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Originally published In Press as doi:10.1074/jbc.M400573200 on April 12, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30579-30587, July 16, 2004
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Prostaglandin E2 Is a Product of Induced Prostaglandin-endoperoxide Synthase 2 and Microsomal-type Prostaglandin E Synthase at the Implantation Site of the Hamster*

Xiaohong Wang{ddagger}, Yan Su{ddagger}, Kaushik Deb{ddagger}, Monika Raposo§, Jason D. Morrow¶||, Jeff Reese{ddagger}, and Bibhash C. Paria{ddagger}**

From the {ddagger}Division of Reproductive and Developmental Biology, the Department of Pediatrics, the Department of Medicine and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2678 and §University of Kansas Medical Center, Kansas City, Kansas 66160-7338

Received for publication, January 20, 2004 , and in revised form, March 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Certain uterine prostaglandins (PGs) are elevated at implantation sites and are needed to trigger the events of blastocyst implantation that include blastocyst-uterine attachment and stromal decidualization with vascular permeability changes. Several decades of investigations showed that treatment with PG synthesis inhibitors, prior to or during the time of implantation, resulted in either complete inhibition or a delay in implantation or reduction in the number of implantation sites with diminished decidual tissue. Consistent with these findings, we observed that whereas a selective PG endoperoxide synthase (Ptgs) 1 inhibitor SC-560 failed to inhibit implantation, a selective Ptgs2 inhibitor SC-236 showed significantly reduced number and size of implantation sites in progesterone-treated ovariectomized pregnant hamsters. It is known that Ptgs2 expression and Ptgs2-derived prostacyclin (PGI2) synthesis at implantation sites are needed for implantation in the mouse (a rodent that needs ovarian estrogen for implantation). However, it is unknown which Ptgs and PG synthases produce which PGs at implantation sites of the hamster (a rodent that does not need ovarian estrogen for implantation). Here we demonstrate that as blastocyst implantation proceeds, a reduction in Ptgs1 expression from uterine luminal epithelial cells and a gradual induction in Ptgs2 expression exclusively in luminal epithelial and adjacent decidual cells occurred at implantation sites of hamsters. Results also reveal that PGE2, but not PGI2, is the major PG at implantation sites where Ptgs2 and microsomal type PGE synthases but not PGI synthases are co-expressed. This elevated uterine PGE2 at implantation sites may serve to initiate or amplify physiological signals required for specific aspects of the implantation process in hamsters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Blastocyst implantation is a required event of pregnancy, and its failure is a leading cause of infertility in females. Since their discovery, prostaglandins (PGs)1 have been implicated as critical regulators of important reproductive events including implantation-associated changes during early pregnancy. This was demonstrated by treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) prior to or during the time of implantation that resulted in either complete or a partial delay in implantation, depending on the species. NSAIDs inhibit implantation in mice (1, 2) and rats (3, 4) but not rabbits (5). However in rabbits, indomethacin did reduce the following: 1) the intensity of the uterine blue dye reaction at the implantation site, 2) the size of the decidual swellings, and 3) the number of implantation sites. When indomethacin is given to progesterone (P4)-treated ovariectomized pregnant hamsters, it caused a delay in implantation (6). These adverse effects of NSAIDs are attributed to their actions on either the uterus, the embryo, or both (5, 6–8).

NSAIDs target PG synthesis by inhibiting the cyclooxygenase (COX) activity of the PG G/H synthase (9). PG G/H synthase, now known as PG-endoperoxide synthase (Ptgs) or COX because of its COX activity, exits in two isoforms Ptgs1 (COX1) and Ptgs2 (COX2). These two enzymes convert arachidonic acid into PGH2, a common precursor of all PGs (9). Ptgs1 is considered a constitutive enzyme. Ptgs2 is an inducible enzyme and is mainly elevated at the sites of inflammation after a plethora of insults. More recently, however, it has been demonstrated that both Ptgs isoforms are inducible in certain tissues. Consistent with this idea, it has been demonstrated in the mouse uterus that although Ptgs1 is inducible by treatment with estrogen and P4 together, Ptgs2 is only inducible by implanting blastocysts at the site of implantation in mice (10). Creation of Ptgs-mutated mice showed distinct functions of Ptgs1 and Ptgs2 in reproductive processes. Mice devoid of Ptgs1 exhibit parturition defects (11). Ptgs2-depleted mice showed implantation and decidualization defects during early pregnancy (12, 13), suggesting the involvement of Ptgs2-directed PG synthesis in the process of implantation. Expression of Ptgs1 is observed in the uterine epithelium prior to implantation in mice. It is then down-regulated at the implantation site. However, expression of Ptgs2 mRNA and protein is observed exclusively in the uterine epithelial and sub-epithelial stromal cells at the implantation site (10, 13). This implantation-related expression of Ptgs2 is consistent with the defects in implantation and decidualization observed in Ptgs2 mutant mice (13).

Studies before the creation of Ptgs-mutated mice suggested various functions of uterine PGs during early pregnancy. Although PGF2{alpha} is responsible for luteolysis, PGE2 exerts actions opposite to PGF2{alpha} to favor establishment of pregnancy with luteoprotective (either luteotrophic or anti-luteolytic) actions (14). PGE2 may also have immunomodulatory roles at the implantation site (15). Recently, it has been demonstrated in the mouse that uterine PGE2 and PGI2 are important for ovulation and initiation of implantation, respectively (16, 17). In contrast, Kennedy (18) showed that in the rat PGE2 but not PGI2 is a key mediator of increased vascular permeability at the implantation site. Considering the fact that the blastocyst either accumulates (19) or synthesizes PGs (20, 21), it has been also shown that PGs affect the glucose metabolism of preimplantation embryos (22) and hatching of the blastocysts (7).

