help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Asselin, E.
Right arrow Articles by Fortier, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Asselin, E.
Right arrow Articles by Fortier, M. A.
Right arrowPubmed/NCBI databases
*Gene*HomoloGene
*Nucleotide*Protein
*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*OXYTOCIN
*PROSTAGLANDIN F2ALPHA
Endocrinology Vol. 138, No. 11 4798-4805
Copyright © 1997 by The Endocrine Society

ARTICLES

Cellular Mechanisms Involved during Oxytocin-Induced Prostaglandin F2{alpha} Production in Endometrial Epithelial Cells in Vitro: Role of Cyclooxygenase-21

Eric Asselin, Patrick Drolet and Michel A. Fortier

Département d’Ontogénie et Reproduction (E.A., P.D., M.A.F.), Centre de Recherches du Centre Hospitalier de l’Université Laval, Centre de Recherche en Biologie de la Reproduction, Ste-Foy, Québec G1V 4G2, Canada; and Département d’Obstétrique et Gynécologie (M.A.F.), Université Laval, Québec G1V 4G2, Canada

Address all correspondence and requests for reprints to: Dr. M. A. Fortier, Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier, de l’Université Laval, 2705 Boulevard Laurier, Sainte-Foy, Québec G1V 4G2, Canada. E-mail: mafortier{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PGs are important regulators of reproductive processes. At the time of luteolysis in vivo, PGF2{alpha} is produced by endometrial cells, in response to oxytocin (OT). The mechanism by which OT induces the release of PGF2{alpha} remains to be defined. We have used 13 different cultures of bovine epithelial endometrial cells to study the effect of OT on the regulation of PGF2{alpha} and to identify the possible involvement of cyclooxygenases (COXs). OT induced a dose-dependent increase of both inositol phosphates (IPs) and [Ca2+]i concentration in epithelial cells labeled with [3H]-myoinositol or loaded with fura-2 (using a fluorescent microscope imaging system), respectively. OT induced a dose-dependent increase of both PGF2{alpha} production and COX-2 gene expression (as demonstrated by RT-PCR and Northern blots). PGF2{alpha} production was increased from 13.3 ± 2.0 to 166.8 ± 22.5 ng/ml (P < 0.0001). On the other hand, COX-2/ß-actin mRNA gene expression (as determined by densitometric analysis) was increased 5.1 ± 0.7-fold (P < 0.001) with OT (10-7 M) treatment, compared with control. Addition of indomethacin (1 µM) and a specific COX-2 inhibitor (NS-398, 1 µM) blocked the OT-induced PGF2{alpha} production. COX-1 and phospholipase A2 mRNA were expressed at steady-state levels, but no effect of OT was detected on their regulation. Combined to OT, 10 µg/ml of recombinant ovine interferon-tau (roIFN-{tau}) was able to decrease significantly (P < 0.0001) the dose-dependent increase of PGF2{alpha} production. Furthermore, partial bovine COX-1 (777 pb) and COX-2 (449 bp) cDNAs were cloned and sequenced. An homology of 83% and 97% was found in relation with rat and sheep, for COX-1, respectively. COX-2 was found to bear 84%, 86%, and 87% of homology in relation to rat, guinea pig, and human, respectively. Collectively, these results demonstrate, for the first time, that COX-2 is involved in the mechanism by which OT regulates PGF2{alpha} production in the endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS, along with sex steroids, are among the most important regulators involved in the success of pregnancy (1). In mammals, it is generally accepted that prostaglandin F2{alpha} (PGF2{alpha}), of uterine origin, is responsible for luteolysis and the return to the estrous cycle. The identification of the uterus as the source of luteolytic PGF2{alpha} has been submitted to strict requirements, which have been met in several species but not in primates. In primates, PGF2{alpha} seems also to be responsible for luteolysis, but its origin is likely within the ovary itself (2). In contrast with PGF2{alpha}, PGE2 may have a luteoprotective action that is either luteotrophic or antiluteolytic (3, 4). PGE2 also is required for changes in vascular permeability that occur in the endometrium before implantation in rodents (5, 6). Inhibitors of prostaglandin synthesis, such as indomethacin, delay or inhibit these changes and prevent implantation from occurring (7). In species such as ruminants, in which PGF2{alpha} of uterine origin is luteolytic, local recognition of the presence of a viable embryo is necessary to prevent the release of PGF2{alpha} and ensure establishment of pregnancy (8). The production of PGF2{alpha} by the uterus is responsible for the luteolytic signal during the estrous cycle, and the administration of PGF2{alpha} is able to terminate pregnancy in several species (9). In ruminants, the pulsatile release of oxytocin (OT) by the neurohypophysis stimulates the production of uterine PGF2{alpha}. The ability of OT to stimulate PGF2{alpha} release is higher at the time of luteolysis (10). The initial released PGF2{alpha} triggers the release of additional OT from the corpus luteum by a positive feedback loop (11), and OT binds to endometrial OT receptors (OTR) to stimulate PGF2{alpha} secretion (12, 13). We and others have previously demonstrated that PGF2{alpha} is regulated by OT in vitro (14, 15, 16). Oxytocin has been shown to activate the inositol-specific phospholipase C in various cell systems (17, 18), generating two second messengers: inositol triphosphate (which increases intracellular calcium) and diacylglycerol (DAG) (which activates protein kinase C) (19). Calcium in human decidual cells (20) or the activation of protein kinase C in human amnion cells (17, 21) and bovine endometrial cells (15) have been shown to increase PG production.

In ruminants, large amounts of interferon (IFN)-{tau} are produced by the trophoblast during the preimplantation period (22, 23) and serve as the embryonic signal, released at the time of maternal recognition of pregnancy, to prevent the luteolytic process from occurring. Recently, we have demonstrated that roIFN-{tau} stimulated PGE2 production and COX-2 gene expression in the bovine endometrium in vitro (24, 25), and others have shown that in the ovine, it inhibits estrogen receptor necessary for induction of OT receptor before luteolysis (26). When no viable embryo is present, a net increase in PGF2{alpha} production is observed in response to OT in vivo. We suggest that OT induces gene expression of cyclooxygenase (COX) and/or phospholipase A2 (27) to effect increased PGF2{alpha} production.

