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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1482
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2308-2313
Copyright © 2005 by The Endocrine Society

Expression of Human Prostaglandin Transporter in the Human Endometrium across the Menstrual Cycle

Jihong Kang, Pierre Chapdelaine, Julie Parent, Eric Madore, Philippe Y. Laberge and Michel A. Fortier

Unité de Recherche en Ontogénie et Reproduction (J.K., P.C., J.P., E.M., M.A.F.), Centre de Recherche du Centre Hospitalier Universitaire de Québec, Ste-Foy, Québec G1V 4G2, Canada; and Département d’Obstétrique et Gynécologie (P.Y.L., M.A.F.), Université Laval, Ste-Foy, Québec G1K 7P4, Canada

Address all correspondence and requests for reprints to: Michel A. Fortier, Ph.D., Unité de Recherche en Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Université Laval, 2705, Boulevard Laurier, Sainte-Foy, Québec G1V 4G2, Canada. E-mail: mafortier{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins (PGs) are important regulators of reproductive function. The mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Prostaglandin transporter (PGT) has been identified as a functional PG carrier. The aim of our study was to outline the expression of PGT in the human endometrium across the menstrual cycle. Quantitative RT-PCR showed human PGT (hPGT) expression to be strong in the proliferative and early secretory phases and low in the middle to late secretory phase. Northern blot analysis revealed hPGT mRNA transcript of 4 kb in the human endometrium. A peptide-directed polyclonal antibody was generated in rabbits against the 22 amino acids forming the C terminus of hPGT. Antibody specificity was demonstrated by Western blot. Immunoblots of endogenous hPGT in the human endometrium revealed a 70-kDa protein in endometrial cells. Endometrial biopsies collected across the menstrual cycle were used to assess hPGT protein expression by immunohistochemistry. hPGT was immunolocalized to luminal, glandular epithelial, and stromal cells. Because it was observed at the mRNA level, semiquantitative analysis showed a higher protein expression in proliferative and early secretory phases than in the mid-late secretory phase. In conclusion, our study revealed that hPGT expression is modulated in epithelial and stromal cells of the human endometrium at both mRNA and protein levels during the menstrual cycle. These findings support a role for hPGT as an important new player in the regulation of PG action in the human endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGS) ARE bioactive lipids playing important roles in female reproduction. In the human endometrium, circumstantial evidence suggests that PGs exert diverse physiological functions in processes including implantation, menstruation, and parturition. PGs appear also to be implicated in endometrial dysfunction and pathology such as carcinomas, menorrhagia, and dysmenorrhea (1).

PGs are synthesized from arachidonic acid, released from phospholipids stored in cell membrane through phospholipase action. Cyclooxygenase (COX) enzymes are the rate-limiting enzymes responsible for the conversion of arachidonic acid to PGH2, the common precursor of various forms of PGs. Three isoforms of COX enzyme have been identified: a constitutively expressed form COX-1; an induced form COX-2; and COX-3, a splice variant of COX-1 (2, 3, 4). The structure and function of COX-1 and COX-2 have been well characterized. The functional role of COX-3 in human physiology and pathology remains unclear (1). Terminal PGs are subsequently produced by specific prostanoid synthase enzymes from PGH2. In the case of PGE2 and PGF2{alpha}, we found that microsomal prostaglandin E synthase-1 and -2, cytosolic prostaglandin E synthase, and prostaglandin F synthase are expressed in the human endometrium (5, 6, 7).

Once synthesized, PGs are released and exert their effects by coupling to cell surface G protein-coupled receptors or nuclear envelope receptors in an autocrine or paracrine manner (8). PGs are charged organic anions at physiological pH. Considerable evidence suggests that the transport of PGs across the plasma membrane occurs through a carrier-mediated process (9). A recent study proposed a two-step model of PG signal termination involving their migration in the cytosol (10).

Prostaglandin transporter (PGT) was identified to be a functional PG carrier. It was first cloned and characterized in the rat (11) and then in the human (12) and mouse (13). PGT transports PGE2, PGF2{alpha}, PGD2, and to a lesser extent TxB2 (14). Previous results showed that PGT mRNA is expressed in reproductive tissues such as testis, ovary, and uterus (11, 12), but it was proposed to be associated mainly with PG clearance and metabolism. Our laboratory has recently cloned bovine PGT and studied the expression and function of PGT in the bovine reproductive system (15). These data demonstrated the spatiotemporal expression of bovine PGT in uterine tissues and proposed a role for PGT in the regulation of reproductive processes. In the human endometrium, temporal expression of PGE2, PGF2{alpha}, and their receptors has been demonstrated and shown to vary with the phase of the menstrual cycle (16, 17). PGT may regulate the local concentrations of PGE2 and PGF2{alpha} by transporting them through the cellular membrane for metabolism in the cytosol or facilitate paracrine actions. However, the expression of PGT in the human endometrium is unknown.

