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Endocrinology Vol. 145, No. 11 5280-5293
Copyright © 2004 by The Endocrine Society

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

J. A. Arosh, S. K. Banu, S. Kimmins, P. Chapdelaine, L. A. MacLaren and M. A. Fortier

Unité d’Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Centre Hospitalier de l’Université de Laval, Centre de Recherche en Biologie de la Reproduction (J.A.A., S.K.B., P.C., M.A.F.), and Département d’Obstétrique et Gynécologie, Université Laval (M.A.F.), Québec, Canada G1K 7P4; and Departments of Plant and Animal Sciences, Nova Scotia Agricultural College (S.K., L.A.M.), Truro, Nova Scotia, Canada B2N 5E3

Address all correspondence and requests for reprints to: Dr. Michel A. Fortier, Unité d’Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Centre Hospitalier de l’Université de Laval, 2705 boulevard Laurier, Ste-Foy, Québec, Canada GIV 4G2. E-mail: mafortier{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recognition and establishment of pregnancy involve several molecular and cellular interactions among the conceptus, uterus, and corpus luteum (CL). In ruminants, interferon-{tau} (IFN{tau}) of embryonic origin is recognized as the pregnancy recognition signal. Endometrial prostaglandin F2{alpha} (PGF2{alpha}) is the luteolysin, whereas PGE2 is considered a luteoprotective or luteotrophic mediator at the time of establishment of pregnancy. The interplay between IFN{tau} and endometrial PGs production, transport, and signaling at the time of maternal recognition of pregnancy (MRP) is not well understood. We have studied the expression of enzymes involved in metabolism of PGE2 and PGF2{alpha}, cyclooxygenase-1 (COX-1) and COX-2, PG synthases (PGES and PGFS), PG 15-dehydrogenase, and PG transporter as well as PGE2 (EP2 and EP3) and PGF2{alpha} receptors. IFN{tau} influences cell-specific expression of COX-2, PGFS, EP2, and EP3 in endometrium, myometrium, and CL in a spatio-temporal and tissue-specific manner, whereas it does not alter COX-1, PGES, PG 15-dehydrogenase, PG transporter, or PGF2{alpha} receptor expression in any of these tissues. In endometrium, IFN{tau} decreases PGFS in epithelial cells and increases EP2 in stroma. In myometrium, IFN{tau} decreases PGFS and increases EP2 in smooth muscle cells. In CL, IFN{tau} increases PGES and decreases EP3. Together, our results show that IFN{tau} directly or indirectly increases PGE2 biosynthesis and EP2-associated signaling in endometrium, myometrium, and CL during MRP. Thus, PGE2 may play pivotal roles in endometrial receptivity, myometrial quiescence, and luteal maintenance, indicating polycrine (endocrine, exocrine, paracrine, and autocrine) actions of PGE2 at the time of MRP. Therefore, the establishment of pregnancy may depend not only on inhibition of endometrial PGF2{alpha}, but also on increased PGE2 production in cattle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) ARE key mediators of several female reproductive functions, including ovulation, fertilization, implantation, and parturition (1, 2, 3). Arachidonic acid, an essential fatty acid stored in membrane phospholipids, is the primary precursor of PGs. Cyclooxygenases (COX-1 and COX-2) convert arachidonic acid into PGH2, the common precursor of the various forms of PGs, including PGE2 and PGF2{alpha}. The downstream enzymes, PGE synthase (PGES) and PGF synthase (PGFS), catalyze the conversion of PGH2 to PGE2 and PGF2{alpha}, respectively (4). Current evidence suggests that there are three forms of PGES, among them microsomal PGES-1 (mPGES-1), is highly inducible along with COX-2 (5). We have identified aldoketoreductase 1B5 (AKR1B5) as the PGFS involved in the production of PGF2{alpha} in bovine endometrium (6). Catabolism of PGs is governed by PG 15-dehydrogenase (PGDH), which converts PGE2 and PGF2{alpha} into inactive metabolites, PGEM and PGFM, respectively (7, 8). Cellular and compartmental transport of PGE2 and PGF2{alpha} are facilitated by the PG transporter (PGT) (9, 10). PGE2 and PGF2{alpha} exert their effects primarily through G protein-coupled receptors, designated EP and PGF2{alpha} receptor (FP), respectively. The EP receptor has four subtypes, EP1, EP2, EP3, and EP4. FP has two subtypes, FPA and FPB. EP2 and EP4 are coupled to adenylate cyclase and generate cAMP activating the protein kinase A signaling pathway. EP1 and FP receptors are coupled to phospholipase C, generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium (Ca2+) and diacylglycerol, an activator of protein kinase C. The bovine EP3 receptor exists in four isoforms A–D, having a wide range of action from inhibition of cAMP production to increases in Ca2+ and IP3 (11, 12).