The COX protein is a rate-limiting enzyme involved in the biosynthesis of PGs using arachidonic acid (AA) as its principal substrate (28, 29). Two genes (COX-1 and COX-2) (30) encode this enzyme. The constitutive isoform, COX-1, is expressed in all tissues (31) and most, if not all, nucleated cells. On the other hand, the inducible form, COX-2, is present only after induction by a variety of factors (such as CG, cytokines, and tumor promoters) (32, 33, 34, 35). Other reports demonstrated that COX-2 also was expressed during the implantation period in the rat endometrium (36, 37). Phospholipase A2 also is a rate-limiting enzyme for PGs production, because it acts directly on phospholipids (phosphatidyl choline) to release AA, necessary for PGs synthesis (1). A recent study, using sheep uterine tissues, demonstrated that COX-2 was highly, but transiently, expressed from days 12–15 of the estrous cycle in vivo (38). In the same study, COX-2 protein was expressed for an extended period in pregnant animals. These results in vivo would support the results obtained in vitro in our laboratory, where roIFN-{tau} induced an up-regulation of COX-2 mRNA and PGE2 production (24, 25). Consistent with results obtained in vivo, the present study was conducted in vitro, to determine the mechanism by which OT regulates PGF2{alpha} production in endometrial epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture plates were purchased from Becton Dickinson (Lincoln Park, NJ); RPMI-1640 and FBS were obtained from ICN Biochemicals Inc. (Missisauga, ONT). Fura-2/AM was purchased from Molecular Probes (Eugene, OR). Human cytosolic PLA2 cDNA was a gift from Dr. Walid Mourad (C.H.U.L research center, Québec). Tracers for PGE2 and PGF2{alpha}, used in the enzyme immunoassay (EIA), were purchased from Cayman Chemical Co. (Ann Arbor, MI). All reagents used for the RT-PCR (MgCl2, DTT, MMLV-RT, Taq polymerase, and respective buffers) were purchased from Gibco BRL (Burlington, ON). Indomethacin was purchased from Sigma (St. Louis, MO) and specific COX-2 inhibitor (NS-398) from Biomol (Plymouth Meeting, PA).

Production of recombinant ovine (ro)IFN-{tau} and antiviral activity assay
The roIFN-{tau} was provided by Dr. Fuller Bazer. It was produced and purified as described previously by Ott et al. (39), and antiviral activity of IFN-{tau} was determined as described by Pontzer et al. (40). In the present study, the dose used was 10 µg/ml. The concentration of roIFN-{tau} used is in the physiological range reported for this IFN to produce antiproliferative effects in vitro (41) and PGE2 regulation in vitro (24). Further, it has been shown that secretion of oIFN-{tau} increases to about 10,000 ng/h on day 16 for sheep conceptuses (42), and intrauterine injections of 100 µg/day on days 11–15 delay luteolysis (39). The antiviral activity of roIFN-{tau} was 1 x 108 U/mg protein.

Isolation of endometrial cells and culture
Bovine uteri were collected at the slaughterhouse within 15 min of death, and the physiological status of the tissue was estimated by examination of ovarian morphology (43). Uteri were transported to the tissue culture laboratory and dissected under a laminar flow hood. In this study, a total of 13 early-cycle uteri (days 1–5) were used: 4 for COXs experiments (Northern blots and RT-PCR), 3 for inhibitor experiments, 3 for roIFN-{tau} experiments, and 3 for IP measurements. Endometrial epithelial cells were cultured in 6-well plates (for RNA extraction) or 24-well plates (inhibitor assays and roIFN-{tau} experiments), as described previously (16). Medium (RPMI-1640 + 10% FBS-DC depleted of steroids by dextran-charcoal extraction) was changed every 2 days until the cells were used. Confluency of epithelial cells, isolated from endometrium in the beginning (days 1–5) of estrous cycle, occurs after 6–7 days in culture.

Experimental protocol
After the cells reached confluency, the medium was replaced with 2.0 ml of fresh serum-free RPMI-1640 containing increasing doses of OT. One plate was used for each concentration tested. The OT vehicle (water) was added to control plates. Cells were then incubated at 37 C in an atmosphere of 5% CO2: 95% air for 24 h. For all experiments, at the end of the incubation period, culture medium was recovered for PG measurements and stored at -20 C until further processing. Cells were recovered for RNA extraction or for DNA measurement (inhibitor assays and roIFN-{tau} experiments).

COX inhibitors assays and roIFN-{tau} experiments
After the cells reached confluency, the medium was replaced with 1.0 ml of fresh serum-free RPMI-1640 containing OT (10-7 M), combined or not with indomethacin (1 µM) or NS-398 (1 µM) in triplicates for 24 h at 37 C in an atmosphere of 5% CO2: 95% air. Using three different epithelial cell cultures, different doses of OT were added combined or not with roIFN-{tau} (10 µg/ml) for 24 h. The OT vehicle (water) was added to control wells. At the end of the incubation period, culture medium was recovered for PG measurement and stored at -20 C until further processing. The plates were rinsed with ethanol, and DNA content was determined for each well by 3,5 diamino benzoic acid (DABA) fluorescence, according to Fiszer-Szafarz et al. (44).

RT-PCR
Total RNA (400 ng) from each OT dose and controls for each cell type were used for preparation of first-strand cDNA by RT. The RNA samples were incubated with 0.5 µg oligo (deoxythymidine) primers at 65 C for 10 min in a final vol of 10 µl and then put on ice. Samples were then incubated 60 min at 37 C in a reaction mixture containing buffer (1X), 10 mM DTT, 1.25 mM deoxynucleotide triphosphate (dNTP), and 200 U Muloney murine leukemia virus RT (MMLV-RT) in a final vol of 20 µl. After incubation, reaction volumes were brought up to 65 µl. A negative control was performed at the same time, using the same reaction mixture, substituting water for the MMLV-RT, to ensure absence of any contaminating genomic DNA in the RNA template.

