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Regular Article |
a Département d'Ontogénie et Reproduction, Centre de Recherche en Biologie de la Reproduction, Centre de Recherche du CHUL, Université Laval, Ste-Foy, Québec, Canada GIV 4G2
b Département d'Obstétrique et Gynécologie, Université Laval, Ste-Foy, Québec, Canada GIV 4G2
c Centre de Recherche en Reproduction Animale, Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2) is responsible for luteolysis and prostaglandin E2 (PGE2) is thought to be involved in maternal recognition of pregnancy. In the present study, healthy uteri were collected from cows at the abattoir, and days of the estrous cycle were determined macroscopically. The uteri were classified into seven groups as Days 1–3, 4–6, 7–9, 10–12, 13–15, 16–18, and 19–21 of the estrous cycle. Endometrial scrapings were collected. The expression of cyclooxygenase (COX)-1 and COX-2 mRNAs and proteins and PGE synthase (PGES) mRNA was analyzed by Northern and Western blot. There was no expression of COX-1, either mRNA or protein, on any day of the estrous cycle. In contrast, COX-2 mRNA and protein were expressed at low and high levels on Days 1–12 and 13–21 of the estrous cycle, respectively. The level of expression of PGES was moderate, low, and high on Days 1–3, 4–12, and 13–21 of the estrous cycle, respectively. There were significant correlations between COX-2 mRNA and protein levels and between COX-2 and PGES mRNA levels. COX-1 mRNA and protein are not expressed on any day of the estrous cycle, whereas COX-2 mRNA and protein and PGES mRNA are differentially expressed and regulated in bovine endometrium during the estrous cycle. COX-2, rather than COX-1, is the primary isoenzyme involved in the endometrial production of prostaglandins, and the COX-2 and PGES pathway is responsible for the endometrial production of PGE2 in the bovine endometrium during the estrous cycle.
corpus luteum, female reproductive tract, mechanisms of hormone action, ovulatory cycle, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The downstream enzymes, PGE synthase (PGES) and PGF synthase (PGFS), catalyze the conversion of PGH2 to PGE2 and PGF2, respectively. It has been postulated that COX-1 is a constitutive enzyme involved in housekeeping functions. In contrast, COX-2 is an inducible enzyme that plays a role in various pathological and some physiological conditions in animal tissues [1, 2]. The expression and regulation of COX-1 and COX-2 are tissue and species specific. Their expression within a given species and tissue varies depending on physiological or pathological status [3–5]. In some tissues, coexpression of COX-2 and PGES has been demonstrated [6]. Very recently, bovine COX-2 and PGES cDNAs were cloned and sequenced. The bovine COX-2 and PGES possess 91%–100% and 75%–85% amino acids homology with those enzymes of other species [7, 8].
In ruminants, PGF2 and PGE2 are the primary PGs produced in the uterine endometrium, but their secretory patterns are different [9, 10]. During the bovine estrous cycle, the period of Days 15–17 is critical for either luteolysis or pregnancy recognition [11–13]. During luteolysis, endometrial PGF2 is secreted in a series of pulses in response to modulation by endogenous oxytocin [11]. In early pregnancy, interferon 
(IFN) acts as the embryonic signal to inhibit the pulsatile secretory pattern of PGF2 and corpus luteum (CL) regression [14]. In addition to IFN, PGE2 probably is also involved in maternal recognition of pregnancy as a temporary luteotrophic signal in ruminants [15–17]. Irrespective of the individual PGs involved, PGF2 or PGE2 synthesis must occur through one of the COX pathways. In ruminants, both in vivo [18–22] and in vitro [7, 23–26] studies have revealed the expression and regulation of COX-1 and COX-2 in uterine tissues under different physiological states such as recognition of pregnancy, luteolysis, pregnancy, and parturition. Only a few studies have been undertaken during the estrous cycle in ovine endometrium. Initially, Husling et al. [27] reported that the increase in the ability of the uterus to synthesize PGs was related to the changes in the PGHS protein. Later, another group using different techniques demonstrated that in ovine endometrium PGHS-2 protein was expressed maximally between Days 10 and 16 of the estrous cycle [9] and the level of expression of PGHS-2 mRNA did not differ significantly throughout the estrous cycle [28]. More recently, Charpigny et al. [21, 26] demonstrated that in ovine endometrium, COX-1 protein was expressed at steady state levels and COX-2 protein was highly and transiently expressed from Day 12 to Day 15 of the estrous cycle and during pregnancy but disappeared at the end of the cycle. Moreover, the results of these studies demonstrated a functional correlation between COX-2 expression and PGE2 production. However, the enzymes involved in endometrial production of PGE2 are poorly understood. Recently, we demonstrated expression of PGES in bovine endometrium [29].
