13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2 ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.
Introduction
UTERINE SYNTHESIS OF prostaglandin (PG) E2 and F2 plays an important role in regulation of the estrous cycle and establishment of pregnancy through autocrine, paracrine, and endocrine actions in many domestic species (1, 2, 3). Because PGE2 and PGF2 differ in many of their actions, the PGE2/PGF2 ratio may integrate information from different sources. The PGE2/PGF2 ratio affects corpus luteum (CL) function, endometrial cell growth and differentiation, blood flow, vascular permeability, embryo migration, and implantation (4).
The estrous cycle of the pig is dependent on the uterus as the source of luteolysin, PGF2 (1), which together with PGE2 reaches the CL by local and/or systemic mechanisms (5). In pigs, the CL is caused to regress on d 15–16 of the estrous cycle by an increase in pulsatile endometrial secretion of PGF2 that occurs after d 13. During this period the PGE2/PGF2 ratio in the utero-ovarian vein reaches 1:3 (6). Moreover, mean concentrations, peak frequency, and peak amplitude of PGF2 in utero-ovarian vein plasma are higher in cyclic than in pregnant gilts on d 12–17 (7, 8, 9). On the other hand, uterine flushings of pregnant gilts contain significantly higher amounts of PGF2 than those from cyclic gilts (10). Bazer and Thatcher (7) proposed that the maternal recognition of pregnancy in the pig involves redirection of the PGF2 secretion from the uterine venous drainage (endocrine) to the uterine lumen (exocrine) by conceptus estrogen secretion. A part of the putative mechanism of CL protection during early pregnancy could also be the retrograde transfer of PGF2 from venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF2 (11). However, the above-mentioned hypotheses do not satisfactorily explain the mechanisms involved in maintaining CL function during early pregnancy in the pig, as discussed below.
Another potential mechanism by which a conceptus could inhibit luteolysis is by changing the PGE2/PGF2 ratio, i.e. in favor of the luteoprotective/antiluteolytic PGE2 (6, 12, 13). On d 11–13 of pregnancy, at approximately the time of maternal recognition of pregnancy, the PGE2/PGF2 ratio in the uterus and uterine vein increases (>1:1) (4, 6), suggesting that PGE2 can overcome the luteolytic effect of PGF2, thus preventing CL regression (6). It is possible that one of the important mechanisms of the maternal recognition of pregnancy could occur by the conceptus alerting the endometrial expression of enzymes involved in PG synthesis. PGs are produced from arachidonic acid, which is further metabolized to an unstable PGH2 by the cyclooxygenase (COX) enzymes COX-1 and COX-2. However, it has been reported that no differences in staining intensity of COX between cyclic and pregnant gilts on d 10, 12, and 15 were observed (14). Thus, it seems unlikely that the porcine conceptus targets COX expression as a means to modulate PGF2 and PGE2 release.
PGH2 is rapidly converted into different prostanoids (PGE2, PGF2, PGD2, PGI2, and TxA2) by specific terminal PG synthases or reductases (15). Additionally, PGE2 could be converted into PGF2 by PG-9-ketoreductase (PG-9-KR). PGF synthase (PGFS) has been primarily studied in the bovine species, and three characterized isoforms (16, 17, 18, 19) belong to the AKR1C subclass of the aldo-keto reductase family. Lung-type PGFS (20, 21) or 20-hydroxysteroid dehydrogenase (AKR1B5), a distinct enzyme with potent PGFS activity recently identified in bovine endometrium (3, 22), is down-regulated in bovine endometrial cells by interferon-. It suggests that the conceptus could target expression of enzymes involved in PGF2 synthesis to decrease PGF2 levels. However, in the available literature, no information could be found on the expression and regulation of PGFS in the pig endometrium.
In many species, endometrium also secretes PGE2, which in contrast to PGF2 exerts a luteoprotective action. Recent evidence suggests the existence of three forms of PGE synthase (PGES); among them, microsomal PGES-1 (mPGES-1) is highly inducible along with COX-2 (23, 24) and was found to be the main enzyme in the bovine endometrium associated with increased PGE2 production in vitro (25). We hypothesize that mPGES-1 may have a supportive role in maintenance of pregnancy by modulating the PGE2/PGF2 ratio on d 11–13 of pregnancy and that it may be involved in increasing uterine PGE2 after initiation of implantation in pigs.
