Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M103709200 on July 11, 2001

J. Biol. Chem., Vol. 276, Issue 36, 34323-34330, September 7, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/36/34323    most recent
M103709200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Filion, F.
Right arrow Articles by Sirois, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Filion, F.
Right arrow Articles by Sirois, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular Cloning and Induction of Bovine Prostaglandin E Synthase by Gonadotropins in Ovarian Follicles Prior to Ovulation in Vivo*

France Filion, Nadine Bouchard, Alan K. Goff, Jacques G. Lussier, and Jean SiroisDagger§

From the Centre de recherche en reproduction animale and the Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada

Received for publication, April 25, 2001, and in revised form, July 10, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prostaglandin E2 (PGE2) is thought to be an ultimate prostaglandin effector during the ovulatory process, and the objectives of this study were to clone bovine PGE synthase (PGES) and to characterize its regulation by gonadotropins in preovulatory follicles in vivo. The bovine PGES complementary DNA (cDNA) was shown to contain a 5'-untranslated region of eight base pairs (bp), an open reading frame of 462 bp and a 3'-untranslated region of 406 bp. The putative bovine PGES open reading frame encodes a 153-amino acid protein that is 85, 78, and 78% identical to the human, rat, and mouse PGES homologs, respectively. The regulation of PGES during ovulation was studied using three different models in vivo: 1) human chorionic gonadotropin (hCG)-induced ovulation during a normal estrous cycle; 2) hCG-induced ovulation following ovarian hyperstimulation; and 3) spontaneous ovulation during natural estrus. Results from semi-quantitative reverse transcription-polymerase chain reaction/Southern blotting analyses showed that the hCG/luteinizing hormone surge caused a significant increase in PGES mRNA. Levels of PGES transcripts were low or undetectable prior to hCG/luteinizing hormone but increased markedly 18-24 h after hCG in models 1 and 2, and 18-24 h after the onset of natural estrus in model 3 (p < 0.05). Analyses on isolated preparations of granulosa and theca interna cells indicated that the granulosa cell layer was the predominant site of follicular PGES expression. The regulation of the protein was studied in the same models using a specific antibody raised against a fragment of bovine protein (Delta PGES; from Glu49 to Val146). Results from immunoblots showed an induction of bovine PGES (Mr = 17,000) 18-24 h after hCG treatment or onset of estrus (p < 0.05). The protein was detected in extracts of granulosa cells but not in theca interna. Collectively, these results demonstrate that the ovulatory process is associated with a gonadotropin-dependent induction of PGES in granulosa cells of ovarian follicles in vivo, thus establishing for the first time the regulation of the enzyme in a physiological context.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ovulation is essential to all mammalian reproductive cycles and involves a complex series of biochemical and biophysical events that ultimately lead to the rupture of the preovulatory follicle and the release of the maternal germ cell. The ovulatory process shows numerous signs of an acute inflammatory reaction, including hyperemia, edema, leukocyte extravasation, and induction of proteolytic and collagenolytic activities (1-3). Prostaglandins (PG),1 which play a central role in the inflammatory reaction, are also key mediators of ovulation (4-7). Intrafollicular levels of PGs are dramatically increased in the hours preceding ovulation in several species (8-13), and inhibitors of PG synthesis were shown to block ovulation in vivo and in vitro in most species tested (14-21). The predominant and obligatory role of PGE2 is beginning to emerge, as evidenced from the anovulatory phenotype observed in mice deficient for the EP2 PGE2 receptor (22) and the ability of exogenous PGE2, but not PGF2alpha , to restore ovulation in prostaglandin G/H synthase-2 (PGHS-2)-deficient mice (23). Although the specific functions of PGs during ovulation remain to be fully characterized, one putative mechanism involves their role in the activation of the proteolytic/collagenolytic cascade leading to follicular rupture (24).

The molecular control of PG synthesis in ovarian cells prior to ovulation has received much attention in recent years, with efforts focusing primarily on the regulation of PGHS, generally considered the first rate-limiting enzyme in the biosynthetic pathway of all PGs from arachidonic acid (25-30). It has now been clearly established that the marked increased in PG synthesis prior to ovulation results from the selective induction of PGHS-2 (12, 13, 30-37). Several studies have shown that the preovulatory surge of gonadotropins causes an induction of PGHS-2, but not PGHS-1, mRNA, and protein in granulosa cells of ovarian follicles prior to ovulation in vivo (12, 13, 30-32). Interestingly, the time course of PGHS-2 induction after exogenous human chorionic gonadotropin (hCG) treatment was shown to vary greatly among species, being very rapid in rats (2-4 h post-hCG; Ref. 32) and relatively delayed in cattle (18 h post-hCG; Ref. 12) and horses (30 h post-hCG; Refs. 13, 37). This difference in timing of PGHS-2 induction appeared directly related to the species-specific length of the ovulatory process, thereby involving PGHS-2 as a potential regulator of the mammalian ovulatory clock (37, 38). Studies in vitro have established that although the gonadotropin-dependent induction of PGHS-2 in granulosa cells works primarily through the cAMP-dependent protein kinase pathway, other kinases such as protein kinase C and tyrosine kinases could be involved (33). Ultimately, the obligatory role of PGHS-2 expression during the ovulatory process was underscored in PGHS-2 deficient mice that were infertile and exhibited ovulation failure (39, 40). However, though the increase in PG synthesis prior to ovulation clearly involves PGHS-2 induction, the potential regulation of the terminal enzyme involved in the conversion of PGH2 into PGE2, i.e. PGE synthase (PGES), has not been addressed.

The cloning and characterization of the first PGES was recently reported from human cells (41) and shown to correspond to a protein previously identified as microsomal glutathione S-transferase 1 like-1 (MGST1-L1, Ref. 41) or a gene product referred to as p53-induced gene (PIG12; Ref. 42). This initial report was rapidly followed by the isolation of the mouse and rat homologs (43, 44, 45). PGES is a member of the MAPEG (membrane-associated proteins in eicosanoids and glutathione metabolism) superfamily, which also includes microsomal glutathione S-transferase 1 (MGST1), MGST2, MGST3, 5-lipoxygenase activating protein, and leukotriene C4 synthase (46). The enzyme is a 16-kDa membrane-bound protein encoded by a transcript of about 2.0 kb and a gene spanning 14.8 kbp and composed of three exons (41, 47). Interestingly, the enzyme was shown to be induced in vitro by various proinflammatory stimuli already known to induce PGHS-2 expression including lipopolysaccharide (LPS), interleukin (IL)-1beta , and tumor necrosis factor (TNF)-alpha (41, 43, 44, 48, 49). The induction of PGES has also been reported recently in two inflammatory models in vivo, including LPS-induced pyresis and adjuvant-induced arthritis (43, 44). However, the regulation of PGES has not been described under physiological conditions. The dramatic and obligatory increase in PGE2 synthesis during the ovulatory process raises the possibility that PGES could act as a gonadotropin-regulated gene in the ovary and be involved in the regulation of this key physiological process. Therefore, the general objective of the present study was to characterize the expression of PGES in ovarian follicles during ovulation. The specific objectives were to clone and determine the primary structure of bovine PGES and to study the regulation of PGES mRNA and protein by gonadotropins in bovine follicles in specific models of ovulation in vivo.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The QuikHyb hybridization solution and the ExAssist/SOLR system were purchased from Stratagene Cloning Systems (La Jolla, CA). [alpha -32P]dCTP was obtained from Mandel Scientific-New England Nuclear Life Science Products (Mississauga, Ontario, Canada). Prime-a-Gene labeling system, Access RT-PCR kit, pGEM-T easy Vector System I were purchased from Promega (Madison, WI). TRIzol total RNA isolation reagent, 1-kbp DNA ladder, and synthetic oligonucleotides were obtained from Life Technologies, Inc. Biotrans nylon membranes (0.2 µm) were purchased from ICN Pharmaceuticals (Montréal, Québec, Canada), and Hybond-P polyvinylidene difluoride membranes, Rainbow molecular weight markers, ECL plus, horseradish peroxidase-linked donkey anti-rabbit secondary antibody, PGEX-2T vector, BL-21 protease-deficient Escherichia coli strain, and glutathione-Sepharose beads were obtained from Amersham Pharmacia Biotech. Folltropin-V (follicle-stimulating hormone) was purchased from Vetrepharm Canada Inc. (Ontario, Canada), and APL (hCG) was obtained from Ayerst laboratories (Montréal, Québec, Canada). Bio-Rad Protein Assay and all electrophoretic reagents were purchased from Bio-Rad Laboratories. Expand High Fidelity DNA polymerase was purchased from Roche Molecular Biochemicals.

