Expression of prostaglandin E synthases in the bovine oviduct

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Theriogenology 73 (2010) 103–111 www.theriojournal.com

Expression of prostaglandin E synthases in the bovine oviduct D. Gauvreau a,b, V. Moisan a,b, M. Roy a,b, M.A. Fortier a,b,c, J-F. Bilodeau a,b,c,* a

Unite´ de Recherche en Ontoge´nie et Reproduction, Centre de Recherche du Centre Hospitalier de l’Universite´ Laval, Que´bec, Canada b Centre de Recherche en Biologie de la Reproduction (CRBR), Universite´ Laval, Que´bec, Canada c De´partement d’Obste´trique et Gyne´cologie, Faculte´ de me´decine, Universite´ Laval, Que´bec, Canada Received 10 May 2009; received in revised form 31 July 2009; accepted 23 August 2009

Abstract The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E2 is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH2. The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH2 to PGE2, the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE2 production were present in the Bos taurus oviduct. # 2010 Elsevier Inc. All rights reserved. Keywords: Cattle; Cyclooxygenases; Prostaglandin E synthases; Prostaglandin H synthases; Uterine tube

1. Introduction In mammals, the oviduct is a specialized organ playing crucial roles in the success of early reproductive events. The oviduct is responsible for the transport and storage of male and female gametes, and it provides an optimal microenvironment for their final maturation. Moreover,

* Corresponding author. Tel.: +418 525 4444x46153; fax: +418 654 2765. E-mail address: [email protected] (J.-F. Bilodeau). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.08.006

this organ constitutes the natural site of fertilization and early embryonic development [1]. In addition to steroid hormones, prostaglandins (PGs) are among the most important modulators for the success of these processes. The PGs have important roles in ovulation and fertilization [2,3]. Indeed, PGs can either contract or relax the muscle layers of the oviduct for the transport of the gametes and zygote [4–6]. Several prostaglandins were found in bovine [7], human [8], and rat oviducts [9]. Among them, PGE2 was the most abundant in the bovine oviduct, where its concentration peaked during proestrus on the side ipsilateral to the site of ovulation [7].

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Prostaglandin E2 is a well characterized prostaglandin with pleiotropic actions. The biosynthesis of PGs is initiated with the release of arachidonic acid (AA) from membrane phospholipids through the action of phospholipases. Thereafter, AA is converted into PGH2, the precursor of all prostaglandins, by prostaglandin endoperoxide synthase or prostaglandin H synthase (PGHS) commonly named cyclooxygenases (COXs) [10]. Prostaglandin H2 is subsequently transformed into PGE2 through the action of prostaglandin E synthases (PGESs). The activity of COX is considered the ratelimiting step in prostaglandin production. Two COX enzymes encoded by two different genes have been described: COX-1 is expressed in a constitutive manner, whereas COX-2 is an inducible enzyme. Furthermore, three isoenzymes of PGES have been detected and characterized. Microsomal prostaglandin E synthase-1 (mPGES-1) is mostly coupled with COX-2 for delayed production of PGE2. The latter coupling was shown in HEK293 cells co-transfected by human COX-2 and mPGES [11]. The second PGES, named cPGES for cytosolic PGES, was found in the cytosol of different rat tissues [12,13]. It has also been characterized as glutathione dependent in rat brain and deferent duct microsomes [12,13] or in lysates from HEK293 cells transfected by recombinant cPGES [13]. Cytosolic PGES is also preferably coupled to COX-1 for immediate PGE2 biosynthesis, as demonstrated in HEK293 cells co-transfected with either COXs and cPGES [13]. Of note, cPGES is a 23-kDa protein identical to p23, a ubiquitous chaperone protein of the Hsp90 hormone-receptor complex [14]. A novel type of membrane-associated PGES, named mPGES-2, was purified from microsome of the bovine heart and characterized as glutathione independent in vitro using the purified enzyme in the presence of various concentrations of thiol agents [15]. Microsomal prostaglandin E synthase-2 is constitutively expressed and coupled to both COX-1 and COX-2 but has a slightly greater affinity for COX-2 [16]. This was demonstrated again in HEK293 co-transfected with COX-1 or COX-2 with mPGES-2 [16]. The PGs have autocrine, paracrine, and even endocrine effects [17,18] through their cell surface G-protein–coupled receptor [19,20]. Gene inactivation studies using null mutation in mice have shown that PGs are necessary for female reproductive function. It was demonstrated that COX-2/ female mice suffered from multiple reproductive failure at several levels, including ovulation, fertilization, and implantation [2]. Interestingly, PGE2 (EP2) receptor null mice exhibited a very similar phenotype [21]. The concentration of PGE2