Achieving an optimal PGE2/PGF2 ratio is essential for luteolysis or maintenance of the CL, which are the critical events in domestic animal female reproduction. We further hypothesized that the enzymes downstream of COX-2 may determine the ratio of PGE2/PGF2 in porcine uterus and have an influence on both PG concentrations in utero-ovarian circulation. Therefore, the objectives of the present study were 1) to clone and characterize porcine PGFS and mPGES-1 cDNA sequences; 2) to determine the temporal expression profiles of PGFS and mPGES-1 in the endometrium during the estrous cycle and early pregnancy in the pig; and 3) to determine the spatial distribution of PGFS and mPGES-1 proteins in the porcine uterus.
Materials and Methods
Tissue collection
Uteri were collected from cyclic crossbred gilts from a local abattoir. Days of the estrous cycle were determined by utero-ovarian morphology (26, 27). Uteri were classified into seven groups corresponding to d 1–4 (n = 7), 5–8 (n = 8), 9–12 (n = 8), 13–15 (n = 4), 16–17 (n = 5), and 18–21 (n = 6) or every 2 d starting from d 10 (n = 3–7) for comparison of enzyme expression in endometrium from cyclic vs. pregnant animals. Gilts randomly assigned to a pregnant group, after exhibiting two estrous cycles of normal length, were bred at the onset of estrus (d 0) and then 12 and 24 h later. Pregnant gilts were slaughtered at a local abattoir on d 10–11 (n = 8), 12–13 (n = 3), 14–15 (n = 7), 16–17 (n = 5), 18–19 (n = 4), 20–21 (n = 4), 22–23 (n = 3), and 24–25 (n = 4) of pregnancy, and uteri were collected. Pregnancy was confirmed by the presence of conceptuses. Endometrium dissected from myometrium was collected from the middle portion of the uterine horn. During the implantation stage, endometrium was also separated from trophoblasts and was collected from implantation sites.
Endometrial and other tissue samples (liver, kidney, lung, CL, 20-d embryo, oviduct, brain, heart, and myometrium) were snap-frozen in liquid nitrogen and stored at –80 C until further use. Cross sections of uterus samples were also fixed for immunohistochemical analyses. All procedures involving animals were approved by the Local Research Ethics Committee and were conducted in accordance with the national guidelines for agricultural animal care.
Total RNA isolation
Total RNA was extracted from endometrial and other tissue samples using the acid guanidinium thiocyanate-phenol-chloroform method (28) and treated with DNase I (Invitrogen Life Technologies Inc., Carlsbad, CA) as described by the supplier’s protocol.
Cloning and sequencing of the porcine PGFS and mPGES-1 cDNAs
The porcine PGFS and mPGES-1 cDNAs were isolated in fragments using a multistep cloning strategy. The 978-bp PGFS and 510-bp mPGES-1 RT-PCR products were initially cloned from porcine endometrial total RNA obtained from a gilt on d 14 of the estrous cycle. Briefly, total RNA (2 μg) was reversed transcribed using an oligo(dT) primer and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Amplification of obtained cDNA was performed with oligonucleotide primers designed according to homologous sequences (Table 1; primers 1/2 for PGFS and primers 5/6 for mPGES-1). The PCR conditions for PGFS and mPGES-1 were 95 C for 35 sec, 55 C for 35 sec, and 72 C for 1 min for 33 cycles and 94 C for 30 sec, 56 C for 30 sec, and 72 C for 45 sec for 30 cycles, respectively. The partial cDNAs were cloned into a pCR 4-TOPO cloning vector using TOPO TA cloning kit, version N (Invitrogen) according to the manufacturer’s protocol. Proper recombinant plasmids were identified and isolated from transformed bacterial colonies using standard techniques (29). Sequencing of the insert was performed by the Institute of Biochemistry and Biophysics of Polish Academy of Sciences (Warsaw, Poland) using vector-based T3 and T7 oligonucleotide primers. The nucleotide sequence of the clone was determined on both strands to verify the clone’s identity.
Sequences obtained from initial RT-PCR products served to design specific oligonucleotides for 5' and 3' rapid amplification of cDNA ends procedures. The 5' and 3' rapid amplification of cDNA ends for PGFS and mPGES-1 was performed using GeneRacer Kit (Invitrogen) as directed by the manufacturer. The primers used for the cloning are shown in Table 1. Endometrial total RNA (2 μg) obtained from a gilt on d 14 of the estrous cycle was reverse transcribed using Superscript III reverse transcriptase and a poly-dT oligonucleotide with anchor sequences at its 5' end. Amplification of the 5'-terminal sequences was performed with primer 2 (for PGFS) or primer 6 (for mPGES-1) and adapter primer 9. The obtained products were used in a second nested PCR with primer 3 (for PGFS) or primer 7 (for mPGES-1) and adapter primer 10. To obtain 3' ends of the porcine sequences, amplification was performed with primer 1 (for PGFS) or primer 5 (for mPGES-1) and adapter primer 11. The first PCR rounds were followed by the second-round PCR with specific primer sets (primers 4/12 for PGFS and primers 8/12 for mPGES-1). At each step, the PCR product was cloned into a pCR 4-TOPO cloning vector (TOPO TA cloning kit, version N; Invitrogen) and sequenced.
