Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print March 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037036
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/1/36    most recent
biolreprod.104.037036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parent, J.
Right arrow Articles by Fortier, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parent, J.
Right arrow Articles by Fortier, M. A.
Agricola
Right arrow Articles by Parent, J.
Right arrow Articles by Fortier, M. A.
BIOLOGY OF REPRODUCTION 73, 36–44 (2005)
DOI: 10.1095/biolreprod.104.037036
© 2005 by the Society for the Study of Reproduction, Inc.

Expression and Contribution of Three Different Isoforms of Prostaglandin E Synthase in the Bovine Endometrium1

Julie Parent 3, and Michel A. Fortier 2 3,4 

Unité de Recherche en Ontogénie et Reproduction,3 Centre Hospitalier Universitaire de Québec (CHUL), Centre de Recherche en Biologie de la Reproduction (CRBR), Département d'Obstétrique et Gynécologie,4 Université Laval, Ste-Foy, Quebec, Canada G1V 4G2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandins are involved in the regulation of several reproductive processes such as ovulation, luteolysis, and establishment of pregnancy. Prostaglandin E2 (PGE2) appears to favor establishment of pregnancy in most mammals studied so far. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases (PGES). Three PGES have been identified in humans, but in the bovine, microsomal PGES2 and cytosolic PGES genes have neither been cloned nor associated to any physiological processes. The present study was undertaken to clone bovine MPGES2 and CPGES and to report on their regulation in the endometrium during the estrous cycle. CPGES mRNA expression declines progressively during the cycle; its protein is not modulated according to a precise pattern. MPGES2 mRNA and protein expression decrease from the beginning of the cycle until Days 13–15 and then increase until ovulation. Immunohistochemical analysis reveals that both enzymes are located in luminal epithelial and glandular epithelial cells and at a lower level in stromal cells. In addition, using the bovine endometrial cell line BEND, where higher accumulation of PGE2 is observed following treatment with phorbol 12-myristate 13-actetate (PMA) and tumor necrosis factor-{alpha} (TNF-{alpha}), we have found an associated increase of MPGES1 and COX2 but not CPGES or MPGES2 protein expression. Together, our results suggest that MPGES1 is not the only PGES present in the bovine endometrium but is the main enzyme associated with increased PGE2 production in vitro.

bovine, endometrium, establishment of pregnancy, female reproductive tract, mechanisms of hormone action, ovulatory cycle, pregnancy, prostaglandin biosynthesis, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandins (PG) are major contributors to reproductive processes, including ovulation, implantation, parturition, luteolysis, and recognition of pregnancy [1, 2]. In ruminants, it is widely accepted that prostaglandin F2{alpha} (PGF2{alpha}), originating from the endometrium, is the luteolytic signal [3]. In contrast, prostaglandin E2 (PGE2) is thought to exert opposite actions to favor establishment of pregnancy, including a luteoprotective action that is either antiluteolytic or luteotropic [4, 5]. PGE2 also modulates the immune system to prevent rejection of the conceptus allograft [6].

PGE2 production begins when phospholipases liberate arachidonic acid (AA) stored in membrane phospholipids. AA is then converted into prostaglandin endoperoxide H2 (PGH2) by cyclooxygenases (COX), also known as prostaglandin synthases. Two isoforms of the COX enzyme, types 1 and 2, are coded by different genes and catalyze the double oxygenation and reduction of AA. PGH2 is then transformed into PGE2 via PGE synthases (PGES). Three forms of PGES have been characterized so far. Microsomal PGES1 (MPGES1), also known as prostaglandin E synthase (PTGES), was the first identified and reported as inducible by agents such as cytokines and LPS [7]. This enzyme is often coupled with COX2 for delayed and sustained production of PGE2 [8]. Its sequence, currently known in bovine, exhibits 85% homology with the corresponding human enzyme [9]. We have described previously the regulation of MPGES1 expression during the bovine estrous cycle and its association with COX2 [10]. A cytosolic PGES (CPGES), also known as cytosolic PTGES or PTGES3, identical to p23, a ubiquitous chaperone protein weakly bound to the steroid hormone receptor/hsp90 complex [11, 12], was characterized and found coupled to COX1 for immediate production of PGE2 [13]. Enzymatic activity from a third PGES, microsomal PGES2 (MPGES2), also known as prostaglandin E synthase 2, was recently purified from bovine heart [14], and cloning of homologous human and monkey sequences was done [15]. This PGES is associated with both isoforms of COX, with a slight preference for COX2 [16]. Very little is known about the physiological actions of MPGES2, and its sequence is not known in the bovine species.