Thus, in the present study, we investigated the expression of human PGT (hPGT) at both mRNA and protein levels in nonpregnant human endometrium across the menstrual cycle. The data in this study report on the human endometrial epithelial and stromal expression of PGT showing a cyclical change of PGT across the menstrual cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Culture medium RPMI 1640 was purchased from ICN Biomedicals, Inc. (Aurora, OH). DMEM/F-12, gentamicin, transferrin, insulin, IL-1ß, and Mayer hematoxylin were purchased from Sigma (St. Louis, MO). Fetal bovine serum was bought from Wisent Inc. (Québec, Canada). Superscript II reverse transcriptase, DNA ladder, dithiothreitol, 5 x forward reaction buffer, 5 x first-strand buffer, TRIzol, and pEF6/V5-His TOPO vector were purchased from Invitrogen (Burlington, Canada). LightCycler FasterStart DNA Master SYBR Green I mix and MgCl2 were bought from Roche Diagnostics (Laval, Québec, Canada). All oligonucleotide primers were chemically synthesized using ABT 394 synthase (Perkin-Elmer, Foster City, CA). [{alpha}-32P]dCTP radioactivity was bought from Perkin-Elmer Life Sciences (Markham, Ontario, Canada). Bright Star-Plus nylon membrane and UltraHyb solution were purchased from Ambion Inc. (Austin, TX). Biotinylated secondary antibody (goat antirabbit IgG) was bought from Dako Diagnostics of Canada, Inc. (Mississauga, Ontario, Canada). Vectastain Elite ABC kit was purchased from Vector Laboratories, Inc. (Burlingame, CA). Goat antirabbit horseradish peroxidase-conjugated IgG was bought from Jackson Immunoresearch Laboratories (West Grove, PA). Enhanced chemiluminescent system (Renaissance) was purchased from NEN Life Science Products (Boston, MA). TNT quick coupled transcription/translation system was purchased from Promega (Madison, WI). [35S]-methionine and Ready-To-Go DNA labeling kit were purchased from Amersham Biosciences (Buckinghamshire, UK). Protein G-Sepharose beads were bought from Pharmacia Biotech AB (Uppsala, Sweden).

Tissue collection

Endometrial biopsies were taken from women with regular menstrual cycles undergoing gynecological investigation for benign conditions. Informed consent for donation of anonymous endometrial samples was obtained before tissue collection. Biopsies were classified first according to the stated last menstrual period and verified by histological examination of the slides prepared for immunohistochemistry by the hospital pathologist using standard criteria (18). The research protocol was approved by Centre Hospitalier Universitaire de Québec Ethics Committee on Human Research. The tissue samples were placed in sterile Hanks’ balanced salt solution containing 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 4 C and transported to the laboratory immediately.

Cell culture

Primary human endometrial nonseparated and separated stromal and epithelial cells were prepared as we described recently (19). Cells were cultured in RPMI 1640 medium containing 50 ng/ml gentamicin, 10 µg/ml insulin, 5 µg/ml transferrin, and 10% fetal bovine serum. As we described recently (19), in separate stromal cell cultures, the contaminating epithelial cells are about 1%. However, in epithelial-rich cultures, the ratio of contaminating stromal cells can reach 5% under our culture conditions.

Treatment

The medium was changed every 2 d. After reaching confluence, cells were treated with fresh, serum-free RPMI 1640 medium containing different concentrations of IL-1ß (0, 1, 10 ng/ml) at 37 C in an atmosphere of 5% CO2-95% air. After 24 h, cells were recovered for protein assay.

RNA extraction and RT-PCR

Total RNA was extracted from endometrial biopsies (n = 55) or cells with TRIzol according to the manufacturer’s instruction. Then 2.5 µg total RNA from each sample was reverse transcribed using random hexamer primers and Superscript II reverse transcriptase in a final volume of 20 µl. Thereafter cDNA samples were used as the template for PCR using the specific primers as designed for real-time PCR. The PCR conditions were 94 C for 30 sec, 55 C for 30 sec, and 72 C for 1 min for 35 cycles, followed by extension at 72 C for 10 min.