The earliest contact between the implantation-competent blastocyst and the receptive uterus in any mammal designates the onset of blastocyst implantation. In mice and rats, maternal estrogen is essential to initiate the process of blastocyst implantation in a P4-primed uterus (23–25). In contrast, maternal estrogen is not required to initiate the process of implantation in the P4-primed uterus in hamsters, rabbits, pigs, guinea pigs, monkeys, and perhaps in humans (26–36). Although the importance of PGs during early pregnancy has been recognized in several species that showed P4-dependent implantation, the process of PG synthesis in the uterus of these species has not been studied in detail. As part of our continuing effort to understand the contribution of Ptgs-derived PGs to the process of implantation in various species, we have studied the expression, regulation, and function of Ptgs and Ptgs products during early pregnancy in hamsters that showed P4-dependent implantation. Our results reveal that Ptgs2-derived PGE2 is involved in the successful establishment of implantation in hamsters.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Adult virgin male and female golden hamsters (Mesocricetus auratus)8–12 weeks-old were purchased from either Sasco, Omaha, NE, or Charles River Laboratories, Wilmington, MA. They were maintained in a 14-h light, 10-h dark cycle in the Animal Facility Laboratory of the Kansas Medical Center (Kansas City, KS) and Vanderbilt University (Nashville, TN) with an unlimited access to water and food according to the institutional guidelines on the care and use of laboratory animals.

Preparation of Pregnant Hamster and Uterine Tissue Collection— Only hamsters with three consecutive 4-day estrous cycles were used in this study. One female was housed with two fertile males overnight on the evening of proestrus. Finding of sperm in the vaginal smear the next morning (estrus) indicated the 1st day (day 1) of pregnancy. Hamsters on days 1–3 of pregnancy were killed at 0900 h, and whole uteri were collected after confirmation of pregnancy by flushing and recovering embryos from oviducts and/or uteri (37). Whereas whole uteri were collected on the morning of day 4 (0900 h), implantation and interimplantation sites were collected on the afternoon (1600 h) of day 4 and the morning (0900 h) of days 5 and 6 after an intravenous injection of Chicago Blue B dye solution (Sigma; 0.25 ml of 1% dye in saline). Implantation sites on these days were visualized by intermittent blue bands along the horns. On days 7–8, implantation sites were distinct and were identified visually without blue dye injection (37). Uterine tissues were immediately frozen in cold Super Friendly Freeze'it (Curtin Matheson Scientific, Houston, TX) and stored at -70 °C until extraction of RNA, in situ hybridization, or immunohistochemistry/immunofluorescence.

Implantation occurs without delay in hamsters ovariectomized or hypophysectomized on day 2 of pregnancy and given P4 daily (29, 33, 35). This suggests that implantation in hamsters is P4-dependent. Thus, it is possible that implantation occurred with correct expression of implantation-specific genes because of the regulation of these genes by either P4 or blastocysts. To address this issue, a group of pregnant hamsters was ovariectomized on day 2 (0900 h) and given a subcutaneous injection of P4 (Sigma; 1 mg in 0.1 ml of sesame seed oil/hamster) on either days 2 and 3 or days 2–4. Control hamsters underwent sham operation and were injected with 0.1 ml of vehicle, sesame seed oil (Sigma). Whole uteri were collected on the morning of day 4 (0900 h). Implantation sites, detected by blue dye injection, were collected on the morning of day 5 (0900 h) for in situ hybridization.

Analysis of Blastocyst Implantation after Treatment of Ptgs Inhibitors in Ovariectomized P4-treated Pregnant Hamsters—The potential of isoform-selective Ptgs inhibitors to inhibit or delay the initiation of implantation was determined in ovariectomized P4-treated pregnant hamsters. Pregnant hamsters were ovariectomized on day 2 (0900 h) and maintained on daily P4 (1 mg/hamster) injections. One group of hamsters was used as control and received vehicle (0.2 ml of 0.5% (w/v) methylcellulose (Sigma) and 0.1% (v/v) polysorbate 80 (Sigma)) through oral gavage. A second group of animals received SC-560 (20 mg/kg oral gavage) twice per day (0900 and 1700 h) from days 2 to 4. SC-560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; Cayman Chemical, Ann Arbor, MI) is a selective inhibitor of Ptgs1. The remaining hamsters received SC-236 (20 mg/kg oral gavage) once per day (0900 h) on days 2–4. SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide; Calbiochem) is a selective inhibitor of Ptgs2 and has a longer half-life in vivo, which allowed for dosing once/day (38). Hamsters were killed on the morning of day 5 (0900 h) 15 min after blue dye injection. The uteri were examined for evidence of local accumulation of the blue dye at the blastocyst implantation sites. The number of blue implantation sites and the intensity of their blue color were visually recorded. Wet weight of the individual implantation site was also recorded. Significant differences in number of implantation sites and their wet weights between vehicle and Ptgs inhibitors were determined by Student's t test.

Measurement of PG Levels at Implantation Site—To determine and compare the PG levels between implantation and interimplantation sites, normal pregnant hamsters were killed on day 5 (0900 h) after blue dye injection. Blue bands (implantation sites) and areas between two blue bands (interimplantation sites) were carefully separated, weighed, and snap-frozen for PG assay. The PG content (PGE2, PGF2{alpha}, PGD2, thromboxane B2 (a stable metabolite of thromboxane A2), and 6-keto-PGF1{alpha}, a stable metabolite of PGI2) of implant and interimplantation sites was then quantified by utilizing gas chromatography/negative ion chemical ionization mass spectrometric assays as described previously (39). Differences in PG levels between implantation and interimplantation sites were compared by Student's t test.