and the expression and distribution of EP receptors mRNA were previously analyzed in the bovine oviduct during the estrous cycle [7,22]. Furthermore, the phospholipase A2 and COX enzymes involved in the first steps of its synthesis were also examined [23]. We have previously studied the expression and regulation of the three PGESs in the bovine uterus [24], but nothing has been reported in the oviduct. Therefore, the objective of the current study was to investigate the spatiotemporal mRNA and protein expression of three PGESs throughout the estrous cycle in the bovine oviduct. We also examined COX-1 and COX-2 mRNA expression involved in the production of the substrate for the PGES (PGH2) to confirm previous findings. 2. Materials and methods 2.1. Collection of tissue samples Bos taurus holstein cow oviducts were collected at an abattoir, placed on ice, transported to the laboratory, and processed within 4 h after animal death. Reproductive tracts with gross evidence of inflammation or anatomic defects were discarded. The stage of the estrous cycle was defined by evaluation of the ovaries (follicle and corpus luteum) and by the morphologic appearance of the endometrium and the cervix, as previously described [25]. The oviducts were then classified into three groups: metestrus (Days 1 to 3), diestrus (Days 12 to 15), and proestrus (Days 18 to 21). Oviducts ipsilateral and contralateral to the corpus luteum (CL) or the dominant follicle were analyzed separately. The contralateral oviduct was the oviduct opposite to the regressing CL or to the ovary bearing the dominant follicle. Only animals that ovulated by ovarian alternations were used. The oviducts were dissected on a chilled glass plate (to remove blood vessels and mesosalpinx) and were then cut into three sections (isthmus, isthmic-ampullary junction, and ampulla). Tissues were frozen in dry ice or processed for immunohistochemistry (see Section 2.6) and kept at –80 8C pending analysis. 2.2. Preparation of RNA and cDNA Total RNA was extracted using TRIzol reagent as described in the manufacturer’s instructions (Invitrogen, Burlington, ON, Canada) [26]. After migration of the samples on an ethidium bromide–treated 0.5% agarose gel, RNA integrity was verified by visual assessment of 18S and 28S rRNA intensities after ultraviolet light exposure. Degraded RNA samples were

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discarded. Briefly, 4 mg of total RNA were reverse transcribed with random hexamer primers and the Superscript II reverse transcriptase (Invitrogen). The reverse-transcribed cDNA was diluted 10-fold (200 mL final volume).

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one peak in the analysis. The quantification analysis of the data was performed by using the LightCycler analysis software, as previously described [26]. Gene expression was expressed as the ratio of gene concentration on 18S rRNA. Each cDNA was quantified in duplicate, and the average value of each sample was used for quantification.

2.3. Real-time quantitative PCR

2.4. Protein extraction

Primers sets used to amplify COX-1, COX-2, cPGES, mPGES-1, and mPGES-2 were designed based on known bovine sequences, as described previously (Table 1) [24,25]. As an internal control, 18S rRNA was amplified (Table 1). The expected PCR products were visualized by agarose gel electrophoresis, eluted, and sequenced (Center of Genomics, CHUL Research Center, QC, Canada) [24]. The quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) reactions were carried out in a LightCycler (Roche Diagnostics, Laval, QC, Canada). Reactions were performed in a 20-mL reaction mixture containing 5 mL diluted cDNA, 0.25 mM of each primer, 3 mM MgCl2 for mPGES-1 and COX-1 and 4 mM of MgCl2 for mPGES-2, cPGES and COX-2, 2 mL FastStart Master SYBRGreen I mix (Roche Diagnostics), and PCR-grade water up to the final volume. The RT-PCR reactions were performed as follows: denaturation at 95 8C for 10 min, followed by multiple numbers of cycles of amplification (95 8C for 0 s, between 58 and 65 8C for 5 s [see Table 1 for details], and 72 8C for 20 s) with single acquisition of fluorescence at the end of the extension step. After amplification, samples were slowly heated at 0.1 8C/sec from 60 to 95 8C with continuous reading of fluorescence to obtain a melting curve. The specificity of each amplicon was determined using the melting curve analysis program of the LightCycler software. The amplicons showed only