Expression of the COX-1 gene was determined by amplification of a 777-bp region of the sheep (45) COX-1 cDNA sequence (370–1144). Amplification was carried out using the antisense downstream sequence 5'-TCC AAC CTT ATC CCC AGC C-3' and the sense upstream sequence 5'-CAT GGC GAT GCG GTT GC-3'. For COX-2, expression of the gene was determined by amplification of a 449-bp region of the human (30) COX-2 cDNA sequence (411–858), and the sequences of the primers were 5'-TCC AGA TCA CAT TTG ATT GAC A-3' and 5'-TCT TTG ACT GTG GGA GGA TAC A-3'. Primers sequences were chosen from homologous nucleotide sequences of published human (30) and rat (46) COX-1 cDNAs and sheep (45) and rat (46) COX-2 cDNAs. After amplification, COX-1 and COX-2 cDNAs were cloned in pCR 2.1 plasmid using a InvitroGene cloning kit. The cDNA were sequenced by the sequencing service (Laval University, Québec) using a dideoxy PCR technique. The bovine COX-1 and COX-2 cDNAs were submitted to the GenBank database (accession numbers AF004943 and AF004944, respectively).

As a positive control for each RNA preparation, a ß-actin sequence was also amplified simultaneously in adjacent tubes. A 458-bp fragment was amplified using bovine ß-actin cDNA primers 5'-GAG GAT CTT CAT GAG GTA GTC TGT CAG GTC-3' and 5'-CAA CTG GGA CGA CAT GGA GAA GAT CTG GCA-3'. All other conditions were identical to those for COX cDNA amplification. All primers were chosen with the aid of the OLIGO 4.01 primer analysis software (National Biosciences, Inc., Plymouth, MN). Each reaction contained 5 µl of RT template or negative control, 1.5 mM MgCl2, 1x Buffer, 0.2 mM dNTPs, 10 µM of each primer, and 1.5 U Taq polymerase in a final volume of 50 µl. The PCR cycling conditions chosen were 30 sec at 94 C, 30 sec at 55 C, and 30 sec at 72 C for 30 cycles, followed by a 10-min extension at 72 C. Reaction products were analyzed on 0.7% agarose gels. Bands were visualized by ethidium bromide staining. Each experiment was performed four times using different epithelial cell preparations.

RNA isolation and Northern blots
Total RNA was prepared according to Chomczynski and Sacchi (47). Briefly, after homogenizing the cells in 4 M guanidium thiocyanate, RNA was extracted with phenol/chloroform-isoamyl alcohol (42:1), precipitated with isopropanol and washed with 70% ethanol. Total RNA (20 µg) was loaded on a 1% agarose-2.2 M formaldehyde gel and transferred to nylon membrane (Quiagen, Chatsworth, CA). After 4 h of prehybridization in a 50% formamide solution at 42 C, the membrane was sequentially hybridized overnight at 42 C in the same solution, to which was added a [32P]dCTP-labeled cDNA probe corresponding to bovine COX-1 and COX-2 (sequences determined above), human PLA2 mRNA, and [32P]dCTP-labeled ß-actin human cDNA. The [32P]-labeled DNA probes were produced by random priming protocol using a T7 Quickprime kit (Pharmacia Biotech, Baie D’Urfé, Québec). Membranes were washed and exposed to x-ray film with an intensifying screen at -80 C for 1 (ß-actin) to 24 h (COX-1, COX-2, and PLA2). Membranes were stripped between hybridizations with a boiling 0.1% SSC, 0.5% SDS solution. Autoradiograms were quantified by densitometry using Whole Band Analysis (WBA) software on BioImage. Each experiment was performed four times using different epithelial cell cultures.

Measurement of [Ca2+]i
Endometrial cells were grown on 25-mm glass coverslips placed in 6-well plates. At confluency, cells were loaded for 30 min at 22 C with 2 µM Fura-2/AM (from a 1 mM stock solution in DMSO) in 1 ml RPMI 1640 culture medium. After the loading period, cells were rinsed with Hanks’ buffered saline solution (HBS), containing (mM): NaCl, 130; KCl, 3.5; CaCl2, 1.1; MgCl2, 0.1; NaHCO3, 5; HEPES, 20; supplemented with 1% glucose. Hydrolysis of Fura-2-loaded cells was continued for 30–60 min at 22 C in the same HBS buffer. All these manipulations were conducted in the dark. Before use, the cells were washed three times with HBS buffer. The coverslips were mounted on a 2-ml chamber and placed over an Axiovert 100 HD/DIC-TV inverted epifluorescence microscope (Zeiss, Germany). The light source was generated by a 100-W mercury-xenon lamp (Hamamatsu Photonics, Japan), and the two wavelengths (340 and 380 nm) were provided by two monochromators. Fluorescent images were collected by an intensified charge-coupled device camera (Hamamatsu Photonics, Model Sony XC-75). A commercial image analysis software (Metafluor v2.0, Universal Imaging Corp., West Chester, PA) was used for data acquisition, monitoring, and image analysis. Stimulation of cells was achieved by adding 10 µl HBS, containing OT (10-7 M final concentration), near the recording site.

Total inositol-phosphates measurement
When cells reached confluency, 24 h before stimulation with OT, medium was replaced with TCM-199 (Gibco BRL, Life Technologies, Inc.) containing 10% FBS-DC and 1 µci/ml myo-3H-inositol (Amersham, Oakville, Ontario, Canada). Inositol-phosphates were measured, as described previously (with a slight modification) (48). Medium was aspirated, and cells were incubated for 15 min at 37 C in the presence of Kreb’s solution gased with 5% CO2, 95% O2 and containing 20 mM lithium chloride (LiCl). The enzymatic assay was conducted at 37 C by adding OT (10-7 M) for 10 min, and the reaction was stopped by addition of an equal volume of ethanol/HCl 0.02 N. Plates were stored at -20 C until further processing. All samples were lyophilized and resuspended in 1 ml of water and eluted on Dowex columns. To remove free 3H-inositol, the columns were rinsed with H2O and 6 ml of 5 mM sodium tetraborate/60 mM ammonium formate (NH4COOH). Then, 6 ml of 1 M NH4COOH/0.1 M HCOOH were added to remove total 3H-inositol-phosphates. Samples were recuperated separately in scintillation vials and counted with a ß counter in 10 ml scintillation liquid. The results were expressed in percent of control.