Cloning of Bovine PGES-- To clone the bovine PGES cDNA, a bovine cDNA library prepared with mRNA isolated from a preovulatory follicle obtained 24 h post-hCG (50) was screened with a human PGES cDNA (42). The probe was labeled with [alpha -32P]dCTP using the Prime-a-Gene labeling system (Promega) to a final specific activity greater than 1 × 108 cpm/µg DNA. Approximately 250,000 phage plaques were screened, and hybridization was performed at 55 °C with QuikHyb hybridization solution (Stratagene). Four positive clones were plaque-purified through secondary and tertiary screenings, and pBluescript phagemids containing the cloned DNA insert were excised in vivo with the Ex-Assist/SOLR system (Stratagene). DNA sequencing was performed commercially (Université Laval, Québec, Canada).

To obtain the missing 5'-end of bovine PGES, a three-step nested PCR approach was designed. Three sense primers specific for sequences located near the multiple cloning site of the pBluescript vector (S1 = 5'-ACAGGAAACAGCTATGACCTTGATTACG-3', S2 = 5'-CTCGAAATTAACCCTCACTAAAGGG-3', S3 = 5'-CCTGCAGGTCGACACTAGTGGATCC-3') and three antisense primers derived from the bovine PGES cDNA clone (AS1 = 5'-TACATCCCTGGATTCAGAAGGTCG-3', AS2 = 5'-TATCAATCGTGACGGTCCGTCTC-3', AS3 = 5'-ATGCCACGGTGTGTACCATACGG-3') were synthesized commercially (Life Technologies). For the first PCR reaction, a sample (5 µl) of the bovine cDNA library was denatured for 15 min at 94 °C, and amplification was performed with 1 µl of 20 µM of external sense (S1) and antisense (AS1) primers, 1 µl of 25 mM of dNTP (Amersham Pharmacia Biotech), 0.5 µl of Expand High Fidelity DNA polymerase and 5 µl of 10× PCR buffer (Roche Molecular Biochemicals) in a total reaction volume of 50 µl. The reaction was run in an Omnigene TR3 SM5 thermal cycler (Hybaid Limited, Franklin, MA) for 20 cycles of 94 °C for 15 s, 62 °C for 45 s, and 68 °C for 1 min. The second PCR reaction was performed using 2 µl from the first PCR reaction, middle sense (S2) and antisense (AS2) primers, and 30 cycles of PCR conditions described above. The final nested PCR reaction was performed using 2 µl from the second PCR reaction, internal sense (S3) and antisense (AS3) primers, and 40 cycles of PCR conditions described above. After electrophoresis on a 1% 0.04 M Tris-acetate, 0.001 M EDTA-agarose gel, the DNA fragment was excised and ligated into pGEM-T easy vector (Promega) according to the manufacturer's protocol. DNA sequencing was performed commercially, and the missing 5'-end of the bovine PGES open reading frame was revealed.

A 850-bp fragment of the bovine GAPDH cDNA was generated by RT-PCR using a sense (5'-TGTTCCAGTATGATTCCACCC-3') and an antisense primer (5'-TCCACCACCCTGTTGCTGTA-3') (36), and the Access RT-PCR System (Promega) according to the manufacturer's protocol. The PCR product was isolated after electrophoresis, subcloned into pGEM-T easy Vector, and sequenced to confirm its identity.

In Vivo Models of Ovulation-- The regulation of PGES expression during the ovulatory process in vivo was studied in three distinct models previously characterized by us (12, 35, 51). For all models, Holstein heifers 2-3 year old that exhibited normal estrous cycles were used, and follicular development was monitored by real-time ultrasonography (12). Briefly, in the first model (hCG-induced ovulation during a normal cycle), bovine preovulatory follicles were obtained after induction of luteolysis on day 7 of the estrous cycle (12). An ovulatory dose of hCG (3000 IU, intravenously) was administered after induction of luteolysis, and the ovary bearing the preovulatory follicle was isolated by ovariectomy (via colpotomy) at various time points after hCG (0-26 h post-hCG) (12). The interval of time from hCG administration to ovulation is 26-28 h post-hCG in this model. In the second model (hCG-induced ovulation following ovarian hyperstimulation), the development of multiple preovulatory follicles was stimulated by the administration of a standard 4-day protocol of exogenous follicle-stimulating hormone (Folltropin-V, Vetrepharm Canada Inc.) (51). After induction of luteolysis, hCG (2500 IU) was administered to induce ovulation, and ovariectomies were performed at 0, 18, and 24 h after hCG treatment (51). In this model, multiple ovulations are expected 28-30 h post-hCG (51). In the third model (spontaneous ovulation during natural estrus), corpus luteum regression was induced on day 7 of the cycle, and animals were allowed to come into estrus (35). The preovulatory follicle was obtained by ovariectomy at specific times after the onset of estrus (0, 18, and 24 h after onset of estrus). In this model, the onset of estrus coincides with the endogenous LH surge, and the interval from the LH surge to ovulation is ~30 h (35). For all three models, the preovulatory follicles were dissected from the ovary with a scalpel, and pieces of follicle wall (i.e. theca interna with attached granulosa cells) were prepared as previously described (12). In selected cases, some pieces of follicle wall were further dissected into isolated preparations of granulosa cells and theca interna (12). All tissue samples were stored at -70 °C. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Université de Montréal.

RNA Extraction and Semi-quantitative RT-PCR/Southern Blotting-- Total RNA was extracted from bovine preovulatory follicles and various tissues using TRIzol (Life Technologies, Inc.) and a Kinematica PT 1200C Polytron Homogenizer. All non-ovarian follicular tissues were obtained from a slaughterhouse. The Access RT-PCR System (Promega) was used for semi-quantitative analysis of PGES and GAPDH mRNA levels. Reactions were performed as directed by the manufacturer using sense (5'-TGATGAACGGCCAGGTGCTC-3') and antisense (5'-ATGCCACGGTGTGTACCATACGG-3') primers specific for bovine PGES, and sense (5'-TGTTCCAGTATGATTCCACCC-3') and antisense (5'-TCCACCACCCTGTTGCTGTA-3') primers specific for bovine GAPDH (36). These reactions resulted in the generation of PGES and GAPDH DNA fragments of 330 and 850 bp, respectively. Each reaction was performed using 100 ng of total RNA, and cycling conditions were one cycle of 48 °C for 45 min and 94 °C for 2 min, followed by a variable number of cycles of 94 °C for 30 s, 59 °C for 1 min, and 68 °C for 2 min. The number of cycles used was optimized for each gene to fall within the linear range of PCR amplification and were 22 and 13 cycles for PGES and GAPDH, respectively. Following PCR amplification, samples were electrophoresed on 2% 0.04 M Tris-acetate, 0.001 M EDTA-agarose gels, transferred to nylon membranes, and hybridized with corresponding radiolabeled PGES and GAPDH cDNA fragments using QuikHyb hybridization solution (Stratagene). Membranes were exposed to a phosphor screen, and signals were quantified on a Storm imaging system using the ImageQuant software version 1.1 (Molecular Dynamics).