Hormonal Effects on Uterine Ptgs2 mRNA Expression—To determine the effect of steroid hormones on uterine Ptgs expression, hamsters were ovariectomized without regard to their stage of estrous cycle and rested for 12 days. These hamsters were treated with a subcutaneous injection of either P4 (500 µg/hamster) or estradiol-17{beta} (E2, Sigma; 1.0 µg/hamster) or E2 plus P4 or the vehicle sesame seed oil. All steroids were dissolved in sesame oil. Hamsters were killed at 2, 6, 12, and 24 h after injection of hormones, and uteri were collected for in situ hybridization. The dosages of P4 and E2 were chosen depending on the previously reported sensitivity of the hamster uterus to these hormones (33, 37). The hamster uterus is very sensitive to P4, and treatment of 500 µg of P4 daily is reported to be sufficient to maintain pregnancy in ovariectomized pregnant hamsters (33). In contrast, sensitivity of the hamster uterus to E2 is poor (40), and single injection of 1 µg of E2 is required to induce genes in the ovariectomized hamster uterus (37).

Cloning of the Hamster Ptgs1, Ptgs2, mPtges, and Ptgis Partial cDNAs—Reverse transcription-PCR was employed to generate the hamster-specific Ptgs1, Ptgs2, mPtges, and Ptgis partial cDNA clones. The mouse complete coding sequences for all four genes were compared for homology with the human and the rat coding sequences using the ClustalW Multiple Sequence Alignment program. The mouse coding region that showed maximum sequence homology between the three species was used to design sense and antisense PCR primers for Ptgs1, Ptgs2, mPtges, and Ptgis. The primers were Ptgs1 (GenBankTM accession number NM008969; spanning nucleotides 1427–1797; size, 371; 5'-CCT TCA ATG AAT ACC GAA AGA GG-3' (sense) and 5'-GTA ATC TGG CAC ACG GAA GG-3' (antisense)), Ptgs2 (GenBankTM accession number NM011198; spanning nucleotides 435–821; size, 387; 5'-CAA CCT CTC CTA CTA CAC C-3' (sense) and 5'-CTA CCT GAG TGT CTT TGA CTG-3' (antisense)), mPtges (GenBankTM accession number AB041997 [GenBank] ; spanning nucleotides 105–514; size, 410; 5'-AGC ACA CTG CTG GTC ATC AAG-3' (sense) and 5'-GCT GCT GGT CAC AGA TGG TG-3' (antisense)), and Ptgis (GenBankTM accession number NM008968; spanning nucleotides 309–701; size, 393; 5'-CCT CAT GGA GAG GAT TTT TGA T-3' (sense) and 5'-ACA CTG CAT GCA TGG TCT TTA T-3' (antisense)).

Total RNAs (1 µg) from day 4 pregnant hamster uterus and kidney were reversed-transcribed with oligo(dT) at 42 °C for 50 min using the SuperScript First Strand Synthesis System (Invitrogen). PCR was carried out with an initial denaturation of 95 °C for 10 min followed by 40 cycles consisting of denaturation at 94 °C for 1 min, annealing at 64 °C for 30 s, and elongation at 72 °C for 30 s. This was followed by a final extension at 72 °C for 8 min. Reverse transcription-PCR generated uterine Ptgs1, Ptgs2, and Ptgis, and kidney mPtges products were subcloned into a pCR®-II-TOPO® cloning vector (3.9 kb) using TOPO TA Cloning kit, version K2 (Invitrogen), and nucleotide sequences of the clone were determined on both strands to verify the identity of the clone. Nucleotide sequences of these partial cDNA clones showed more than 85% sequence similarity with that of the GenBankTM nucleotide data base for rats, mice, and humans. The GenBankTM accession numbers for hamster Ptgs1 and Ptgs2 are AF414605 [GenBank] and AF345331 [GenBank] , respectively.

RNA Probe Preparation—For in situ hybridization, plasmids bearing hamster cDNA were extracted, purified, and linearized to generate antisense and sense riboprobes, which were transcribed using appropriate RNA polymerases (Ptgs1, SP6/NotI for antisense and T7/HindIII for sense; Ptgs2, SP6/XhoI for antisense and T7/HindIII for sense; mPtges, T7/BamHI for antisense and SP6/NotI for sense; Ptgis, SP6/NotI for antisense and T7/HindIII for sense) and labeled with 35S (41). All labeled sense and antisense cRNA probes used for hybridizations had specific activities of ~2 x 109 dpm/µl.

In Situ Hybridization—The protocol was followed as described by Das et al. (41). Briefly, uterine cryosections were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 15 min on ice. After prehybridization, sections were hybridized to 35S-labeled antisense probes at 45 °C for 4 h in 50% formamide hybridization buffer. Parallel sections were hybridized with 35S-labeled sense probes as negative control. After hybridization and washing, sections were incubated with RNase A (20 µg/ml) at 37 °C for 20 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co.). The slides were poststained with hematoxylin and eosin.

Immunohistochemistry—Rabbit polyclonal antibody to Ptgs2 was a generous gift from Dr. S. K. Dey, Vanderbilt University, Nashville, TN. Rabbit polyclonal antibody to mPtges was purchased from Cayman Chemicals, Ann Arbor, MI. Immunocytochemical localization of these proteins was performed as described previously (42). In brief, frozen sections (10 µm) were mounted onto poly-L-lysine-coated glass slides and fixed in 2% paraformaldehyde for Ptgs2 for 15 min and in Bouin's solution for mPtges for 30 min. Sections were then washed with phosphate-buffered saline (PBS), treated with 10% nonimmune goat serum (Zymed Laboratories Inc., San Francisco, CA) for 10 min at room temperature, and incubated with respective primary antibodies (antibody dilution, Ptgs2 (0.022 µg/µl) and mPtges (0.02 µg/µl)) overnight at 4 °C. After removal of the primary antibody, the sections were washed with PBS and incubated with biotin-goat anti-rabbit IgG (Zymed Laboratories Inc.) for 10 min at room temperature. PBS-washed sections were treated with periodic acid (Sigma; 0.23% in PBS) for 45 s to block the endogenous peroxidase activity. PBS-washed sections were next incubated with streptavidin-peroxidase conjugate (Zymed Laboratories Inc.) for 10 min at room temperature. Sections were then washed with PBS and incubated with AEC single solution (Zymed Laboratories Inc.) to obtain final color product. Sections were lightly counterstained with hematoxylin (Zymed Laboratories Inc.). Red deposits indicated the sites of immunoreactive proteins. The specificity of the staining was confirmed with primary antibody either preabsorbed with the antigen peptide (1 µM) or replaced with equal amounts of nonimmune rabbit IgG (Sigma).