We have previously demonstrated that, in bovine endometrial epithelial and stromal cells in vitro, an increase in PGE2 accumulation is observed following treatment with the embryonic signal, IFN-{tau} [17]. This increase was accompanied by increased COX2 and MPGES1 mRNA, but not COX1 or phospholipase A2 expression [17, 18]. However, MPGES2 and CPGES were never studied in bovine in relation with PGE2 production in any reproductive processes, despite proven importance of PGE2 effects in reproduction, especially in recognition of pregnancy [1–6]. Therefore, the objectives of the present study were to 1) clone bovine CPGES and MPGES2; 2) study their expression in the bovine endometrium during the estrous cycle; and 3) evaluate the contribution of each PGES in PGE2 production in vitro in a bovine endometrial cell line.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Topo pEF6/V5 6xHis TA Cloning Kit, TRIzol, and reagents used for the reverse-transcription (RT) (dNTPs, Superscript II, buffer, and random hexamers) were obtained from Invitrogen Corporation (Carlsbad, CA). Taq polymerase and buffer used for the polymerase chain-reaction (PCR), DNA labeling kit (-dCTP), and Hyperfilm ECL were purchased from Amersham Pharmacia Biotech (Baie d'Urfé, QC, Canada). All restriction enzymes were purchased from New England Biolabs (Pickering, ON, Canada). QIAquick gel extraction kit was bought from Qiagen (Mississauga, ON, Canada). BrightStar Plus membrane and ULTRAhyb solution were purchased from Ambion Inc. (Austin, TX). Western Lightning Chemiluminescence Reagent Plus and [{alpha}-32P] dCTP were purchased from PerkinElmer Life Sciences (Boston, MA). Interleukin-1ß (IL-1ß), tumor necrosis factor-{alpha} (TNF-{alpha}), phorbol 12-myristate 13-actetate (PMA), and Mayer hematoxylin were purchased from Sigma (St. Louis, MO). Prestained protein markers were purchased from Mandel Scientific, New England Nuclear Life Science Products (Mississauga, ON, Canada). Trans-Blot Transfer Medium (nitrocellulose membranes) were purchased from Bio-Rad Laboratories (Hercules, CA). Tracers for PGE2 used in the enzymatic immunoassay (EIA), and rabbit antibodies against human MPGES1 and MPGES2 were purchased from Cayman Chemical (Ann Arbor, MI). Mouse antibody against human CPGES was purchased from Affinity BioReagents Inc. (Golden, CO). Rabbit antibody against sheep COX2 was kindly provided by Stacia Kargman (Merck Frosst, Kirkland, QC, Canada). The goat anti-rabbit and the goat anti-mouse antibodies conjugated to horseradish peroxidase were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). BioMax films are from Eastman Kodak Corporation (New York, NY). Vectastain Elite ABC Kit was purchased from Vector Laboratories Inc. (Burlingame, CA). Goat anti-rabbit biotinylated Ig was obtained from Dako Diagnostics Inc. (Mississauga, ON, Canada).

Collection of Tissues

All bovine tissues used in the present study were collected at a local abattoir immediately after slaughter of animals. Tissues were placed on ice and transported to the laboratory within 1.5 h. The physiological status of the tissue was estimated by examination of ovarian morphology as we described previously [10]. Tissues were cut into small pieces, snap-frozen in liquid nitrogen, and stored at –80°C until used.

RNA Isolation

Samples were thawed on ice and RNA isolation was done with TRIzol reagent according to the manufacturer's instructions. RNA samples were resuspended in water containing diethyl pyrocarbonate (0.05% v:v) and stored at –80°C. Before use, RNA concentration was measured by absorbance at 260 nm.

Molecular Cloning of Bovine MPGES2 and CPGES

RNA from bovine endometrium (Day 5) was used as a template and was reverse transcribed using random primers and Superscript II reverse transcriptase. Bovine MPGES2 partial coding sequence (172 base pairs [bp]) and CPGES complete coding sequence (483 bp) were generated by RT-PCR using the following primers: MPGES2, sense 5'-GCAGGGCTGAGATCAAGTTC-3' and antisense 5'-GCCTTCATGGCTGGGTAGTA-3'; CPGES, sense 5'-ATGCAGCCTGCTTCTGCA-3' and antisense 5'-TTACTCCAGATCTGGCAT-3'. The primers were deduced from homologous sequences in mouse, rat, and human for CPGES and human and monkey for MPGES2. The PCR conditions were 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec for 35 cycles. The RT-PCR products were cloned into Topo cloning pEF6/v5 and sequenced.

DNA Sequencing and Sequence Analyses

The plasmid DNA was isolated using the Qiagen plasmid purification system. After using the appropriate restriction enzymes, the clones were sequenced by the sequencing service (CHUL, Québec, Canada). Multiple sequence alignments were obtained using the LFASTA program, version v2.lu00 [19].

Northern Blot Analysis

Equal amounts (15 µg) of total RNA were loaded on a 1.2% w:v formaldehyde agarose gel, electrophoresed, and transferred onto a nylon membrane. The cDNA probes for bovine MPGES2 and bovine CPGES were generated by PCR amplification with specific primers (MPGES2, sense 5'-GCAGGGCTGAGATCAAGTTC-3' and antisense 5'-GCCTTCATGGCTGGGTAGTA-3'; CPGES, sense 5'-ATGCAGCCTGCTTCTGCA-3' and antisense 5'-TTACTCCAGATCTGGCAT-3'). The cDNA fragments of bovine MPGES2 and CPGES were obtained by Eco R1 digestion of recombinant clones, thus liberating a 172-bp fragment for MPGES2 and a 483-bp fragment for CPGES, ready to be labeled. These recombinant clones came from a cloning in Topo Cloning kit (Invitrogen). The same membrane was used for both messengers, one at a time. The blots were stripped off by boiling in 1% SDS for 30 min and rehybridized. Probes were labeled with DNA labeling kit (-dCTP) and [{alpha}32P]-dCTP and purified by precipitation [20]. Prehybridization was performed at 45°C in UltraHyb solution for 4–5 h, then the labeled probe was added and hybridization was performed overnight at 45°C. Washings were done at room temperature 3 x 10 min, and at 60°C 2 x 15 min in SSC 0.2x supplemented with 0.1% SDS. Signals were detected by autoradiography on Kodak X-omat at –80°C after exposure for 2–5 days before development. Bands were quantified by BioImage Visage 110s from Genomic Solutions Inc. (Ann Harbor, MI). Intensity of each band was normalized to the intensity of corresponding 18S RNA, as seen on the gel.