Production of an Anti-bovine PGES Antibody-- A pair of sense (5'-GATGGATCCGAGGACGCTCAGAGACATGGA-3') and antisense (5'-TCAGAATTCGACAATCTGCAGGGCCATGGA-3') primers that incorporated a BamHI and an EcoRI restriction site, respectively, were designed from the bovine PGES open reading frame to generate a fragment (Delta PGES) spanning the region from Glu49 to Val146. The fragment was amplified by PCR using the Expand High Fidelity polymerase (Roche Molecular Biochemicals) and following the manufacturer's protocol. The fragment was isolated after electrophoresis, digested with BamHI and an EcoRI, subcloned into pGEX-2T in frame with the GST coding region (Amersham Pharmacia Biotech), and sequenced to confirm its integrity. Protease-deficient E. coli BL-21 (Amersham Pharmacia Biotech) were transformed with the Delta PGES/pGEX-2T construct, expression of recombinant Delta PGES/GST fusion protein was induced with isopropyl-1-thio-beta -D-galactopyranoside, and bacterial protein extracts were obtained after sonication and centrifugation (52). The Delta PGES/GST fusion protein was purified by affinity on glutathione-Sepharose beads (Amersham Pharmacia Biotech), digested with thrombin to release the Delta PGES, resolved by one-dimensional SDS-PAGE, transferred on nitrocellulose, and stained with Ponceau S Red (52). The Delta PGES band (Mr = 11,000) was cut and used to immunize rabbits as previously described (52).

Cell Extracts and Immunoblot Analysis-- Cell extracts were prepared as previously described (12). Briefly, tissues were homogenized in TED buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1 mM diethyldithiocarbamic acid) containing 0.1% Tween 20 and centrifuged at 30,000 × g for 1 h at 4 °C. The crude pellets (membranes, nuclei, mitochondria) were sonicated in TED sonication buffer (20 mM Tris, pH 8.0, 50 mM EDTA, 0.1 mM DEDTC) containing 1.0% Tween 20. The sonicates were centrifuged at 16,000 × g for 15 min at 4 °C. The recovered supernatant (cell extract) was stored at -80 °C until electrophoretic analyses were performed. Protein concentration was determined by the method of Bradford (53) (Bio-Rad Protein Assay). Samples (5-100 µg of proteins) were resolved by one-dimensional SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (12). Membranes were incubated with the polyclonal anti-bovine PGES antibody (1:1000), and immunoreactive proteins were visualized by incubation with the horseradish peroxidase-linked donkey anti-rabbit secondary antibody (1:3000 dilution) and the enhanced chemiluminescence system (ECL plus) following the manufacturer's protocol (Amersham Pharmacia Biotech).

Statistical Analyses-- One-way ANOVA was used to test the effect of time after hCG or after the onset of estrus on levels of PGES mRNA levels. When ANOVAs indicated significant differences (p < 0.05), Dunnett's test was used for multiple comparisons with the control (0 h post-hCG or post-estrus). Statistical analyses were performed using JMP software (SAS Institute, Inc., Carry, NC).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation and Characterization of Bovine PGES-- Two strategies were used to clone the bovine PGES cDNA. In the first approach, four positive clones were isolated from the screening of 250,000 phage plaques of a bovine follicular cDNA library with a human PGES cDNA probe. Sequencing results showed that the longest clone (clone Bo2-4) did not contain the full-length open reading frame and started at a region corresponding to the amino acid Ala43 of human PGES. Thus, a second approach involving nested PCR was used and allowed to obtain the missing 5'-end of bovine PGES. The deduced bovine PGES cDNA was shown to include a 5'-untranslated region of 8 bp, an open reading frame of 462 bp (including the stop codon), and a 3'-untranslated region of 406 bp (Fig. 1).


View larger version (53K):
[in this window]
[in a new window]
  		Fig. 1.   Nucleotide sequence of the bovine PGES cDNA. The bovine PGES cDNA was cloned by library screening, as described under "Experimental Procedures". The bovine PGES cDNA is composed of a 5'-untranslated region of 8 bp (lowercase letters), an open reading frame of 462 bp (uppercase letters), and a 3'-untranslated region of 406 bp (lowercase letters). The translation initiation (ATG) and stop (TGA) codons are shown in bold types, and numbers on the right refer to the last nucleotide on that line. The nucleotide sequence was submitted to GenBankTM (accession number AY032727).

The amino acid sequence of bovine PGES was deduced from the cDNA, and comparisons were made with other mammalian homologs (Fig. 2). The coding region of the bovine PGES cDNA encodes a 153-amino acid protein, which is similar in length to rat and mouse PGES but one residue longer than human PGES. Overall comparisons between bovine PGES and the human, rat, and mouse homolog revealed a 85, 78 and 78% identity at the amino acid level, respectively. The central region of the protein appeared well conserved, whereas the 5'- and 3'-ends were more divergent (Fig. 2).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 2.   Deduced amino acid sequence of bovine PGES and comparison with other mammalian homologs. The amino acid sequence of bovine (bov) PGES is aligned with the human (hum), rat, and mouse (mou) homologs. Identical residues are marked with a printed period, hyphens indicate gaps in protein sequences created to optimize alignment, and numbers on the right refer to the last amino acid on that line.

Tissue Distribution of Bovine PGES-- The distribution of PGES mRNA expression was studied in various bovine tissues by RT-PCR/Southern blot. Results showed that levels of PGES transcripts varied across tissues (Fig. 3). Levels of PGES mRNA were highest in the seminal vesicle and in a preovulatory follicle obtained 24 h after hCG treatment, moderate to high in the stomach, intestine, pituitary and liver, and relatively low in other tissues tested. In contrast, levels of GAPDH mRNA were relatively constant in the same tissues (Fig. 3).


View larger version (69K):
[in this window]
[in a new window]
  		Fig. 3.   Expression of PGES mRNA in bovine tissues. Total RNA was extracted from various bovine tissues, and samples (100 ng) were analyzed for PGES and GAPDH (control gene) content by a semiquantitative RT-PCR/Southern blotting technique, as described under "Experimental Procedures". A, expression of PGES mRNA in bovine tissues. B, expression of GAPDH mRNA in bovine tissues. Numbers of PCR cycles for each gene were within the linear range of amplification, and they represented 22 and 13 cycles for PGES and GAPDH, respectively. Numbers on the right indicate the size of the PCR fragment. The sample denoted Follicle was obtained from a preovulatory follicle isolated 24 h after an ovulatory dose of hCG.

Induction and Cellular Localization of PGES mRNA in Bovine Follicles Prior to Ovulation-- To study the potential regulation of PGES mRNA in bovine follicles during the ovulatory process, preovulatory follicles were isolated every 6 h between 0 and 24 h after hCG treatment, and samples of total RNA were extracted from the follicle wall (theca interna with attached granulosa cells) and analyzed by semi-quantitative RT-PCR/Southern blotting. Results showed a marked regulation of steady state levels of PGES transcripts in bovine follicles after hCG treatment in vivo. Whereas low or undetectable levels of PGES mRNA were present at 0 h, a marked and progressive induction was observed after hCG treatment, with levels being maximal at 18 and 24 h post-hCG (Fig. 4A). When results from several follicles isolated at 0 (n = 4), 6 (n = 2), 12 (n = 4), 18 (n = 4), and 24 h (n = 4) post-hCG were expressed as ratios of PGES to GAPDH, a significant increase was observed in levels present at 18-24 h as compared with 0 h post-hCG (p < 0.05; Fig. 4C).