Double Immunostaining—In case of double immunostaining of Ptgs2 and mPtges, a slide having sections from several day 5 implantation sites was fixed in a mixture (1:1) of 4% paraformaldehyde and Bouin's solution. After fixation, the slide was first incubated with rabbit polyclonal antibodies of mPtges (1:500 dilutions) overnight at 4 °C and visualized with a fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody (Zymed Laboratories Inc.). The same slide was then incubated with a rabbit polyclonal Ptgs2 antibody (1:500 dilutions) at room temperature for 2 h and visualized with a rhodamine-labeled secondary antibody (Zymed Laboratories Inc.). Primary antibodies were replaced with nonimmune rabbit IgG in control sections. Fluorescence images were captured in a fluorescence microscope. The staining was also performed in reverse order, and in both cases inappropriate cross-reactions were negligible.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isoform-specific Ptgs Inhibition Alters Embryo Implantation in Ovariectomized P4-treated Pregnant Hamsters—Studies in various species demonstrated that pharmacological inhibition of Ptgs by NSAIDs leads to either complete or partial delay in implantation (1–5, 43). These observations were supported by an infertility phenotype of Ptgs2 null mice (13). The most commonly studied NSAID is indomethacin, which inhibits activity of both Ptgs1 and Ptgs2 (44). Recently, compounds that selectively inhibit the activity of each Ptgs isoform have been produced. Thus, we used a pharmacological approach to perturb Ptgs1 or Ptgs2 functions during early pregnancy, and we examined their separate effects on implantation process in hamsters. Compared with the vehicle treatment, hamsters that received the Ptgs1 inhibitor SC-560 (15 or 30 mg/kg) showed no implantation failure on day 5 (Table I). We observed no or little differences in the number of implantation sites as well as their wet weight between the vehicle-treated control group and the SC-560-treated group. Treatment with the Ptgs2 inhibitor SC-236 (15 mg/kg) significantly (p < 0.05) reduced the number of normal implantation sites when compared with controls. One of seven SC-260-treated animals showed no implantation. Three degenerated blastocysts were recovered upon flushing the uteri from this animal. The remaining SC-236-treated hamsters showed less blue dye accumulation (visual observation) and reduced wet weight (p < 0.05) of implantation sites on day 5. The dosages of these drugs were reported not to be toxic to rodents (45). We also observed no maternal deaths, although the outcome of pregnancy on later days was unknown. The results of this experiment suggest that whereas inhibition of Ptgs1 activity has no effect on normal implantation process, inhibition of Ptgs2 activity might lead to impairment of the embryo implantation process. These observations lead to an investigation of Ptgs1 and Ptgs2 expression patterns during early pregnancy in hamsters.


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  		TABLE I
Effects of Ptgs inhibitors on implantation in ovariectomized progesterone (P4)-treated hamsters Pregnant hamsters were ovariectomized on day 2 (0800 h) and maintained with daily injections of P4 (1 mg/hamster). Drugs were given from days 2 to 4 of pregnancy. Implantation sites were determined by blue dye injection on day 5 at 0900 h. Wet weights of the implantation sites were recorded. Numbers in parentheses indicate number of observations.

 
Ptgs1 and Ptgs2 mRNAs Are Differentially Expressed in Uterine Cells during the Peri-implantation Period—Ptgs1 and -2 are rate-limiting enzymes for PG synthesis from arachidonic acid (9). Uterine expression patterns and functions of Ptgs1 and -2 in the mouse (10) suggest that PG synthesis requires both ovarian P4 and estrogen for the initiation of the implantation process. However, there are no studies in species such as the hamster, which requires only ovarian P4 for implantation. The uterus is composed of heterogeneous cell types that undergo rapid and dynamic changes during early pregnancy under the control of ovarian steroids (46). Thus to define the expression of Ptgs1 and Ptgs2 in uterine cells during normal pregnancy, we first investigated Ptgs1 and -2 mRNA expressions by in situ hybridization analysis using hamster-specific Ptgs1 and Ptgs2 cDNA probes, respectively. Figs. 1 and 2 showed autoradiographic images of the distribution of Ptgs1 and Ptgs2, respectively. On day 1 of pregnancy, Ptgs1 and Ptgs2 mRNA expressions were observed only in luminal epithelial cells but not in glandular epithelial, stromal, myometrial, or vascular endothelial cells. The autoradiographic signals of both Ptgs1 and Ptgs2 in the luminal epithelium were reduced on day 2 of pregnancy. On day 3 of pregnancy, both signals increased in the entire luminal epithelium. Although autoradiographic signals for Ptgs1 persist in the entire luminal epithelium, Ptgs2 signals were localized mainly in uterine luminal epithelial cells surrounding the blastocyst as the uterus prepares to initiate the process of implantation on the morning of day 4 (0800–0900 h). During the time of implantation on the afternoon of day 4, Ptgs1 mRNA expression was reduced in the luminal epithelial cells surrounding the implanting blastocyst as compared with its expression away from the blastocyst. In contrast, Ptgs2 mRNA signal was observed in high intensity in luminal epithelial cells as well as in several layers of stromal cells only surrounding, but not away from, the implanted blastocyst. These patterns of Ptgs1 and Ptgs2 mRNA signals were also maintained in implantation sites on days 5 and 6. Whereas Ptgs1 expression (Fig. 1) was reduced in luminal epithelial cells surrounding the implanted blastocyst, increased expression of Ptgs2 mRNAs (Fig. 2) was noticed both in the luminal and stromal cells in the immediate vicinity of implanting blastocyst on day 5 of pregnancy. On day 6 of pregnancy, Ptgs1 was expressed in low levels only in the remaining mesometrial epithelial cells. Ptgs1 was not expressed in the stromal or decidual cells. However, cells of the entire primary decidual zone (PDZ), but not the secondary decidual zone, expressed Ptgs2 mRNA on day 6. No Ptgs1 and Ptgs2 mRNA expressions were observed in embryonic cells. On days 7 and 8 of pregnancy, Ptgs1 expression was not observed in any cell types at the implantation site (Fig. 1). However, whereas Ptgs2 signal was reduced in the PDZ on day 7, it started appearing in the trophoblast cells (Fig. 2). Trophoblast cells of the day 8 embryo showed prominent expression of Ptgs2. No Ptgs2 mRNA expression was observed in other cells beside trophoblast cells. Sections of day 5 implantation sites showed no signals when hybridized with sense cRNA probes (data not shown).