Cell Culture and Experimental Protocol

The commercial bovine endometrial cell line (BEND, ATCC# CRL-2398) was purchased from American Type Culture Collection (Manassas, VA). The culture and propagation of BEND cells was done as described in the instructions provided by ATCC. Briefly, cells were grown in a 1:1 mixture of Ham F12 and Eagle minimal essential medium with Earle balanced salt solution (D-valine modification) with 1.5 mM L-glutamine, 1.5 g/L sodium bicarbonate containing 0.034 g/L D-valine, 10% fetal bovine serum (FBS), and 10% horse serum (HS). In all four experiments, BEND cells were plated in 24-well plates at a 1:3 split ratio and grown at 37°C under a humidified atmosphere containing 95% O2 and 5% CO2. When the cells were fully attached (24 h), the culture medium was replaced with fresh medium supplemented with 10% FBS and HS. Under those conditions, cells reached confluence 2 days later. BEND cells were grown to confluence and the medium was replaced with RPMI-1640 without FBS and HS for 24 h before stimulation. Cells were treated for 24 h with TNF-{alpha} (10–9 M) or PMA (10–7 M) to stimulate PG production or with human IL-1ß (1 ng/ml), which has no effect. Three wells were used for each treatment by experiment. For all four experiments, at the end of incubation time, the culture medium was recovered for PGE2 measurement and stored at –20°C until further processing. For protein extraction, cells were lysed with 200 µl of lysis buffer (10 mM Tris-HCl, pH 7.4, 1% SDS, 1 mM dithiothreitol [DTT], and 1 mM phenylmethylsulfonylfluoride [PMSF]) and extraction was done immediately.

Protein Extraction

All bovine tissues kept at –80°C were homogenized in homogenization buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 1 mM PMSF, and 1 mM DTT) and resulting proteins were extracted and measured as described previously [21]. Protein samples from BEND cells were suspended in 25 µl SDS-PAGE loading buffer (0.06 M Tris-HCl, pH 6.8, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol, and 0.025% bromophenol blue), and proteins from bovine tissues were resuspended in 50 µl SDS-PAGE loading buffer. All samples were then boiled for 5 min and protein content was estimated using 1 µl of the sample.

Western Blot Analysis

Aliquots (10 µg) of total protein were loaded in each lane and electrophoresed through 13% SDS-polyacrylamide gels followed by electrotransfer onto a nitro-cellulose membrane. Prestained protein markers were used as molecular weight standard for each analysis. After staining with Ponceau Red to ensure that the same amount of protein was transferred onto the membrane, blocking was done in 5% fat-free dry milk powder in PBS and 0.05% Tween-20 (PBS-T) overnight at 4°C. The membrane was then incubated with the antibody raised against COX2 (dilution 1/3000), MPGES1 (dilution 1/500), or MPGES2 (dilution 1/250), or CPGES (dilution 1/1500), or ß-actin (dilution 1/5000) in 2% fat-free dry milk powder in PBS-T for 1 h at room temperature. Washings were done for 30 min in PBS-T. The second antibody, goat anti-rabbit (COX2, MPGES1, and MPGES2 analysis) or goat anti-mouse (CPGES and ß-actin analysis) conjugated to horseradish peroxidase (dilution 1/10 000 in 2% fat-free dry milk powder in PBS-T) were then incubated for 45 min at room temperature. The membrane was washed for another 30 min in PBS-T. Bands were revealed by addition of a chemiluminescent substrate according to the manufacturer's instructions. The blots were exposed to Hyperfilm ECL with intensifying screen. Bands were quantified by BioImage Visage 110s from Genomic Solutions Inc.

Immunohistochemistry

Immunohistochemical staining was performed as described in the instruction manual of the Vectastain kit (Paraffin section) using 1/500 dilution of CPGES and MPGES1 antibodies or dilution 1/250 of MPGES2 antibody and Mayer hematoxylin as counterstain. For negative control, preimmune rabbit serum was used instead of PGES antibodies. Photos were captured using the Spot program (Carsen Group Inc., Markham, ON, Canada).

ELISA for Prostaglandins

For PGE2 measurement, an EIA was used, using acetylcholinesterase-linked PG tracers as described previously [22]. We have used a fully characterized rabbit anti-PGE2 [22, 23]. The inter- and intraassay coefficients of variation (n = 12) were 16% and 10%, respectively.

Statistical Analysis

Data for expression of enzymes and PGE2 levels obtained are presented as the means ± SEM of PGE2 (pg/ml) or ratio of target enzyme/ 18S or ß-actin. The data were treated by analysis of variance using Super ANOVA Software (Abacus Concepts Inc., Berkeley, CA). Individual comparison of means was made using Student-Newman-Keuls test. Sources of variation included experiments, treatments, and their interactions. Differences were considered statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Bovine CPGES and MPGES2

The complete coding sequence of bovine CPGES (GenBank/EBI accession no. AY692440) is presented in Figure 1. For this sequence, consisting of 483 bp, the homology is more than 97% with human sequence in nucleic acids, and the deduced amino acids sequence is 99% homologous to the human CPGES protein. The only differences are glycine12 and glycine13 replacing arginine12 and aspartic acid13 in the human sequence. It is worth noting that tyrosine9, which is involved in enzymatic activity and is conserved among species [13, 24], is also present in the bovine CPGES sequence.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 1. Molecular cloning and sequencing of bovine CPGES cDNA. Bovine nucleic acids sequence is shown at the top and predicted amino acids sequence at the bottom. Numbers on the left refer to the first nucleic acid or amino acid on that line, and numbers on the right refer to the last nucleic acid or amino acid on that line. The bovine CPGES sequence was cloned and sequenced as described in Material and Methods

The partial coding sequence of bovine MPGES2 (GenBank/EBI accession no. AY692441) is shown in Figure 2. The amplified sequence of bovine MPGES2 consists of 172 bp (approximately 15% of the human MPGES2 complete coding sequence), with a homology of 93% with human MPGES2 at the nucleic acids level. The deduced amino acids sequence of 56 amino acids is 95% homologous to the corresponding human MPGES2 (amino acids 138–193). In comparison with the corresponding human sequence, three amino acids are different in the partial bovine sequence: serine159, glutamic acid183, and glutamic acid184.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Molecular cloning and sequencing of bovine MPGES2 cDNA. Bovine nucleic acids sequence is shown at the top and predicted amino acids sequence at the bottom. Numbers on the left refer to the first nucleic acid or amino acid on that line, and numbers on the right refer to the last nucleic acid or amino acid on that line. The bovine MPGES2 sequence was cloned and sequenced as described in Material and Methods