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4.   Time-dependent induction of PGES mRNA by hCG in bovine preovulatory follicles. Total RNA extracts were prepared from bovine preovulatory follicles isolated 0, 6, 12, 18, and 24 h after the administration of an ovulatory dose of hCG, and samples (100 ng) were analyzed for PGES and GAPDH (control gene) content by a semiquantitative RT-PCR/Southern blotting technique, as described under "Experimental Procedures". A, regulation of PGES mRNA in bovine follicles (one representative follicle per time point). B, constitutive expression of GAPDH mRNA in the same bovine follicles. Numbers on the right indicate the size of the PCR fragment. C, relative changes in PGES transcripts in bovine follicles after hCG treatment. The intensity of PGES and GAPDH signals were quantified using a Storm imaging system (Molecular Dynamics), and data from PGES mRNAs were normalized with the control gene GAPDH (n = four distinct follicles, i.e. animals, per time point at 0, 12, 18, and 24 h post-hCG, and n = two distinct follicles at 6 h post-hCG). Bars marked with an asterisk are significantly different from 0 h post-hCG (p < 0.05).

To establish the cellular localization of PGES mRNA expression in bovine preovulatory follicles, isolated preparations of granulosa cells and theca interna were obtained from follicles isolated at 6, 12, 18, 20, 22, and 24 h post-hCG and analyzed by RT-PCR/Southern blotting (Fig. 5). The results indicated that the granulosa cell layer was the predominant site of PGES transcripts in bovine follicles obtained between 12 and 24 h post-hCG (Fig. 5). The presence of very low levels of PGES mRNA in granulosa cells at 6 h post-hCG (Fig. 5) is in keeping with results obtained with the intact follicle wall at the same time point (Fig. 4). Levels of PGES mRNA in theca interna remained low and relatively constant (Fig. 5).


View larger version (49K):
[in this window]
[in a new window]
  		Fig. 5.   Cellular localization of PGES mRNA in bovine preovulatory follicles. Bovine preovulatory follicles were isolated 6, 12, 18, 20, 22, and 24 h after the administration of an ovulatory dose of hCG. Each follicle was dissected into preparations of granulosa cells and theca interna, and samples (100 ng) of total RNA were analyzed for PGES and GAPDH (control gene) content by a semiquantitative RT-PCR/Southern blotting technique, as described under "Experimental Procedures". A, expression of PGES mRNA in granulosa cells (GC) and theca interna (TI). B, expression of GAPDH in the same follicular tissues. Numbers on the right indicate the size of the PCR fragment.

Gonadotropin-dependent Induction of PGES mRNA in Bovine Follicles after Ovarian Hyperstimulation and during Natural Estrus-- The cow is generally a monoovulatory species, thus producing a single preovulatory follicle per animal during each reproductive cycle, as observed in the model described above (Figs. 4 and 5). However, the development of multiple preovulatory follicles can be induced in this species with proper exogenous gonadotropin treatments. To further investigate the regulation of PGES mRNA in bovine follicles in vivo and to determine whether PGES is expressed during induced multiple ovulations, the expression of PGES mRNA was studied in follicles isolated 0, 18, and 24 h post-hCG in animals subjected to a conventional ovarian hyperstimulation protocol (51). Results showed a dramatic regulation of PGES transcripts during hCG-induced multiple ovulations (Fig. 6). Levels of PGES mRNA were undetectable at 0 h but markedly increased at 18 and 24 h post-hCG (Fig. 6A). When results from multiple follicles isolated at 0 (n = 7), 18 (n = 8), and 24 h (n = 9) post-hCG were expressed as ratios of PGES to GAPDH a significant induction was observed at 18 and 24 h post-hCG (p < 0.05; Fig. 6C).


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 6.   Induction of PGES mRNA by gonadotropins in bovine preovulatory follicles after ovarian hyperstimulation and during spontaneous ovulation. Total RNA extracts were prepared from bovine preovulatory follicles, and samples (100 ng) were analyzed for PGES and GAPDH (control gene) content by a semiquantitative RT-PCR/Southern blotting technique, as described under "Experimental Procedures". A, regulation of PGES mRNA in bovine preovulatory follicles isolated during hCG-induced ovulation following ovarian hyperstimulation (one representative follicle per time point). B, constitutive expression of GAPDH mRNA in the same bovine follicles. Numbers on the right indicate the size of the PCR fragment. C, relative changes in PGES transcripts in bovine follicles after hCG treatment and ovarian hyperstimulation. The intensity of the PGES and GAPDH signal was quantified using a Storm imaging system (Molecular Dynamics), and data from PGES mRNAs were normalized with the control gene GAPDH (n = 7, 8, and 9 follicles per time point for 0, 18 ,and 24 h, respectively). Bars marked with an asterisk are significantly different from 0 h post-hCG (p < 0.05). D, regulation of PGES mRNA in bovine preovulatory follicles isolated 0, 18, and 24 h after the onset of a natural estrus (one representative follicle per time point). E, constitutive expression of GAPDH mRNA in the same bovine follicles. F, relative changes in PGES transcripts in bovine follicles isolated during natural estrus. Results from PGES were normalized with the control gene GAPDH (n = 2, 3, and 4 follicles per time point for 0, 18, and 24 h, respectively). Bars marked with an asterisk are significantly different from 0 h post-estrus (p < 0.05).

In the two previous models, i.e. hCG-induced ovulation during a normal cycle (Figs. 4 and 6A) and hCG-induced ovulation following ovarian hyperstimulation (Fig. 6), exogenous human CG was administered to animals to trigger the ovulatory process. To clearly establish that the induction of PGES in bovine preovulatory follicles is a physiological event triggered by the natural endogenous LH surge, the regulation of PGES mRNA was studied in follicles isolated 0, 18, and 24 h after the onset of estrus. In cattle, the onset of estrus coincides with the endogenous LH surge (35). Results showed that levels of PGES mRNA were low at 0 h and significantly increased 18 and 24 h after the onset of estrus (p < 0.05; Fig. 6, D-F).

Gonadotropin-dependent Induction and Cellular Localization of PGES Protein in Bovine Follicles during Ovulation-- To determine whether the gonadotropin-dependent induction of PGES mRNA in bovine follicles prior to ovulation was associated with changes in levels of PGES protein, a specific antibody was generated against a fragment of recombinant bovine PGES and used in immunoblot analyses. Fig. 7A shows the reactivity of the antibody against the purified PGES fragment (Delta PGES; Mr = 11,000) that served as immunizing antigen. In the subsequent experiment, an immunoblot analysis was performed on extracts prepared from various bovine tissues and from a preovulatory follicle obtained 24 h after hCG treatment. Results revealed the presence of a very strong immunoreactive PGES signal in the seminal vesicle extract, with the signal appearing as a Mr = 17,000 band (Fig. 7B), in keeping with the predicted size of the protein. Among other tissues tested, a weak but detectable PGES signal (Mr = 17,000) was detected only in the preovulatory follicle sample (Fig. 7B).