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  		FIG. 1.
Peri-implantation (days 1–8) hamster uterus showed cell-specific autoradiographic localization of Ptgs1 mRNA as detected by in situ hybridization. Photographs were captured under dark field at x20. Uterine tissues were collected on the morning (0900 h) of each day from days 1 to 8 (D1–8). However, on day 4 implantation sites were also collected at 1600 h (D4 pm). am, antimesometrial side; bl, blastocyst; em, embryo; le, luminal epithelium; m, mesometrial side; myo, myometrium; s, stroma.

 



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  		FIG. 2.
Peri-implantation (days 1–8) hamster uterus showed cell-specific localization of Ptgs2 mRNA as detected by in situ hybridization. Photographs were captured under dark field at x20. Uterine tissues were collected on the morning (0900 h) of each day from days 1–8 (D1–8). However, on day 4 implantation sites were also collected at 1600 h (D4 pm). am, antimesometrial side; bl, blastocyst; em, embryo; le, luminal epithelium; m, mesometrial side; myo, myometrium; s, stroma.

 
Because of the restricted Ptgs2 expression pattern at the implantation site and because Ptgs2 is an early inducible gene in response to cytokines, growth factors, and blastocysts (47), we next compared the expression of Ptgs2 in the uterus of ovariectomized P4-treated pregnant and normal pregnant hamsters only on days 4 and 5.

Uterine Ptgs2 mRNA Expression Is Similar in Ovariectomized P4-treated Pregnant and Normal Pregnant Hamsters on Days 4 and 5—Because hamsters do not need ovarian estrogen for implantation, delayed implantation does not occur in ovariectomized P4-treated pregnant hamsters (33). The observed expression patterns of Ptgs2 (Fig. 3) in the ovariectomized P4-treated pregnant uterus were similar to its expression during normal pregnancy on days 4 and 5. As the time of implantation approached on day 4, Ptgs2 mRNAs were tending to accumulate in the presumptive implantation site. The Ptgs2 mRNA expression pattern in day 5 implantation sites of ovariectomized P4-treated pregnant hamsters showed very similar expression patterns as observed during normal day 5 of pregnancy. These results indicate that in the absence of ovarian estrogen, the influence of P4 is enough to bring about implantation-related uterine changes in Ptgs2 expression in the presence of a blastocyst. To clarify further whether ovarian steroids influence Ptgs2 in uterine cells, we examined Ptgs2 expression in ovariectomized hamsters treated with P4 and/or E2 or the vehicle (oil).



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  		FIG. 3.
Cell-specific Ptgs2 mRNA expression as detected by in situ hybridization in ovariectomized progesterone-treated (OVX+P4) and sham-operated pregnant (days 4–5) hamsters. Photographs were captured under dark field at x20. Uterine tissues were collected on the morning (0900 h) of days 4 and 5 (D4 and D5). am, antimesometrial side; bl, blastocyst; le, luminal epithelium; m, mesometrial side; myo, myometrium; s, stroma.

 
Ptgs2 Is Regulated by Steroid Hormones in the Ovariectomized Hamster Uterus—Control animals treated with oil show no expression of Ptgs2 mRNAs. Ovariectomized hamsters treated with P4 and E2 alone showed little increase in the levels of Ptgs2 in uterine luminal epithelial cells as compared with the oil-injected control (Fig. 4). However, a substantial amount of Ptgs2 mRNA accumulation was observed in the epithelial cells of ovariectomized hamsters treated with both P4 and E2 by 6 h followed by a gradual decrease by 24 h. The expression of Hgfl mRNA expression was studied as a positive control (data not shown) in these ovariectomized uterine tissues after various hormone treatments and matched previously reported patterns (37). The day 1 pregnant uterus is used as a positive control for Ptgs2.



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  		FIG. 4.
In situ hybridization of Ptgs2 mRNAs in steroid-treated adult ovariectomized hamsters. Hamsters were given a single injection of sesame seed oil (vehicle for steroids), P4 (500 µg/hamster), or E2 (1 µg/hamster) or a co-injection of P4 with E2 and killed 2, 6, 12, and 24 h later. Dark field photographs were shown at x20 magnification. A section from a day 1 pregnant hamster uterus was used as a positive control. Because oil-treated uteri did not show positive hybridization at any time point, a representative oil-treated uterine section at 6 h was presented. le, luminal epithelium; myo, myometrium; s, stroma.