Bovine tissues stored at 80 8C were thawed and homogenized with an Ultra-turrax T25 (Janke & Kundel Labortechnik, Staufen, Germany) in a homogenization buffer (10 mM K2HPO4 at pH 7.8, 1 mM ethylenediamine tetraacetic acid, 0.5 mM phenylmethanesulfonyl fluoride, 120 mg/mL leupeptin). The resulting protein homogenates from isthmus, isthmic-ampullary junction, and ampulla sections were diluted 1:1 in 2X Laemmli’s sample buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl at pH 6.8) containing 5% (v:v) 2-mercaptoethanol then boiled for 10 min. Each sample protein content was determined by the BCA assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. 2.5. Western blot analysis Aliquots of total protein (50 mg/well) were loaded and separation carried out on 12% wt/vol (for cPGES and mPGES-2) and 15% wt/vol (for mPGES-1) sodium dodecyl sulfate (SDS)-polyacrylamide gels followed by transfer onto an 0.2-mm nitrocellulose membrane (BioRad, Hercules, CA, USA). Prestained full-range rainbow molecular weight markers (GE Healthcare Biosciences Inc., Baie d’Urfe, QC, Canada) were used as molecular weight standard for each analysis. Protein

Table 1 Specific oligonucleotide primers used for quantitative RT-PCR analysis of bovine oviductal tissues. Genes

Primers

Fragment length (bp)

Annealing temperature (8C)

GenBank accession no.

COX-1

F: 50 -tcccacctacaacgtagc-30 R: 50 -tgagtttcccatccttaaag-30 F: 50 -tcgagatcacatttgattgaga-30 R: 50 -tctttgactgtgggaggataca-30 F: 50 -gtattgccggaacgacccag-30 R: 50 -aatctcaaagggccatcggtc-30 F: 50 -atcaagttctcctcctacag-30 R: 50 -gggtagtaggtgatgatgtc-30 F: 50 -ttctgcaaagtggtacgatc-30 R: 50 -atcatcttcccagtctttcc-30 F: 50 -gtaacccgttgaaccccatt-30 R: 50 -ccatccaatcggtagtagcg-30