Many studies from our laboratory indicate that PG metabolic enzymes, COX-2, PGFS, PGES, PGDH, and EP2 receptors, are highly expressed in endometrium during mid- and late luteal phases of the bovine estrous cycle (6, 13, 14, 15, 16), whereas information on contractile FP and EP3 receptors is lacking. PGT is expressed and regulated in endometrium, myometrium, and utero-ovarian plexus (UOP) and may play pivotal roles in cellular transport of PGs within uterine tissues and from the uterus to the ovary/corpus luteum (CL) during the estrous cycle (17). It has been suggested that endometrial/extraluteal PGs could influence luteal PG production in ruminants (18, 19, 20). In a recent study we have shown that the bovine CL expressed the complete PG biosynthesis, transport, and signaling components that may determine its life span and function (21).

Recognition and establishment of pregnancy involve several molecular and cellular interactions among the conceptus, uterus, and CL (22). During the bovine estrous cycle (d 21 ± 1), d 15–17 are considered the critical period for either luteolysis, followed by a new estrous cycle, or establishment of pregnancy in the presence of a viable embryo (23, 24). The bovine endometrium secretes PGF2{alpha} and PGE2 throughout the estrous cycle, but the secretory pattern is different (25, 26, 27). Endometrial PGF2{alpha} is secreted in a pulsatile manner and is considered the luteolytic mediator. The luteolytic mechanism involves a sequence of events involving positive feedback loops between endometrium and CL/ovary (22, 28). It has been shown that endometrial production of PGE2 is higher during the mid and late luteal phases of the estrous cycle in the bovine (26). PGE2 has been proposed to have multiple roles as a temporary luteotrophic, luteostatic, or luteoprotective signal and as an important mediator in endometrial receptivity, myometrial quiescence, and immune function at the fetal-maternal interface during the establishment of pregnancy (29, 30, 31, 32). PGE2 also acts as a mitogenic, antiapoptotic, and angiogenic factor in different cell types (33, 34).

Interferon-{tau} (IFN{tau}) secreted by embryonic trophoblast cells is recognized as the pregnancy recognition signal (22). Secretion of IFN{tau} by bovine blastocyst is highest between d 15 and 17, but is observed up to d 28 of pregnancy (22). The mechanism of action of IFN{tau} in ruminant endometrium has been reviewed extensively (22, 23, 35, 36, 37, 38). Inadequate response of the endometrium to IFN{tau} is likely to be one of the major reason for improper production and action of PGF2{alpha} and PGE2, leading to embryonic loss and pregnancy failure in cattle. Accumulating evidence indicates that IFN{tau} could decrease COX-2 expression and PGF2{alpha} production (39) or increase COX-2 expression and PGE2 production (40, 41). Therefore, achieving an optimal PGE2 to PGF2{alpha} ratio is essential for endometrial receptivity, myometrial quiescence, and maintenance of CL and progesterone (P4) secretion, which are the prerequisites for successful establishment of pregnancy (22). However, the net production and action of PGF2{alpha} and PGE2 depend not only on COX-2, but also on PG synthases, transporter, and specific EP and FP receptors. Our objectives were to study the expression of 1) PGs biosynthetic enzymes COX-1, COX-2, PGES, and PGFS (AKR1B5); 2) PG catabolic enzyme PGDH; 3) PG transporter PGT, and 4) PG receptors EP2, EP3, and FP in endometrium, myometrium, and CL after treatment with exogenous IFN{tau} at the time of maternal recognition of pregnancy (MRP). An additional objective was to study the expression patterns of EP2, EP3, and FP mRNA in endometrium and myometrium on different days of the estrous cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The numerous reagents necessary for this study were purchased from the following suppliers: Superscript II RT, DNA and RNA ladders, dithiothreitol, T4 kinase, 5x forward reaction and first strand buffers, and TRIzol (Invitrogen Life Technologies, Inc., Burlington, Canada); Random Primer-pd(N)6, deoxy-NTPs, RNA Guard, rTaq DNA polymerase, PCR 10x buffer, and Ready-To-Go DNA labeling kit (Amersham Biosciences, Montréal, Canada); prestained protein markers (New England Biolabs, Inc., Mississauga, Canada); Bright Star-Plus nylon membrane and UltraHyb (Ambion, Inc., Austin, TX); Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA); [{gamma}-32P]ATP and deoxy-CTP (PerkinElmer, Markham, Canada); Renaissance (Life Science Products, Inc., Boston, MA); BioMax film (Eastman Kodak Co., Rochester, NY); plasmid and mRNA purification kits (Qiagen, Mississauga, Canada); Mayer’s hematoxylin (Sigma-Aldrich Canada Ltd., Oakville, Canada); LightCycler FasterStart DNA Master SYBR Green I mix and MgCl2 (Roche, Laval, Canada); and Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). All oligonucleotide primers were chemically synthesized using ABT 394 synthase (PerkinElmer, Foster City, CA). The other chemicals used were molecular biological grade available from Laboratoire Mat or Fisher Biotech (Quebec, Canada). Goat antirabbit biotinylated immunoglobulin (Ig; Dako Diagnostics of Canada, Inc., Mississauga, Canada); goat antirabbit or antimouse IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA); and monoclonal antimouse ß-actin antibody and antihuman rabbit EP2 polyclonal antibody (Cayman Chemicals, Ann Arbor, MI) were used in this study. Antibodies against bovine PGFS (6), PGDH (16), and PGT (17) were produced in our laboratories as described previously. Antisheep COX-1 and COX-2 antibodies were donated by Dr. Stacia Kargman (Merk-Frost, Montréal, Canada). Antibovine PGES (42) was a gift from Dr. Jean Sirois (Centre de Recherche en Reproduction Animale, University of Montréal, Ste-Hyacinthe, Canada). Recombinant ovine IFN{tau} was donated by Drs. F. W. Bazer and T. E. Spencer (Animal Biotechnology Laboratory, Texas A&M University, College Station, TX).