Tissue Distribution of Bovine MPGES1, MPGES2, and CPGES

Western blot analyses were performed in various tissues taken from one cow (Day 5 of the estrous cycle) and one bull. At that day of the estrous cycle, as shown in Figure 3, MPGES1 protein (Fig. 3A) is expressed mainly in the lung, ovary, and kidney and to a lesser extent in the spleen, testis, and myometrium. MPGES2 protein (Fig. 3B) is expressed in all tissues tested, albeit at different levels in the following order: heart > kidney > muscle > testis > endometrium = ovary > myometrium = spleen = lung. CPGES protein (Fig. 3C) is most abundant in the testis and ovary and is expressed at lower levels in the endometrium, myometrium, kidney, and lung, and only faintly in the spleen, heart, and muscle.



View larger version (72K):
[in this window]
[in a new window]
  		FIG. 3. Expression of MPGES1, MPGES2, and CPGES protein in bovine tissues. Tissues were collected from one cow at Day 5 of the estrous cycle and one bull and were frozen in liquid nitrogen. Proteins were extracted, submitted to electrophoresis, and transferred onto a nitrocellulose membrane. Western blot hybridization was done with antibodies raised against MPGES1, MPGES2, and CPGES. Blots were exposed for 30–120 sec. E, Endometrium; my, myometrium; O, ovary; T, testis; H, heart; mu, muscle; S, spleen; K, kidney; Lu, lung

Localization of MPGES1, MPGES2, and CPGES Proteins in Bovine Endometrium

Immunohistochemical analysis (Fig. 4) was performed on six different cows (Day 18) and shows that, in bovine endometrium, the three PGES are located in all cell types (epithelial, glandular, and stromal cells). Even if some staining is present in luminal and glandular epithelial cells in control, we can see that MPGES1 protein (Fig. 4A) is generally expressed in glandular epithelial cells more than in luminal epithelial cells and to a lower extent in stromal cells. We observed that stromal cells surrounding epithelial cells are stained more intensely than other stromal cells. MPGES2 protein (Fig. 4B) is mainly expressed in luminal epithelial cells followed by glandular epithelial cells, but staining is also present in stromal cells at a lower level. Here again, stromal cells adjacent to luminal epithelial cells stain more intensely than distant stromal cells. CPGES protein (Fig. 4C) is expressed at high levels in glandular epithelial cells and luminal epithelial cells, but expression is also detected in stromal cells.



View larger version (132K):
[in this window]
[in a new window]
  		FIG. 4. Localization of MPGES1, MPGES2, and CPGES proteins in bovine endometrium. Immunohistochemical analysis was performed on six different cows at Day 18 of the estrous cycle as described in Material and Methods. One representative immunohistochemical analysis is shown per enzyme. All three enzymes were localized in epithelial cells, glandular epithelial cells, and stromal cells at various levels. Magnification x200

Expression of MPGES2 and CPGES mRNA in the Endometrium During the Estrous Cycle

We have previously reported MPGES1 mRNA expression during the estrous cycle [10]. Therefore, only MPGES2 and CPGES mRNA expression were studied by Northern blot analysis (Fig. 5). MPGES2 mRNA expression (Fig. 5A) appears higher at the beginning and at the end of the cycle (Days 1–6 and 19–21) in comparison with midcycle, but it is not statistically significant (Days 13–15). CPGES mRNA expression (Fig. 5B) is higher at the beginning of the cycle (Days 1–9) and then follows a progressive decline to reach its lowest point at the follicular phase of the cycle (Days 19–21) (P < 0.05).



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 5. Expression of bovine MPGES2 and CPGES mRNA in the endometrium during the estrous cycle. Tissues were collected from 14 cows at different stages of the estrous cycle. Total RNA was extracted with TRIzol and 15 µg of total RNA were loaded on a formaldehyde gel, then transferred onto a nylon membrane. Northern hybridization was done with cDNA probes specific for bovine MPGES2 (A) and bovine CPGES (B). 18S is shown to standardize RNA quantity on the membrane. Representative blots of one experiment are presented; ratios represent means ± SEM of two different experiments. Results are expressed as ratios of spot density MPGES2/18S and CPGES/18S (arbitrary units). Columns not sharing the same superscript are significantly different

Figure 6 shows MPGES1 (A), MPGES2 (B), and CPGES (C) protein expression throughout the estrous cycle. MPGES1 protein expression is not modulated significantly during the cycle. MPGES2 protein expression is lowest on Days 10–12 and then increases rapidly to reach higher expression at the time of luteolysis (Days 16–18) (P < 0.05). CPGES protein expression is highly variable and does not follow any specific pattern.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 6. Expression of bovine MPGES1, MPGES2, and CPGES proteins in the endometrium during the estrous cycle. Tissues were collected from 21 cows at different stages of the estrous cycle (three per group). Proteins were extracted as described in Material and Methods, and 10 µg of proteins were loaded on a 13% polyacrylamide gel and transferred onto a nitrocellulose membrane. Western hybridization was done with antibodies raised against MPGES1 (A), MPGES2 (B), or CPGES (C). ß-Actin is shown to standardize protein quantity on the membrane. Numbers on the right indicate size of the protein. Representative blots of one experiment are presented; ratios represent means ± SEM of three different experiments. Results are expressed as ratios of spot density MPGES1/ß-actin, MPGES2/ß-actin, and CPGES/ß-actin (arbitrary units). Columns not sharing the same superscript are significantly different

In Vitro Modulation of MPGES2, CPGES, and MPGES1 Proteins Following Treatment with IL-1ß, PMA, and TNF-{alpha} in BEND cells