No information has been available concerning the expression of COX-1 and COX-2 in bovine endometrium during the estrous cycle. Hence, the main objectives of the present study were to document the expression of COX-1 and COX-2 mRNA and protein in bovine endometrial tissue on different days of the estrous cycle and to demonstrate coexpression of COX-2 and PGES in bovine endometrium during the estrous cycle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Over the years, we have relied extensively on material from the abattoir to conduct in vitro studies. At present, we and others follow the method described by Ireland et al. [30] for classifying the four stages of the estrous cycle based on ovarian morphology. Abattoir/slaughterhouse material is a convenient and economical source of animal tissues, but the pathological conditions and physiological states of tissues obtained from this source are unknown. Therefore, we have included some characteristics of uterine and ovarian morphology to allow selection of healthy tissues on specified days of the estrous cycle. The proposed method for selection and classification of uterine tissues integrates criteria described by Ireland et al. [30] and by Miyamoto et al. [31].
Apparently healthy uteri at different days of the estrous cycle were collected at the local abattoir. Uteri were obtained within 10 min of slaughter, immediately placed on ice, and transported to the laboratory within 1–1.5 h. Uteri were dissected from the surrounding tissues, washed with physiological saline (PBS), and cut open on their longitudinal axes along the greater curvature. The endometrium was scraped using a surgical blade and processed according to the protocols described below. The tissues were maintained on ice at all times. The days of the estrous cycle were determined by macroscopic examination of both ovaries and uteri according to criteria in Table 1 and Figure 1. Uteri were classified into seven groups as Days 1–3 (n = 4), 4–6 (n = 3), 7–9 (n = 3), 10–12 (n = 3), 13–15 (n = 6), 16–18 (n = 8), and 19–21 (n = 7). Careful examinations were done so that infected uteri and uteri in first postpartum estrus could be eliminated. External and internal characteristics of the CL, presence or absence of follicles, follicle diameter, uterine tonicity, and characteristics of the endometrium and cervix were examined carefully. To indentify infected and first postpartum uteri, uterine consistency, vascularization, thickness of blood vessels at the lesser curvature, and consistency of caruncles were examined externally. Internally, the entire mucous membrane and surface of the caruncles were examined for signs of inflammation.
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Total Protein Extraction
The endometrial scraping (500 mg wet weight) was thoroughly homogenized in 3 volumes of buffer (0.02 M Tris-HCl pH 7.5, 0.5 M EDTA, 1 mM dithiothreitol, 0.1 M PMSF, 10% glycerol) using a Polytron homogenizer. Total proteins were extracted and quantified using a procedure recently described [32]. Homogenized material was stored at -20°C until use.