Real-time PCR (LightCycler)

Primers were designed from the known sequences of human PGT and 18S. The sequences of hPGT primers were: forward, 5'-GGA TGC TGT TTG GAG GAA TCC TCA-3'; reverse, 5'-GCA CGA TCC TGT CTT TGC TGA A-3'. The sequences of 18S primers were: forward, 5'-GTA ACC CGT TGA ACC CCA TT-3'; reverse, 5'-CCA TCC AAT CGG TAG TAG CG-3'. 18S was used as an internal standard. Standard RT-PCR products of hPGT and 18S were ligated into pEF6/V5-His TOPO vector. The plasmids containing the appropriate cDNA inserts were used as standards in LightCycler. Real-time quantitative RT-PCR (LightCycler) was performed using fluorescent SYBR Green I according to the manufacturer’s instruction. Each reaction mixture contained FastSTART DNA Master SYBR Green 1, MgCl2 (3 mM), forward and reverse primers, and 2 µl of template (cDNA or standard) in a total volume of 20 µl. The reaction mixture was denatured for 5 min at 95 C and subjected to 50 cycles for hPGT or 32 cycles for 18S in a three-step PCR consisting of a denaturation step (95 C for 10 sec), annealing step (57 C for 5 sec), and extension step (72 C for 20 sec). The amplified products were verified by agarose gel electrophoresis and showed single bands of predicted sizes for each sample and no products for the negative controls. hPGT mRNA level was expressed as a ratio of hPGT to 18S.

Northern blot

Northern blot was performed as described recently (20). Briefly, approximately 20 µg of total RNA was loaded on 1.2% formaldehyde-agarose gel, electrophoresed, and transferred onto a nylon membrane. Full-length hPGT cDNA (2 kb) was used as the probe. The cDNA probe was labeled with [{alpha}-32P]dCTP using Ready-To-Go DNA labeling kit. Prehybridization and hybridization were carried out at 45 C in UltraHyb solution. Signals were detected by autoradiography on BioMax film (Kodak, Rochester, NY) after exposure overnight at –80 C.

Antibody preparation, competition study, and Western blot

A rabbit polyclonal anti-hPGT antiserum was generated against a synthetic peptide consisting of amino acids 622–643 of hPGT (CFISWRVKKNKEYNVQKAAGLI). The 22-amino acid sequence was blasted against the protein databases, and no protein was found to cross-react. The peptide was dissolved in PBS and used to perform antibody competition. Zero- and 10-fold excess amounts of peptide were incubated together with the antibody overnight at 4 C and then used as primary antibody for Western blot. Protein extraction, quantification, and Western blot analysis were performed as described recently (19). Aliquots of 10 µg protein of each sample were separated on 8% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were blocked overnight at 4 C in PBS containing 5% fat-free dry milk and 0.05% Tween 20. The membrane was subsequently probed with rabbit anti-hPGT (1:10,000) and goat antirabbit horseradish peroxidase-conjugated IgG (1:10,000). Immunoreactive proteins were visualized with an enhanced chemiluminescent system.

In vitro transcription/translation

The cDNA encoding hPGT C terminus was synthesized by PCR using the specific primers: forward, 5'-GAT CAT GCA TCC TAA TAC GAC TCA CTA TAG GGA ACA GCC ACC ATG ATC CTT TGT GTT CCT TTG TTC TTC AT-3'; reverse, 5'-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTA GAT GAG GCC TGC CGC CTT CTG CAC-3'. The cDNA was then used in the TNT quick coupled transcription/translation system in the presence of [35S]-methionine according to the manufacturer’s protocol.

Immunoprecipitation

Four microliters of the translation product from TNT quick coupled transcription/translation system was incubated with preimmune and anti-hPGT sera, respectively, for 1 h at 4 C. The complexes were then precipitated by incubation with Protein G-Sepharose beads for 1 h at room temperature. After a brief centrifugation, supernatant was removed from the beads. The beads were then washed three times with PBS and resuspended in SDS-PAGE sample buffer.