As additional confirmation, clones containing the entire coding region were isolated by RT-PCR (not shown) and found to correspond with the deduced primary PGFS and mPGES-1 transcripts reported herein. Nucleotide and amino acid sequence comparisons were performed online by standard BLAST analyses at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov) and by ClustalW multiple sequence alignment (30).
RT-PCR
RT was used to generate cDNA for real-time PCR. RT-PCR was used to determine PGFS and mPGES-1 mRNA expression in porcine tissues. Briefly, 2 μg of total RNA sample was reverse transcribed in a total reaction volume of 25 μl containing 5.5 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP mix, 1 U/μl ribonuclease inhibitor, 1 μg oligo(dT) primer, and 15 U/μl avian myeloblastosis virus reverse transcriptase (all from Promega). RNA was first denatured at 70 C for 10 min, followed by 42 C for 60 min for RT, and then 94 C for 5 min to terminate the reaction and chilled on ice. Resulting cDNA was amplified in PCR with 200 μM dNTP mix, 0.5 μM of the appropriate pair of primers, and 0.04 U/μl Taq DNA Polymerase (Sigma-Aldrich Co., St. Louis, MO).
The amplification for PGFS was performed with oligonucleotide primers designed according to the cloned porcine sequence: sense primer, 5'-GGACTTGGCACTCTCGTCTC-3', and antisense primer, 5'-AAACCCTCTTCACAGCCCTA-3'. The PCR conditions for PGFS were 95 C for 35 sec, 55 C for 35 sec, and 72 C for 1 min for 29 cycles. For mPGES-1 amplification, 24 cycles (94 C for 30 sec, 56 C for 30 sec, and 72 C for 45 sec) were performed with primers designed according to the cloned porcine cDNA: sense primer 13, 5'-ATCAAGATGTACGTGGTGGC-3', and antisense primer 14, 5'-GAGCTGGGCCAGGGTGTAGG-3'. -Actin amplification was performed with sense primer 15 (5'-ACATCAAGGAGAAGCTCTGCTACG-3') and antisense primer 16 (5'-AGGGGCGATGATCTTGATCTTCA-3') in the same conditions as for mPGES-1.
PCR products were run on 1.2% agarose gels containing 0.5 μg/ml ethidium bromide and photographed under UV light. To check for the specificity of RT-PCR products, three controls were set: 1) RNA samples were directly amplified without RT; 2) RT was done without adding reverse transcriptase followed by PCR amplification; and 3) RNA samples were replaced by nuclease-free water in RT-PCR.
Real-time PCR quantitation
Real-time PCR was performed with a DNA Engine Opticon continuous fluorescence detection system (MJ Research, Inc., San Francisco, CA) using QuantiTect SYBR Green PCR master mix (QIAGEN GmbH, Hilden, Germany), following the manufacturer’s instructions. Briefly, total RNA was reverse transcribed as described above. Real-time PCR (50 μl) included 25 μl QuantiTect SYBR Green PCR master mix, 0.5 μM sense and antisense primers each, and reverse-transcribed cDNA (3 μl of diluted RT product). To evaluate mRNA levels of both terminal synthases, specific primers were used: sense 5'-ACGCTGCTGGTCATCAAGA-3' and antisense 5'-GAACAGCTCCTCCCTCTTCA-3' for PGFS, primers 13/14 for mPGES-1, and primers 15/16 for -actin, respectively. For quantification, standard curves consisting of serial dilutions of the appropriate purified cDNA were included. Before amplification, an initial denaturation (15 min at 95 C) step was used. The PCR programs for each gene were performed as follows: 38 cycles of denaturation (15 sec at 95 C), annealing (30 sec at 52.5 C for PGFS and at 55 C for mPGES-1 and -actin), and elongation (60 sec at 72 C). After PCR, melting curves were acquired by stepwise increases in the temperature from 50–95 C to ensure that a single product was amplified in the reaction. Data obtained from the real-time PCR for PGFS and mPGES-1 were normalized against -actin. Intraassay coefficients of variations for single PGFS and mPGES-1 assays were 8.7 and 6.3%, respectively. Sensitivity was at least 0.5 ng/ml (PGFS) and 0.1 ng/ml (mPGES-1). Control reactions in the absence of reverse transcriptase were performed to test for genomic DNA contamination. Furthermore, specificity of RT-PCR products was confirmed by gel electrophoresis and sequencing.