377

60

AF004943

449

60

AF031698

177

65

NM_174443

152

62

AY692441

285

61

AY692440

151

58

DQ222453

COX-2 mPGES-1 mPGES-2 cPGES 18S

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from bovine ovaries and bovine lung for mPGES-1 and bovine endometrium for mPGES-2 and cPGES were loaded in each gel as an internal positive control. Blocking was done in 5% fat-free dry milk powder in TBS and 0.01% Tween-20 (TBS-T) for 1 h at room temperature. The membrane was then incubated with a rabbit polyclonal anti-cPGES (dilution 1/1500), a rabbit polyclonal anti-mPGES-2 (dilution 1/1000) (Cayman Chemical, Ann Arbor, MI, USA), and a mouse monoclonal anti-b-actin (1/2500) (Sigma-Aldrich, Oakville, ON, Canada) for 2 h at room temperature. Of note, separate membranes were used for each PGES. The membranes were not stripped but reprobed for bactin assessment. For mPGES-1 analysis, the membrane was incubated with a rabbit polyclonal anti-mPGES-1 (dilution 1/200) (Cayman Chemical) overnight at 4 8C. Each antibody was diluted in 2% fat-free dry milk powder in TBS-T. The secondary antibodies goat antirabbit (for cPGES, mPGES-1, mPGES-2 analysis) or goat anti-mouse (for b-actin) conjugated to horseradish peroxidase (dilution 1/10,000 in 2.5% fat-free dry milk powder in TBS-T) were then incubated for 45 min at room temperature. Bands were revealed by the addition of an enhanced chemiluminescent substrate according to the manufacturer’s instructions (GE Healthcare BioSciences). Bands were analyzed by Agfa Arcus II scanner (Afga, Toronto, ON, Canada) and quantified by ImageJ software (NIH, Bethesda, MD, USA) at the local genomic center (CHUL Research Center, QC, Canada). Densitometry values obtained from the Western blot analysis were corrected for the time of exposition using an internal standard (positive control) and normalized to b-actin protein. 2.6. Immunohistochemistry Oviduct sections were fixed overnight in 4% paraformaldehyde (Sigma-Aldrich). Optimum cutting temperature medium (OCT; Canemco, St. Laurent, QC, Canada) was used to embed the sections before freezing in liquid nitrogen, as previously described [27]. Paraformaldehyde-fixed, OCT-embedded bovine oviduct sections (8 mm) were dried, washed 5 min in phosphate buffer saline (PBS), treated for 30 min in 0.3% H2O2/methanol, and rehydrated in graded alcohols (95% and 70%) (Sigma-Aldrich). After equilibration for 5 min in PBS, sections were blocked for 2 h with 10% horse serum and incubated overnight at 4 8C with antibody against mPGES-1, mPGES-2, and cPGES (Cayman Chemical) in PBS containing 0.1% bovine serum albumin. The next day, the slides were washed in PBS and incubated for 1 h with the

corresponding biotinylated secondary antibody (1:1500; Vector Laboratories, Burlington, ON, Canada). After washing in PBS, sections were submitted to an avidin-biotin complex (ABC) solution for 20 min at room temperature (Vectastain ABC Elite Kit, Vector Laboratories). The signal was detected using a solution of 3-amino-9-ethylcarbazole (AEC; Sigma-Aldrich), 50 mM acetate buffered to pH 5.2 (0.2 M sodium acetate; 0.2 M acetic acid), and 0.002% H2O2. Sections were then counterstained with Gill #1 hematoxylin and mounted in Mowiol mounting solution (SigmaAldrich). 2.7. Statistical analysis A three-way ANOVA with repeated measures was used to study the effect of the factors stage of the estrous cycle, oviduct side, and oviduct section on mRNA and protein levels. To account for the possible correlations between observations made on the same cow, we considered the factors oviduct side and oviduct section as repeated measures. The MIXED procedure of SAS software (SAS OnlineDoc 9.1.3; SAS Institute Inc., Cary, NC, USA) was used with a repeated statement and the covariance structure that minimized the Akaike criterion. A logarithm transformation for mRNA values was used to reach the normality and the homogeneity of the variance assumptions. The graphical representation of mRNA means for each prostaglandin synthase (Fig. 1) represents a transformation of the logarithmic value returned to the original scale according to the smearing estimate [28]. The normality and the homogeneity of the variance assumptions were met for the Western blot analysis. Pairwise comparisons were then made using the protected Fisher least significant difference (LSD). For all analyses, a P value < 0.05 was considered significant. 3. Results 3.1. Expression of COXs mRNA in the bovine oviduct The mRNA analysis was done on six pairs of oviducts. All quantitative RT-PCR measurements were done in duplicate for a given oviduct section. The COX1 and COX-2 mRNAs were detected in both ipsilateral and contralateral oviducts at every stage of the estrous cycle and in all sections (data not shown). Statistical analysis for COXs mRNA expression revealed no interactions between the three fixed factors studied; stage of estrous cycle, side of the oviduct, and sections.

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Fig. 1. PGES mRNA expression in the bovine oviduct expressed as ratios of PGES mRNA/18S rRNA (internal standard). Oviduct sections: I, isthmus; I-A, isthmic-ampullary junction; A, ampulla. Estrous cycle: black bars, metestrus; gray bars, diestrus; white bars, proestrus. (A) Expression of mPGES-1 mRNA in oviduct sections according to the estrous cycle. (B) Expression of mPGES-2 mRNA in oviduct sections according to the estrous cycle. (C) Expression of cPGES mRNA in oviduct sections according to the estrous cycle. Data are means  SEM of six animals for each estrous cycle time (one pair of oviduct per animal). a–eColumns without a common superscript differed (P < 0.05).