Experiment 1
Animal management and treatment protocols were described previously (43, 44). Briefly, beef heifers (19 ± 1 d cycle length) were used. All procedures performed were in accordance with the guidelines of the Canadian Council on Animal Care 1993 animal protocols and were reviewed and approved by the Nova Scotia Agricultural College animal care and use committee. Estrus was synchronized with a double PGF2{alpha} regimen at 11-d intervals. One regular estrous cycle was observed. Again estrus was synchronized with a single injection of PGF2{alpha} on d 12 of the estrous cycle. The induced estrus was considered d 0. On d 14, the animals were divided into control (n = 3) and treatment (n = 3) groups. Recombinant ovine IFN{tau} (5 ml; 0.25 mg/dose = biological activity of 5 x 107 antiviral units/d) and 0.1% BSA (5 ml) in saline were infused intrauterine (body of the uterus) transcervically in treatment and control groups, respectively. A total of four doses at 12-h intervals were given. On d 16, all animals were slaughtered, and reproductive tracts were collected. Uterine horns were identified as ipsi- or contralateral to the CL. The genital tracts were separated to isolate the endometrium, myometrium (only from ipsilateral horn), CL, and UOP (from both ipsi- and contralateral horns) (15, 17). IFN{tau} was used to mimic the pregnancy recognition-associated events in the presence of viable embryo, as described in sheep (45).

Experiment 2
Bovine uteri on different days of the estrous cycle were collected at a local abattoir. Days of the estrous cycle were determined by utero-ovarian morphology (15). Uteri were classified into seven groups: d 1–3 (n = 4), d 4–6 (n = 3), d 7–9 (n = 3), d 10–12 (n = 3), d 13–15 (n = 6), d 16–18 (n = 7), and d 19–21 (n = 5). Endometrium and myometrium were separated and collected.

Cross-sections of tissues were prepared and processed for immunohistochemistry as described below. Tissues were cut into small pieces, snap-frozen in liquid nitrogen, and stored at –80 C until used. Total RNA was isolated using TRIzol according to the manufacturer’s protocol. Total proteins were extracted and quantified as described previously (15). The expressions of COX-1, COX-2, and PGT mRNA were studied using Northern blot. The expressions of COX-1, COX-2, PGES (mPGES-1), PGFS (AKR1B5), PGDH, PGT, and EP2 proteins were studied by Western blot. EP2, EP3, and FP mRNAs were studied using real-time quantitative RT-PCR (LightCycler). Cellular localization of PGFS, PGES, and EP2 proteins was performed by immunohistochemistry.

Quantitative RT-PCR (LightCycler)
LightCycler reaction using SYBR Green I (Roche) and quantification were performed as we previously described (13). In brief, total RNA (1 µg) was reverse transcribed using random primer and Superscript II RT. Sets of specific primers were deduced from the known sequences of bovine EP2, EP3, EP4, and FP as we previously described (13). LightCycler reactions were performed as previously described (32) in a total volume of 20 µl in microcapillary tubes according to the manufacturer’s instructions. Recombinant plasmids containing specific inserts of EP2, EP3, and FP and the purified PCR product for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as templates. Plasmid DNAs or PCR products were quantified and serially diluted from 100 to 0.01 pg/2 µl. Each reaction mixture contained 2 µl cDNAs, 2 µl FasterStart DNA Master SYBR Green I mix, 2 µl sense and antisense primers each (0.5 µM), 1.6 µl 25 mM MgCl2, and 10.4 µl PCR grade H2O. The LightCycler programs for each gene were as follows: denaturation (95 C for 10 min), PCR amplification and quantification (95 C for 10 sec, 60 C for 5 sec, and 72 C for 20 sec) with single fluorescence measurement at specific temperature (acquisition) for 5 sec repeated for 30–50 cycles depending on the gene studied (Fig. 2Go), a melting program (70–95 C at the rate of 0.1 C/sec with continuous fluorescence measurement), and finally a cooling step to 40 C. At all steps, the transition temperature was 20 C/sec.