 
Expression of Ptgs2 Protein in the Peri-implantation Uterus Is Cell- and Implantation-specific—The above-described expression patterns of Ptgs2 mRNAs in the peri-implantation uterus and hormonal regulation of this gene in ovariectomized hamsters suggest that Ptgs2 is most likely an inducible gene in the hamster uterus. Thus, we examined the cellular distribution of Ptgs2 gene product Ptgs2 protein in the peri-implantation uterus. Uterine Ptgs2 expression as detected by immunohistochemistry follows the similar pattern of Ptgs2 mRNA expression observed during the first 8 days of pregnancy. This protein is mainly found in uterine epithelial cells on day 1 and the morning of day 4 of pregnancy (Fig. 5). Immunoreactive Ptgs2 protein was detected in epithelial and stromal cells adjacent to implanting the blastocyst on the afternoon of day 4 and the morning of day 5. Whereas only a few stromal cells showed Ptgs2 expression on the afternoon of day 4, it is expressed in several layers of subepithelial stromal cells on day 5 of pregnancy. At the implantation site on day 5, several stromal cells also showed perinuclear localization of Ptgs2 protein. There were no such signals observed when the day 5 implantation sites were incubated with only IgG (data not shown). A set of decidual cells that forms the PDZ around the implanted embryo was positive for Ptgs2 on day 6. Expression of Ptgs2 protein overlapped with Ptgs2 mRNA expression in the implantation sites on days 7 and 8. Ptgs2 protein is mainly expressed in trophoblast cells of the embryo on these days. These data suggest that uterine Ptgs2 mRNA is faithfully transcribed to Ptgs2 protein in all days during early pregnancy in hamsters. As we have noticed the elevated expression of Ptgs2 in the implantation site, we attempted to identify which PG levels elevated at the implantation site.



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  		FIG. 5.
Peri-implantation (days 1–8) hamster uterus showed cell-specific localization of Ptgs2 protein as detected by immunohistochemistry. Photographs were captured under bright field. Magnification is indicated in each panel. Uterine tissues were collected on the morning (0900 h) of each day from days 1 to 8 (D1–8). However, on day 4 implantation sites were also collected at 1600 h (D4 pm). bl, blastocyst; em, embryo; ge, glandular epithelium; le, luminal epithelium.

 
PGE2 Is the Most Abundant PG at the Implantation Sites— The reduced expression of Ptgs1 mRNA and the increased expression of Ptgs2 mRNA and protein at the implantation site, as compared with their expression patterns in uterine tissue away from the implantation site, prompted us to measure and compare the levels of PGs at the implantation site versus interimplantation site on day 5 of pregnancy. Fig. 6 shows the levels of PGs assayed by gas chromatography/mass spectrometry. Among various PGs, only PGE2 showed significantly (p < 0.05) higher levels at implantation sites compared with the interimplantation sites. PGF2{alpha}, thromboxane B2 (a stable metabolite of thromboxane A2) and 6-keto-PGF1{alpha} (a stable metabolite of PGI2) did not exhibit any significant change. PGD2 is undetectable in hamster uterine tissues. Thus, it is assumed that PGE2 is primarily formed by the enzymatic action of Ptgs2, as Ptgs1 expression is low at the implantation sites on day 5 of pregnancy.



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  		FIG. 6.
Comparison of level of PGs at implantation versus interimplantation sites on day 5 of pregnancy in the hamster. Numbers in parentheses indicate number of observations. The error bars indicate the S.E. Statistical analysis was performed using Student's t test (*, p < 0.05).

 
There still remains a question as to how a Ptgs2-derived product is converted to PGE2 at the implantation site. It is also unknown whether or not Ptgis is also expressed at the implantation site of hamsters as reported previously in mice (17). One way to address these concerns is by co-localization of Ptgs2 and PGE2- or PGI2-synthesizing enzymes at the same location of implantation sites. Although PGI synthase converts PGH2 to PG12, PGE synthase converts PGH2 to PGE2. To date, two Ptges genes (mPtges and cPtges) have been cloned. Whereas cPtges expression is constitutive (48), mPtges expression is inducible in various tissues by cytokines (49). However, we lack evidence whether inducible mPtges is present in the same location of the implantation site where Ptgs2 is induced. These reported data led us to investigate the expression patterns of mPtges mRNA and protein levels around the time of implantation in hamsters.

mPtges, but Not Ptgis, Is Localized in the Same Location of Implantation Where Ptgs-2 Is Expressed—We investigated the expression mPtges and Ptgis in implantation sites on days 5 and 6 of pregnancy. Close similarity was observed in the expression Ptgs2 mRNA and mPtges mRNA, but not Ptgis mRNA, at the implantation site on days 5 and 6 of pregnancy. mPtges expression occurred in luminal epithelial and stromal cells around the implanting blastocyst on day 5 (Fig. 7). With the progression of implantation to day 6, mPtges was mainly expressed in the cells of the PDZ (Fig. 7). Ptgis mRNA expression was not observed in uterine epithelial, stromal, and decidual cells at the implantation sites on days 5 and 6. However, we noticed Ptgis mRNA expression in the outer longitudinal muscle layer of the uterus on both days 5 and 6 and in the developing embryo on day 6 but not on day 5 (Fig. 7). Immunocytochemistry confirmed the presence of mPtges protein at the implantation site on days 5 and 6 of pregnancy. mPtges protein was co-localized with mPtges mRNA. In a subset of cells, mPtges was most densely expressed in the perinuclear region.



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  		FIG. 7.
Implantation sites (days 5 and 6) of hamsters showed cell-specific autoradiographic and immunohistochemical localizations of mPtges and Ptgis mRNAs and proteins, respectively. Autoradiographic (in situ hybridization) photographs were captured under dark field at x40 for day 5 and x20 for day 6. Immunohistochemical photographs were captured under bright field at x40. am, antimesometrial side; bl, blastocyst; em, embryo; le, luminal epithelium; m, mesometrial side; myo, myometrium; pdz, primary decidual zone; sdz, secondary decidual zone.

 
Because both Ptgs2 and mPtges, but not Ptgis, are expressed in the same location of the implantation site, we investigated the possible co-expression of Ptgs2 and mPtges proteins by double immunostaining on day 5 implantation sections. As shown in Fig. 8, these two enzymes were co-expressed in a subset of stromal cells at the implantation site. The distinct staining pattern and more abundant expression of Ptgs2 exclude the possibility that there was cross-reaction between these two antibodies.



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  		FIG. 8.
Co-localization of Ptgs2 (red) and mPtges (green) proteins as detected by double immunofluorescence staining in day 5 implantation sites of pregnant hamsters. Photographs were captured under bright field at x40. am, antimesometrial side; bl, blastocyst; le, luminal epithelium; m, mesometrial side.