Figure 7 shows MPGES2 (A), CPGES (B), MPGES1 (C), and COX2 (D) protein expression and PGE2 accumulation (E) in BEND cells following treatment with IL-1ß, PMA, and TNF-{alpha}. In BEND cells, IL-1ß did not affect PGE2 accumulation or PGES or COX2 protein expression. PMA and TNF-{alpha} increased PGE2 accumulation approximately 15-fold and 9-fold, respectively (P < 0.05). Expression of CPGES and MPGES2 proteins was not modulated by any treatment. By contrast, MPGES1 protein expression was increased by PMA (2.5-fold) and by TNF-{alpha} (3.5-fold) (P < 0.05). COX2 protein expression was also significantly increased by PMA (13-fold) and by TNF-{alpha} (10-fold) (P < 0.05).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 7. In vitro modulation of MPGES2, CPGES, MPGES1, and COX2 protein expression in BEND cells. The commercially available endometrial cell line BEND was used in this experiment. Cells were grown until confluence and stimulated or not for 24 h with human IL-1ß (1 ng/ml), PMA (10–7 M), or TNF-{alpha} (10–9 M). Supernatant was kept for PGE2 measurement by ELISA (E). Proteins were extracted and Western blot analysis was performed as described in Material and Methods with antibody raised against MPGES2 (A), CPGES (B), MPGES1 (C), or COX2 (D). ß-Actin is shown as a control of protein quantity. Numbers on the right indicate size of the protein. Representative blots of one experiment are presented; ratios represent means ± SEM of four different experiments. Results are expressed as ratios of spot density MPGES2/ß-actin, CPGES/ß-actin, MPGES1/ß-actin, and COX2/ß-actin (arbitrary units), and PGE2 levels in pg/ml. *, P < 0.05 compared with control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we present the partial sequence of bovine MPGES2 and the complete sequence of bovine CPGES. We also show here for the first time their presence in bovine endometrium, their modulation during the estrous cycle, and the relationship between PGE2 production and expression of the three known PGES in a bovine endometrial cell line.

Bovine MPGES1 was already cloned and characterized [9], but we have cloned here two other enzymes known to possess PGES activity, for which we show the complete sequence of CPGES and partial sequence of MPGES2. The molecular cloning of bovine CPGES and bovine MPGES2 (Figs. 1 and 2, respectively) shows that both enzymes are highly conserved among species with homologies of 99% and 94% in amino acids for CPGES and MPGES2, respectively, compared with their human counterpart. By comparison, bovine MPGES1 is also well conserved, with 85% homology with human MPGES1 at the amino acid level [9]. Therefore, it is not surprising that antibodies raised against human MPGES1 and MPGES2 peptides and human CPGES recombinant protein cross-react with bovine homologous enzymes, both in immunohistochemical and Western blot analyses.

In the first reports of MPGES1 tissue distribution and regulation in bovine [9, 18], only mRNA expression was studied. To evaluate further its potential contribution as an enzyme involved in PGE2 production in bovine, we report here MPGES1 protein expression (Fig. 3A). Interestingly, the results obtained here show the exact same pattern of expression previously shown for mRNA [9, 18]. The results obtained for mRNA expression of MPGES2 and CPGES in bovine tissues are in line with what was found in other species [13, 15].

We also show here modulation of MPGES2 and CPGES during the estrous cycle (Figs. 5 and 6). We have already reported on the coexpression of MPGES1 mRNA with COX2 mRNA during the estrous cycle [10], but protein was not studied at that time. Even if the present results do not show statistical differences, the pattern of protein expression follows what was observed for mRNA, higher at the middle and the end of the cycle (Fig. 3A). While CPGES mRNA expression declines progressively during the cycle, its protein levels do not follow any significant pattern and thus could not be linked with modulation in hormonal status. MPGES2 mRNA expression is fairly high during the first phase of the estrous cycle and decreases to its lowest level at Days 13–15, but no statistical difference is found. Minimal MPGES2 protein expression precedes the minimal expression in mRNA at Days 10–12 and then increases rapidly to its highest expression at Days 16–18, when progesterone levels are high, and then increases with levels of estrogen. If the pattern for MPGES1 expression is globally similar between mRNA and protein, there are differences in patterns of expression for both MPGES2 and CPGES when we compare mRNA and protein. It suggests that those enzymes may be regulated at the posttranscriptional level. It is impossible to draw any conclusion on coexpression of either CPGES or MPGES2 with COX1 during the estrous cycle in the tissues studied because, as we have shown previously, COX1 mRNA and protein are not expressed in the bovine endometrium [10]. Interestingly, when we consider that the bovine endometrium produces PGE2 throughout the estrous cycle, but that this production is higher at mid- and late-luteal phases [25] when progesterone levels decrease, the patterns of expression obtained here demonstrate that PGE2 accumulation follows closely MPGES1 and COX2 expression [10]. Interestingly, the highest point of expression for MPGES2 protein is also in midluteal phase. It is, therefore, difficult to evaluate the contribution of each enzyme to PGE2 production during the estrous cycle without a measurement of their activity and access to their PGH2 substrate.