The only significant change in mRNA expression was detected for COX-1 in relation with the oviduct section investigated (P = 0.0246). Indeed, a progressive increase from the uterine to the ovarian pole of the oviduct was observed where COX-1 mRNA in the ampulla was 12-fold higher than that in the isthmus (data not shown). 3.2. Expression of three PGESs mRNA in the bovine oviduct As observed for COXs, all three PGESs were present in each phase of the estrous cycle and in each oviduct section (Fig. 1). There was one interaction, between stage and section for mPGES-1 mRNA expression (n = 6 animals or pairs of oviducts; P = 0.0161). Only the stage of the estrous cycle affected the expression of mPGES-1 in all sections of the oviduct (Fig. 1A). Indeed, the highest levels of mRNA expression occurred during the periovulatory period. Globally, the mPGES-1 mRNA expression was higher at proestrus (5.6-fold) and the metestrus (7.2-fold) compared with that at diestrus (Fig. 1A). The expression of mPGES-2 and cPGES were similar, with a progressive increase from isthmus to ampulla (Fig. 1B, C). The expression of mPGES-2 mRNA was 3.1-fold higher in the isthmic-ampullary junction (P = 0.0013) and 5.3-fold higher in the ampulla (P < 0.0001) than that in the isthmus (Fig. 1B). Similarly, cPGES mRNA levels were also higher in the isthmic-ampullary junction (3.5-fold; P = 0.0195) and in the ampulla (7.1-fold; P < 0.0001) than that in the isthmus. Additionally, in the case of cPGES, the mRNA level was higher in the ampulla than that in the isthmic-ampullary junction (Fig. 1C).

3.3. Expression of three PGESs in the bovine oviduct The protein expression for mPGES-1, mPGES-2, and cPGES was also analyzed in five animals or pairs of oviducts (Fig. 2). All three PGESs were detected in both oviducts in all sections of the oviduct, as expected from mRNA analysis above. There was no interaction among the three factors studied (stage of the estrous cycle, side relative to the CL, and section of the oviducts) for the three PGESs. Protein expression was clearly higher toward the ovaries (ampulla > isthmic-ampullary junction > isthmus) for mPGES-2 and cPGES as expected from mRNA analysis (Figs. 1B, C and 3B, C). More

Fig. 2. Protein levels of mPGES-1, mPGES-2, and cPGES in the isthmus (I), isthmic-ampullary junction (I-A), and ampulla (A) regions of the ipsilateral and contralateral bovine oviduct at metestrus, diestrus, and proestrus stages. C: positive control from bovine endometrium. 1C: positive control from bovine lung. The loading control was b-actin. These results are a single replicate and are representative of five animals (pairs of oviducts).

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Fig. 3. Analysis of PGES protein expression in the bovine oviduct. Western blots were analyzed by densitometry. b-actin was the loading control. (A) mPGES-1 protein levels along the oviduct. (B) mPGES-2 protein levels along the oviduct. (C) cPGES protein levels along the oviduct. (D) mPGES-1 protein levels in ipsilateral and contralateral oviducts (E) mPGES-2 protein levels in ipsilateral and contralateral oviducts. IODn, normalized integrated optical density. I, isthmus; I-A, isthmic-ampullary junction; A, ampulla. Data from five animals at each estrous cycle time (one pair of oviducts per animal). a–cColumns without a common superscript differed (P < 0.05).