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  		FIG. 2. Expression of PGES, PGFS, and PGDH in endometrium, myometrium, and CL at the time of MRP. A, Western analysis. B, Densitometric values of PGES, PGFS, and PGDH, expressed as the mean ± SEM ratio relative to ß-actin individually as an internal control. C, PGES to PGFS ratio. In each group, three different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis. P < 0.05: CONT vs. IFN{tau}, a and b, PGFS; c, PGES; d–f, PGES to PGFS ratio.

 
Northern blot analysis
Northern blotting and hybridization were performed as we described previously (15). Briefly, total RNA (~20 µg) was loaded in each lane and electrophoresed on 1.2% formaldehyde-agarose gel. RNA was transferred overnight onto a nylon membrane in 10x standard saline citrate. The cDNA probes for COX-1, COX-2, and PGT were labeled with [{alpha}-32P]deoxy-CTP (3000 Ci /mmol) using Ready-To-Go DNA labeling kit. Prehybridization (2–3 h) and hybridization (overnight) were carried out at 45 C using UltraHyb. The blots were stripped off by boiling in 1% sodium dodecyl sulfate for 30 min and rehybridized with [{gamma}-32P]deoxy-ATP-labeled oligoprobe specific to 18S ribosomal RNA as an internal control for normalization. The blots were exposed to BioMax film, and densitometry of autoradiograms was performed using an {alpha} Imager ({alpha} Innotec Corp., Montréal, Canada). Densitometry values are expressed as the mean ± SEM between individual mRNA levels and 18S based on the integrated density value. Bovine COX-1 and COX-2 (15) and PGT cDNAs (17) were obtained and used as probes as described previously.

Western blot analysis
Western blot analysis was performed as we previously described (15). Briefly, total proteins (~20 µg) were loaded in each lane and electrophoresed on 10% SDS-PAGE, followed by electrotransfer onto nitrocellulose membrane. The following primary antibodies (raised in rabbit) were used for the respective protein: antisheep COX-1 and COX-2 (1:3,000); antibovine PGES (1:2,000), PGFS (1:3,000), PGDH (1:2,000), and PGT (1:1,000); and antihuman EP2 (1:500). Goat antirabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (1:20,000). Chemiluminescent substrate was applied according to the manufacturer’s instructions. The blots were exposed to BioMax films, and densitometry was performed using an {alpha} Imager. ß-Actin (1:5,000) was measured as an internal control for normalization. Densitometry values are expressed as the mean ± SEM between individual protein levels and ß-actin based on integrated density value.

Immunohistochemistry
Cross-sections were made in the middle portion of the uterine horn and CL. Tissues were fixed in 4% buffered formaldehyde for 4 h at 4 C and processed using standard procedures. Paraffin sections (3 µm) were made. Immunohistochemistry was performed using the Vectastain Elite ABC kit according to the manufacturer’s protocols (Vector Laboratories, Inc.) and as described previously (14). Endogenous peroxidase activity was removed by fixing sections in 0.3% hydrogen peroxide in methanol. Tissue sections were blocked in 10% goat serum for 1 h at room temperature. The primary antibodies were the same as described above. The following concentrations were used: PGFS, 1:1000; PGES, 1:500; and EP2, 1:500. Incubation with the primary antibodies was performed overnight at 4 C. The sections were also incubated with the secondary antibody (goat antirabbit IgG biotinylated, 1:200) for 30 min at room temperature. For the negative control, preimmune or control rabbit serum was used at the respective dilution used for primary antibodies. Between each step, tissue sections were washed in PBS. Finally, tissues were stained with Mayer’s hematoxylin. Photos were captured using the Spot program (Carsen Group, Inc., Markham, Canada), and quantification was performed using Image-Pro-Plus (Media Cybernetics, Cabin John, MD). Preimmune serum was used for PGFS, and control serum was used for commercial and donated antibodies EP2 and PGES (control serum was collected without immunization from the same species in which the antibody was raised).

Statistical analysis
All numerical data are presented as the mean ± SEM. Data were analyzed using ANOVA, followed by Scheffé’s test (Super ANOVA, Abacus Concepts, Inc., Berkeley, CA). Differences were considered statistically significant at the 95% confidence level (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of COX-1 and COX-2
IFN{tau} increased (P < 0.05) COX-2 mRNA and protein expression in endometrium (~1.4-fold), but not in myometrium. No influence of IFN{tau} on luteal expression of COX-2 was evident (Fig. 1Go). COX-1 mRNA and protein were expressed at constant low levels, and IFN{tau} did not influence the expression.



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  		FIG. 1. Expression of COX-1 and COX-2 in endometrium, myometrium, and CL at the time of MRP. A, Northern analysis. B, Densitometric values of COX-1 and COX-2 mRNAs, expressed as the mean ± SEM ratio relative to 18S RNA individually as an internal control. C, Western analysis. D, Densitometric values of COX-1 and COX-2 proteins, expressed as the mean ± SEM ratio relative to ß-actin individually as an internal control. In each group, three different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis. P < 0.05: CONT vs. IFN{tau}, a, COX-2 mRNA; b, COX-2 protein.

 
Expression of PGFS, PGES, and PGDH
IFN{tau} decreased (P < 0.05) PGFS expression in endometrium (~1.5-fold) and myometrium (~3.5-fold), but not in CL. By contrast, PGES was up-regulated (P < 0.05) in CL (~2.8-fold), whereas no modulation was observed in endometrium or myometrium. PGDH was expressed at constant low levels. As an indication of the relative importance of the PGE2 and PGF2{alpha} pathways, we analyzed the PGES to PGFS ratio. The data indicate that IFN{tau} increased (P < 0.05) the PGES to PGFS ratio in endometrium (~1.5-fold), myometrium (~2.0-fold), and CL (~2.7-fold; Fig. 2Go). Immunohistochemistry showed that PGFS and PGES proteins were localized in luminal epithelium and stroma of endometrium and in circular and longitudinal smooth muscle cells of myometrium (Fig. 3AGo). Quantification of PGES and PGFS staining (Fig. 3BGo) revealed that PGFS was down-regulated (P < 0.05) in epithelial cells of endometrium (~1.8-fold), and in both smooth muscle cell types of myometrium (~3.1-fold), but not in stroma or glandular epithelium of endometrium. No modulation was observed for PGES in any of the cell types of uterus. In CL, PGES and PGFS were preferentially localized in large luteal cells (LLCs; Fig. 3AGo), and IFN{tau} increased (P < 0.05) PGES expression (~2.1-fold), but not PGFS (Fig. 3BGo).



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  		FIG. 3. A, Cellular localization of PGES, PGFS, and EP2 proteins in endometrium, myometrium, and CL at the time of MRP. All of these proteins are expressed in luminal epithelium, stroma, and glandular epithelium of endometrium and in circular and longitudinal smooth muscle cells of myometrium in a distinctive manner. In CL, they are preferentially expressed in LLCs. B, Quantitative analysis is based on integrated OD (IOD). Values are expressed as the mean ± SEM. In each group, three different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis (P < 0.05). CONT vs. IFN{tau}, a–c, PGFS; d, PGES; e–g, EP2. Immunohistochemistry was performed using the Vectastain Elite ABC kit as described in Materials and Methods.

 
Expression of PGT
PGT mRNA and protein were expressed in endometrium, myometrium, and CL (Fig. 4Go). IFN{tau} did not influence PGT expression in any of the tissues. The level of PGT expression was high in endometrium, moderate in CL, and low in myometrium (P < 0.05). In UOP, PGT mRNA and protein were more highly expressed (P < 0.05) on the ipsilateral side to CL (Fig. 5Go). IFN{tau} did not alter PGT expression patterns in UOPs.



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  		FIG. 4. Expression of PGT in endometrium, myometrium, and CL at the time of MRP. A, Northern analysis. B, Densitometric values of PGT mRNA expressed as the mean ± SEM ratio with 18S RNA as an internal control. C, Western analysis. D, Densitometric values of PGT protein, expressed as the mean ± SEM ratio relative to ß-actin as an internal control. In each group, three different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis.

 


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  		FIG. 5. Expression of PGT in UOP at the time of MRP. A, Northern analysis. B, Densitometric values of PGT mRNA expressed as the mean ± SEM ratio with 18S RNA individually as an internal control. C, Western analysis. D, Densitometric values of PGT protein, expressed as the mean ± SEM ratio relative to ß-actin individually as an internal control. In each group, three different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis.

 
Expression of EP2, EP3, and FP
In experiment 1, our data illustrate that in endometrium, IFN{tau} increased (P < 0.05) EP2 mRNA and protein expression (~1.2-fold), but not EP3 or FP mRNAs. In myometrium, IFN{tau} increased EP2 mRNA (~4-fold) and protein (~2.7-fold); in contrast, it decreased EP3 (2.8-fold), but had no effect on FP expression (P < 0.05). In CL, IFN{tau} decreased EP3 (1.8-fold; P < 0.05), but did not affect FP or EP2 significantly (Fig. 6Go). Quantification of immunohistochemistry indicated that IFN{tau} up-regulated (P < 0.05) EP2 in endometrial stroma (2.0-fold), but not in luminal or glandular epithelium. In myometrium, IFN{tau} increased EP2 expression 1.5-fold in circular muscle layers and 3-fold in longitudinal muscle layers. In CL, EP2 was preferentially expressed in LLCs (Fig. 3Go).



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  		FIG. 6. Expression of EP2, EP3, and FP in endometrium, myometrium, and CL at the time of MRP. A, LightCycler quantification of EP2, EP3, and FP mRNA, expressed as the mean ± SEM ratio with GAPDH individually as an internal control. B, Western analysis of EP2. C, Densitometric values of EP2, expressed as the mean ± SEM ratio relative to ß-actin individually as an internal control. In each group, three different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis, P < 0.05: CONT vs. IFN{tau}, a and b, EP2 mRNA; c and d, EP3 mRNA; e and f, EP2 protein.