Western Blots
Approximately 20-µg aliquots of total proteins were loaded in each lane and electrophoresed on 10% SDS polyacrylamide gels followed by electrotransfer onto nitrocellulose membranes (Amersham Pharmacia Biotech, Montreal, PQ, Canada). Prestained protein markers (New England Biolabs, Mississauga, ON, Canada) were used as molecular weight standards for each analysis. As positive controls, endometrial epithelial cells from primary culture were used for COX-1 analysis and an endometrial scraping from Day 16 of the estrous cycle was used for COX-2 analysis. The blots were stained with 0.5% ponceau-s (Fisher Biotech, Montreal, PQ, Canada) in 1% acetic acid for evaluating the quality of protein transfer. Blots were performed simultaneously and separately (duplicate) for COX-1 and COX-2. Proteins were blocked overnight at 4°C with 5% fat-free dry milk powder in PBS and 0.05% Tween-20 (PBS-T). The blots were incubated with primary antibody (rabbit polyclonal antisera raised against purified sheep seminal vesicle COX-1 [lot no. 241] or rabbit polyclonal antisera raised against purified sheep placental COX-2 [lot no. 243]; Merk-Frost, Montreal, ON, Canada) for 1 h at room temperature at a dilution of 1:3000 in 1% fat-free dry milk powder in PBS-T. The blots were washed 3 times at 10-min intervals in PBS-T and then incubated with secondary antibody (goat anti-rabbit IgG conjugated with horseradish peroxidase; Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at room temperature at a dilution of 1:20 000 in 3% fat-free dry milk powder in PBS-T. Blots were then washed 3 times at 10-min intervals in PBS-T. Chemiluminescent substrate was applied according to the manufacturer's instructions (Renaissance; NEN, Life Science Products, Boston, MA). The blots were exposed to BioMax film (Eastman Kodak, Rochester, NY) with an intensifying screen for 1–5 min at room temperature. The intensity of the signal was quantified by the densitometry of autoradiograms using Alpha Imager 2000 (Alpha Innotec Corp., Montreal, ON, Canada).
Total RNA Extraction
The endometrial scraping (500 mg wet weight) was homogenized in 10 parts (5 ml) of TRIzol (Life Technologies, Burlington, ON, Canada) using a Polytron homogenizer. Total RNA was extracted following the manufacturer's protocol. RNA was quantified using a ultraviolet spectrophotometer at 260 and 280 nm absorbance and stored at -80°C until use.
Northern Blots
Approximately 20 µg of total RNA was loaded in each lane and electrophoresed on a 1.2% formaldehyde-agarose gel. An RNA ladder (High Range; Life Technologies) was used as a molecular weight standard for each analysis. As positive controls, endometrial epithelial cells from primary culture were used for COX-1 analysis and an endometrial scraping from Day 16 of the estrous cycle was used for COX-2 analysis. RNA quality was evaluated by visualizing the ethidium bromide-stained rRNA bands (28S and 18S) under ultraviolet light. After electrophoresis, RNA was transferred overnight onto nylon membranes (Bright Star-plus; Ambion, Austin, TX) in 10x saline sodium citrate (SSC; 1x SSC is 3 M NaCl and 0.3 M sodium citrate, pH 7.0). The blots were made in duplicate for mRNA analysis of COX-1 and COX-2. Completion and uniformity of blotting were assessed by determining transfer of 28S and 18S rRNA from the gel. The blots were cross-linked with ultraviolet light for 5 min, baked at 80°C for 1 h, and stored at -207°C until use. A commercial hybridization solution (UltraHyb; Ambion) was used as prehybridization and hybridization solution. Prehybridization was carried out for 1 h at 45°C. The cDNA probes for COX-1 and COX-2 were labeled with (32P-dCTP, 3000 Ci/mmol) using a Ready-To-Go DNA labeling kit (Amersham) to a specific activity of approximately 109 cpm/µg and used at a final concentration of 106 cpm specific probe/ml of hybridization solution. The hybridization was carried out overnight at 45°C. After hybridization, the blots were washed in solution I (2x SSC and 0.1% SDS) twice for 5 min each at room temperature and further washed in solution II (0.1x SSC and 0.1% SDS) twice for 15 min each at 68°C. Blots were exposed to BioMax film with intensifying screen at -80°C until good signals were observed (<7 days). The intensity of the signal was quantified by densitometry of autoradiograms using Alpha Imager. The blots were stripped of COX-1 and COX-2 probes by boiling in 1% SDS for 30 min and were rehybridized with a [
-32P]ATP-labeled oligoprobe specific to 18S rRNA to normalize each level of COX-1 and COX-2 mRNA [33]. To analyze the coexpression of COX-2 and PGES, blots were prepared separately. Approximately 15 µg of total RNA was loaded in each lane, and blotting and hybridization were performed as described above. The blots were probed with PGES cDNA and then stripped off and reprobed with COX-2 cDNA. The intensity of the bands was quantified, and each band was normalized with ethidium bromide-stained 18S rRNA on the gel.