SDS-PAGE and autoradiography

The translation product and precipitated complex were boiled for 2 min before loading on 10% SDS-PAGE. The gel was fixed in 50% methanol/10% acetic acid for 15 min, washed in 10% methanol/10% acetic acid for 10 min, and then dried. Dry gel was exposed to Kodak BioMax film for 14–16 h at room temperature.

Immunohistochemistry

Endometrial tissues [n = 24, 10 from proliferative phase (d 4–14), six from periovulatory-early secretory (d 15–18), and eight from mid-late secretory phase (d 19–30)] were fixed in 4% paraformaldehyde and prepared as paraffin-embedded sections. Slides were deparaffinized in xylene and washed in ethanol. Endogenous peroxidase activity was quenched with 3% (vol/vol) H2O2 in methanol. Antigen retrieval was performed by heating sections in 10 mM citrate buffer for 15 min. Tissue sections were then blocked with 10% goat serum for 1 h at room temperature followed by overnight incubation at 4 C with rabbit anti-hPGT serum (1:4000). Nonimmune rabbit serum was used as the negative control. Sections were further incubated with biotinylated secondary antibody and ABC reagent. Immunostaining was revealed by using 3-amino-9-ethylcarbazole, and Mayer hematoxylin was used for counterstaining. Immunostaining intensity was evaluated and interpreted as absent (0), weak (1), moderate (2), or intense (3). Scoring was done by three independent observers not involved with the study who were asked to rate the intensity of the staining without knowing the stage of the cycle or the identity of the protein studied. Individual scores for each slide were averaged and expressed as relative expression level.

Statistical analysis

Data are presented as the mean ± SEM. Statistical analysis was performed using ANOVA followed by Fischer’s protected least significant differences, Duncan new multiple range, and Student-Newman-Keuls multiple comparison tests (Super ANOVA; Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Quantification of hPGT mRNA expression in human endometrial biopsies

Figure 1AGo shows the quantification of hPGT mRNA expression in endometrial biopsies. hPGT mRNA expression was higher in proliferative phase (d 4–14, mean ± SEM = 109.4 ± 30.4) and early secretory phase (d 15–18, mean ± SEM = 142.6 ± 32.9) than in the mid-late secretory phase (d 19–30, mean ± SEM = 31.87 ± 7.29). A significant difference was observed between proliferative (d 4–14), early secretory phase (d 15–18), and middle and late secretory phases (d 19–30) (P < 0.05). Amplification quality was validated by analysis of agarose gel electrophoresis (Fig. 1BGo, upper section) and melting curve (Fig. 1BGo, lower section). Both melting curves and gel analysis showed a single peak or band at the expected size, respectively.



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  		FIG. 1. A, Real-time PCR (LightCycler) quantification of hPGT in the endometrium across the menstrual cycle. Total RNA was extracted from human endometrial biopsies. A total of 55 samples were used, 24 samples on d 4–14, 12 on d 15–18, and 19 on d 19–30. Quantification was performed with LightCycler using SYBR Green I. 18S was used as an internal standard. Results were expressed as the mean ± SEM of relative hPGT to 18S mRNA expression levels. Columns with different superscripts are significantly different (P < 0.05). B, Amplification quality was validated by analysis of melting curves expressed as the first derivative of fluorescence over time –d(F1)/dT and agarose gel electrophoresis. The two methods showed a single peak or band at the expected temperature and size, respectively.

 
Human PGT mRNA expression in primary endometrial cells

RT-PCR analysis showed that hPGT mRNA is present in both primary endometrial stromal and epithelial cells (Fig. 2AGo, lanes 1 and 2). Northern blot analysis revealed a 4-kb transcript in human endometrial stromal cells (Fig. 2BGo).



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  		FIG. 2. hPGT mRNA expression in primary endometrial cells and Northern blot analysis. A, hPGT mRNA expression was analyzed by RT-PCR. Lanes 1 and 2 represent human primary endometrial stromal (Stro) and epithelial (Epi) cells. B, Northern blot analysis of hPGT in endometrial stromal cells.

 
Validation of anti-hPGT antibody and expression of hPGT protein in primary endometrial cells

We first demonstrated the specificity of the antiserum for the peptide. Immunoreactive bands were present in primary stromal and epithelial cells (Fig. 3AGo, lanes 1 and 2), corresponding to the predicted hPGT molecular mass of 70 kDa. Moreover, preincubation of the antiserum with the peptide resulted in the disappearance of the signals (Fig. 3AGo, lanes 3 and 4), confirming that this antibody was specific to the peptide. In addition, the sequence coding for hPGT C terminus was synthesized by PCR using specific primers. A segment of hPGT protein (amino acids 409–643) encoded by this sequence was then expressed using TNT quick coupled transcript/translation system in the presence of this sequence and [35S]-methionine. Thereafter the translation products were both directly loaded on SDS-PAGE and used for immunoprecipitation. Positive signals were observed by autoradiography at the predicted molecular mass of 25 kDa from the direct addition of the translation products (Fig. 3BGo, lane 3) and the antiserum in immunoprecipitation (Fig. 3BGo, lane 2) but not from the preimmune serum in immunoprecipitation (Fig. 2BGo, lane 1). The different expressions between the antiserum and preimmune serum in immunoprecipitation further indicated that the antibody was specific to hPGT C terminus.



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  		FIG. 3. Competition study, immunoprecipitation, and effect of IL-1ß on hPGT protein expression. A, Competition study. Primary human endometrial stromal (Stro) and epithelial (Epi) cells showed hPGT immunostaining (lanes 1 and 2). The signals disappeared when anti-hPGT antiserum was preincubated with the peptide antigen (lanes 3 and 4), demonstrating the specificity of the antibody to the peptide. B, In vitro transcription/translation and immunoprecipitation. A segment of hPGT protein encoded by the hPGT C terminus was expressed using TNT quick coupled transcript/translation system. The translation product was then incubated with preimmune (lane 1), anti-hPGT sera (lane 2), or directly put on SDS-PAGE (lane 3). Signals were observed in lanes 2 and 3 at the expected sizes, further demonstrating the specificity of the antibody to hPGT. C, Nonseparated human endometrial cells were seeded in 24-well plates. After reaching confluence, cells were incubated with IL-1ß [0 (Ctrl), 1, and 10 ng/ml] for 24 h. Proteins were extracted for hPGT assay (lanes 1–3).

 
Effect of IL-1ß on hPGT protein level

The effect of IL-1ß on hPGT protein level was determined in nonseparated endometrial cells. Confluent cells were incubated with different concentrations of IL-1ß (0, 1, 10 ng/ml) for 24 h. Western blot analysis showed that IL-1ß did not significantly increase hPGT protein level (Fig. 3CGo, lanes 1–3).

Immunohistochemistry of hPGT in the human endometrium

Immunohistochemical staining for hPGT was performed in human endometrial biopsies collected across the menstrual cycle. Positive staining was detected in tissue sections throughout the cycle (Fig. 4AGo). hPGT protein is present in luminal and glandular epithelial and stromal cells. The staining was then evaluated in epithelial and stromal cells, respectively, by semiquantitative analysis. The intensity of staining did not vary between epithelial and stromal cells, and thus the overall hPGT protein expression was used to compare the different phases of the menstrual cycle. hPGT protein was found to be significantly higher in proliferative and early secretory phases than the mid-late secretory phase (Fig. 4BGo). The pattern of hPGT protein expression follows that of mRNA expression (Fig. 1AGo) during the menstrual cycle.



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  		FIG. 4. Immunohistochemical analysis of hPGT in human endometrial tissues. A, Positive staining was detected in the luminal, glandular epithelial, and stromal cells. hPGT protein staining is present in all biopsies throughout the menstrual cycle. B, Semiquantitative analysis of immunohistochemistry. Designations of 0 (absent), 1 (weak), 2 (moderate), and 3 (intense) indicate the relative intensities of the signals averaged. Columns with different superscripts are significantly different (P < 0.05). Magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PGs have been discovered and studied for decades, but the mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Members of organic anion transporting polypeptide and organic anion transporter gene families have been suggested to play a role in the transport of PGs but not with the same affinity and/or specificity (9, 21, 22). The mechanism by which newly synthesized PGs efflux from cells is still in dispute. Previous thinking held that PGs efflux by simple diffusion (9). However, recent data have shown that multidrug resistance protein 4 functions as a PG efflux transporter (23). PGT is the first cloned PG transporter (14), and it emerges as a functional uptake-carrier of PGs with high affinity for PGE2, PGF2{alpha}, and PGD2. The role of PGT in mediating PG clearance and degradation was first proposed in 1996 (12). Several experiments were performed thereafter for functional studies, such as influx measurements using Hela cells and Xenopus oocytes as experimental models. More recently Nomura et al. (10) proposed a two-step model in which coexpression of the PG uptake carrier PGT along with oxidizing enzyme 15-hydroxyprostaglandin dehydrogenase is required for PG oxidation. Extracellular PGs are transported by a PGT-mediated process across the plasma membrane followed by cytoplasmic oxidation.

In animals, we and others (20, 24, 25) have shown that PGs are key regulators of female reproductive functions. PGs appear also to play important roles in the human endometrium. Aberrant ratios of PGE2/PGF2{alpha} are associated with menstrual disorders such as dysmenorrhea and menorrhagia (26). Therefore, keeping proper local concentrations of PGE2 and PGF2{alpha} is important to maintain a normal physiological status. Previous studies reported on the concentrations of PGE2 and PGF2{alpha} in the human endometrium (26, 27). PG, especially PGF2{alpha}, release was higher in the mid-, late secretory, and menstrual phases. COX-2 enzyme, which catalyzes the committed step in PG biosynthesis, plays a crucial role in reproductive processes. Ablation of the COX-2 gene in mice results in multiple reproductive failures in females (24, 28). COX-2 immunoreactivity was observed to be significantly higher in the late secretory phase by immunohistochemistry (29). In the present study, we demonstrate the expression of PGT in the human endometrium across the cycle. hPGT is significantly higher in the menstrual, proliferative, and early secretory phases than in the mid-late secretory phase. Based on the role of PGT in PG clearance and degradation, the data presented here support previous observations of PG release in the endometrium. PG levels are regulated both by the synthetic and degradation processes. The elevated expression of COX-2 in the late secretory phase and the associated reduction in hPGT expression can explain the higher levels of PG released in the endometrium at the middle and late secretory phases. Milne et al. (16) and Milne and Jabbour (17) demonstrated the expression of PGE2 receptors EP2, EP4, and PGF2{alpha} receptor (FP) in the nonpregnant human endometrium. There was no significant change in EP2 receptor mRNA expression across the menstrual cycle. EP4 receptor mRNA expression, in contrast, was significantly higher in late proliferative biopsies than at other phases. FP receptor mRNA expression was also shown to be significantly higher in mid- to late-proliferative endometrium. Provided that PGs efflux by simple diffusion, COX, terminal PG synthases, PG receptors, PGT, and 15-hydroxyprostaglandin dehydrogenase would constitute a complex integrating system for PG production, function, transport, and metabolism, thus regulating local PG concentrations to orchestrate reproductive events in the endometrium. Prostacyclin expression and function have also been confirmed in the human endometrium (30). However, PGT does not mediate the uptake of prostacyclin (14). This highly unstable PG may not need the same clearance system because it is inactivated spontaneously within seconds.

It was found that immunostaining for COX was localized mainly in the luminal and glandular epithelial cells (31, 32). EP2 and EP4 receptors were localized to the epithelial cells (16) and FP receptor predominantly to the epithelial cells (17). The present study demonstrates that hPGT is localized in luminal and glandular epithelial as well as stromal cells during the cycle. Furthermore, PG dehydrogenase activity was shown to be localized in the glands (33). Taken together, these data suggest that in the human endometrium, epithelial cells are likely to be the main site of PG production, function, and metabolism.

The factors that regulate the expression of PGT in the human endometrium are still unknown. Previous studies suggested that PGT is coordinately regulated with COX (9). However, IL-1ß significantly increased COX-2 protein level in cultured human endometrial cells (19), but it had no effect on hPGT protein level (Fig. 3CGo). This result is consistent with the observation of Topper et al. (34), who showed that various biochemical stimuli, such as IL-1ß and TNF{alpha}, did not significantly influence hPGT.

In summary, this study demonstrates the expression of hPGT in the human endometrium throughout the cycle. Immunohistochemistry analysis revealed the localization of PGT to both epithelial and stromal cells. PGT may contribute to the regulation of the local concentrations of PGs by transporting PGs across the cellular membrane for metabolism. These findings provide important new information to understand the role of PGs in the human endometrium.


   Footnotes
 
This work was supported by a grant from the Canadian Institute of Health Research (to M.A.F.).

First Published Online January 18, 2005

Abbreviations: COX, Cyclooxygenase; FP, PGF2{alpha} receptor; hPGT, human PGT; PG, prostaglandin; PGT, PG transporter.

Received July 27, 2004.

Accepted January 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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