Preparation of cytosol and membrane fractions for Western blot
Protein fractions for immunoblotting were obtained using the following procedure. Briefly, endometrial and other tissues were homogenized on ice in homogenization buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 μg/ml aprotinin, 52 μM leupeptin, 1 mM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). Homogenates were then centrifuged for 10 min at 1000 x g at 4 C. The supernatant was centrifuged for 1 h at 105,000 x g at 8 C, and the resulting supernatant and precipitate were used as the cytosol and membrane fraction, respectively. The fractions were stored at –70 C for further analysis. The protein concentration was determined by the Bradford (31) method.
Western blot analysis
Equal amounts (30 μg) of cytosol (for PGFS) and membrane (for mPGES-1) fractions were dissolved in SDS gel-loading buffer [50 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 2% -mercaptoethanol], heated to 95 C for 4 min, and separated on 12% (for PGFS) and 15% (for mPGES-1) SDS-PAGE. Separated proteins were electroblotted onto 0.2-μm nitrocellulose membrane in transfer buffer [20 mM Tris-HCl buffer (pH 8.2), 150 mM glycine, and 20% methanol). After blocking in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1.5 h at 25.6 C, the membranes were incubated overnight with 1:2000 anti-lung-type PGFS antiserum (17) , 1:1500 anti-liver-type PGFS antiserum (16), or 1:1000 polyclonal anti-mPGES-1 antibodies (Cayman Chemical, Ann Arbor, MI) at 4 C. Subsequently, PGFS and mPGES-1 were detected by incubating the membrane with 1:20,000 dilution of secondary polyclonal antirabbit alkaline phosphatase-conjugated antibodies (Sigma-Aldrich) for 1.5 h at 25.6 C. Immune complexes were visualized using a standard alkaline phosphatase visualization procedure (29). Western blots were quantitated using Kodak 1D program (Eastman Kodak, Rochester, NY). Interassay coefficients of variations for PGFS and mPGES-1 Western blots assays were 8.1 and 13.2%, respectively. Intraassay coefficients of variations of PGFS and mPGES-1 assays were 5.6 and 9.4%, respectively.
Immunohistochemistry
Immunohistochemical localization of PGFS and mPGES-1 was performed using Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, uterine tissues were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 4 h at room temperature and then rinsed three times for 10 min with PBS. Fixed tissues were stored in 18% sucrose in PBS containing 0.01% sodium azide and then frozen at –80 C. Cryostat cross-sections (6 μm in thickness) were dehydrated in ethanol ascending concentrations series (50, 70, and 96% absolute ethanol). After blocking of endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 30 min, the sections were treated in 0.75% glycine in TBS for 30 min and incubated in 10% normal goat serum. Incubations with the anti-lung type PGFS antiserum (1:500), anti-liver type PGFS antiserum (1:250), or polyclonal anti-mPGES-1 antibodies (1:50; Cayman Chemical) were performed overnight at 4 C. For the negative control, normal goat serum (1:10) was used instead of primary antibodies. The sections were further incubated with secondary antibodies (biotinylated goat antirabbit IgG, 1:200) for 30 min at room temperature and then with AB complex (Vectastain ABC kit; Vector) for 30 min. Between each step, sections were washed in TBS. The immunoreaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as a substrate and then examined under a light microscope (Olympus BX 60; Olympus Optical Co. Ltd., Tokyo, Japan) and photographed with an Olympus digital camera.
Statistical analysis
Statistical analyses were performed using ANOVA, followed by Tukey multiple comparison test (Graphpad Prism 4.0; Graphpad Software, Inc., San Diego, CA). All numerical data are presented as the mean ± SEM, and differences were considered as statistically significant at the 95% confidence level (P < 0.05).