View larger version (44K):
[in this window]
[in a new window]
  		Fig. 7.   Time-dependent induction of PGES protein by hCG in bovine preovulatory follicles. A specific polyclonal antibody against a fragment of the bovine PGES protein (Delta PGES; from amino acid residue Glu49 to Val146; Fig. 2) was generated as described under "Experimental Procedures". A, a sample of the recombinant PGES fragment (Delta PGES) used as immunizing antigen was analyzed by one-dimensional SDS-PAGE and immunoblotting. The size of Delta PGES PGES was approximately Mr = 11,000, as expected. B, protein extracts (10 µg/lane) prepared from various bovine tissues and a preovulatory follicle isolated 24 h post-hCG were analyzed by one-dimensional SDS-PAGE and immunoblotting. A very strong PGES signal (Mr = 17,000) was observed in the seminal vesicle, whereas only a very weak band was present in the preovulatory follicle sample. C, extracts were prepared from bovine preovulatory follicles isolated 0, 6, 12, 18, 20, 22, 24, and 26 h after hCG treatment, and protein samples (100 µg/lane; 10 times more than in panel B) were analyzed by one-dimensional SDS-PAGE and immunoblotting. A sample of seminal vesicle (5 µg) was included as a positive control. Markers on the right indicate the migration of molecular weight standards (panels A and B), or the size of immunoreactive PGES (panel C).

To study the regulation of PGES protein in vivo, extracts of preovulatory follicles isolated between 0 and 26 h after hCG were analyzed by Western blots. Results showed a marked induction of PGES protein (Mr = 17,000) after hCG treatment (Fig. 7C). Whereas levels of PGES protein were low or undetectable at 0, 6, and 12 h, they first increased at 18 h and reached their maximum at 24 h post-hCG. Similar results were obtained when the analysis presented in Fig. 7 was repeated with another set of extracts prepared from eight different preovulatory follicles (data not shown).

In the last series of experiments, the regulation of PGES protein was studied in bovine preovulatory follicles during natural estrus. Results showed that immunoreactive PGES (Mr = 17,000) was not present at the onset of estrus (0 h), but was induced 18 h and 24 h later (Fig. 8A). The cellular localization of PGES protein was studied in isolated preparations of granulosa cells and theca interna obtained from three preovulatory follicles collected 24 h after the onset of estrus. Results revealed that immunoreactive PGES was detected only in extracts of granulosa cells (Fig. 8B).


View larger version (56K):
[in this window]
[in a new window]
  		Fig. 8.   Regulation and cellular localization of PGES protein in bovine preovulatory follicles during spontaneous ovulation. A, bovine preovulatory follicles were isolated 0, 18, and 24 h after the onset of estrus (n = two follicles per time point). In this model, the onset of estrus (0 h) coincides with the endogenous LH surge. Extracts were prepared, and protein samples (100 µg/lane) were analyzed by one-dimensional SDS-PAGE and immunoblotting using the anti-bovine PGES antibody. B, isolated preparations of granulosa cells (GC) and theca interna (TI) were obtained from three preovulatory follicles (i.e. animals; 845, 842, 849) isolated 24 h after the onset of estrus, and samples of protein extracts (100 µg/lane) were analyzed by SDS-PAGE and immunoblotting. Markers on the right indicate the size of immunoreactive PGES.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study is the first to demonstrate the regulation of PGES in a physiological context in vivo. Previous studies have documented the effects of different proinflammatory agonists on the regulation of PGES expression in vitro, including induction by IL-1beta , LPS, TNF-alpha , beta-amyloid, and phorbol 12-myristate 13-acetate in A549 human lung adenocarcinoma cells (46, 48), rat osteoblasts, peritoneal macrophages and astrocytes (43, 54), and human dermal fibroblasts and vascular smooth muscle cells (49). Also, recent reports showed that PGES induction occurs in vivo in various rat tissues following an LPS challenge (43, 44) and in the paws of rats subjected to a pharmacological model of adjuvant-induced arthritis (44). However, the regulation of PGES under physiological conditions had not been described. Using the bovine preovulatory follicle model, the present study provides the first evidence of a marked regulation of PGES in the hours just prior to ovulation, a process during which PG synthesis is obligatory (7, 40). Our results indicate that PGES expression is not constitutive in follicular cells but is induced by gonadotropins prior to follicular rupture. One could predict that the control of PGES expression is likely involved in other reproductive processes in which PGE2 is known to play a role, including fertilization, decidualization, and implantation (40, 55, 56).

One important outcome of the present study is the demonstration that the induction of PGES by gonadotropins in bovine preovulatory follicles directly parallels the induction of PGHS-2 previously described in the same three models in vivo (12, 35, 51). As observed for PGES, induction of PGHS-2 protein occurred between 18-26 h post-hCG in this model of induced ovulation during a normal estrous cycle (12). Similarly, PGHS-2 expression was shown to be up-regulated 18 and 24 h after hCG-induced ovulation following ovarian hyperstimulation (51), as noted for PGES in the present study. Lastly, the physiological nature of this phenomenon was confirmed in a previous report where PGHS-2 induction was observed 18 and 24 h after the onset of estrus (i.e. the LH preovulatory surge) (35), as observed for PGES. It should also be noted that in all three models the induction of PGHS-2 (and PGES) is associated with a dramatic increase in intrafollicular concentrations of PGE2 (12, 35, 51). Thus, collectively, these results suggest that the preovulatory rise in follicular prostaglandin synthesis is dependent on a coordinate induction of both PGHS-2 and PGES gene expression by gonadotropins. Future studies should establish whether common or distinct molecular mechanisms are responsible for the striking similarities observed in the control of these two genes. The functional coupling of PGHS-2 and PGES in the ovary appears as an efficient mean to regulate the conversion of arachidonic acid into PGE2, as both enzymes are inducible isoforms of membrane-bound proteins acting sequentially in the PG biosynthetic pathway (57). The apparent link between PGHS-2 and PGES induction after gonadotropin treatment in preovulatory follicles in vivo compares with the effect of LPS and other proinflammatory stimuli on the co-induction of both enzymes in various tissues and cell types in vivo and in vitro (43, 44, 45, 48). Such similarities are in keeping with the established parallel between the physiological process of ovulation and an acute inflammatory reaction (1-3).

The level of PGES mRNA expression in preovulatory follicles isolated 24 h post-hCG was very high when compared with other non-ovarian tissues tested, being equaled only by levels observed in the seminal vesicle. However, despite the equivalent amount of PGES transcript in both tissues, the level of PGES protein in the seminal vesicle far surpassed the level present in the preovulatory follicle. Although the basis for this important difference remains to be clearly established, the very transient nature of PGES transcript induction in preovulatory follicles, as compared with its high constitutive expression in seminal vesicles, is likely to account, at least in part, to differences in protein accumulation. The detection of high levels of PGES in bovine seminal vesicles compares with results observed in humans (41) and rats (44), but this is the first report to document the localization of PGES in ovarian cells. Our results indicate that the first important rise in PGES mRNA expression occurred 12 h post-hCG, whereas increase in the protein was detected only after 18 h of treatment, which could relate to the sequential nature of transcription and translation events. However, the ability to detect the transcript several hours prior to the protein could be due, at least in part, to the higher sensitivity of the technique used to study the message (RT-PCR/Southern blotting) as compared with the one employed for the protein (immunoblotting). The preovulatory follicle contains two major cell layers, the granulosa and the theca interna, and this study establishes that the granulosa cell layer is the predominant site of follicular PGES expression. In contrast, the PGES transcript was present only at very low levels in theca interna, and our inability to detect low levels of PGES protein in this layer may again relate to the limit of sensitivity of the analysis. Because previous studies showed that PGHS-2 is selectively induced by gonadotropins in granulosa cells of bovine follicles (12, 35), as well as in other species (13, 31, 37), the present study suggests that the increase in PGE2 synthesis prior to ovulation could also involve a concomitant induction of PGES in the same cell type (12). Future studies will be needed to characterize the subcellular localization of PGES expression in granulosa cells, but one could predict abundant perinuclear localization of the enzyme, as observed for PGHS-2 in these cells (35) and based on the recent perinuclear co-localization of PGES and PGHS-2 in HEK293 cells (43).