 
In experiment 2, EP2, EP3, and FP mRNAs were expressed and modulated in endometrium and myometrium during the estrous cycle (Fig. 7Go). The level of expression of EP2 was higher (P < 0.05) between d 10–18 of the estrous cycle in both endometrium and myometrium. The level of expression of EP3 was higher (P < 0.05) on d 13–21 of the estrous cycle in myometrium, but was expressed at a constant low level in endometrium throughout the estrous cycle. The level of FP expression was slightly increased on d 16–21 of the estrous cycle in myometrium, whereas no modulation was evident in endometrium. Comparatively, EP3 and FP expressions were higher (P < 0.05) in myometrium than in endometrium.



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  		FIG. 7. Expression of EP2, EP3, and FP receptors in endometrium and myometrium during the bovine estrous cycle based on real-time RT-PCR (LightCycler) quantification. As an internal standard, GAPDH mRNA was measured. Values are expressed as the mean ± SEM ratio between EP2 or EP3 or FP and GAPDH mRNAs. In each group, three to six different uteri were analyzed. Data were analyzed using ANOVA, followed by Scheffé’s post hoc analysis (P < 0.05). a and b, EP2 mRNA, d 10-18 vs. others; c, EP3 mRNA, d 13-21 vs. others.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We present here an integrated view of the biosynthesis, transport. and signaling of PGE2 and PGF2{alpha} in endometrium, myometrium, and CL at the time of MRP in cattle. The net production and action of PGE2 and PGF2{alpha} in a given tissue are governed by several components, such as metabolic enzymes, transporter, and receptors. Among them, cPLA2, COX-1, COX-2, and PGDH are involved in nonselective production of PGs, and PGT is involved in nonselective transport of PGs, whereas PG synthases (PGES and PGFS) and receptors (EP and FP) control the selective production and associated specific biological actions of PGE2 and PGF2{alpha}. We have characterized the expression of both selective and nonselective components in the uterus and CL and found that PGE2 and PGF2{alpha} components are expressed in a tissuespecific and spatio-temporal manner. The PGE2 and PGF2{alpha} biosynthetic and signaling pathways are selectively activated at the time of pregnancy recognition, suggesting polycrine (endocrine, exocrine, paracrine, and autocrine) actions of PGE2 in uterus and CL.

Relative expression of PG metabolic enzymes indicates that IFN{tau} decreases PGFS in endometrium and myometrium, whereas there is no effect on PGES. Cell-specific expression shows that the decrease in PGFS is more prominent in endometrial luminal epithelial and myometrial smooth muscle cells. This key alteration leads to an increase in the PGES to PGFS ratio in endometrium as well as myometrium. IFN{tau} increases COX-2 expression in endometrium, but not in myometrium. COX-1 and PGDH expressions are not altered by IFN{tau} in any of the tissues studied. It has been demonstrated, based on the Kcat/Km ratio, that the conversion of PGH2 to PGE2 by mPGES-1 is 150-fold higher than the conversion of PGH2 to PGF2{alpha} by PGFS (AKR1B5) (5, 6). This suggests that although PGES and PGFS are expressed at the same level in a given tissue, PG biosynthesis would be favorably directed toward PGE2, rather than PGF2{alpha}, production. IFN{tau} appears to regulate COX-2 and PG synthases in a cell-specific manner and preferentially direct the biosynthetic pathway toward PGE2 in endometrium as well as myometrium. Recently, we have shown that COX-2-PGES and COX-2-PGFS pathways are responsible for PGE2 and PGF2{alpha} production, respectively, during implantation and/or the luteolytic window in bovine endometrium in vivo (6, 15). Our previous in vitro studies have shown that IFN{tau} increases COX-2 expression and favors PGE2 compared with PGF2{alpha} production in bovine endometrial cells (40, 41).

The newly synthesized PGs must be effluxed to exert most of their biological effects (9, 10). In ruminants, endometrial PGE2 and/or PGF2{alpha} are transferred from the uterine to the ovarian compartment through UOP to bring forth their endocrine actions, luteolysis, or luteostasis. The present study shows that IFN{tau} does not alter PGT expression in endometrium, myometrium, or UOP, suggesting that the PG transport system is present, but not modulated, at the time of MRP. We have shown in our previous study that PGT is highly expressed in endometrium and UOP at the luteolytic/pregnancy recognition window during the bovine estrous cycle, and we proposed a role for PGT in cellular and compartmental transport of PGE2 and PGF2{alpha} (17).

The data presented in this report demonstrate that during the estrous cycle, FP is expressed at a very low level throughout the estrous cycle in the endometrium, whereas a slight increase is observed in myometrium at the end of the cycle. IFN{tau} does not influence FP expression in either tissue. EP2 mRNA is highly expressed at mid and late luteal phases in both endometrium and myometrium, in agreement with our previous observations for EP2 protein (14). EP3 mRNA is expressed at a constant, but very low, level in endometrium throughout the cycle, whereas it increases in myometrium during the second half of the estrous cycle. Relative expression of PG receptors indicates that IFN{tau} increases EP2 expression in endometrium and myometrium. By contrast, IFN{tau} decreases EP3 in myometrium, but not in endometrium. Cell-specific expression reveals that the increase in EP2 is more evident in endometrial stroma and myometrial smooth muscle cells, a pattern consistent with our previous observations during early pregnancy (44). Together, the present findings indicate that IFN{tau} increases EP2- and decreases EP3-associated signaling in a tissue-specific manner at the time of MRP in cattle. The PGE2-cAMP pathway is involved in mitogenesis, angiogenesis, vasodilatation, endometrial receptivity, decidualization, and myometrial quiescence during the establishment of pregnancy in a variety of species (33, 34, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55). PGE2 increases COX-2, EP2, and its own production and action through cAMP in different cell types (46, 47, 56). EP2 receptor has been considered a relaxant receptor in myometrium of different species (48, 49, 57, 58). Butaprost, an agonist of EP2, increases cAMP production, reduces myometrial contraction (59), and abolishes oxytocin-induced myometrial activity (58). In human uterus, abundant expression of EP3 is associated with increased myometrial contraction, and loss of EP3 seems to be an important regulatory mechanism to maintain the quiescence of myometrium (60).

Rescue of CL and maintenance of P4 secretion are the final goals of the pregnancy recognition process. The present findings indicate that IFN{tau} significantly increases luteal expression of PGES, decreases EP3, and slightly increases EP2. PGES and EP2 are primarily localized in LLCs. No modulation is evident on luteal expression of COX-1, COX-2, PGFS, PGDH, PGT, and FP. The increase in luteal PGES suggests its role in preferential production of luteal PGE2, as shown in other systems. Contrasting patterns of EP2 and EP3 suggest that luteal PGE2 probably uses both EP2 and EP3 receptor subtypes to accomplish distinct purposes during the CL life span, as described in other systems (61). Together, the presence of PGE2 biosynthetic, transporting, and EP2-associated signaling systems in LLCs strongly suggest that PGE2 is a potential candidate to rescue the CL at the time of pregnancy recognition in cattle. In our recent study we have shown the dynamic temporal expression of PG components in CL throughout the bovine estrous cycle and explained the role of luteal PGs in CL life and function (21).

It is established in cyclic cows that endometrial PGF2{alpha} initiates the luteolytic process between d 15–17 of the estrous cycle, whereas intrauterine administration of PGE2 (28, 29, 30, 31, 62, 63) or IFN{tau} (22) or the presence of a viable embryo (28, 62) during this window counteracts the luteolytic effect of PGF2{alpha} and increases the life span of the CL. Early studies have shown that PGE2 is a potential luteoprotective mediator in ruminants. PGE2 originating from the uterus and/or conceptus is the potential factor involved in maintenance of the CL (64, 65, 66). The concentration of PGE2 in endometrium and uterine venous plasma is increased during early pregnancy in sheep (67). Studies involving surgical separation of UOP suggest that a secretory product(s) from the gravid uterus is transported through UOP to rescue the CL at the time of establishment of pregnancy in cattle (68, 69). Infusion of PGE2 from d 10–17 in the uterine horn adjacent, but not opposite, to CL maintains its function in sheep (31). It is reported that PGE2 stimulates P4 secretion in bovine and ovine CL (66, 70, 71, 72, 73, 74). PGE2 increases luteal P4 secretion (luteal maintenance) by activating the EP2-cAMP-protein kinase A pathway in human, rabbit, and ruminant CLs (70, 75, 76, 77, 78). Moreover, a positive correlation between luteal PGE2 and P4 has been demonstrated during the menstrual cycle in humans (79) and the estrous cycle in cows (76). Others have reported that EP3 is not expressed in LLCs of ovine CL (80). The existence of different EP3 subtypes does not permit us at present to relate our results to a specific signaling pathway in CL. However, based on circumstantial evidence, it is presumed that EP3 is preferentially associated with luteal regression. Taken together, the present findings and previous observations by us and others indicate that endometrial PGE2 may direct luteal PG production preferentially toward PGE2 at the time of MRP. This suggests that the CL life span depends on endometrial/extraluteal PGs at the critical period between d 15–17 of the estrous cycle in cattle. Collectively, our observations on the expression of luteal PGE2 biosynthetic and EP2-associated signaling systems support a pivotal role for luteal PGE2 in the rescue of the CL (luteal maintenance) at the time of MRP. Thus, our findings support new emerging concepts that endometrial/extraluteal PGs influence intraluteal PG production in ruminants (19, 20).

The PGFS (AKR1B5) we evaluated in this study also possesses 20{alpha}-hydroxysteroid dehydrogenase activity (6), converting P4 into inactive progestin, a phenomenon first described in rat and rabbit CL (81, 82, 83). A high intraluteal P4 concentration down-regulates 20{alpha}-hydroxysteroid dehydrogenase activity, whereas PGF2{alpha} stimulates it in rat CL (82, 83). Recent evidence suggests that in ruminants, in addition to the well recognized production of P4 by the CL, the endometrium produces P4, which may contribute to the local concentrations within the uterus (84, 85). P4, as the primary regulator, provides the conducive intrauterine environment for embryo/conceptus survival (22, 24, 84, 85). The present findings showing the presence of AKR1B5 in CL and uterus suggest that this enzyme might be a key regulator of P4 action in both tissues.