Results
Cloning and sequencing of the porcine PGFS and mPGES-1 cDNAs
We have cloned and characterized the primary structures of porcine PGFS and mPGES-1 (GenBank accession nos. AY863054 and AY857634, respectively). The 1129-bp PGFS and 1175-bp mPGES-1 cDNAs contained 966-bp and 459-bp open reading frames that encoded proteins of 322 and 153 amino acids, respectively (Fig. 1, A and B). Calculated molecular mass for porcine PGFS was approximately 36.8 kDa, and for porcine mPGES-1, it was approximately 17.3 kDa. The deduced amino acid sequence of porcine PGFS possessed 73% identity with all the bovine isoforms, lung-type PGFS (18), liver-type PGFS (16), and PGFS II (19), and 75, 72, 71, and 67% with ovine (32), human (33), horse (34), and canine (Kowalewski, M. P., and B. Hoffman, unpublished; GenBank no. AAW69917) PGFS, respectively. PGFS consisted of a single conserved aldo-keto reductase domain (Fig. 1A), which was supposed to have an (/)8-barrel three-dimensional structure (35). Furthermore, critical amino acid residues that are required for catalytic activity, NADP+ cofactor binding, and the substrate pocket (35) were found to be conserved in porcine PGFS (Fig. 1A). The predicted amino acid sequence of porcine mPGES-1 possessed 91% identity with the homologs of cow (36) and horse (Sirois, J., N. Bouchard, and F. Filion, unpublished; GenBank no. AY057096), 86% with human (23), and 77% with mouse (37) and rat homologs (37). Porcine mPGES-1 showed about 40% similarity to microsomal glutathione S-transferase 1, another membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily member. Arg110, the residue strictly conserved in all MAPEG superfamily members and essential for catalytic function (37, 38), and the putative MK-886-binding motif were present in porcine mPGES-1 (Fig. 1B). The motif ERXXXAXXNXXD/E has been proposed to represent a consensus sequence for interaction with arachidonic acid and/or several of its oxygenation products (39).
Expression of PGFS and mPGES-1 in different porcine tissues
RT-PCR analysis demonstrated high abundance of PGFS mRNA in liver, kidney, CL, lung, oviduct, and embryo; an intermediate abundance in endometrium and heart; very low expression in myometrium; and no detectable expression in brain (Fig. 2A). Western blot analyses showed a similar tissue distribution pattern of PGFS protein using both anti-liver-type PGFS antiserum and anti-lung-type PGFS antiserum (Fig. 2B). The highest levels of PGFS protein were detected in liver. An intermediate amount of PGFS protein was found in CL, kidney, brain, and heart, and the lowest levels were found in lung, endometrium, myometrium, embryo, and oviduct (Fig. 2B).
The abundance of mPGES-1 mRNA and protein was the highest in CL and intermediate in lung, kidney, myometrium, and embryo (Fig. 2, A and B). Low mPGES-1 mRNA and high mPGES-1 protein expression was observed in endometrium. Although an intermediate abundance of mPGES-1 protein was detected in oviduct, mPGES-1 mRNA could not be detected in this tissue. Neither mPGES-1 mRNA nor protein was detected in liver, brain, and heart.
Endometrial PGFS expression during the estrous cycle and during early pregnancy
To evaluate PGFS expression levels during the estrous cycle and early pregnancy, real-time RT-PCR and Western blotting were performed (Fig. 3). The PGFS protein expression profiles were identical for both anti-liver-type and anti-lung-type antisera in Western blotting analyses. Quantification of PGFS expression (Fig. 3, A and C) revealed significant up-regulation on d 13–15 in both mRNA (vs. metestrus and the follicular phase; P < 0.05) and protein levels (vs. all other days of the estrous cycle; P < 0.001). However, induction of PGFS mRNA expression occurred earlier, on d 5–8 of the estrous cycle (Fig. 3A). The increase of PGFS mRNA (d 5–15) and protein levels (d 13–15) was 8.5-fold and 3.7-fold, respectively, in comparison with the low mRNA and protein levels on d 18–21 of the estrous cycle.
In early pregnancy, no significant variation in PGFS mRNA levels was observed (Fig. 3B). However, PGFS protein levels were significantly lower on d 10–11 and d 12–13 when compared with d 14–25 and d 14–17 and 22–25, respectively (Fig. 3D). Protein expression was intermediate on d 14–23 and the highest on d 24–25 of pregnancy (P < 0.001).
Comparison of PGFS and mPGES-1 expression in the endometrium at the corresponding stages of the estrous cycle and early pregnancy required regrouping cyclic gilts every 2 d starting from d 10 of the estrous cycle (Table 2). Interestingly, the levels of PGFS mRNA on d 10–15 of the estrous cycle and the corresponding days of pregnancy were comparable; however, PGFS protein tended to decrease (P = 0.057) on d 10–13 of pregnancy when compared with respective days of the estrous cycle (Table 2). Furthermore, in pregnancy, up-regulation of PGFS at both the mRNA and protein levels was observed on d 16–21 when compared with respective days of the estrous cycle (P < 0.05).
Endometrial mPGES-1 expression during the estrous cycle and during early pregnancy
Quantification of mPGES-1 mRNA revealed no significant variation throughout the estrous cycle (Fig. 4A); however, an increase in protein expression on d 13–15, when compared with d 18–21, was observed (P < 0.05; Fig. 4C).
During pregnancy, higher levels of mPGES-1 mRNA occurred on d 10–11 when compared with d 12–17 (P < 0.05). Intermediate levels of mPGES-1 protein were observed on d 10–13. Afterward, mPGES-1 mRNA and protein expression decreased, followed by an increase from d 18–19 or 22–23 of pregnancy, respectively (Fig. 4, B and D). The highest mPGES-1 mRNA and protein levels were reached on d 24–25 of pregnancy (vs. d 10–21, P < 0.001; and vs. d 22–23, P < 0.05).
Comparison of the expression patterns between pregnancy and the estrous cycle (Table 2) revealed no significant differences on d 10–13 in expression of mPGES-1 in pregnancy vs. the corresponding stage of the estrous cycle. When comparing the expression patterns in pregnancy with the estrous cycle, the protein and mRNA levels in pregnancy were lower on d 14–15 and on d 16–17 (P < 0.05 vs. the corresponding days of the estrous cycle), respectively, but the protein levels were significantly higher on d 18–19 (P < 0.05 vs. the corresponding days of the estrous cycle).
The mPGES-1:PGFS ratio
The paired expression data (mRNA and protein) analyzed as the mPGES-1:PGFS ratio are presented in Fig. 5. Except for the periovulatory stages (d 1–4 and 18–21), patterns of both mRNA and protein ratios during the estrous cycle were parallel (Fig. 5, A and C). The mRNA and protein ratio of mPGES-1 to PGFS were decreased on d 13–15 of the estrous cycle when compared with d 16–21 and d 5–12 and 16–17 of the cycle, respectively. The most dynamic changes occurred in the ratio of protein expression between d 13–15 (low) and 16–17 of the estrous cycle (high) (P < 0.001). The pattern of mPGES-1:PGFS mRNA ratio during early pregnancy generally corresponded to the pattern of mPGES-1:PGFS protein ratio, except d 24–25 (Fig. 5, B and D). Moreover, significantly higher levels of mPGES-1:PGFS protein ratio (4–7.5 fold higher than on d 14–21 and 24–25; P < 0.05) were observed on d 10–13 of pregnancy.
Immunohistochemical localization of endometrial PGFS and mPGES-1 expression
Cyclic and pregnant uteri were used to study PGFS and mPGES-1 protein expression immunohistochemically. The strongest immunostaining of both terminal PG synthases was observed in the luminal and glandular epithelium (Fig. 6). PGFS and mPGES-1 staining was also detected in myometrium, vascular endothelium, and stroma.
Discussion
In the present study, we have cloned and characterized the porcine PGFS and mPGES-1 cDNAs. There is no existing report or data on porcine PGFS and mPGES-1 cDNA cloning or their characterization. Comparative analysis of the amino acid sequences of the porcine terminal PG synthases revealed a high percentage (67–75% for PGFS and 77–91% for mPGFS-1) of identity with corresponding known mammalian homologs. By contrast, porcine mPGES-1 exhibited only about 40% identity to microsomal glutathione S-transferase 1, another member of the MAPEG family. These results are in line with what has been reported for mPGES-1 of other species (23, 37). Porcine mPGES-1 possessed the conserved residue for the MAPEG family members, Arg110, which is critical for catalytic function (38) and the putative MK-886-binding motif (Fig. 1B). The motif ERXXXAXXNXXD/E in mPGES-1 has been proposed to represent a consensus sequence for interaction with arachidonic acid and/or several of its oxygenation products (39). Porcine PGFS (Fig. 1A) contained the aldo-keto reductase domain with highly conserved, critical amino acid residues that were required for catalytic activity, NADP+ cofactor binding, and the substrate pocket (35).
We found that PGFS and mPGES-1 represented distinct tissue distribution patterns and were widely expressed in various tissues in the pig. PGFS was abundant in liver, kidney, lung, and CL, whereas expression of mPGES-1 was high in CL and intermediate in lung, kidney, embryo, and myometrium. The fact that we found no detectable mRNA and protein levels of mPGES-1 in liver, brain, or heart is consistent with very low mRNA expression in the same tissues in the rat (40).
The immunohistochemistry data demonstrated the localization of PGFS and mPGES-1 mostly in the epithelial cells of endometrium, indicating that this type of cell is the main source of PG synthesis. This finding is in agreement with in vitro studies showing high PGF2 release from porcine epithelial cells (41, 42, 43). The present results are consistent with immunohistochemical localization of terminal PG synthases in uterus in other species (3, 25, 34, 44).
This study provides the first demonstration that endometrial PGFS is up-regulated around the time of luteolysis in the pig. High expression of PGFS in the endometrium corresponds to high levels of luteolytic uterine secretion of PGF2 (6, 45) and significant up-regulation of PGF2 receptors in the CL (46, 47). Interestingly, the temporal increase of PGFS protein expression also correlates with the period of luteolytic capacity of porcine CL, which occurs after d 13 (9, 48). During this period, PGF2, reaching the CL, acts through luteal PGF2 receptors and causes a decrease of expression of many genes involved in the progesterone biosynthesis process (49).
Our findings are in contrast with similar studies in the horse, which revealed no increase of PGFS expression at the time around luteolysis (34). However, similar to our research, an increase of PGFS protein levels in diestrus has been reported in mice (50). Accordingly, in bovine species, there is up-regulation of the enzyme AKR1B5 possessing potent PGFS activity in diestrus but also on later days of the estrous cycle (22).
In pigs, just before implantation, the conceptuses undergo rapid elongation (51) and signal their presence to the maternal system by estrogen synthesis and secretion, which is a prerequisite for maintaining the CL function (7, 52, 53). Until now, there has been no clear explanation for the mechanism protecting CL from the luteolytic action of PGF2 in pigs. Interestingly, during the time of maternal recognition of pregnancy (about d 10–13) and at a period corresponding to the time around luteolysis (d 14–15) in cyclic gilts, endometrial PGFS mRNA expression was not affected by pregnancy; however, PGFS protein levels tended to decrease on d 10–13 of pregnancy when compared with the corresponding days of the estrous cycle. Pregnancy does not appear to have an effect on the overall amount of PGF2 released by the uterus at a period corresponding to the time around luteolysis in cyclic gilts; rather, it may affect the pulsatility of its secretion (8, 9), as has been suggested in ruminants (1, 54). On the other hand, the lack of effect of pregnancy on porcine PGFS expression in the endometrium on d 14–15 post estrus is consistent with results of a study of PGFS expression carried out in the mare (34). However, unlike the pig, uterine PGF2 production in the mare is significantly reduced in pregnant compared with nonpregnant mares (55).
Continued synthesis of PGs is required for implantation and maintenance of pregnancy (56, 57). As anticipated, in pregnant gilts, induction of PGFS mRNA and protein expression occurred on d 16–21 when compared with the corresponding stage of the estrous cycle. Furthermore, PGFS protein expression increased markedly after d 22 of pregnancy. These findings indicate a significant contribution of endometrial PGFS to the increase of PGF2 in uterine lumen during the progression of implantation (4).
Our results revealed that mRNA levels of mPGES-1, the second studied enzyme, did not vary significantly throughout the estrous cycle. Only a slight increase of mPGES-1 protein expression during the late luteal phase was observed. These findings can be supported by previous reports showing that the secretion of PGE2 also increases from d 13–16 of the estrous cycle; however, it remains 3-fold lower than those of PGF2 (6). In bovine and equine endometrium, mPGES-1 expression was not modulated significantly during the estrous cycle (25, 34), showing a similar pattern of mPGES-1 expression as that described in this report. However, in humans, abundant levels of mPGES-1 protein were shown to be expressed in endometrium during the proliferative phase, whereas a very low expression was observed during the late secretory phase of the menstrual cycle (44).
The uterine PGE2/PGF2 ratio plays an important role on d 11–15 post estrus, the critical period either for luteolysis initiating a new estrous cycle or for the establishment of pregnancy in pigs. Our findings show low mRNA and protein ratios of mPGES-1 to PGFS on d 13–15 of the estrous cycle (Fig. 5, A and C) that correlate with decreased PGE2/PGF2 ratio observed in uterine vein just before luteolysis (6). During maternal recognition of pregnancy, the protein ratio of mPGES-1 to PGFS was significantly higher (4- to 7.5-fold) when compared with d 14–21 and 24–25 of pregnancy (Fig. 5D). In pregnancy, mPGES-1 mRNA expression was also relatively high on d 10–11, and the protein levels were intermediate on d 10–13. These findings correspond to the peak of PGE2 in endometrium, which occurs earlier in pregnancy than in the estrous cycle (6). Interestingly, two periods of intermediate/high endometrial mPGES-1 expression correlate with reported previously biphasic estrogen secretion synthesis by the conceptus (53, 58). Indeed, estrogen secreted by the conceptus may stimulate mPGES-1 expression in the endometrium as it increases endometrial PGE2 production (59); for this reason, this steroid may be a key factor in increasing the PGE2/PGF2 ratio (43, 60, 61). Estradiol may also be responsible for increased mPGES-1:PGFS mRNA ratio we found around the follicular and periovulatory stage of the estrous cycle. The luteoprotective action of elevated levels of uterine PGE2 reaching the CL is additionally supported by elevated levels of luteal binding sites for PGE2 on d 14 of pregnancy, in contrast to d 14 of the estrous cycle (62), coupled with reduced luteal PGF2 receptors vs. cyclic pigs at the corresponding stage (46). However, we expected a much higher increase of mPGES-1 expression around the time of maternal recognition of pregnancy and significantly higher expression levels of mPGES-1 on d 10–13 when compared with the corresponding stage of the estrous cycle. Therefore, the question remains as to whether the minimal changes seen in mPGES-1 and PGFS expression in the endometrium on d 10–13 provide a sufficient stimulus to modulate the PGE2/PGF2 ratio in the uterus and systemic circulation during the maternal recognition of pregnancy.