Lastly, this report is the first to describe the cloning and characterization of bovine PGES, and sequencing results further underscore emerging similarities and differences among the primary structure of the enzyme across species. Comparative analyses show that the amino acid sequence of the bovine protein is relatively similar to other known mammalian homologs, being more than 77% identical to human (41), mouse (43), and rat (43, 44, 54) PGES. These multiple comparisons may also help define putative functional domains. The Arg110 amino acid residue shown to be crucial for catalytic function in human PGES (43) is conserved in the bovine enzyme (Arg111). Also, two internal regions (Arg39 to Ala51 and Asp63 to Pro97) showing 100% identity across species may contain important structural/regulatory domains. In contrast, the amino-terminal (Met1 to Leu14) and carboxyl-terminal (Lys121 to Leu153) portions of the protein appear much less conserved, with an average of 59 and 61% identity, respectively, when these regions of bovine PGES are compared with the corresponding domains of the rodent enzymes. The PGES isoform isolated in the present study represents one the two PGES isoforms cloned thus far and has been referred to as mPGES for membrane-bound PGES (43). The enzyme is glutathione-dependent, appears as an inducible isoform functionally linked to PGHS-2, and has been proposed as an important regulator of the inflammatory process (41, 43-45, 48). Interestingly, a second glutathione-dependent PGES isoform, referred to as cPGES for cytosolic PGES, has recently been identified (58). Besides its distinct localization (cytosolic versus membrane-bound), cPGES is constitutively expressed in various tissues, appears functionally coupled to PGHS-1, and is thought to be involved in the synthesis of PGE2 needed to maintain tissue homeostatic functions. Although the absence of PGHS-1 expression and of constitutive PG synthesis in granulosa cells does not suggest that cPGES is present in this cell type, future studies will be needed to specifically investigate this issue.

In summary, this report demonstrates for the first time that the ovulatory process is accompanied by a gonadotropin-dependent induction of PGES in granulosa cells of ovarian follicles in vivo, thus establishing the regulation of the enzyme in a physiological context. Combined with results from previous work showing the induction of follicular PGHS-2 in preovulatory follicles (12, 35, 51), the present study suggests that the increase in PGE2 synthesis prior to ovulation is accompanied by the coordinate induction of both enzymes. Because the cAMP-dependent protein kinase pathway is the primary second messenger system involved in transducing gonadotropin action (59), the present study also adds to the emerging variety of agonists and signaling pathways able to control PGES gene expression. Ovarian follicles and granulosa cells represent interesting models to study the fine molecular mechanisms involved in the regulation of the PGES gene, and considering the obligatory role of PGE2 synthesis during the ovulatory process, they could also provide a valuable approach in the testing of new PG synthesis inhibitors.

    ACKNOWLEDGEMENT

We thank Dr. B. Vogelstein (Johns Hopkins University) for providing the human PIG12 (PGES) cDNA.

    FOOTNOTES

* This work was supported by Canadian Institutes of Health Research (CIHR) Grant MT-13190 (to J. S.) and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche Grant 99-ER-3016 (to J. G. L. and J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AY032727.

Dagger Supported by a CIHR Investigator Award.

§ To whom correspondence should be addressed: Tel.: 450-773-8521 (ext. 8542); Fax: 450-778-8103; E-mail: siroisje@medvet.umontreal.ca.