Enzyme immunoassays (EIA) of prostaglandins
For PGE2 and PGF2{alpha} measurements, an enzyme immunoassay technique (EIA) was used, which used acetylcholinesterase-linked PG tracers, as described previously (16). We have used fully characterized rabbit anti-PGE2 (49) and sheep anti-PGF2{alpha} (Bio Quant, Ann Arbor, MI). The inter- and intraassay coefficients of variation (n = 12) were 16% and 10%, respectively.

Statistical analysis
Data were analyzed by ANOVA using super ANOVA software (ABACUS Concepts, Inc., Berkeley, CA). Sources of variation included effects caused by treatments and their interactions. The effect of the different dose-responses was determined using orthogonal contrasts.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PGF2{alpha} regulation by OT in epithelial cells
As previously demonstrated (15, 16, 24, 50), PGF2{alpha} (13.3 ± 2.0 vs. 1.8 ± 0.5 ng/ml for PGE2) is the major prostaglandin produced in nonstimulated epithelial cells (Fig. 1Go). OT increased (P < 0.0001) PGF2{alpha} production up to 166.8 ± 22.5 ng/ml in a dose-dependent manner, whereas PGE2 production also was stimulated in a dose-dependent manner, but proportionately at a lower level (23.3 ± 3.6 ng/ml at the highest dose used).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Effect of OT on prostaglandin production in epithelial cells. The culture medium of confluent cultures was replaced with fresh RPMI-1640 without serum, and increasing doses of OT were added in triplicate wells. After 24 h, the medium was recovered and kept for assay of PGs. PGE2 (open squares) and F2{alpha} (solid circles) were measured by EIA, as described in Materials and Methods. The results are presented as mean ± SEM of triplicate determinations for four experiments. *, P < 0.0001.

 
Generation of second messengers
To demonstrate the effect of OT on the generation of second messengers in bovine epithelial cells, total IPs and [Ca2+]i were measured. OT induced a dose-dependent increase of total IP accumulation, which reached 326 ± 37% (P < 0.0001) stimulation at the highest dose used (Fig. 2Go). Because IP production is thought to mediate mobilization of [Ca2+]i, we next evaluated, using a fluorescent imaging system, the effects of OT on [Ca2+]i concentrations (Fig. 3Go). OT (10-7 M) caused a rapid increase in [Ca2+]i in epithelial cells. This increase was rapid in onset, with a spike-type increase achieved within 5 sec of stimulation, followed by a gradual reduction to near basal levels.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. Influence of OT on inositol-phosphates production. Epithelial cells from confluent cultures were incubated 10 min at 37 C in the presence of a gased Kreb’s solution containing LiCl 20 mM. The enzymatic assay was conducted at 37 C for 10 min with increasing doses of OT (10-9 to 10-5 M), and the reaction was stopped by aspirating the medium and by the addition of an equal volume of ethanol/HCl. Data are expressed in percent of control and represent the mean ± SEM of three experiments run in triplicate. **, P < 0.0001; *, P < 0.001, compared with control.

 


View larger version (12K):
[in this window]
[in a new window]
  		Figure 3. Release of intracellular calcium ([Ca2+]i) in response to OT in a single endometrial epithelial cell. Epithelial cells were grown to confluency on glass coverslip placed at the bottom of 6-well plates. At the time of the experiments, the cells were washed 3 times with HBS and loaded for 30–60 min at 22 C in the presence of 2 µM fura-2/AM. The coverslip was rinsed, mounted on a 2-well chamber, and placed over an image analysis, inverted fluorescence microscope equipped with an intensified charge-coupled device camera for single-cell measurement. The release of [Ca2+]i was measured using 340- and 380-nm wavelengths, and results are presented as the ratio 340/380 nm. The figure illustrates 1 representative recording out of 10 experiments.

 
Effect of OT on steady-state COXs and PLA2 mRNA levels in epithelial cells
To document the mechanism by which the modulation of PGF2{alpha} production described above is effected, steady-state levels of the mRNAs for COX-1, COX-2, and PLA2 were determined (Fig. 4Go). In epithelial cells, a 2.8-kb transcript was detected by hybridization with the COX-1 cDNA probe. The abundance of this transcript was not altered by OT treatment consistent with its housekeeping function. However, after exposure to OT, the 4.1-kb COX-2-associated mRNA was increased in a dose-dependent fashion (P < 0.001), as determined by densitometric analysis, and was increased 508 ± 71%, compared with control. Under unstimulated conditions, COX-2 transcript was slightly detectable. The apparent increase of the 3.4-kb transcript associated with PLA2 mRNA was not significant (P > 0.1). Similarly, COX-1 expression was not affected. OT had no effect on ß-actin mRNA gene expression used as control.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. COX-1, COX-2, and PLA2 mRNA regulation in response to OT in epithelial cells. Confluent cells were cultured 24 h in serum-free RPMI-1640 medium in the absence or presence of increasing concentration of OT; and the COX-1, COX-2, PLA2, and ß-actin mRNA levels were measured by Northern blotting. The densitometric COX-2/ß-actin mRNA ratio is shown and represents the mean ± SEM of four different experiments (P < 0.001). The COX-1 and PLA2/b-acin mRNA ratios were not affected by treatment (histograms not shown). Representative blots of one experiment are presented.

 
RT-PCR analysis of COX-1 and COX-2 mRNA
RT-PCR analysis of bovine COXs in epithelial cells identified the appropriate 777-bp band for COX-1 and a 449-bp band for COX-2 (Fig. 5Go). As for Northern blot analysis, ß-actin was used as a control, and a 349-bp band was observed in all samples, and no variation was observed. Results demonstrate that COX-1 is present in epithelial cells and is not regulated by OT. On the other hand, OT induced a dose-dependent increase of COX-2 mRNA in epithelial cells. These fragments were cloned respectively in pCR 2.1 plasmid, sequenced, and used as cDNA probes for Northern blots. Figure 6Go shows the sequences of both bovine COX-1 and COX-2 mRNA. An homology of 83% and 97% was found in relation with rat (46) and sheep (45) for COX-1, respectively. On the other hand, COX-2 was found to bear 84%, 86%, and 87% of homology in relation to rat (46), guinea pig (51), and human (30), respectively.