 
These results suggest that elevation of PGE2 at the implantation site involves the Ptgs2/mPtges system in hamsters.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Prostaglandins trigger a variety of blastocyst-uterus interactions associated with implantation. These responses include the blastocyst-uterine attachment reaction, uterine vascular permeability changes, and stromal decidualization (23). Evidence for these PG responses was further supported by mutation of Ptgs2 in the mouse (12, 13), where implantation occurs in response to ovarian estrogen (24, 25). Ovarian estrogen is not a requirement for initiation of implantation in certain species including hamsters, rabbits, pigs, monkeys, and perhaps humans (26–36). However, it is unknown which PGs are produced and how they are synthesized at the implantation site under such hormonal regulation of implantation. This information would be of value in understanding some defective implantation processes. To address some of these questions, the uterine expression of enzymes responsible for PG synthesis was studied in an animal model that does not require ovarian estrogen for implantation. As discussed below, the present study highlights that blastocyst implantation in hamsters is associated with an increase in PGE2 that is synthesized at the implantation site through co-expressed Ptgs2 and mPtges enzymatic actions.

At the beginning of this study we sought to determine whether uterine PG synthesis has any role in implantation in hamsters. Ovariectomized P4-treated pregnant animals were used to avoid any effects ovarian estrogen might have in initiation of implantation in hamsters. Our results show that the Ptgs1-selective inhibitor SC-560 has no effect on implantation compared with the vehicle treatment group. This result is similar to the results obtained in mice treated with either SC-560 or aspirin that preferentially blocks Ptgs1 (13, 43), indicating that selective Ptgs1 inhibition alone has little or no influence on implantation. However, inhibition of PG synthesis with indomethacin at or around the time of implantation either partially or completely prevents implantation in rats, mice, hamsters, and rabbits (1, 4, 6, 44, 50, 51). Evidence has also accumulated that PGs may be required for decidualization (52). Indomethacin blocks both Ptgs1 and Ptgs2 (53). Therefore, reduction in Ptgs1 at implantation sites and lack of effect of Ptgs1 inhibitor SC-560 on implantation formed the basis for investigating the role of Ptgs2. Females treated with SC-236 showed reduction in the number and size of implantation sites, and diminished blue dye accumulation compared with vehicle-treated controls. Faint blue color at the implantation site could be the result of reduced uterine capillary permeability. Overall reduction in the size of implantation could be due to delay in the initiation of implantation and decidualization. A short delay in implantation process has been reported in mice with mutated cytosolic phospholipase A2{alpha} that supplies arachidonic acid for the PG synthesis (54). A delay in implantation and decidualization could result from the effect of this drug either on the blastocyst, the uterus, or both. However, our results2 suggest that Ptgs2 is not expressed in the hamster blastocyst. Thus, it is assumed that SC-236 inhibits uterine Ptgs2 activity in hamsters. Our observations that SC-236 adversely affects the implantation process in hamsters are similar to the previous reports in mice treated with Ptgs2 inhibitor celecoxib or DuP97 (13, 43). Overall, it appears that Ptgs2-derived PGs play an important role during the process of implantation in hamsters. However, the uterine site of PG production has not been established previously in this species.

One of the ways of studying PG synthesis is to analyze the uterine expression patterns of both Ptgs1 and Ptgs2. It was observed that before implantation (days 1–4) uterine luminal epithelial cells are the major source of Ptgs1 and Ptgs2. The reason for the expression of both enzymes in the same cells is unknown. It is possible that both of these enzymes play the same role in the uterine epithelium on these preimplantation days. Most interesting, however, when blastocysts undergo implantation on days 5 and 6, Ptgs1 is gradually decreased from epithelial cells around the implantation sites. It only persists in luminal epithelial cells away from the implantation site. The reverse is true for Ptgs2 expression on these days. Ptgs2 gradually disappears from the epithelial cells away from the blastocysts but concentrates exclusively in both epithelial and stromal or PDZ cells surrounding the implanting embryos. Ptgs2 expression at day 5 implantation sites is in agreement with the Ptgs2 expression in mice (10). However, unlike mice that showed only mesometrial stromal expression of Ptgs2 on the day 6 implantation site, hamsters showed Ptgs2 expression in decidual cells (PDZ cells) immediately surrounding the implanting embryo. Expression of Ptgs2 in cells of the PDZ of days 5 and 6 and its expression in trophoblast cells of the growing embryo on later days of pregnancy suggest that Ptgs2 could not only be associated with inflammatory aspects of implantation but also be involved in protection of the embryo proper by its immunomodulatory and cell-cell barrier formation properties (55, 56). In rodents, implantation is followed by loss of uterine luminal epithelial cells and their barrier functions just around the implanted embryo (42). This loss of epithelial cells together with local inflammation at the implantation site may pose a threat to the semiallogenic embryo from maternal toxic materials including immunoglobulins. In this situation, the ability of the decidual cells to act as a barrier is critically important in terms of embryonic loss. Ptgs2-derived PGs have been suggested to be important in restoring epithelial barrier functions with healing of colonic ulcers (57). Thus, the induction of Ptgs2 in the decidual cells immediately surrounding the embryo demonstrates a potential role of Ptgs2-derived PGs in induction of temporary barrier functions in these cells. In this regard we have already demonstrated that the cells of the PDZ surrounding the implanted embryo express tight junctional molecules (42, 58).