Based on findings from present and previous studies from our laboratory (6, 8, 14, 15, 21, 40, 41, 44), we propose a model integrating the polycrine actions of PGE2 at the time of establishment of pregnancy in cattle (Fig. 8Go). The antiluteolytic effect of IFN{tau}, the luteotropic and immunomodulatory effect of PGE2, EP2-associated signaling, and the related changes in uterus eventually lead to the establishment of pregnancy. In contrast, increased PGF2{alpha} production, FP-associated signaling, and activation of EP3 subtypes in uterus increase Ca2+ and IP3 and may produce uterine contractility and, in CL, bring forth luteal regression. Together, uterine contractility and luteal regression culminate in pregnancy failure and/or a return to a new estrous cycle.



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  		FIG. 8. A proposed model integrating polycrine actions of PGE2 at the time of establishment of pregnancy in cattle based on our findings from this and previous studies (6 8 14 15 21 40 41 44 ). High physiological levels of IFN{tau} selectively decreased endometrial PGF2{alpha} pulsatile secretion (serves as an antiluteolytic signal) and increased PGE2 production. Selective localization of PGT mediated the cellular and compartmental transport of PGE2 in uterine tissues. Activation of EP2 increased cAMP generation and the associated uterine and vascular changes (endometrial angiogenesis, vasodilation and receptivity, myometrial quiescence, and immunomodulation). Embryonic PGE2 may produce its synergistic effect with endometrial PGE2. The high level PGE2 was competitively transported through UOP by PGT to CL, where it increased luteal PGE2 biosynthesis and EP2-associated signaling, thereby favoring luteal maintenance (serves as a luteotrophic mediator). The antiluteolytic effect of IFN{tau}, the luteotropic and immunomodulatory effect of PGE2, and EP2-associated signaling and the related changes in uterus eventually led to the establishment of pregnancy. In contrast, increased PGF2{alpha} production, FP signal, and activation of EP3 subtypes in uterus resulted in increased Ca2+ and IP3, leading to uterine contractility. Transport of endometrial PGF2{alpha} to CL increased luteal PGF2{alpha} production and thereby initiated luteal regression. Together, uterine contractility and luteal regression culminated in pregnancy failure and/or a return to a new estrous cycle.

 
Early embryonic mortality remains the major single cause of infertility in the bovine. Up to 40% of early embryonic losses occur between d 15–17 of the estrous cycle (24). Treatments targeting PG biosynthetic enzymes, transporters, and receptors could represent novel therapeutic strategies to improve the conception rate in cattle. It has been shown that dietary eicosapentenoic acid decreases endometrial secretion of PGF2{alpha} and increases the conception rate in cows (24). Not much information is available on the regulation of PG systems in uterus and CL. Future studies are required in this arena to unravel the molecular mechanisms involved in the regulation of uterine and luteal production and the action of PGs.

Collectively, our study shows that IFN{tau} selectively and specifically activates PGE2 and PGF2{alpha} biosynthesis, transport, and signaling in endometrium, myometrium, and CL in a tissue-specific and spatio-temporal manner. Our results indicate the polycrine actions of PGE2 in endometrial receptivity, myometrial quiescence, and luteal maintenance at the time of recognition and establishment of pregnancy in cattle. They also suggest that establishment of pregnancy may depend not only on inhibition of endometrial PGF2{alpha}, but also on increased PGE2 production in cattle.



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  		FIG. 3A. Continued

 

   Acknowledgments
 
We thank staff of Nova Scotia Agricultural College for the help rendered during management of animals and experimental procedures; our colleagues, Christian Villeneuve, Jerome Lapointe, and Gerry Russell, for the assistance with collection of tissues; and Eric Madore for technical advice on the PGFS and PGDH analyses.


   Footnotes
 
This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada (to M.A.F. and L.A.M.).

J.A.A. and S.K.B. contributed equally to this work.

Abbreviations: AKR-1B5, Aldoketoreductase 1B5; CL, corpus luteum; COX, cyclooxygenase; FP, prostaglandin F2{alpha} receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN{tau}, interferon-{tau}; m, microsomal; IgG, immunoglobulin G; IP3, inositol triphosphate; LLC, large luteal cell; MRP, maternal recognition of pregnancy; P4, progesterone; PGDH, prostaglandin 15-dehydrogenase; PGES, prostaglandin E synthase; PGE2, prostaglandin E2; PGF2{alpha}, prostaglandin F2{alpha}; PGFS, prostaglandin F synthase; PGT, prostaglandin transporter; UOP, utero-ovarian plexus.

Received May 10, 2004.

Accepted for publication August 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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