View larger version (97K):
[in this window]
[in a new window]
  		Figure 5. RT-PCR identification of COX-1 and COX-2 mRNA in epithelial cells after OT treatment. Total cellular RNA was reverse transcribed, and the resulting cDNA was used for PCR amplification. COX-1 gene expression was analyzed by amplification of a 777-bp sequence of the sheep COX-1 cDNA. PCR products were electrophoresed and stained with ethidium bromide. DNA size markers are presented in lane 1. COX-2 gene expression was analyzed by amplification of a 449-bp sequence of the human COX-2 cDNA. The gels were exposed to UV (1.0 sec for photographic purposes). The results presented are from one representative, out of four separate experiments.

 


View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. PCR cloning and sequencing of bovine COX-1 and COX-2 cDNAs. RT-PCR fragments of COX-1 and COX-2 obtained in Fig. 5Go were used. A, Dideoxy-sequencing results of COX-1 are shown with inferred amino acids listed beneath their codons. Sequences are presented in rows of 20 codons or amino acid, beginning with amino acid no. 370 of sheep sequence. B, Bovine COX-2 dideoxy-sequencing and deduced amino acid sequence displayed as in A. Sequences start with amino acid no. 411 of human sequence. COX-1 and COX-2 sequences are available in GenBank database (accession nos. AF004943 and AF004944, respectively).

 
Effect of COXs inhibitors on OT-induced PGF2{alpha} production
In epithelial cells, addition of OT (10-7 M) resulted in a significant stimulation of PGF2{alpha} production (Fig. 7Go), as described previously in Fig. 1Go. Addition of indomethacin (1 µM) alone inhibited significantly (P < 0.0001) PGF2{alpha} synthesis, compared with control (1903 ± 390 vs. 342 ± 35 pg/µg DNA). Combined with OT, indomethacin inhibited the stimulation of PGF2{alpha} production (P < 0.0001). Addition of a specific COX-2 inhibitor (NS-398, 1 µM) also blocked the effect of OT on PGF2{alpha} production (P < 0.0001) but had no effect on basal PGF2{alpha} production.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 7. Effect of indomethacin and NS-398 on OT-induced PGF2{alpha} production. Epithelial cells were cultured 24 h in serum-free RPMI-1640 medium in the absence or presence of OT, combined or not with indomethacin (1 µM) or NS-398 (10 µM). PGF2{alpha} was measured by EIA, as described in Materials and Methods. The results are presented as mean ± SEM of three different experiments run in triplicate. Columns with different superscripts are significantly different from control (P < 0.0001).

 
Effect of roIFN-{tau} on OT-induced PGF2{alpha} production
The influence of roIFN-{tau} on PGF2{alpha} production induced by OT is shown in Fig. 8Go. As demonstrated in Fig. 1Go, OT stimulated PGF2{alpha} production in a dose-dependent manner. However, in the presence of roIFN-{tau} (10 µg/ml), the stimulation of PGF2{alpha} production was decreased significantly (P < 0.0001), at 10-7, to 10-5 M of OT.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 8. Effect of roIFN-{tau} on OT-induced PGF2{alpha} production in epithelial cells. Confluent epithelial cells were stimulated with increasing concentrations of OT, in the presence or absence of roIFN-{tau} (10 µg/ml), for 24 h in serum-free RPMI-1640. PGF2{alpha} was measured by EIA, as described in Materials and Methods. The results are presented as mean ± SEM of triplicate determinations for three experiments. *, P < 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the luteolytic process, which causes the destruction of the corpus luteum (CL) for the return to a new estrous cycle, OT plays a key role by stimulating pulsatile release of PGF2{alpha} (52, 53). The source of PGF2{alpha} is from the endometrium in vivo. Thus, cultured epithelia have been used to study the effect of OT on the regulation of prostaglandins. The binding of OT to its receptor, coupled to a G protein, activates a phospholipase C, which in turn, activates phosphoinositide turnover and generates IP3 (54) and DAG. IP3 is known to cause mobilization of intracellular Ca2+ ([Ca2+]i) stores, whereas DAG can activate a protein kinase C (PKC). The first part of this study was to confirm that OT could regulate IPs and Ca2+ in cultured endometrial epithelial cells. A recent study, using gonadotrope-derived {alpha}T3 cell line, demonstrated that OT was able to induce IP production and increase cytosolic Ca2+ (55). Using a fluorescent microscope imaging system, we were able to demonstrate a direct effect of OT on [Ca2+]i mobilization only at the epithelial level. The results obtained in the present study were comparable with results obtained with the {alpha}T3 cell line, suggesting that in the endometrium, OT acts also on a G protein-coupled receptor. We have previously demonstrated that activation of PKC is necessary for OT response (15), and now we demonstrate that IPs and Ca2+ are second messengers for OT action in the regulation of PGF2{alpha} production.

In terms of PGs production, our previous results (15, 16) are in agreement with the literature suggesting that OT response is limited to epithelial cells of the endometrium, inducing a 12.5-fold increase of PGF2{alpha} production. However, the intracellular mechanisms involved in the regulation of PGF2{alpha} in the endometrium are poorly understood. In ovine, a recent study has proposed a possible role of phospholipase A2 (PLA2) in the OT-induced PGF2{alpha} production (27). The exclusive role of PLA2 in the process is unlikely because, in that study, an inhibitor and a stimulator of PLA2 activity were used to characterize the effect of OT. Stimulation of PLA2 with fluoride induced the release of AA in all cases, but an inhibition of the PGF2{alpha} production was reached only at the highest dose of aristolochic acid (10-4 M) used as a PLA2 inhibitor. Furthermore, in the present study, the results demonstrate that OT had no significant effect on the regulation of PLA2 gene expression. It still is possible, however, that OT may stimulate the activity of the PLA2 protein. However, to support our findings, a more recent study showed that COX-2 was expressed during days 12–15 during the estrous cycle in the ewe uterus, showing at least that PLA2 is not the only factor regulated around the time of luteolysis (38).