Probe Synthesis
Bovine COX-1 and COX-2 partial cDNAs were obtained by RT-PCR amplification according to previously published procedures [23]. To obtain the bovine PGES partial cDNA, 1 µg total RNA from endometrium was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and oligo (dT) primers (Life Technologies). PCR amplification was done using specific primers (sense, 5'-AAACATATGCCTGCCCACAGCCTGGTGATG-3'; antisense, 5'-AAACATATGTCACAGGTGGCG-GGCTGCCTC-3') deduced from the recently published sequence of bovine PGES [8]. The amplified products were then cloned into pCR 3.1 TA (COX-1 and COX-2) and pCR 2.1 TOPO (PGES) vectors (Invitrogen, Life Technologies) and confirmed by sequencing with T7 DNA polymerase kit (USB Corporation, Montreal, ON, Canada). BamHI and XhoI cloning sites (Amersham) were used for COX-1 and COX-2; EcoRI (Amersham) was used for PGES. The fragments (COX-1: 777 base pairs [bp]; COX-2: 449 bp; PGES: 466 bp) were isolated using a QIAquick gel extraction kit (Qiagen, Mississauga, ON, Canada).
Statistical Analysis
Expression levels were expressed as relative integrated densitometry values (IDV). All numerical data were summarized as the mean ± SEM. Correlation between the expression of COX-2 mRNA and that of COX-2 protein and between the expression of COX-2 mRNA and PGES mRNA during different days of the estrous cycle was determined. Variations in the level of expression of COX-1, COX-2, and PGES among the days of the estrous cycle were determined using ANOVAs followed by Fischer protected least squares difference, Duncan new multiple range, and Student-Newman-Keuls multiple comparison tests (Super ANOVA; Abacus Concepts, Berkeley, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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COX-1 Expression
Neither COX-1 mRNA nor COX-1 protein was expressed in bovine endometrium on any day(s) of the estrous cycle (Fig. 2).
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COX-2 Expression
Multiphasic expression of COX-2 mRNA and protein was detected in the bovine endometrium at different days of the estrous cycle (Figs. 2 and 3). At all days of the estrous cycle studied, the expression of COX-2 mRNA and protein were significantly correlated (r = 0.87, P < 0.01). COX-2 mRNA and protein were expressed at low levels between Days 1 and 12 but at significantly higher levels (P < 0.01) between Days 13 and 21 of the estrous cycle. Between Days 13 and 21, COX-2 mRNA levels did not vary significantly (P > 0.05). In contrast, COX-2 protein levels were highly regulated. COX-2 protein levels peaked between Days 16 and 18 at levels significantly higher (P < 0.01) than those observed on Days 13–15 and 19–21 of the estrous cycle. There was no significant difference, however, in COX-2 protein levels between Days 13–15 and Days 19–21 of the estrous cycle.
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COX-2 and PGES Coexpression
During the estrous cycle, PGES mRNA levels were moderate between Days 1 and 3, low between Days 4 and 12, and significantly higher (P < 0.05) between Days 13 and 21 of the estrous cycle (Fig. 4). The level of PGES and COX-2 expression was significantly correlated (r = 0.85, P < 0.01) between Days 13 and 21 of the estrous cycle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiphasic expression of COX-2 mRNA and protein in the bovine endometrium during different days of the estrous cycle is consistent with the inducible nature of COX-2 enzyme observed in other tissues [1–5]. COX-2 mRNA and protein were expressed at low levels between Days 1 and 12 and at high levels between Days 13 and 21 of the estrous cycle. Moreover, the expressions of COX-2 mRNA and protein were positively correlated throughout the estrous cycle. This finding indicates that during the bovine estrous cycle expression of COX-2 is regulated at both the mRNA and protein levels.