specifically, mPGES-2 protein signal was higher in the ampulla than that in the isthmic-ampullary junction by 23% (P = 0.0028) and that in the isthmus by 50% (P < 0.0001). The mPGES-2 protein signal was also higher in the isthmic-ampullary junction than that in the isthmus by 35% (P = 0.0006). Similarly, the expression of cPGES protein was higher in the ampulla than that in the isthmic-ampullary junction by 27% (P = 0.0160) and that in the isthmus by 60% (P < 0.0001). The cPGES protein expression also had a higher level in the isthmic-ampullary junction than that in the isthmus by 48% (P = 0.0003). Conversely, mPGES-1 level was higher only in the ampulla compared with that in the isthmic-ampullary section (Fig. 3A). Notably, mPGES-1 protein expression level was higher in the contralateral than in the ipsilateral oviduct by 41% (Fig. 3D; P = 0.0215). In contrast, mPGES-2 protein expression level was higher in the ipsilateral than in the contralateral oviduct by 27% (Fig. 3E; P = 0.0007). 3.4. Immunolocalization of three PGESs in the oviduct Immunohistochemistry was used to determine the cells that specifically expressed the three PGESs in the

oviduct. Protein expression was determined in three oviductal sections—isthmus, isthmic-ampullary junction, and ampulla—throughout the estrous cycle. Because there was no relative staining differences between cell types for the PGESs during the estrous cycle, we show only one stage in Fig. 4. Immunolocalization revealed a similar expression pattern for each PGES protein in the isthmus, the isthmic-ampullary junction, and the ampulla section of the oviduct. The PGESs were found everywhere in the oviductal epithelium, lamina propria, and smooth muscle cells of all sections. However, the staining was clearly more intense in the oviductal epithelial cells in all sections. Interestingly, PGES signal intensities were principally localized on the luminal surface of the epithelial cells in all sections (Fig. 4). 4. Discussion In this study, we detected in the bovine oviduct a modulation of the spatiotemporal expression of mRNA of one of the two COXs. The mRNA for COX-1 was maximally expressed in the ampulla at the ovarian pole of the oviduct, but not modulated during the estrous cycle. This result was different from a previously published study [23], where COX-1 mRNA expression

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Fig. 4. Immunolocalization of mPGES-1, mPGES-2, and cPGES in various sections of the bovine oviduct. Three animals were analyzed at each stage of the estrous cycle. As no difference in relative staining was observed throughout the estrous cycle, only the three sections are shown. I, isthmus; I-A, isthmic-ampullary junction; A, ampulla. Nonspecific IgG was used as negative control (–). The red color represents positive results. The counterstain was Mayer’s hematoxylin. Final magnification 200 and  400.

was higher during the periovulatory period in oviductal epithelial cells. However, the current results for COX-2 mRNA were similar to those of Odau et al. [23], with no significant effects of stage, side, or section for COX-2 mRNA in the bovine oviduct. These results were surprising, as COX-1 is known as a constitutive enzyme expressed at constant levels in many physiologic processes, in contrast with COX-2 shown to be potentially regulated by the estrous cycle in bovine endometrial tissues [25]. In contrast with the bovine uterus, COX-1 appeared to play a strategic role in the oviduct. Indeed, Odau et al. [23] reported that COX-1

protein was approximately four- to fivefold more abundant than COX-2 in flushed oviductal cells. Moreover, COX-1 activity predominated over that of COX-2 in oviductal cell lysates, as shown using selective COX inhibitors. Finally, COX-1 was expressed ubiquitously in the oviduct, whereas COX2 was restricted to the epithelial layer [23]. We present here apparently the first study of spatiotemporal expression of the three known PGESs in the bovine oviduct during the estrous cycle. All PGESs were expressed with cPGES and mPGES-2 following a pattern very close to that of COX-1. In