In the biosynthetic pathway of PG production, COX is a rate-limiting suicide enzyme, which uses AA as a substrate (56). The results in the present study demonstrate the first evidence that COX-2 is expressed in bovine endometrium and that OT up-regulates COX-2 mRNA gene expression. The constitutive form of COX (COX-1) did not vary with OT treatment but seems to be more abundant than COX-2, even at its maximally stimulated level. The abundance of the COX-1 transcript is not surprising, given the suicidal nature of the catalytic process and its housekeeping function (56). The results of the present study, therefore, would suggest regulation of COX-2, not only at the transcription but also at the translation level, and this is supported by results obtained by Charpigny et al. (38) in the ovine in vivo. Another study in the ovine demonstrated that mRNA for PGHS (prostaglandin synthase or COX) did not vary during the cycle (57). However, the mRNA detected was 2.8 Kb and probably corresponds to the constitutive COX-1, as demonstrated in the present study. The presence of a specific inhibitor of COX-2 (NS-398) was able to block the OT-induced PGF2{alpha} production, which supports the hypothesis that COX-2 is necessary for the stimulation of PGF2{alpha} production. In epithelial cells, under unstimulated conditions, the major PG produced is PGF2{alpha}. Thus, by inducing COX-2 gene expression, the resulting OT effect is a net increase of PGF2{alpha} production in these cells. This preferential production of PGF2{alpha} in response to OT may be simply an amplification of the conditions prevailing at the basal level. The present study was conducted only in epithelial endometrial cells because we have shown that OT response was not present in stromal cells (16, 24, 25). Similarly, in a preliminary experiment, we have found that OT receptors (OTR) were present in epithelial cells but not in stromal cells or in the circular or longitudinal cells of the myometrium in vitro (data not shown). In epithelial cells, we previously have demonstrated that IFN-{tau} was able to change the primary PG produced from PGF2{alpha} to PGE2, also via an up-regulation of COX-2 gene expression (24, 25). Therefore, in these cells, IFN-{tau} probably acts also farther down in the PGs biosynthesis cascade. On the contrary, OT induced a stimulation of both PGE2 and PGF2{alpha}, but the ratio is maintained in favor of PGF2{alpha}. In the present study, addition of IFN-{tau} was able to reduce the effect of OT on PGF2{alpha} production. The mechanism responsible for this inhibition by IFN-{tau} is not known, but it may involve a down-regulation of OTR in vitro, as demonstrated by others in the ovine in vivo (26). A recent study published by Charpigny et al. (38) strongly supports the present results and those obtained with IFN-{tau} (25). They have demonstrated in vivo that the COX-2 protein was transiently expressed in high concentrations close to the time when OT is released to induce luteolysis. During early pregnancy, COX-2 also was expressed, indicating that PGs are necessary for establishment of pregnancy. We have previously reported that PGE2 production and COX-2 gene expression were regulated by roIFN-{tau}. Because PGE2 possesses antiluteolytic and luteotrophic properties, it can be proposed that the IFN-{tau} regulation of PGE2 and COX-2 is a mechanism necessary for establishment of pregnancy. However, in the absence of a conceptus signal, OT stimulate PGF2{alpha} production via COX-2.

In conclusion, the present results demonstrate, using an integrated in vitro system, that OT stimulation, which stimulates PGF2{alpha} production, also: 1) stimulates the generation of inositol phosphates; 2) induces the release of [Ca2+]i; and 3) up-regulates COX-2 gene expression. These results in vitro support and complement the results obtained in vivo by Charpigny et al. (38), suggesting that COX-2 may be a key enzyme in the regulation of PGF2{alpha} production by OT during luteolysis.


   Acknowledgments
 
The authors would like to thank Dr. Fuller W. Bazer for provision of roIFN-{tau}, Dr. Thomas G. Kennedy for generously donating the PGE2 antiserum for the ELISA technique, and Lise Lacouline for her help in PG measurements.


   Footnotes
 
1 This work has been supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Grant OGPIN030 (to M.A.F.) and an NSERC scholarship (to E.A.). Back