The induction of Ptgs2 at the implantation site is not surprising because similar induction of Ptgs2 occurs at the implantation site of mice (10). What is surprising is that in the absence of ovarian estrogen, implantation sites of P4-treated pregnant hamsters showed a similar pattern of Ptgs2 expression suggesting that either P4 or blastocysts are involved in the induction of Ptgs2. By using ovariectomized hamsters, it was noted that neither P4 nor E2 is a strong inducer of Ptgs2 in the uterus. However, simultaneous treatment with both hormones to ovariectomized hamsters strongly induced Ptgs2. The need for estrogen stimulation of Ptgs2 in the hamster uterus is somewhat paradoxical. One intriguing possibility is that preimplantation embryos of hamsters may have the capacity to produce estrogen that acts locally on the uterus. In this context, we have preliminary evidence that preimplantation embryos of hamsters express the Cyp19 gene that encodes aromatase protein.2

The differential expression of Ptgs1 and Ptgs2 at hamster implantation sites suggests that PGs produced at the implantation site are mainly the contributed products of Ptgs2. We reported previously (17) that PGI2 rather than PGE2 is the major prostanoid in the mouse implantation site. In contrast, Kennedy (3, 18) showed that in the rat and hamster PGE2 is a key mediator at the implantation site. The data presented here support the later observations and show increased PGE2 content in the implantation site as compared with its content in the interimplantation site of hamsters. It is unknown, however, which Ptges is involved in the synthesis of PGE2. There are two isoforms of Ptges (mPtges and cPtges) that catalyze the conversion of PGH2 to PGE2. In 1999, Jakobsson et al. (59) identified a human mPtges that was inducible by interleukin-1{beta},an inflammatory cytokine, suggesting the involvement of mPtges in inflammation and in the acute-phase response. Because inflammatory changes occur at the implantation site with reduced expression of Ptgs1 mRNA, it is likely that mPtges is involved in the Ptgs2-derived PGE2 synthesis. Indeed, we observed that Ptges2 and mPtges were co-expressed in the uterus in a temporally and spatially similar manner, suggesting that they are functionally linked. Preferential coupling of Ptgs2 and mPtges was demonstrated recently in a cell line that was transfected with mPtges together with either Ptgs1and Ptgs2 (60). In this experiment cells co-transfected with mPtges and Ptgs2 produced PGE2 from much lower concentrations of arachidonic acid compared with those transfected with mPtges and Ptgs1. This group also demonstrated that cPtges is functionally linked to Ptgs1 (48). It has been demonstrated previously (61) that preferential coupling of Ptgs2 with mPtges is needed to generate PGE2 in brain endothelial cell. mPtges expression in stromal and epithelial cells around the implanting embryo has also been reported in implantation sites of mice (52). Our observation of Ptgs2 and mPtges co-expression around the implantation site suggests that Ptgs2 may be preferentially coupled with mPtges for PGE2 production at the implantation site of hamsters (Fig. 9).



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  		FIG. 9.
A model of PGE2 synthesis at the implantation site of hamster. An embryonic signal(s) at the uterine cell surface leads to transcription and translation of Ptgs1 and mPtges genes to make PGE2. Both cytosolic and perinuclear cPLA2, Ptgs2, and mPtges are involved in the synthesis of PGE2.

 
The specific role of PGs in human implantation is not clear. Previous studies investigating expression of the components of the PGE2 biosynthetic and signaling pathways in the human endometrium have outlined temporal variation across the menstrual cycle. Expression of COX/PGE synthase enzymes and synthesis of PGE2 are highest in the perimenstrual and proliferative phases of the menstrual cycle, and the sites of expression/synthesis are localized to multiple cellular compartments within the endometrium including epithelial, stromal, and perivascular cells (62). Both Ptgs1 and Ptgs2 are expressed in the uterine luminal epithelial, but not in stromal, cells during peri-implantation phases in the human endometrium (63). Thus, in the uterus, Ptgs1 and Ptgs2 functions are not also mutually exclusive because one isoform can also compensate for the lack of another isoform (64, 65). In the current study, we demonstrated that PGE2 is the preferential product of Ptgs2 and mPtges at the implantation site, and inhibition of Ptgs2 activity by using Ptgs2-selective inhibitor causes both the reduction in the number and the size of the implantation sites on day 5 of pregnancy in hamsters. Implantation together with stromal decidualization, i.e. cell proliferation, edema, and increased vascular permeability, has been recognized as a proinflammatory event, and identification of its actual mechanism is still an important issue in modern research. Lipid mediator PGs have been implicated in triggering inflammatory processes. Because PGE2 is a potent inflammatory PG, our finding suggests that this lipid mediator is involved in local implantation associated changes in the uterus at the site of implantation. As discussed above PGE2 could be also involved in the protection of embryo proper from the harmful materials originating from the mother. In general, complete success of the blastocyst implantation process involves sequential maternal uterine changes to nurture further growth and protection of the embryo. Ptgs2-derived PGE2 at the implantation site helps to complete the implantation process without any adverse effects.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HD 42636, HD 44741, HD 40193, HD 37394 (to B. C. P.), GM 15431, CA 77839, DK 48831, RR 00095 (to J. D. M.), and HD 40221 (to J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. Back

** To whom correspondence should be addressed: Division of Reproductive and Developmental Biology, Dept. of Pediatrics, Vanderbilt University Medical Center, 1161 21st Ave. South, Nashville, TN 37232-2678. Tel.: 615-322-8640; Fax: 915-322-4704; E-mail: bc.paria{at}vanderbilt.edu.

1 The abbreviations used are: PGs, prostaglandins; Ptgs1, prostaglandin-endoperoxide synthase 1; Ptgs2, prostaglandin-endoperoxide synthase 2; COX, cyclooxygenase; Ptges, PGE synthase; mPtges, microsomal-type Ptges; Ptgis, PGI synthase; P4, progesterone; E2, estradiol-17{beta}; NSAIDs, nonsteroidal anti-inflammatory drugs; PDZ, primary decidual zone; PBS, phosphate-buffered saline. Back

2 X. Wang and B. C. Paria, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. S. K. Dey for comments on the manuscript and for unlimited laboratory access to conduct these studies. We are grateful to Dr. Sanjoy K. Das for expertise in gene cloning and useful discussions in preparing this manuscript.



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 DISCUSSION
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