An increase of the uterine PGE2 levels and increase of the PGE2/PGF2 ratio on d 11–13 of pregnancy could also be a result of the direct contribution of the conceptus to PGE2 secretion (63), correlating with the increased expression of PG biosynthetic enzymes in the conceptus around the time of elongation (64, 65). Another potential mechanism that may be responsible for the increase of the PGE2/PGF2 ratio during the establishment of pregnancy could be the inhibition of the activity of endometrial PG-9-KR (66). Although PG-9-KR has been identified in endometrium in other species (66, 67), the presence of this enzyme has not been reported in the porcine uterus yet.
It has been observed that products secreted by the porcine conceptus stimulate uterine production of PGF2 and PGE2 in vivo (68) and in vitro (69). In agreement, we found induction of mPGES-1 protein on d 18–19 in pregnant gilts when compared with the respective days of the estrous cycle. Furthermore, similar to PGFS, significant up-regulation of mPGES-1 protein expression was detected after d 22, with the maximum on d 24–25 of pregnancy. Up-regulation of both PG synthases affected by pregnancy could be a possible reason for the increase of PG secretion in uterus observed in early pregnancy (4). High levels of both terminal PG synthases after initiation of implantation may indicate a potential role of these enzymes in placentation and the establishment of pregnancy. The high endometrial expression of mPGES-1 that we found is in agreement with other reports on the significant role of mPGES-1 in implantation in other species (70, 71). Although PGE2 receptors (EP) have not been cloned in the pig, the PGE2-binding sites were reported in porcine endometrium (72). On the basis of knowledge about other species, we can speculate that PGE2 produced in uterus could act in an autocrine/paracrine role via EP2 and/or EP4, resulting in a local increase of endometrial vascular permeability and preparing for angiogenesis and placentation (73, 74). Endometrial up-regulation of mPGES-1 after initiation of implantation may be involved also in fetal allograft survival by suppressing maternal immune responses in an immunoregulatory role of PGE2 (75, 76).
To our knowledge, this is the first report of the cloning and the characterization of the two key PG synthases, PGFS and mPGES-1, in the pig. We have also shown simultaneous functional changes of expression of both PGFS and mPGES-1 in the porcine endometrium during the estrous cycle and early pregnancy. The spatiotemporal expression of PGFS throughout the estrous cycle indicates a significant role of PGFS in the regulation of luteolysis in this species. The comparison of endometrial PGFS and mPGES-1 expression on d 10–13 of the estrous cycle and pregnancy suggests a supportive rather than a major role of these enzymes in the increase of the uterine PGE2/PGF2 ratio during the period of maternal recognition of pregnancy. However, high endometrial expression of both terminal PG synthases after initiation of implantation may indicate their involvement in placentation and could be a result of local changes that occur in the uterus during the establishment of pregnancy.
Acknowledgments
We are grateful to Jan Klos, Katarzyna Gromadzka-Hliwa, and Michal Blitek for technical assistance. We also thank Dr. Wioletta Blaszczak for advice during the making of microscopy pictures.
Footnotes
This work was supported by Grant 2 P06D 041 26 from the State Committee for Scientific Research in Poland and by the Academy of Finland. M.M.K. was awarded the Domestic Grant for Young Scientists from the Foundation for Polish Sciences.
The PGFS and mPGES-1 sequences reported in this paper have been submitted to the GenBank database under accession numbers AY863054 and AY857634, respectively.
First Published Online October 13, 2005
Abbreviations: CL, Corpus luteum; COX, cyclooxygenase; EP, PGE2 receptors; MAPEG, membrane-associated proteins involved in eicosanoid and glutathione metabolism; mPGES-1, microsomal prostaglandin E synthase-1; PG, prostaglandin; PG-9-KR, PG-9-ketoreductase; PGES, PGE synthase; PGFS, PGF synthase; TBS, Tris-buffered saline.
Accepted for publication October 3, 2005.
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