Received May 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Poyser NL 1995 The control of prostaglandin production by the endometrium in relation to luteolysis and menstruation. Prostaglandins Leukot Essent Fatty Acids 53:147–195[CrossRef][Medline]
  2. Hearn JP, Webley GE, Gidley Baird AA 1991 Chorionic gonadotrophin and embryo-maternal recognition during the peri-implantation period in primates. J Reprod Fertil 92:497–509[Abstract]
  3. Pratt BR, Butcher RL, Inskeep EK 1977 Antiluteolytic effect of the conceptus and of PGE2 in ewes. J Anim Sci 45:784–791
  4. Magness RR, Huie JM, Hoyer GL, Huecksteadt TP, Reynolds LP, Seperich GJ, Whysong G, Weems CW 1981 Effect of chronic ipsilateral or contralateral intrauterine infusion of prostaglandin E2 (PGE2) on luteal function of unilaterally ovariectomized ewes. Prostaglandins Med 6:389–401[CrossRef][Medline]
  5. Kennedy TG 1983 Prostaglandin E2, adenosine 3':5'-cyclic monophosphate and changes in endometrial vascular permeability in rat uteri sensitized for the decidual cell reaction. Biol Reprod 29:1069–1076[Abstract]
  6. Kennedy TG 1980 Prostaglandins and the endometrial vascular permeability changes preceding blastocyst implantation and decidualization. Prog Reprod Biol 7:234–243
  7. Smith SK, Kelly RW 1988 Prostaglandins and establishment of pregnancy. In: Chapman M, Grudzunskasw G, Chard T (eds) Implantation, Biological and Clinical Aspects. Springer-Verlag, New York, pp 147–160
  8. McCracken JA, Schramm W, Okulicz WC 1984 Hormone receptor control of pulsatile secretion of PGF2a from the ovine uterus during luteolysis and its abrogation in early pregnancy. Anim Reprod Sci 7:31–55
  9. Horton EW, Poyser NL 1976 Uterine luteolytic hormone: a physiological role for prostaglandin F2{alpha}. Physiol Rev 56:595–651[Abstract/Free Full Text]
  10. Lafrance M, Goff AK 1985 Effect of pregnancy on oxytocin-induced release of prostaglandin F2{alpha} in heifers. J Reprod Fertil 33:1113–1119
  11. Flint AP, Sheldrick EL, McCann TJ, Jones DS 1990 Luteal oxytocin: characteristics and control of synchronous episodes of oxytocin and PGF2{alpha} secretion at luteolysis in ruminants. Domest Anim Endocrinol 7:111–124[CrossRef][Medline]
  12. Roberts JS, McCracken JA, Gavagan JE, Soloff MS 1976 Oxytocin-stimulated release of prostaglandin F2{alpha} from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endocrinology 99:1107–1114[Abstract]
  13. Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson Jr L 1991 Hormonal regulation of uterine secretion of prostaglandin F2{alpha} during luteolysis in ruminants. Biol Reprod 45:655–663[Abstract]
  14. Lafrance M, Goff AK 1990 Control of bovine uterine prostaglandin F2{alpha} release in vitro. Biol Reprod 42:288–293[Abstract]
  15. Kim JJ, Fortier MA 1995 Cell type specificity and protein kinase C dependency on the stimulation of prostaglandin E2 and prostaglandin F2{alpha} production by oxytocin and platelet-activating factor in bovine endometrial cells. J Reprod Fertil 103:239–247[Abstract]
  16. Asselin E, Goff A, Bergeron H, Fortier M 1996 Influence of sex steroids on the production of prostaglandins F2{alpha} and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biol Reprod 54:371–379[Abstract]
  17. Moore JJ, Dubyak GR, Moore RM, Vander Kooy D 1988 Oxytocin activates the inositol-phospholipid-protein kinase C system and stimulates prostaglandin production in human amnion cells. Endocrinology 123:1771–1777[Abstract]
  18. Flint APF, Sheldrick EL 1986 Ovarian oxytocin and the maternal recognition of pregnancy. J Reprod Fertil 76:831–839[Abstract]
  19. Nishizuka Y 1988 The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 334:661–665[CrossRef][Medline]
  20. Mitchel MD 1991 The regulation of decidual prostaglandin biosynthesis by growth factors, phorbol esters, and calcium. Biol Reprod 44:871–874[Abstract]
  21. Zakar T, Olsen DM 1988 Stimulation of human amnion prostaglandin E2 production by activators of protein kinase C. J Clin Endocrinol Metab 67:915–923[Abstract]
  22. Roberts RM, Leaman DW, Cross JC 1992 Role of interferons in maternal recognition of pregnancy in ruminants. Proc Soc Exp Biol Med 200:7–18[Abstract]
  23. Bazer FW 1992 Mediators of maternal recognition of pregnancy in mammals. Proc Soc Exp Biol Med 199:373–384[Medline]
  24. Asselin E, Bazer FW, Fortier MA 1997 Recombinant ovine and bovine interferons tau regulate prostaglandin production and oxytocin response in cultured bovine endometrial cells. Biol Reprod 56:402–408[Abstract]
  25. Asselin E, Lacroix D, Fortier MAIFN-{tau} increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro. Mol Cell Endocrinol, in press
  26. Spencer TE, Bazer FW 1996 Ovine interferon tau suppresses transcription of the estrogen receptor and oxytocin receptor genes in the ovine endometrium. Endocrinology 137:1144–1147[Abstract]
  27. Lee JS, Silvia WJ 1994 Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F2{alpha} in response to oxytocin: role of phospholipase A2. J Endocrinol 141:491–496[Abstract]
  28. Smith WL 1992 Prostanoid biosynthesis and mechanisms of action. Am J Physiol 263:F181–F191
  29. DeWitt DL 1991 Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim Biophys Acta 1083:121–134[Medline]
  30. Hla T, Neilson K 1992 Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA 89:7384–7388[Abstract/Free Full Text]
  31. Needleman P, Turk J, Jakschik BA, Morrison AR, Lefkowith JB 1986 Arachidonic acid metabolism. Annu Rev Biochem 55:69–102[CrossRef][Medline]
  32. Han SW, Lei ZM, Rao CV 1995 Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. Endocrinology 137:1791–1797[Abstract]
  33. Sirois J, Richards JS 1992 Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles. J Biol Chem 267:6382–6388[Abstract/Free Full Text]
  34. Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR 1991 TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 266:12866–12872[Abstract/Free Full Text]
  35. O’Banion MK, Winn VD, Young DA 1992 cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase. Proc Natl Acad Sci USA 89:4888–4892[Abstract/Free Full Text]
  36. Jacobs AL, Hwang D, Julian J, Carson DD 1994 Regulated expression of prostaglandin endoperoxide synthase-2 by uterine stroma. Endocrinology 135:1807–1815[Abstract]
  37. Chakraborty I, Das SK, Wang J, Dey SK 1996 Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the periimplantation mouse uterus and their differential regulation by the blastocyst and ovarian-steroids. J Mol Endocrinol 16:107–122[Abstract]
  38. Charpigny G, Reinaud P, Tamby JP, Créminon C, Martal J, Maclouf J, Guillomot M 1997 Expression of cyclooxygenase-1 and -2 in ovine endometrium during estrous cycle and early pregnancy. Endocrinology 138:2163–2171[Abstract/Free Full Text]
  39. Ott TL, Van Heeke G, Johnson HM, Bazer FW 1991 Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-I trophoblast interferon ovine trophoblast protein-1:purification and antiviral activity. J Interferon Res 11:357–364[Medline]
  40. Pontzer CH, Torres BA, Vallet JL, Bazer FW, Johnson HM 1988 Antiviral activity of the pregnancy recognition hormone ovine trophoblast protein-1. Biochem Biophys Res Comm 152:801–807[CrossRef][Medline]
  41. Pontzer CH, Bazer FW, Johnson HM 1991 Antiproliferative activity of a pregnancy recognition hormone, ovine trophoblast protein-1. Cancer Res 51:5304–5307[Abstract/Free Full Text]
  42. Ashworth CJ, Bazer FW 1989 Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone. Biol Reprod 40:425–433[Abstract]
  43. Ireland JJ, Coulson PB, Murphee RL 1979 Follicular development during four stages of the estrous cycle of beef cattle. J Anim Sci 49:1261–1269
  44. Fiszer-Szafarz B, Szafarz D, Guevara DMA 1981 A general, fast, and sensitive micromethod for DNA determination application to rat and mouse liver, rat hepatoma, human leukocytes, chicken fibroblasts, and yeast cells. Anal Biochem 110:165–170[CrossRef][Medline]
  45. Merlie JP, Fagan D, Mudd J, Needleman P 1988 Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem 263:3550–3553[Abstract/Free Full Text]
  46. Feng L, Sun W, Xia Y, Tang WW, Chanmugam P, Soyoola E, Wilson CB, Hwang D 1993 Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys 307:361–368[CrossRef][Medline]
  47. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  48. Asselin E, Fortier MA 1996 Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells. J Cell Physiol 168:600–607[CrossRef][Medline]
  49. Evans CA, Kennedy TG, Challis JRG 1982 Gestational changes in prostanoid concentrations in intrauterine tissues and fetal fluids from pregnant sheep and the relation to prostanoid output in vitro. Biol Reprod 27:1–11[CrossRef][Medline]
  50. Fortier MA, Guilbault LA, Grasso F 1988 Specific properties of epithelial and stromal cells from the endometrium of cows. J Reprod Fertil 83:239–248[Abstract]
  51. Bracken KE, Elger W, Jantke I, Nanninga A, Gellersen B 1997 Cloning of guinea pig cyclooxygenase-2 and 15-hydroxyprostaglandin dehydrogenase complementary deoxyribonucleic acids: steroid-modulated gene expression correlates to prostaglandin F2{alpha} secretion in cultured endometrial cells. Endocrinology 138:237–247[Abstract/Free Full Text]
  52. Zarco L, Stabenfeltd GH, Basu S, Bradford GE, Kindahl H 1988 Modification of prostaglandin F2{alpha} synthesis and release in the ewe during initial establishment of pregnancy. J Reprod Fertil 83:527–535[Abstract]
  53. Flint AP, Stewart HJ, Lamming GE, Payne JH 1992 Role of the oxytocin receptor in the choice between cyclicity and gestation in ruminants. J Reprod Fertil [Suppl] 45:53–58[Medline]
  54. Mirando MA, Ott TL, Vallet JL, Davis M, Bazer FW 1990 Oxytocin-stimulated inositol phosphate turnover in endometrium of ewes is influenced by stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins. Biol Reprod 42:98–105[Abstract]
  55. Evans JJ, Forrest-Owen W, McArdle CA 1997 Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived aT3–1 cell line. Endocrinology 138:2049–2055[Abstract/Free Full Text]
  56. Pan Chen YN, Bienkowski MJ, Marnett LJ 1987 Controlled tryptic digestion of prostaglandin H synthase. J Biol Chem 262:16892–16899[Abstract/Free Full Text]
  57. Salamonsen LA, Hampton AL, Clements JA, Findlay JK 1991 Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus. J Reprod Fertil 92:393–406[Abstract]