In ruminants, endometrial PGF2 is considered the luteolytic hormone. Bovine endometrium becomes responsive to PGF2-releasing factors after Day 13 [11]. In the bovine endometrium, maximal production of PGF2 (estimated by levels of its PGFM [13,14-dihydro-15-keto PGF2] metabolite in circulation) is observed between Days 13 and 16 [34]. The first endometrial PGF2 pulse appears on Day 14 of the bovine estrous cycle [35], and series of PGF2 pulses occur between Days 16 and 17 [11]. However, the basic mechanisms underlying increased PG production at the cellular level in the bovine endometrium have been poorly described. In the ovine endometrium, evaluation of PG biosynthesis revealed that maximal levels of PGHS-2 protein appear between Days 10 and 16 of the estrous cycle [9] while maximal PGF2 production occurs with an 8-fold increase in the concentration of uterine PGHS [27]. COX-2 was later found to be highly and transiently expressed from Day 12 to Day 15 of the ovine estrous cycle [21]. All the studies conducted in the ovine endometrium demonstrated that the expression of COX-2 was coincident with the expected time of luteolysis, i.e., Days 13–16 of the estrous cycle (average length of estrous cycle is 16–17 days). In the present study, the maximal expression of COX-2 mRNA and protein between Days 13 and 21 and the peak expression of COX-2 protein between Days 16 and 18 of the estrous cycle also matches the expected time of luteolysis (Days 16–17) in the cow. Furthermore, the present findings strongly support the accepted concept that in ruminants, endometrial production of PGs during the middle and late luteal phase of the estrous cycle is consequent to an increase in COX-2 enzyme and is a prerequisite for the endometrial production of PGs.
The level of COX-2 mRNA and protein between Days 1 and 12 of the estrous cycle is surprising. In most tissues and cells, COX-2 has been described as an inducible enzyme, which is not expressed under normal or resting conditions [1–5]. However, some tissues such as neurons of the central nervous system and macula densa cells of the juxtaglomerular apparatus in the renal cortex also expressed COX-2 at low level. However, expression in these areas does not represent constitutive expression because COX-2 levels are dynamically regulated in these tissues [36, 37]. The bovine endometrium appears to be another tissue expressing low basal levels of COX-2 from Day 1 to Day 12 and an induced level from Day 13 to Day 21 of the estrous cycle. We and others have shown that the expression of COX-2 mRNA could be modulated by oxytocin [19, 24], progesterone and estradiol [20, 25], LH and FSH [38], and IFN [23] under different physiological conditions in bovine uterine tissues and cells. The bovine endometrium secretes PGF2 and PGE2 throughout the estrous cycle [31, 39], but the secretory pattern is different [39]. In the present study, the multiphasic expression of COX-2 suggests that COX-2 is the enzyme responsible for both the basal and pulsatile secretion of endometrial PGs at different days of the bovine estrous cycle.
The maximal expression of COX-2 mRNA and protein between Days 13 and 21 of the estrous cycle coincides with both the expected time of luteolysis (Days 16–17) and the mid-luteal (Days 13–15) and follicular (Days 19–21) phases of the estrous cycle. The expression of COX-2 mRNA and protein after the expected time of luteolysis, i.e., during the follicular phase, suggests that endometrial PGs (PGF2 and/or PGE2) also play a role in structural luteolysis (luteal cell apoptosis), uterine tonicity, cervical relaxation, and preovulatory events, which are the normal physiological phenomena occurring during the follicular phase of the estrous cycle.
Further, the expression of COX-2 mRNA and protein on Days 13–15, before the expected time of luteolysis, indicates that the endometrial PG production machinery is present as early as Day 13 of the estrous cycle. COX-2 expression is normally induced immediately before the increase in PG production. Therefore, early expression of COX-2 between Days 13–15 of the bovine estrous cycle must be required for both luteolysis and the other reproductive events taking place during the same period. During the bovine estrous cycle, luteolysis and recognition of pregnancy occur at the same period on a competitive basis. In ruminants, the presence of a viable embryo prevents pulsatile PGF2 secretion through the release of IFN [14], but IFN could also stimulate COX-2 expression and PGE2 production in vitro [23]. In the 1970s, the proposal was made that PGE2 might serve as a temporary luteotrophic hormone and immunomodulator during establishment of pregnancy in ruminants [15–18, 40].
During biosynthesis of PGs, COX-2 is only able to control the conversion of arachidonic acid to PGH2. Biologically active PGE2 and PGF2 are catalyzed from PGH2 by PGES and PGFS, respectively. In the present study, PGES was coexpressed with COX-2 at a high level between Days 13 and 21 of the estrous cycle. These results suggest that the machinery for endometrial PGE2 production is readily present as early as Day 13 of the estrous cycle. We therefore infer that the bovine endometrium becomes responsive to produce PGE2 after Day 13 of the estrous cycle. Days 13–15 may represent optimal uterine receptivity. The presence of a viable embryo (IFN) may switch on the COX-2 and PGES pathway and PGE2 production and effect establishment of pregnancy. In the absence of an embryonic signal, other factors may switch on the COX-2 and PGFS pathway, PGF2 production, and luteolysis. However, the factors that regulate this functional coupling remain to be determined. In the ovine endometrium, the levels of expression of COX-2 mRNA [28] and protein [21, 26] did not differ between Days 12 and 16 of the estrous cycle and early pregnancy. Moreover, Charpigny et al. [26] found a functional correlation between COX-2 expression and PGE2 production on Day 12 of the estrous cycle and early pregnancy; at that time, PGF2 secretion was low and only reached a peak concentration later (Days 15–16). The study also suggested that expression of COX-2 and COX-2-dependent PGs was both related to luteolysis and required for the establishment of pregnancy in sheep. Our present findings combined with previous results [21, 23, 26, 29] suggest that the COX-2 and PGES pathway might be responsible for the endometrial production of PGE2 during the estrous cycle and/or early pregnancy in ruminants. These findings further support the concept that COX-2 might be involved in several and even opposed physiological events. The low and moderate levels of PGES expression between Days 1 and 12 of the estrous cycle also coincide with the low basal level expression of COX-2. The physiological significance of this coexpression, however, remains to be determined. Nonetheless, a recent study showed that the bovine endometrium is able to produce PGE2 along with PGF2 at low and elevated levels throughout the estrous cycle [39].
Several years ago it was proposed that PGE2 of embryonic origin might play a role in the establishment of pregnancy in ruminants. Recently, Charpigny et al. [41] demonstrated that COX-2 was highly expressed in the trophoblastic cells of the ovine embryo between Days 10 and 17 of pregnancy, whereas COX-1 was undetectable. These authors also suggested that embryonic production of PG during establishment of pregnancy might be through the COX-2 pathway. Furthermore, the pattern of expression of COX-2 in ovine embryo closely matches the pattern of IFN expression between Days 10 and 21 of pregnancy [42]. Therefore, the available evidence strongly suggests that COX-2, rather than COX-1, is responsible for the production of PGE2 of both embryonic and endometrial origin in ruminants during the establishment of pregnancy [21, 23, 26, 41].
COX-1 is expressed constitutively in most tissues, and expression of this enzyme does not vary greatly in the adult animal. There is considerable information on the regulation of COX-2 gene expression but little information about how the expression of COX-1 is controlled [2]. The absence of COX-1 mRNA and protein during the estrous cycle in the present study was unexpected. Similar results were observed in pregnant and parturient cows, where COX-1 was not expressed in the endometrium and was only sporadically expressed in the myometrium [18]. In agreement with these data, only minimal COX-1 expression was detected in the myometrium during the estrous cycle (data not shown). Taken together, these results demonstrate that COX-1 is not expressed in endometrium and is expressed at very low levels in myometrium during the bovine estrous cycle, pregnancy, and parturition. Further, in knockout studies with COX-1 null mice, females had difficulties with parturition but not with ovulation, fertilization, and implantation, whereas COX-2 null female mice had reproduction failures at multiple levels [43–46]. In recent reviews [1, 2, 46] different properties of COX-1 and COX-2 have been described to explain how COX-2 can operate independently of COX-1.
Unlike the results for COX-2, the expression of COX-1 appears quite different between cows and ewes. In pregnant cows, COX-1 was not expressed in the endometrium and cotyledons and was only sporadically expressed in the myometrium and caruncles [18]. In pregnant ewes, COX-1 was expressed in the endometrium, myometrium, cotyledon, and amnion [47]. In recent studies in the ewe, the constitutive expression of COX-1 protein in the endometrium during the estrous cycle was described [21, 26], whereas no expression of COX-1 mRNA and protein was found in bovine endometrium during the estrous cycle. Moreover, in ovine endometrium there was no expression of COX-2 during the early stage of estrous cycle (Days 1–11) [21, 26], whereas we did find COX-2 expression at similar stages in the cow. These results suggest that species variation exists between ewes and cows in the expression of COX-1 and COX-2 in uterine tissues. However, both in vivo and in vitro studies in ovine and bovine uterine tissues have shown that COX-2 expression is modulated in response to various stimuli and is well correlated with the production of PGs during luteolysis [19, 21, 26], maternal recognition of pregnancy [21, 23, 26], pregnancy [18], and parturition [18, 22].
In this study, bovine endometrial epithelial cells in primary culture were used as a positive control for analyzing COX-1 expression. Irrespective of the days of the estrous cycle, epithelial cell cultures at confluency expressed COX-1 mRNA and protein at relatively high levels. COX-1 expression is neither modulated in response to stimuli oxytocin (OT, phorbol 12-myristste 12-acetate, lipopolysaccharide, IFN) nor correlated with the production of PGE2 and PGF2. In contrast, COX-2 expression is modulated and correlated with PGE2 and PGF2 production [23, 48] (unpublished data).
COX-1 was not expressed on any day of the bovine estrous cycle, whereas COX-2 and PGES were expressed and regulated in bovine endometrial tissue during different days of the estrous cycle. COX-2, rather than COX-1, is the primary isoenzyme involved in the endometrial production of PGs in bovine endometrium during the estrous cycle. Therefore, the COX-2 and PGES pathway might be responsible for the endometrial production of PGE2 during the bovine estrous cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to M.A.F. and in part by a Canadian Institute of Health Research (CIHR) grant (MT-13190) to J.S. J.S. is supported by a CIHR Investigator Award. 
2 Correspondence. FAX: 418 654 2765; mafortier.crchul.ulaval.ca
Accepted: January 30, 2002.
Received: September 18, 2001.
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 J. Lapointe, M. Roy, I. St-Pierre, S. Kimmins, D. Gauvreau, L. A. MacLaren, and J.-F. Bilodeau Hormonal and Spatial Regulation of Nitric Oxide Synthases (NOS) (Neuronal NOS, Inducible NOS, and Endothelial NOS) in the Oviducts Endocrinology, December 1, 2006; 147(12): 5600 - 5610. [Abstract] [Full Text] [PDF]
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 S. Odau, C. Gabler, C. Holder, and R. Einspanier Differential expression of cyclooxygenase 1 and cyclooxygenase 2 in the bovine oviduct. J. Endocrinol., October 1, 2006; 191(1): 263 - 274. [Abstract] [Full Text] [PDF]
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T. R. Bilby, A. Guzeloglu, L. A. MacLaren, C. R. Staples, and W. W. Thatcher Pregnancy, Bovine Somatotropin, and Dietary n-3 Fatty Acids in Lactating Dairy Cows: II. Endometrial Gene Expression Related to Maintenance of Pregnancy. J Dairy Sci, September 1, 2006; 89(9): 3375 - 3385. [Abstract] [Full Text] [PDF]
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M. Parent, E. Madore, L. A MacLaren, and M. A Fortier 15-Hydroxyprostaglandin dehydrogenase in the bovine endometrium during the oestrous cycle and early pregnancy. Reproduction, March 1, 2006; 131(3): 573 - 582. [Abstract] [Full Text] [PDF]
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S. Herath, D. P. Fischer, D. Werling, E. J. Williams, S. T. Lilly, H. Dobson, C. E. Bryant, and I. M. Sheldon Expression and Function of Toll-Like Receptor 4 in the Endometrial Cells of the Uterus Endocrinology, January 1, 2006; 147(1): 562 - 570. [Abstract] [Full Text] [PDF]
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S.K. Banu, J.A. Arosh, P. Chapdelaine, and M.A. Fortier Expression of Prostaglandin Transporter in the Bovine Uterus and Fetal Membranes During Pregnancy Biol Reprod, August 1, 2005; 73(2): 230 - 236. [Abstract] [Full Text] [PDF]
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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]
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