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addition, the three PGESs were mostly expressed in oviductal epithelial cells close to the lumen. The analysis comparing expression on ipsilateral and contralateral oviducts revealed inverse preferential expression between mPGES-1 and mPGES-2. Indeed, mPGES-1 protein levels were primarily expressed in the contralateral oviduct, whereas mPGES-2 was mainly expressed in the ipsilateral oviduct. The reason for this specific phenomenon is obscure because it was observed only at the protein level and not influenced by the stage of the estrous cycle. The mechanism behind this phenomenon remains to be elucidated. The combined analysis of the expression pattern of COXs and PGESs led to interesting conclusions. Indeed, preferential coexpression of COX-1 and cPGES and mPGES-2 in epithelial cells takes all its physiologic relevance, as these are known to preferentially couple with COX-1 for PGE2 production [13,16]. As mentioned earlier [23], COX-1 appeared to play a more predominant role in the oviduct epithelial cells where PGESs are mostly present. Because mPGES-1 was exclusively coupled to COX-2, its role in the production of PGE2 in the oviduct might be lesser for PGE2 production than that for the other PGESs coupled to COX-1. According to our results, we anticipated a higher production of PGE2 in the ipsilateral oviduct throughout the estrous cycle, as mPGES-2 was mostly expressed on that side of the oviduct. Interestingly, total tissue PGE2 levels were higher in the ipsilateral oviduct than that in the contralateral oviduct at the follicular stage (metestrus) and postovulatory stage (proestrus) of the bovine estrous cycle [7]. Because there were more PGES enzymes in the ampulla, we expected higher levels of PGE2 in the ampulla than in the isthmus. In that regard, there was a higher level of PGE2 in the ampulla section than in the isthmus section in human fallopian tubes [29,30]. However, Wijayagunawardane et al. reported constant PGE2 levels all along the bovine oviduct [7]. The latter observations corresponded with what we observed with antioxidant systems in the bovine oviduct [31]. Indeed, oviductal fluid glutathione peroxidase activity remained constant throughout the oviduct, in spite of its relatively large variations of the corresponding gene mRNA expression [31]. We believe that the mixing of the oviductal fluid that occurred naturally in response to the movements of the oviduct muscle layer and ciliated cells in the infundibulum and ampulla [6] may have masked the local changes in both antioxidants and PGE2 levels. Some PGESs are glutathione-S-transferases and require thiols (–SH) for their catalytic activity [13]. The glutathione (GSH) conferred stability and pro-

moted the activity of mPGES-1 and was also required by cPGES for isomerization of PGH2 into PGE2 [13]. In contrast, mPGES-2 activity was GSH independent [15]. Interestingly, we reported previously that GSH levels were highest in the oviductal fluid of the ampulla just before ovulation [31]. Because the oviductal fluid is in the proximity of PGES on the luminal side of epithelial cells, perhaps GSH modulates PGES activity and the production of PGE2. The latter correlates with higher PGE2 levels observed during the follicular phase [7]. Furthermore, even if PGES levels remained stable throughout the estrous cycle, the GSH cofactor may play a major role in the regulation of PGE2 production, as its level in the oviductal fluid varies up to three- to fivefold during the estrous cycle [31]. In conclusion, we conducted an integrated study showing simultaneously the expression of COX and PGES enzymes in the oviduct. We inferred that there was a preferential association between COX-1 and its known partners cPGES and mPGES-2 to regulate PGE2 production. Coupled with our previous observations of similar regulation of GSH availability within the oviduct [31], we propose that GSH acts as a modulator of PGES activity and contributes to the modulation of PGE2 levels in the oviduct during the estrous cycle. Acknowledgments This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC grant no. 238570-02 to J-F.B.). The authors thank Dr. Julie Parent for the cloning of PGESs and express gratitude to Pierre Chapdelaine for his technical help. J-F. B. was the recipient of a Canadian Institutes of Health Research (CIHR), Institute of Aging, New Investigators award. M.R. was the recipient of a scholarship from Fonds de Recherche sur la Nature et les Technologies (FQRNT). We also thank Ms. He´le`ne Cre´peau (Laval University, Statistical Service) for assistance with statistical analysis. References [1] Croxatto HB. Physiology of gamete and embryo transport through the fallopian tube. Reprod Biomed Online 2002;4:160–9. [2] 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. [3] Ushikubi F, Sugimoto Y, Ichikawa A, Narumiya S. Roles of prostanoids revealed from studies using mice lacking specific prostanoid receptors. Jpn J Pharmacol 2000;83:279–85. [4] Wanggren K, Stavreus-Evers A, Olsson C, Andersson E, Gemzell-Danielsson K. Regulation of muscular contractions in the

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[5] [6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

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