This article has been cited by other articles:


Home page
 ReproductionHome page
Y. Chen, E. Antoniou, Z. Liu, L. B Hearne, and R M. Roberts
A microarray analysis for genes regulated by interferon-{tau} in ovine luminal epithelial cells
Reproduction, July 1, 2007; 134(1): 123 - 135.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Chen, J. A. Green, E. Antoniou, A. D. Ealy, N. Mathialagan, A. M. Walker, M. P. Avalle, C. S. Rosenfeld, L. B. Hearne, and R. M. Roberts
Effect of Interferon-{tau} Administration on Endometrium of Nonpregnant Ewes: A Comparison with Pregnant Ewes
Endocrinology, May 1, 2006; 147(5): 2127 - 2137.
[Abstract] [Full Text] [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
 J EndocrinolHome page
Z Cheng, M Elmes, S E Kirkup, E C Chin, D R E Abayasekara, and D C Wathes
The effect of a diet supplemented with the n-6 polyunsaturated fatty acid linoleic acid on prostaglandin production in early- and late-pregnant ewes
J. Endocrinol., January 1, 2005; 184(1): 165 - 178.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S.-Z. Wang and R. M. Roberts
Interaction of Stress-Activated Protein Kinase-Interacting Protein-1 with the Interferon Receptor Subunit IFNAR2 in Uterine Endometrium
Endocrinology, December 1, 2004; 145(12): 5820 - 5831.
[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
 Biol. Reprod.Home page
A. K. Goff
Steroid Hormone Modulation of Prostaglandin Secretion in the Ruminant Endometrium During the Estrous Cycle
Biol Reprod, July 1, 2004; 71(1): 11 - 16.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
V. Emond, L. A. MacLaren, S. Kimmins, J. A. Arosh, M. A. Fortier, and R. D. Lambert
Expression of Cyclooxygenase-2 and Granulocyte-Macrophage Colony-Stimulating Factor in the Endometrial Epithelium of the Cow Is Up-Regulated During Early Pregnancy and in Response to Intrauterine Infusions of Interferon-{tau}
Biol Reprod, January 1, 2004; 70(1): 54 - 64.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
K. Okuda, Y. Kasahara, S. Murakami, H. Takahashi, I. Woclawek-Potocka, and D. J. Skarzynski
Interferon-{tau} Blocks the Stimulatory Effect of Tumor Necrosis Factor-{alpha} on Prostaglandin F2{alpha} Synthesis by Bovine Endometrial Stromal Cells
Biol Reprod, January 1, 2004; 70(1): 191 - 197.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page