Published, JBC Papers in Press, July 11, 2001, DOI 10.1074/jbc.M103709200

    ABBREVIATIONS

The abbreviations used are: PG, prostaglandins; PGE2, prostaglandin E2; PGHS, prostaglandin G/H synthase; hCG, human chorionic gonadotropin; PGES, PGE synthase; ANOVA, analysis of variance; kb, kilobase(s); bp, base pair(s); LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; PCR, polymerase chain reaction; RT, reverse transcription; LH, luteinizing hormone; PAGE, polyacrylamide gel electrophoresis; hCG, human chorionic gonadotropin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Espey, L. L. (1980) Biol. Reprod. 22, 73-106
2. Goetz, F. W., Berndtson, A. K., and Ranjan, M. (1991) in Vertebrate Endocrinology: Fundamentals and Biomedical Implications (Pang, P. K. T. , Schreibman, M. P. , and Jones, R., eds) , pp. 127-203, Academic Press, New York
3. Espey, L. L., and Lipner, H. (1994) in Physiology of Reproduction (Knobil, E. , and Neill, J. D., eds) , pp. 725-781, Raven Press, New York
4. Armstrong, D. T. (1981) J. Reprod. Fertil. 62, 283-291
5. Priddy, A. R., and Killick, S. R. (1993) Prostaglandins Leukotrienes Essent. Fatty Acids 49, 827-831
6. Murdoch, W. J., Hansen, T. R., and McPherson, L. A. (1993) Prostaglandins 46, 85-115
7. Sirois, J., Liu, J., Boerboom, D., and Antaya, M. (2000) in Ovulation: Evolving Scientific and Clinical Concepts (Adashi, E. Y., ed) , pp. 208-218, Springer-Verlag, New York
8. LeMaire, W. J., Yang, N. S. T., Behrman, H. H., and Marsh, J. M. (1973) Prostaglandins 3, 367-376
9. LeMaire, W. J., Leidner, R., and Marsh, J. M. (1975) Prostaglandins 9, 221-229
10. Tsang, B. K., Ainsworth, L., Downey, B. R., and Armstrong, D. T. (1979) Prostaglandins 17, 141-148
11. Lau, I. F., Saksena, S. K., and Chang, M. C. (1974) J. Reprod. Fertil. 40, 467-446
12. Sirois, J. (1994) Endocrinology 135, 841-848
13. Sirois, J., and Doré, M. (1997) Endocrinology 138, 4427-4434
14. Armstrong, D. T., and Grinwich, D. L. (1972) Prostaglandins 1, 21-28
15. O'Grady, J. P., Caldwell, B. V., Auletta, F. J., and Speroff, L. (1972) Prostaglandins 2, 97-106
16. Ainsworth, L., Tsang, B. K., Downey, B. R., Baker, R. D., Marcus, G. J., and Armstrong, D. T. (1979) Biol. Reprod. 21, 401-411
17. Murdoch, W. J., and Dunn, T. G. (1983) J. Reprod. Fertil. 69, 671-675
18. De Silva, M., and Reeves, J. J. (1985) J. Reprod. Fertil. 75, 547-549
19. Algire, J. E., Srikandakumar, A., Guibault, L. A., and Downey, B. R. (1992) Can. Vet. Res. 56, 67-69
20. Killick, S., and Elstein, M. (1987) Fertil. Steril. 47, 773-777
21. Mikuni, M., Pall, M., Peterson, C. M., Peterson, C. A., Hellberg, P., Brannstrom, M., Richards, J. S., and Hedin, L. (1998) Biol. Reprod. 59, 1077-1083
22. Tilley, S. L., Audoly, L. P., Hicks, E. H., Kim, H. S., Flannery, P. J., Coffman, T. M., and Koller, B. H. (1999) J. Clin. Invest. 103, 1539-1545
23. Davis, B. J., Lennard, D. E., Lee, C. A., Tiano, H. F., Morham, S. G., Wetsel, W. C., and Langenbach, R. (1999) Endocrinology 140, 2685-2695
24. Reich, R., Daphna-Iken, D., Chun, S. Y., Popliker, M., Slager, R., Adelman-Grill, M. C., and Tsafriri, A. (1991) Endocrinology 129, 1869-1875
25. Hedin, L., Gaddy-Kurten, D., Kurten, R., DeWitt, D. L., Smith, W. L., and Richards, J. S. (1987) Endocrinology 121, 722-731
26. Huslig, R. L., Malik, A., and Clark, M. R. (1987) Mol. Cell. Endocrin. 50, 237-246
27. Curry, T. E., Malik, A., and Clark, M. R. (1987) Am. J. Obstet. Gynecol. 157, 537-543
28. Curry, T. E., Bryant, C., Haddix, A. C., and Clark, M. R. (1990) Biol. Reprod. 42, 307-316
29. Wong, W. Y. L., DeWitt, D. L., Smith, W. L., and Richards, J. S. (1989) Mol. Endocrinol. 3, 1714-1723
30. Wong, W. Y. L., and Richards, J. S. (1991) Mol. Endocrinol. 5, 1269-1279
31. Sirois, J., and Richards, J. S. (1992) J. Biol. Chem. 267, 6382-6388
32. Sirois, J., Simmons, D. L., and Richards, J. S. (1992) J. Biol. Chem. 267, 11586-11592
33. Morris, J. K., and Richards, J. S. (1993) Endocrinology 133, 770-779
34. Ando, M., Kol, S., Kokia, E., Ruutiainen-Altman, K., Sirois, J., Rohan, R. M., Payne, D. W., and Adashi, E. Y. (1998) Endocrinology 139, 2501-2508
35. Liu, J., Carrière, P., Doré, M., and Sirois, J. (1997) Biol. Reprod. 57, 1524-1531
36. Tsai, S. J., Wiltbank, M. C., and Bodensteiner, K. J. (1996) Endocrinology 137, 3348-3355
37. Boerboom, D., and Sirois, J. (1998) Endocrinology 139, 1662-1670
38. Richards, J. S. (1997) Endocrinology 138, 4047-4048
39. Dinchuk, J. E., Car, B. D., Focht, K. J., Johnston, J. J., Jaffee, B. D., Covington, M. B., Contel, N. R., Eng, V. M., Collins, R. J., Czerniak, P. M., Gorry, S. A., and Trazskos, J. M. (1995) Nature 378, 406-409
40. Lim, H., Paria, B. C., Das, S. K., Dinchuk, J. E., Langenbach, R., Trzaskos, J. M., and Dey, S. K. (1997) Cell 91, 197-208
41. Jakobsson, P. J., Thoren, S., Morgenstern, R., and Samuelsson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7220-7225
42. Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W., and Vogelstein, B. (1997) Nature 389, 300-305
43. Murakami, M., Naraba, H., Tanioka, T., Semmyo, N., Nakatani, Y., Kojima, F., Ikeda, T., Fueki, M., Ueno, A., Oh, S., and Kudo, I. (2000) J. Biol. Chem. 275, 32783-32792
44. Mancini, J. A., Blood, K., Guay, J., Gordon, R., Claveau, D., Chan, C. C., and Riendeau, D. (2001) J. Biol. Chem. 276, 4469-4475
45. Yamagata, K., Matsumura, K., Inoue, W., Shiraki, T., Suzuki, K., Yasuda, S., Sugiura, H., Cao, C., Watanabe, Y., and Kobayashi, S. (2001) J. Neurosci. 21, 2669-2677
46. Jakobsson, P. J., Morgenstern, R., Mancini, J., Ford-Hutchinson, A., and Persson, B. (1999) Protein Sci. 8, 689-692
47. Forsberg, L., Leeb, L., Thoren, S., Morgenstern, R., and Jakobsson, P. (2000) FEBS Lett. 471, 78-82
48. Thoren, S., and Jakobsson, P. J. (2000) Eur. J. Biochem. 267, 6428-6434
49. Soler, M., Camacho, M., Escudero, J. R., Iniguez, M. A., and Vila, L. (2000) Circ. Res. 87, 504-507
50. Liu, J., Antaya, M., Goff, A. K., Boerboom, D., Silversides, D. W., Lussier, J. G., and Sirois, J. (2001) Biol. Reprod. 64, 983-991
51. Liu, J., and Sirois, J. (1998) Biol. Reprod. 58, 1527-1532
52. Brûlé, S., Rabahi, F., Faure, R., Beckers, J. F., Silversides, D. W., and Lussier, J. G. (2000) Biol. Reprod. 62, 642-654
53. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254
54. Satoh, K., Nagano, Y., Shimomura, C., Suzuki, N., Saeki, Y., and Yokota, H. (2000) Neurosci. Lett. 283, 221-223
55. Gurevich, M., Harel-Markowitz, E., Marcus, S., Shore, L. S., and Shemesh, M. (1993) Reprod. Fertil. Dev. 5, 281-283
56. Hizaki, H., Segi, E., Sugimoto, Y., Hirose, M., Saji, T., Ushikubi, F., Matsuoka, T., Noda, Y., Tanaka, T., Yoshida, N., Narumiya, S., and Ichikawa, A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10501-10506
57. Smith, W. (1997) Adv. Exp. Med. Biol. 400, 989-1011
58. Tanioka, T., Nakatani, Y., Semmyo, N., Murakami, M., and Kudo, I. (2000) J. Biol. Chem. 275, 32775-32782
59. Richards, J. S., Russell, D. L., Robker, R. L., Dajee, M., and Alliston, T. N. (1998) Mol. Cell. Endocrinol. 145, 47-54

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


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
 ReproductionHome page
M Mihm, P J Baker, L M Fleming, A M Monteiro, and P J O'Shaughnessy
Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes
Reproduction, February 1, 2008; 135(2): 253 - 265.
[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
 Biol. Reprod.Home page
T. Fayad, R. Lefebvre, J. Nimpf, D. W. Silversides, and J. G. Lussier
Low-Density Lipoprotein Receptor-Related Protein 8 (LRP8) Is Upregulated in Granulosa Cells of Bovine Dominant Follicle: Molecular Characterization and Spatio-Temporal Expression Studies
Biol Reprod, March 1, 2007; 76(3): 466 - 475.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
K. Sayasith, K. A Brown, and J. Sirois
Gonadotropin-dependent regulation of bovine pituitary adenylate cyclase-activating polypeptide in ovarian follicles prior to ovulation
Reproduction, February 1, 2007; 133(2): 441 - 453.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
K. Sayasith, N. Bouchard, M. Dore, and J. Sirois
Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process
Reproduction, February 1, 2007; 133(2): 455 - 466.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
K. A Brown, K. Sayasith, N. Bouchard, J. G Lussier, and J. Sirois
Molecular cloning of equine 17{beta}-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo
J. Mol. Endocrinol., January 1, 2007; 38(1): 67 - 78.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
K. F. Rodriguez, L. A. Blomberg, K. A. Zuelke, J. R. Miles, J. E. Alexander, and C. E. Farin
Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression
Physiol Genomics, November 21, 2006; 27(3): 318 - 327.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
K. Sayasith, K. A Brown, J. G Lussier, M. Dore, and J. Sirois
Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation.
J. Mol. Endocrinol., October 1, 2006; 37(2): 239 - 250.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. A. Brown, M. Dore, J. G. Lussier, and J. Sirois
Human Chorionic Gonadotropin-Dependent Up-Regulation of Genes Responsible for Estrogen Sulfoconjugation and Export in Granulosa Cells of Luteinizing Preovulatory Follicles
Endocrinology, September 1, 2006; 147(9): 4222 - 4233.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
K A Brown, D Boerboom, N Bouchard, M Dore, J G Lussier, and J Sirois
Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20{alpha}-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo.
J. Mol. Endocrinol., June 1, 2006; 36(3): 449 - 461.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. N. Diouf, K. Sayasith, R. Lefebvre, D. W. Silversides, J. Sirois, and J. G. Lussier
Expression of Phospholipase A2 Group IVA (PLA2G4A) Is Upregulated by Human Chorionic Gonadotropin in Bovine Granulosa Cells of Ovulatory Follicles
Biol Reprod, June 1, 2006; 74(6): 1096 - 1103.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
T. Sun, W.-B. Deng, H.-L. Diao, H. Ni, Y.-Y. Bai, X.-H. Ma, L.-B. Xu, and Z.-M. Yang
Differential expression and regulation of prostaglandin E synthases in the mouse ovary during sexual maturation and luteal development.
J. Endocrinol., April 1, 2006; 189(1): 89 - 101.
[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. Clin. Endocrinol. Metab.Home page
D. M. Duffy, C. L. Seachord, and B. L. Dozier
An Ovulatory Gonadotropin Stimulus Increases Cytosolic Phospholipase A2 Expression and Activity in Granulosa Cells of Primate Periovulatory Follicles
J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5858 - 5865.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
F. Masse, S. Guiral, L.-J. Fortin, E. Cauchon, D. Ethier, J. Guay, and C. Brideau
An Automated Multistep High-Throughput Screening Assay for the Identification of Lead Inhibitors of the Inducible Enzyme mPGES-1
J Biomol Screen, September 1, 2005; 10(6): 599 - 605.
[Abstract] [PDF]

Home page
 Biol. Reprod.Home page
J. Parent and M. A. Fortier
Expression and Contribution of Three Different Isoforms of Prostaglandin E Synthase in the Bovine Endometrium
Biol Reprod, July 1, 2005; 73(1): 36 - 44.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
D. M. Duffy, C. L. Seachord, and B. L. Dozier
Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle
Hum. Reprod., June 1, 2005; 20(6): 1485 - 1492.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. L. Seachord, C. A. VandeVoort, and D. M. Duffy
Adipose Differentiation-Related Protein: A Gonadotropin- and Prostaglandin-Regulated Protein in Primate Periovulatory Follicles
Biol Reprod, June 1, 2005; 72(6): 1305 - 1314.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
D. M. Duffy, B. L. Dozier, and C. L. Seachord
Prostaglandin Dehydrogenase and Prostaglandin Levels in Periovulatory Follicles: Implications for Control of Primate Ovulation by Prostaglandin E2
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1021 - 1027.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
Y. Shinji, T. Tsukui, A. Tatsuguchi, K. Shinoki, M. Kusunoki, K. Suzuki, T. Hiratsuka, K. Wada, S. Futagami, K. Miyake, et al.
Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts
Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G308 - G315.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. A. Arosh, S. K. Banu, S. Kimmins, P. Chapdelaine, L. A. MacLaren, and M. A. Fortier
Effect of Interferon-{tau} on Prostaglandin Biosynthesis, Transport, and Signaling at the Time of Maternal Recognition of Pregnancy in Cattle: Evidence of Polycrine Actions of Prostaglandin E2
Endocrinology, November 1, 2004; 145(11): 5280 - 5293.
[Abstract] [Full Text] [PDF]

Home page
 J DAIRY SCIHome page
A. Guzeloglu, T. R. Bilby, A. Meikle, S. Kamimura, A. Kowalski, F. Michel, L. A. MacLaren, and W. W. Thatcher
Pregnancy and Bovine Somatotropin in Nonlactating Dairy Cows: II. Endometrial Gene Expression Related to Maintenance of Pregnancy
J Dairy Sci, October 1, 2004; 87(10): 3268 - 3279.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
J. Sirois, K. Sayasith, K. A. Brown, A. E. Stock, N. Bouchard, and M. Dore
Cyclooxygenase-2 and its role in ovulation: a 2004 account
Hum. Reprod. Update, September 1, 2004; 10(5): 373 - 385.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Kamei, K. Yamakawa, Y. Takegoshi, M. Mikami-Nakanishi, Y. Nakatani, S. Oh-ishi, H. Yasui, Y. Azuma, N. Hirasawa, K. Ohuchi, et al.
Reduced Pain Hypersensitivity and Inflammation in Mice Lacking Microsomal Prostaglandin E Synthase-1
J. Biol. Chem., August 6, 2004; 279(32): 33684 - 33695.
[Abstract] [Full Text] [PDF]

Home page
 J DAIRY SCIHome page
A. Guzeloglu, F. Michel, and W. W. Thatcher
Differential Effects of Interferon-{tau} on the Prostaglandin Synthetic Pathway in Bovine Endometrial Cells Treated with Phorbol Ester
J Dairy Sci, July 1, 2004; 87(7): 2032 - 2041.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. A. Arosh, S. K. Banu, P. Chapdelaine, E. Madore, J. Sirois, and M. A. Fortier
Prostaglandin Biosynthesis, Transport, and Signaling in Corpus Luteum: A Basis for Autoregulation of Luteal Function
Endocrinology, May 1, 2004; 145(5): 2551 - 2560.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. A. Brown, D. Boerboom, N. Bouchard, M. Dore, J. G. Lussier, and J. Sirois
Human Chorionic Gonadotropin-Dependent Regulation of 17{beta}-Hydroxysteroid Dehydrogenase Type 4 in Preovulatory Follicles and Its Potential Role in Follicular Luteinization
Endocrinology, April 1, 2004; 145(4): 1906 - 1915.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Sayasith, N. Bouchard, M. Sawadogo, J. G. Lussier, and J. Sirois
Molecular Characterization and Role of Bovine Upstream Stimulatory Factor 1 and 2 in the Regulation of the Prostaglandin G/H Synthase-2 Promoter in Granulosa Cells
J. Biol. Chem., February 20, 2004; 279(8): 6327 - 6336.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
T J Jang, S K Min, J D Bae, K H Jung, J I Lee, J R Kim, and W S Ahn
Expression of cyclooxygenase 2, microsomal prostaglandin E synthase 1, and EP receptors is increased in rat oesophageal squamous cell dysplasia and Barrett's metaplasia induced by duodenal contents reflux
Gut, January 1, 2004; 53(1): 27 - 33.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Murakami, K. Nakashima, D. Kamei, S. Masuda, Y. Ishikawa, T. Ishii, Y. Ohmiya, K. Watanabe, and I. Kudo
Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and -2
J. Biol. Chem., September 26, 2003; 278(39): 37937 - 37947.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Kamei, M. Murakami, Y. Nakatani, Y. Ishikawa, T. Ishii, and I. Kudo
Potential Role of Microsomal Prostaglandin E Synthase-1 in Tumorigenesis
J. Biol. Chem., May 23, 2003; 278(21): 19396 - 19405.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Naraba, C. Yokoyama, N. Tago, M. Murakami, I. Kudo, M. Fueki, S. Oh-ishi, and T. Tanabe
Transcriptional Regulation of the Membrane-associated Prostaglandin E2 Synthase Gene. ESSENTIAL ROLE OF THE TRANSCRIPTION FACTOR Egr-1
J. Biol. Chem., August 2, 2002; 277(32): 28601 - 28608.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Parent, P. Chapdelaine, J. Sirois, and M. A. Fortier
Expression of Microsomal Prostaglandin E Synthase in Bovine Endometrium: Coexpression with Cyclooxygenase Type 2 and Regulation by Interferon-{tau}
Endocrinology, August 1, 2002; 143(8): 2936 - 2943.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. A. Arosh, J. Parent, P. Chapdelaine, J. Sirois, and M. A. Fortier
Expression of Cyclooxygenases 1 and 2 and Prostaglandin E Synthase in Bovine Endometrial Tissue During the Estrous Cycle
Biol Reprod, July 1, 2002; 67(1): 161 - 169.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Lazarus, C. J. Munday, N. Eguchi, S. Matsumoto, G. J. Killian, B. K. Kubata, and Y. Urade
Immunohistochemical Localization of Microsomal PGE Synthase-1 and Cyclooxygenases in Male Mouse Reproductive Organs
Endocrinology, June 1, 2002; 143(6): 2410 - 2419.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/36/34323    most recent
M103709200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Filion, F.
Right arrow Articles by Sirois, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Filion, F.
Right arrow Articles by Sirois, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement