Title: Molecular Cloning and Induction of Bovine Prostaglandin E Synthase by Gonadotropins in Ovarian Follicles Prior to Ovulationin Vivo
Abstract: 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 (ΔPGES; 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. 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 (ΔPGES; 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. prostaglandins prostaglandin E2 prostaglandin G/H synthase human chorionic gonadotropin PGE synthase analysis of variance kilobase(s) base pair(s) lipopolysaccharide interleukin tumor necrosis factor polymerase chain reaction reverse transcription luteinizing hormone polyacrylamide gel electrophoresis human chorionic gonadotropin 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. 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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 (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar, 13Sirois J. Doré M. Endocrinology. 1997; 138: 4427-4434Crossref PubMed Scopus (137) Google Scholar,30Wong W.Y.L. Richards J.S. Mol. Endocrinol. 1991; 5: 1269-1279Crossref PubMed Scopus (142) Google Scholar, 31Sirois J. Richards J.S. J. Biol. Chem. 1992; 267: 6382-6388Abstract Full Text PDF PubMed Google Scholar, 32Sirois J. Simmons D.L. Richards J.S. J. Biol. Chem. 1992; 267: 11586-11592Abstract Full Text PDF PubMed Google Scholar). 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. 32Sirois J. Simmons D.L. Richards J.S. J. Biol. Chem. 1992; 267: 11586-11592Abstract Full Text PDF PubMed Google Scholar) and relatively delayed in cattle (18 h post-hCG; Ref. 12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar) and horses (30 h post-hCG; Refs. 13Sirois J. Doré M. Endocrinology. 1997; 138: 4427-4434Crossref PubMed Scopus (137) Google Scholar, 37Boerboom D. Sirois J. Endocrinology. 1998; 139: 1662-1670Crossref PubMed Scopus (46) Google Scholar). 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 (37Boerboom D. Sirois J. Endocrinology. 1998; 139: 1662-1670Crossref PubMed Scopus (46) Google Scholar, 38Richards J.S. Endocrinology. 1997; 138: 4047-4048Crossref PubMed Google Scholar). Studiesin 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 (33Morris J.K. Richards J.S. Endocrinology. 1993; 133: 770-779Crossref PubMed Scopus (72) Google Scholar). 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 (39Dinchuk 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. Trazskos J.M. Nature. 1995; 378: 406-409Crossref PubMed Scopus (894) Google Scholar, 40Lim H. Paria B.C. Das S.K. Dinchuk J.E. Langenbach R. Trzaskos J.M. Dey S.K. Cell. 1997; 91: 197-208Abstract Full Text Full Text PDF PubMed Scopus (1248) Google Scholar). 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 PGH2into PGE2, i.e. PGE synthase (PGES), has not been addressed. The cloning and characterization of the first PGES was recently reported from human cells (41Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (890) Google Scholar) and shown to correspond to a protein previously identified as microsomal glutathioneS-transferase 1 like-1 (MGST1-L1, Ref. 41Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (890) Google Scholar) or a gene product referred to as p53-induced gene (PIG12; Ref. 42Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2234) Google Scholar). This initial report was rapidly followed by the isolation of the mouse and rat homologs (43Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 44Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 45Yamagata K. Matsumura K. Inoue W. Shiraki T. Suzuki K. Yasuda S. Sugiura H. Cao C. Watanabe Y. Kobayashi S. J. Neurosci. 2001; 21: 2669-2677Crossref PubMed Google Scholar). 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 (46Jakobsson P.J. Morgenstern R. Mancini J. Ford-Hutchinson A. Persson B. Protein Sci. 1999; 8: 689-692Crossref PubMed Scopus (298) Google Scholar). 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 (41Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (890) Google Scholar, 47Forsberg L. Leeb L. Thoren S. Morgenstern R. Jakobsson P. FEBS Lett. 2000; 471: 78-82Crossref PubMed Scopus (101) Google Scholar). Interestingly, the enzyme was shown to be induced in vitroby various proinflammatory stimuli already known to induce PGHS-2 expression including lipopolysaccharide (LPS), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α (41Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (890) Google Scholar, 43Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 44Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 48Thoren S. Jakobsson P.J. Eur. J. Biochem. 2000; 267: 6428-6434Crossref PubMed Scopus (190) Google Scholar, 49Soler M. Camacho M. Escudero J.R. Iniguez M.A. Vila L. Circ. Res. 2000; 87: 504-507Crossref PubMed Scopus (99) Google Scholar). The induction of PGES has also been reported recently in two inflammatory modelsin vivo, including LPS-induced pyresis and adjuvant-induced arthritis (43Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 44Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). 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. The QuikHyb hybridization solution and the ExAssist/SOLR system were purchased from Stratagene Cloning Systems (La Jolla, CA). [α-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-deficientEscherichia 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. To clone the bovine PGES cDNA, a bovine cDNA library prepared with mRNA isolated from a preovulatory follicle obtained 24 h post-hCG (50Liu J. Antaya M. Goff A.K. Boerboom D. Silversides D.W. Lussier J.G. Sirois J. Biol. Reprod. 2001; 64: 983-991Crossref PubMed Scopus (22) Google Scholar) was screened with a human PGES cDNA (42Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2234) Google Scholar). The probe was labeled with [α-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 excisedin 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, 0.001m 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′) (36Tsai S.J. Wiltbank M.C. Bodensteiner K.J. Endocrinology. 1996; 137: 3348-3355Crossref PubMed Scopus (100) Google Scholar), 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. The regulation of PGES expression during the ovulatory process in vivo was studied in three distinct models previously characterized by us (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar, 35Liu J. Carrière P. Doré M. Sirois J. Biol. Reprod. 1997; 57: 1524-1531Crossref PubMed Scopus (44) Google Scholar, 51Liu J. Sirois J. Biol. Reprod. 1998; 58: 1527-1532Crossref PubMed Scopus (27) Google Scholar). 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 (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar). 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 (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar). 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) (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar). 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.) (51Liu J. Sirois J. Biol. Reprod. 1998; 58: 1527-1532Crossref PubMed Scopus (27) Google Scholar). 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 (51Liu J. Sirois J. Biol. Reprod. 1998; 58: 1527-1532Crossref PubMed Scopus (27) Google Scholar). In this model, multiple ovulations are expected 28–30 h post-hCG (51Liu J. Sirois J. Biol. Reprod. 1998; 58: 1527-1532Crossref PubMed Scopus (27) Google Scholar). 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 (35Liu J. Carrière P. Doré M. Sirois J. Biol. Reprod. 1997; 57: 1524-1531Crossref PubMed Scopus (44) Google Scholar). 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 (35Liu J. Carrière P. Doré M. Sirois J. Biol. Reprod. 1997; 57: 1524-1531Crossref PubMed Scopus (44) Google Scholar). 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 (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar). In selected cases, some pieces of follicle wall were further dissected into isolated preparations of granulosa cells and theca interna (12Sirois J. Endocrinology. 1994; 135: 841-848Crossref PubMed Scopus (162) Google Scholar). 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. 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 (36Tsai S.J. Wiltbank M.C. Bodensteiner K.J. Endocrinology. 1996; 137: 3348-3355Crossref PubMed Scopus (100) Google Scholar). 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 mTris-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). A pair of sense (5′-GATGGATCCGAGGACGCTCAGAGACATGGA-3′) and antisense (5′-TCAGAATTCGACAATCTGCAGGGCCATGGA-3′) primers that incorporated aBamHI and an EcoRI restriction site, respectively, were designed from the bovine PGES open reading frame to generate a fragment (ΔPGES) 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 ΔPGES/pGEX-2T construct, expression of recombinant ΔPGES/GST fusion protein was induced with isopropyl-1-thio-β-d-galactopyranoside, and bacterial protein extracts were obtained after sonication and centrifugation (52Brûlé S. Rabahi F. Faure R. Beckers J.F. Silversides D.W. Lussier J.G. Biol. Reprod. 2000; 62: 642-654Crossref PubMed Scopus (23) Google Scholar). The ΔPGES/GST fusion protein was purified by affinity on glutathione-Sepharose beads (Amersham Pharmacia Biotech), digested with thrombin to release the ΔPGES, resolved by one-dimensional SDS-PAGE, transf