We have previously reported numerous times that in vitro, stromal cells are responsible for the majority of PGE2 production [17, 22, 26–30]. Therefore, the results obtained here by immunohistochemical analysis are surprising (Fig. 4). Indeed, MPGES1, MPGES2, and CPGES were all expressed at higher levels in luminal and glandular epithelial cells compared with stromal cells. Our results confirm those of Sun et al. [31], who reported recently COX1, COX2, MPGES1, and CPGES immunostaining in the rhesus monkey endometrium during the menstrual cycle. Both PGES were expressed mainly in glandular and luminal epithelial cells, and MPGES1, COX1, and COX2 were not detected at all in stromal cells. In contrast, we detected all three PGES by Western blot and Northern blot analysis in the Macaque endometrium [32]. One explanation could be that luminal and glandular epithelial cells are closer to one another than stromal cells, thus permitting a higher signal intensity. However, we should note that the slides used in the present study were obtained from a nontreated cycling cow at Day 18 of the estrous cycle; it could be interesting to see if slides from cows treated with an agent known to increase PGE2 production (IFN-{tau}, for example) would show a different pattern of expression. Another study from our group showed that EP2 receptors, also known as PTGER2, the main cAMP-generating PGE2 receptors, were expressed in uterine tissues during the estrous cycle and in early pregnancy [33]. Interestingly, their pattern of expression in glandular, luminal epithelial, and stromal cells throughout the estrous cycle, reaching maximal expression between Days 10 and 18, closely matches that of PGES expression [33]. PGH synthase expression without distinction of the two isoforms was first studied in bovine endometrium in vitro, and it was found to be located mainly in epithelial cells and in stromal cells located near the endometrial surface [34]. Very recently, another study from our group demonstrated that COX1 immunoreactive protein was very weak or nonexistent in bovine uterus and that COX2 staining was higher in epithelial cells and in glandular epithelial cells but was also present in stroma [35], which is in correlation with MPGES1 expression reported here (Fig. 4). Interestingly, in bovine endometrium, high expression of COX2 [10], MPGES1 (present results and [10]) and EP2 [33] on Days 10–18 closely follows PGE2 production [25]. One may say that COX2, instead of PGES, should be the rate-limiting step for the production of PGE2 in the endometrium, and, therefore, that the presence of the three PGES at basal level is sufficient to produce PGE2 when PGH2 is formed. In support of that hypothesis, EP2 knock-out mice [36], like COX2 knock-out mice [37], suffer from ovulation, fertilization, and peri-implantation defects, but MPGES1 knock-out mice do not experience any reproductive defects [38]; CPGES and MPGES2 knock-out mice have not been reported yet.

One aim of the current study was to evaluate the contribution of each PGES to PGE2 production in endometrial cells. A recent study by Sweeney et al. [39] showed that antisense oligonucleotides directed against MPGES1 in A549 cells were able to abolish 70% of MPGES1 mRNA and 50% of protein, but led only to a decrease of 40% of PGE2 production following IL-1ß treatment. This raised questions about the contribution of CPGES and MPGES2 for PGE2 production. Results presented in Figure 7, showing higher expression of MPGES1 and COX2, suggest that these are the primary enzymes responsible for increased PGE2 production in the endometrial cell line (BEND cells). Indeed, MPGES1 is the only PGES of which expression is increased concomitantly with PGE2 production following treatment with PMA and TNF-{alpha}. The results presented here, at the protein level, are consistent with our previous observations of an increased MPGES1 mRNA expression and increased PGE2 production in primary cultures of stromal cells following treatment with TNF-{alpha} and in BEND cells following treatment with PMA [18]. We also reported at that time on the coexpression of MPGES1 mRNA with COX2 mRNA, that we confirm here at the protein level. In the present study, MPGES1 is the only PGES that follows PGE2 production in vitro. Indeed, CPGES and MPGES2 show no significant difference of expression following any treatment when compared with control. We can hypothesize that those enzymes contribute primarily to basal PGE2 production. A constitutive expression of CPGES in relation with COX1 has already been hypothesized in other tissues [13, 40, 41]. To speculate more on that result, one may think that increased PGE2 production at the time of recognition of pregnancy may also be due to increased MPGES1 production. In fact, we previously reported on increased MPGES1 and COX2 mRNA expression following IFN-{tau} stimulation in primary epithelial and stromal cells in culture [18]. Another group also showed in the mouse uterus that MPGES1 is increased at implantation sites and in decidualized cells [42]. However, because we report here the presence of both CPGES and MPGES2 in the bovine endometrium and in the bovine endometrial cell line BEND, we cannot exclude the possibility that some PGE2 production comes from the action of CPGES and MPGES2. Indeed, MPGES1 is not the only enzyme responsible for all PGE2 production in endometrium because MPGES1 null mice have been studied recently and showed no reproductive problems [38]. Another study has shown the presence and regulation of CPGES in mouse endometrium during implantation and decidualization [43]. However, in light of the present results, increased MPGES1 expression is clearly associated with increased PGE2 production in bovine endometrial cells following stimulation with PMA and TNF-{alpha}. The exact contribution of each PGES in a reproductive context in bovine remains to be elucidated.

In conclusion, the present study reports for the first time the presence of MPGES2 and CPGES in the bovine endometrium. However, in light of our results in vitro, we hypothesize that MPGES1, in association with COX2, is the main PGES responsible for increased PGE2 production in endometrial cells.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. T.G. Kennedy for generously donating the anti-PGE2 for the ELISA technique, Dr. Stacia Kargman (Merck Frosst, Kirkland, QC, Canada) for kindly providing antibodies raised against COX2, and Pierre Chapdelaine, Joe A. Arosh, and Marianne Parent for technical assistance.


   FOOTNOTES
 
1 Supported by grants from CIHR and NSERC of Canada (MAF). J.P. is a holder of a studentship from NSERC of Canada. Back

2 Correspondence: Michel A. Fortier, Unité de Recherche en Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec (CHUL), Université Laval, 2705 boulevard Laurier, Ste-Foy, QC, Canada G1V 4G2. FAX: 418 654 2765; mafortier{at}crchul.ulaval.ca Back

Received: 8 October 2004.

First decision: 26 October 2004.

Accepted: 21 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Poyser NL. The control of prostaglandin production by the endometrium in relation to luteolysis and menstruation. Prostaglandins Leukot Essent Fatty Acids 1995 53:147-195[CrossRef][Medline]
  2. Dubois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van De Putte LB, Lipsky PE. Cyclooxygenase in biology and disease. FASEB J 1998 12:1063-1073[Abstract/Free Full Text]
  3. McCracken JA, Custer EE, Lamsa JC. Luteolysis: a neuroendocrine-mediated event. Physiol Rev 1999 79:263-323[Abstract/Free Full Text]
  4. Pratt BR, Butcher RL, Inskeep EK. Antiluteolytic effect of the conceptus and of PGE2 in ewes. J Anim Sci 1977 45:784-791
  5. Magness RR, Huie JM, Hoyer GL, Huecksteadt TP, Reynolds LP, Seperich GJ, Whysong G, Weems CW. Effect of chronic ipsilateral or contralateral intrauterine infusion of prostaglandin E2 (PGE2) on luteal function of unilaterally ovariectomized ewes. Prostaglandins Med 1981 6:389-401[CrossRef][Medline]
  6. Lala PK. Interruption of murine pregnancy by activation of antigen-non-specific killer cells in the endometrium with indomethacin, high dose IL-2 or a combination. Res Immunol 1990 141:159-167[CrossRef][Medline]
  7. Jakobsson PJ, Thorén S, Morgenstern R, Samuelsson B. Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci U S A 1999 96:7220-7225[Abstract/Free Full Text]
  8. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 2000 275:32783-32792[Abstract/Free Full Text]
  9. Filion F, Bouchard N, Goff AK, Lussier JG, Sirois J. Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles before ovulation in vivo. J Biol Chem 2001 276:34323-34330[Abstract/Free Full Text]
  10. Arosh JA, Parent J, Chapdelaine P, Sirois J, Fortier MA. Expression of cyclooxygenase 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biol Reprod 2002 67:161-169[Abstract/Free Full Text]
  11. Johnson JL, Beito TG, Krco CJ, Toft DO. Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes. Mol Cell Biol 1994 14:1956-1963[Abstract/Free Full Text]
  12. Hutchison KA, Stancato LF, Owens-Grillo JK, Johnson JL, Krishna P, Toft DO, Pratt WB. The 23-kDa acidic protein in reticulocyte lysate is the weakly bound component of the hsp foldosome that is required for assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90. J Biol Chem 1995 270:18841-18847[Abstract/Free Full Text]
  13. Tanioka T, Nakatani Y, Semmyo N, Murakami M, Kudo I. Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem 2000 275:32775-32782[Abstract/Free Full Text]
  14. Watanabe K, Kurihara K, Suzuki T. Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart. Biochim Biophys Acta 1999 1439:406-414[Medline]
  15. Tanikawa N, Ohmiya Y, Ohkubo H, Hashimoto K, Kangawa K, Kojima M, Ito S, Watanabe K. Identification and characterization of a novel type of membrane-associated prostaglandin E synthase. Biochem Biophys Res Commun 2002 291:884-889[CrossRef][Medline]
  16. Murakami M, Nakashima K, Kamei D, Masuda S, Ishikawa Y, Ishii T, Ohmiya Y, Watanabe K, Kudo I. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. J Biol Chem 2003 278:37937-37947[Abstract/Free Full Text]
  17. Asselin E, Lacroix D, Fortier MA. IFN-{tau} increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro. Mol Cell Endocrinol 1997 132:117-126[CrossRef][Medline]
  18. Parent J, Chapdelaine P, Sirois J, Fortier MA. Expression of microsomal prostaglandin E synthase in bovine endometrium: coexpression with cyclooxygenase type 2 and regulation by interferon-tau. Endocrinology 2002 143:2936-2943[Abstract/Free Full Text]
  19. Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 1988 85:2444-2448[Abstract/Free Full Text]
  20. Chapdelaine P, Ho-Kim MA, Tremblay RR, Dubé JY. Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes. Int J Biochem 1990 22:75-82[CrossRef][Medline]
  21. Chapdelaine P, Vignola K, Fortier MA. Protein estimation directly from SDS-PAGE loading buffer for standardization of samples from cell lysates or tissue homogenates before Western blot analysis. Biotechniques 2001 31:478 480, 482 [Medline]
  22. Asselin E, Goff AK, Bergeron H, Fortier MA. Influence of sex steroids on the production of prostaglandin F2{alpha} and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biol Reprod 1996 54:371-379[Abstract]
  23. Evans CA, Kennedy TG, Challis JR. Gestational changes in prostanoid concentrations in intrauterine tissues and fetal fluids from pregnant sheep, and the relation to prostanoid output in vitro. Biol Reprod 1982 27:1-11[CrossRef][Medline]
  24. Murakami M, Kudo I. Recent advances in molecular biology and physiology of the prostaglandin E2-biosynthetic pathway. Prog Lipid Res 2004 43:3-35[CrossRef][Medline]
  25. Miyamoto Y, Skarzynski DJ, Okuda K. Is tumor necrosis factor {alpha} a trigger for the initiation of endometrial prostaglandin F2{alpha} release at luteolysis in cattle?. Biol Reprod 2000 62:1109-1115[Abstract/Free Full Text]
  26. Fortier MA, Guilbault LA, Grasso F. Specific properties of epithelial and stromal cells from the endometrium of cows. J Reprod Fertil 1988 83:239-248
  27. Kim JJ, Fortier MA. Cell type specificity and protein kinase C dependency on the stimulation of prostaglandin E2 and prostaglandin F2 alpha production by oxytocin and platelet-activating factor in bovine endometrial cells. J Reprod Fertil 1995 103:239-247
  28. Parent J, Villeneuve C, Alexenko AP, Ealy AD, Fortier MA. Influence of different isoforms of recombinant trophoblastic interferons on prostaglandin production in cultured bovine endometrial cells. Biol Reprod 2003 68:1035-1043[Abstract/Free Full Text]
  29. Asselin E, Bazer FW, Fortier MA. Recombinant ovine and bovine interferons tau regulate prostaglandin production and oxytocin response in cultured bovine endometrial cells. Biol Reprod 1997 56:402-408[Abstract]
  30. Asselin E, Drolet P, Fortier MA. Cellular mechanisms involved during oxytocin-induced prostaglandin F2alpha production in endometrial epithelial cells in vitro: role of cyclooxygenase-2. Endocrinology 1997 138:4798-4805[Abstract/Free Full Text]
  31. Sun T, Li SJ, Diao HL, Teng CB, Wang HB, Yang ZM. Cyclooxygenases and prostaglandin E synthases in the endometrium of the rhesus monkey during the menstrual cycle. Reproduction 2004 127:465-473[Abstract/Free Full Text]
  32. Parent J, Chapdelaine P, Fortier MA. Molecular cloning and tissue distribution of microsomal-1 and cytosolic prostaglandin E synthases in macaque. Prostaglandins Other Lipid Mediat; (in press)
  33. Arosh JA, Banu SK, Chapdelaine P, Emond V, Kim JJ, MacLaren LA, Fortier MA. Molecular cloning and characterization of bovine prostaglandin E2 receptors EP2 and EP4: expression and regulation in endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology 2003 144:3076-3091[Abstract/Free Full Text]
  34. Boos A. Immunohistochemical assessment of prostaglandin H-synthase in bovine endometrial biopsy samples collected throughout the oestrous cycle. Anim Reprod Sci 1998 51:261-273[CrossRef][Medline]
  35. Emond V, MacLaren LA, Kimmins S, Arosh JA, Fortier MA, Lambert RD. Expression of cyclooxygenase-2 and granulocyte-macrophage colony-stimulating factor in the endometrial epithelium of the cow is upregulated during early pregnancy and in response to intrauterine infusions of interferon-tau. Biol Reprod 2004 70:54-64[Abstract/Free Full Text]
  36. Tilley SL, Audoly LP, Hicks EH, Kim HS, Flannery PJ, Coffman TM, Koller BH. Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor. J Clin Invest 1999 103:1539-1545[Medline]
  37. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997 91:197-208[CrossRef][Medline]
  38. Trebino CE, Stock JL, Gibbons CP, Naiman BM, Wachtmann TS, Umland JP, Pandher K, Lapointe JM, Saha S, Roach ML, Carter D, Thomas NA, Durtschi BA, McNeish JD, Hambor JE, Jakobsson PJ, Carty TJ, Perez JR, Audoly LP. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc Natl Acad Sci U S A 2003 100:9044-9049[Abstract/Free Full Text]
  39. Sweeney FJ, Wachtmann TS, Eskra JD, Verdries KA, Lambalot RH, Carty TJ, Perez JR, Audoly LP. Inhibition of IL-1beta-dependent prostaglandin E2 release by antisense microsomal prostaglandin E synthase 1 oligonucleotides in A549 cells. Mol Cell Endocrinol 2003 205:151-157[CrossRef][Medline]
  40. Naraba H, Yokoyama C, Tago N, Murakami M, Kudo I, Fueki M, Oh-Ishi S, Tanabe T. Transcriptional regulation of the membrane-associated prostaglandin E2 synthase gene. Essential role of the transcription factor Egr-1. J Biol Chem 2002 277:28601-28608[Abstract/Free Full Text]
  41. Ivanov AI, Pero RS, Scheck AC, Romanovsky AA. Prostaglandin E(2)-synthesizing enzymes in fever: differential transcriptional regulation. Am J Physiol Regul Integr Comp Physiol 2002 283:R1104-R1117[Abstract/Free Full Text]
  42. Ni H, Sun T, Ding NZ, Ma XH, Yang ZM. Differential expression of microsomal prostaglandin E synthase at implantation sites and in decidual cells of mouse uterus. Biol Reprod 2002 67:351-358[Abstract/Free Full Text]
  43. Ni H, Sun T, Ma XH, Yang ZM. Expression and regulation of cytosolic prostaglandin E synthase in mouse uterus during the peri-implantation period. Biol Reprod 2003 68:744-750[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ReproductionHome page
F. Nuttinck, B. M.-L. Guienne, L. Clement, P. Reinaud, G. Charpigny, and B. Grimard
Expression of genes involved in prostaglandin E2 and progesterone production in bovine cumulus-oocyte complexes during in vitro maturation and fertilization
Reproduction, May 1, 2008; 135(5): 593 - 603.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
B. Samuelsson, R. Morgenstern, and P.-J. Jakobsson
Membrane Prostaglandin E Synthase-1: A Novel Therapeutic Target
Pharmacol. Rev., September 1, 2007; 59(3): 207 - 224.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
P. Chapdelaine, J. Kang, S. Boucher-Kovalik, N. Caron, J. P. Tremblay, and M. A. Fortier
Decidualization and maintenance of a functional prostaglandin system in human endometrial cell lines following transformation with SV40 large T antigen
Mol. Hum. Reprod., May 1, 2006; 12(5): 309 - 319.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Waclawik, A. Rivero-Muller, A. Blitek, M. M. Kaczmarek, L. J. S. Brokken, K. Watanabe, N. A. Rahman, and A. J. Ziecik
Molecular Cloning and Spatiotemporal Expression of Prostaglandin F Synthase and Microsomal Prostaglandin E Synthase-1 in Porcine Endometrium
Endocrinology, January 1, 2006; 147(1): 210 - 221.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
S. Chandrasekharan, N. A. Foley, L. Jania, P. Clark, L. P. Audoly, and B. H. Koller
Coupling of COX-1 to mPGES1 for prostaglandin E2 biosynthesis in the murine mammary gland
J. Lipid Res., December 1, 2005; 46(12): 2636 - 2648.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/1/36    most recent
biolreprod.104.037036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parent, J.
Right arrow Articles by Fortier, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parent, J.
Right arrow Articles by Fortier, M. A.
Agricola
Right arrow Articles by Parent, J.
Right arrow Articles by Fortier, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS