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Endocrinology, doi:10.1210/en.2007-1087
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Endocrinology Vol. 149, No. 1 219-231
Copyright © 2008 by The Endocrine Society

Molecular Cloning and Characterization of Prostaglandin (PG) Transporter in Ovine Endometrium: Role for Multiple Cell Signaling Pathways in Transport of PGF2{alpha}

S. K. Banu, J. Lee, M. C. Satterfield, T. E. Spencer, F. W. Bazer and J. A. Arosh

Reproductive Endocrinology and Cell Signaling Laboratory (S.K.B., J.L., J.A.A.), Department of Veterinary Integrative Biosciences (S.K.B., J.L., F.W.B., J.A.A.), College of Veterinary Medicine and Biomedical Sciences, Center for Animal Biotechnology and Genomics (M.C.S., T.E.S., F.W.B.), Department of Animal Sciences, Texas A&M University, College Station, Texas 77483

Address all correspondence and requests for reprints to: Joe A. Arosh, D.V.M., M.V.Sc., Ph.D., Department of Veterinary Integrative Biosciences, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, Mail Stop: TAMU 4458, College Station, Texas 77843-4458. E-mail: jarosh{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In ruminants, endometrial prostaglandin F2{alpha} (PGF2{alpha}) is the luteolytic hormone. Cellular transport of PGF2{alpha} in the uterine endometrium is critical for regulation of the estrous cycle. Molecular mechanisms responsible for control of PGF2{alpha} transport in endometrium during luteolysis are largely unknown. In the present study, we characterized the prostaglandin transporter (PGT) in ovine endometrium. Ovine PGT cDNA consists of 1935 nucleotides that encode 644 amino acids. In ovine endometria, PGT is highly expressed during the period of luteolysis, between d 14 and 16 of the estrous cycle, in luminal and glandular epithelia. Pharmacological and genomic inhibition of PGT indicates that it is responsible for influx and efflux of PGF2{alpha} in ovine endometrial epithelial cells. Inhibition of PGT during the period of luteolysis prevents the release of oxytocin-induced PGF2{alpha} pulses, and maintains functional corpus luteum and its secretion of progesterone. In ovine endometrial epithelial cells, protein kinase A and protein kinase C pathways are involved in regulating the influx of PGF2{alpha}, whereas epidermal growth factor receptor pathways are implicated in regulation of influx and efflux of PGF2{alpha}. The ERK1/2 pathway is associated with efflux of PGF2{alpha}, whereas Jun-amino-terminal kinase/stress-activated protein kinase pathways are involved in both efflux and influx of PGF2{alpha}. Phosphatidylinositol 3-kinase pathways are not involved in either influx or efflux of PGF2{alpha} in ovine endometrial epithelial cells. These are the first results to demonstrate a functional role for PGT in regulation of PGF2{alpha} efflux and influx in ovine endometrial cells that influence luteolytic mechanisms in ruminants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) are key mediators of several female reproductive functions, including luteolysis, ovulation, fertilization, implantation, pregnancy, and parturition (1, 2, 3, 4). Improper production and action of PGs lead to multiple deficiencies in the reproductive processes (2, 5, 6). PG metabolism and cell signaling mechanisms are complex. Arachidonic acid, an essential fatty acid stored in membrane phospholipids, is the primary precursor of PGs. Cyclooxygenases 1 and 2 convert arachidonic acid into PGH2, the common intermediate metabolite for biosynthesis of various forms of PGs, including PGF2{alpha}, PGE2, PGD2, PGI2, and TxA2. The downstream specific synthases convert PGH2 into selective PGs (7, 8, 9, 10, 11). PGF synthase catalyzes the conversion of PGH2 to PGF2{alpha}. Catabolism of PGF2{alpha} is governed by prostaglandin dehydrogenase, which converts PGF2{alpha} into 13, 14-dihydro-15-keto PGF2{alpha} (PGFM), a stable metabolite of PGF2{alpha} (12). PGF2{alpha} exerts its effects primarily through cell surface G-protein coupled receptors, designated as isoforms FPA and FPB (5, 6).

PGs predominate as charged anions and diffuse poorly through plasma membranes despite their lipophilic nature (13, 14, 15). The transport of PGs through plasma membranes is poorly understood with proposed mechanisms, including simple diffusion, passive transport, active transport, and counter-current and carrier-mediated transport. Although anions such as PGs can cross cell membranes by simple diffusion, the estimated flow rate is too low to maintain a biological function (13, 14, 15). Prostaglandin transporter (PGT) is a member of the 12-transmembrane Solute Carrier Organic Anion Transporter 2A1 (14, 15), and mediates both efflux and influx of PGF2{alpha} (14, 15, 16). The PGT transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), also known as anion transport inhibitor (14, 15) or PGT inhibitor (17), inhibits PGT-mediated transport of PGF2{alpha} in a dose-dependent manner (16, 18, 19). Available evidence indicates that cellular transport of PGF2{alpha} is facilitated by PGT (16).

In ruminants, transport of PGF2{alpha} is critical for regulation of the estrous cycle and establishment of pregnancy (16). Cellular transport of PGF2{alpha} at luteolysis and establishment of pregnancy are not completely understood. Oxytocin-induced pulsatile release of PGF2{alpha} from endometrial luminal and superficial glandular epithelia induces luteolysis (20, 21, 22). PGF2{alpha} is released from endometrial epithelial cells, effluxed into the uterine venous system and transported into the ovarian artery via the utero-ovarian plexus to induce luteal regression. During establishment of pregnancy, interferon {tau} suppresses transcription of estrogen receptor {alpha} directly to prevent expression of oxytocin receptors by uterine epithelia, which abrogates pulsatile release of luteolytic PGF2{alpha} (21, 22); however, basal concentrations of PGF2{alpha} in the uterine lumen and uterine venous blood increase (21, 22, 23). These events likely require carrier-mediated active transport of PGF2{alpha} that cannot be mediated by passive transport mechanisms (16). Although development of the endometrial luteolytic mechanisms is regulated by ovarian steroids and oxytocin, the molecular and cellular events associated with transport of PGF2{alpha} are likely to play a key role in controlling both basal and pulsatile release of PGF2{alpha} from ovine endometrial epithelia. We have reported expression of PGT in bovine endometria during the estrous cycle and pregnancy (16, 24), but functional aspects of PGT-mediated transport of PGF2{alpha} in ruminant endometrial cells remain to be determined.

The objectives of the present study were to: 1) clone and characterize ovine PGT, 2) determine temporal and cell-specific changes in expression of PGT in the ovine endometrium during the estrous cycle, 3) elucidate the role of PGT in luteolysis, and 4) unravel molecular mechanisms underlying PGT-mediated cellular transport of PGF2{alpha} in ovine endometrial epithelial cells. Our results indicate that PGT is expressed and regulated in a spatial and temporal pattern in the ovine endometrium during the estrous cycle, and that inhibition of PGT prevents luteolysis to maintain secretion of progesterone and extends interestrus intervals in ewes. Furthermore, we provide the first evidence for a role for protein kinase A (PKA), protein kinase C (PKC), and epidermal growth factor receptor (EGFR), ERK1/2, and Jun-amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathways, in cellular transport of PGF2{alpha} in ovine endometrial epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The reagents used in this study were purchased from the following suppliers: prestained protein markers and Bio-Rad assay reagents and standards (Bio-Rad Laboratories, Hercules, CA); Protran BA83 Nitrocellulose membrane (Whatman Inc., Sanford, ME); Pierce ECL (Pierce, Rockford, IL); protease inhibitor (Roche Applied Biosciences, Indianapolis, IN); DNA ladder, Trizol, antibiotic-antimycotic, and Trypsin-EDTA (Invitrogen Life Technologies Inc., Carlsbad, CA); one-step RT-PCR and plasmid purification kits (QIAGEN Inc., Valencia, CA); pGEMT-easy vector system 1, Prime-a-gene labeling system and EcoRT restriction enzyme (Promega, Madison, WI); Bright Star-plus nylon membrane and UltraHyb (Ambion Inc., Austin, TX); [3H]PGF2{alpha}, [{alpha}32P]dCTP (PerkinElmer Life Sciences, Wellesley, MA); DMEM/F12, DIDS, and antihuman mouse β-actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO); antihuman rabbit PGT polyclonal antibody and PGT blocking peptide (Cayman Chemicals, Ann Arbor, MI); goat antirabbit or antimouse IgG conjugated with horse radish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD); Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, CA); BioMax film (Eastman Kodak Corp., New York, NY); fetal bovine serum (Hyclone, Logan, UT); tissue culture dishes and plates (Corning Inc., Corning, NY); and inhibitors for PKA (H-89), PKC (GF109203), EGFR (AG1478), ERK1/2 (U0126), P38MAPK (SB203580), JNK/SAPK (SP600125), and phosphatidylinositol 3-kinase (PI3K) (LY294002) (EMD Biosciences, San Diego, CA). PGT small interfering RNA (siRNA), siGLORISC-free siRNA, and DharmaFect-1 were obtained from Dharmacon Inc. (Lafayette, CO). The other chemicals used were molecular biological grade from Fisher (Pittsburgh, PA) or Sigma-Aldrich. All oligonucleotide primers were commercially synthesized by Integrated DNA Technologies Inc. (Coralville, IA).

Study 1: molecular cloning and characterization of ovine PGT.
Cloning of ovine PGT was performed in two steps, using total RNA from ovine endometrium as template. In step 1, RT-PCR was performed using specific sense 5'-TTTCGGGTACCTGCTGGGCTC-3' (660–680) and antisense 5'-TCAGATGAGGCTGGCAGCCTTC-3' (1914–1935) primers and one-step RT-PCR kit (QIAGEN). The primers were deduced from known homologous bovine and human sequences. The RT-PCR cycling conditions were: one cycle of 42 C for 45 min and 94 C for 2 min, followed by 40 cycles at 94 C for 30 sec, 60 C for 1 min, and 72 C for 1 min. The amplified PCR product (1275 bp) was cloned into pGEMT-easy vector and sequenced. Step 2 was performed as described previously using specific sense 5'-ATGGGGCTCCTGCCCAAGCT-3' (1–20) and antisense 5'-AGCTGTGTCCACCCTGC CATAG-3' (705–726) primers. The sense primer was deduced from known homologous bovine and human sequences, and the antisense primer was an ovine-specific sequence obtained from step 1. The amplified PCR product (726 bp) was cloned into pGEMT-easy vector and sequenced. Repeated sequencing of the overlapping PCR products from both sides was performed to confirm the accuracy of sequences of ovine PGT. Plasmid DNA was isolated using the QIAGEN plasmid purification kit. The clones were sequenced at GenTech Laboratory, Texas A&M University, and sequences were analyzed using DNASIS MAX 2.5 software (Hitachi Software Engineering America, Ltd., MiraiBio Division, San Francisco, CA). The deduced amino acid sequence data were compared with entries in the GenBank/European Bioinformatics Institute using Basic Local Alignment Search Tool. Multiple sequence alignments were obtained with the CLUSTALX program. Hydropathy analysis of ovine PGT and consensus for phosphorylation and glycosylation sites in ovine PGT were predicted using the Expasy tool (www.expasy.org).

RNA extraction and semiquantitative RT-PCR/Southern blotting
Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions. PCR was performed using the one-step RT-PCR kit as directed by the manufacturer using sense 5'-AGCTACTTTGGCAGCCGGGTC-3' (265–285) and antisense 5'-GGACCACCAGGACGAAGAGTGAG-3' (972–994) primers specific for ovine PGT, and sense 5'-AGAGAGGCATCCTGACCCTCAAGTACC-3' (181–208) and antisense 5'-CACGTAGCAGAGCTTCTCCTTGATGTC-3' (630–658) primers specific for ovine β-actin. These reactions resulted in the amplification of PGT and β-actin DNA fragments of 629 and 467 bp, respectively. Each reaction was performed using 100 ng total RNA in 50 µl total volume. PCR cycling conditions were one cycle of 42 C for 45 min and 94 C for 2 min, followed by a variable number of cycles at 94 C for 30 sec, 60 C for 1 min, and 72 C for 1 min. The number of cycles used was optimized (25) for each gene to fall within the linear range of PCR amplification. Briefly, during the PCR, 5 µl amplified product was collected at specific cycles from nine to 36 and electrophoresed on 2% agarose gel. The gel was then denatured in buffer I (0.5 M NaOH, 1.5 M NaCl) for 45 min and neutralized in buffer II [l M Tris-HCl (pH 8.0), 1.5 M NaCl] for 30 and 15 min. The DNA was transferred overnight onto a nylon membrane in 10x sodium chloride-sodium citrate (SSC) (26). The cDNA probe for PGT (629 bp) or β-actin (467 bp) was labeled with [{alpha}32P]dCTP (1 x 106 cpm/ml) using the DNA labeling kit as per manufacturer’s instructions. Prehybridization and hybridization were performed for 1 and 3 h, respectively, at 45 C using UltraHyb. Membranes were exposed to a phosphor screen, and signals were quantified using the ImageQuant software (Typhoon 8600; Molecular Dynamics, Sunnyvale, CA). Blots were then exposed to Kodak BioMax film.

Validation of antibody to human PGT in ovine tissues
Rabbit antihuman PGT polyclonal antibody and PGT blocking peptide were purchased from Cayman Chemicals. The synthetic peptide derived from the NH2 terminus of human PGT amino acid sequence (NH2-KLGVSQGSDTSTSRA-COOH, positions 6–20) was used to generate the antibody. Protein extracts (50 µg) from ovine endometrial epithelial and stromal cells were resolved using 10% SDS-PAGE, and immunoblotting was performed as described (26). Specificity of the PGT antibody was confirmed using a PGT blocking peptide along with PGT antibody, per manufacturer’s directions.

Study 2: expression of PGT in ovine endometrium.
All experiments and surgical procedures were in accordance with the Guide for Care and Use of Agriculture Animals and approved by Texas A&M University’s Laboratory Animal Care and Use Committee. Ewes (Suffolk crossbred) that had exhibited at least two estrous cycles of normal duration (17–18 d) were used in this study. Estrus (d 0) was detected using vasectomized rams. Ewes were hysterectomized (n = 5 ewes/d) on d 3, 6, 10, 12, 14, and 16 of the estrous cycle. Uterine cross-sections were taken for histology, and then endometria and myometria were separated physically by dissection. Endometrial tissues were cut into small pieces, snap frozen in liquid nitrogen, and stored at –80 C. Expression of PGT mRNA and protein was determined by RT-PCR/Southern blot and immunoblotting, respectively. Immunohistochemistry was used to determine cellular localization of PGT protein. Numerical data are expressed as mean ± SEM.

Study 3: effects of inhibition of PGT on the estrous cycle.
Ewes (Suffolk crossbred) (n = 12) were checked daily for estrus (d 0) using a vasectomized ram. On d 6 of the estrous cycle, uterine horns were catheterized as described previously (27, 28, 29). On d 11 of the estrous cycle, ewes were assigned randomly to the following treatments (n = 4 ewes per group) from d 11–15 postestrus at 12-h intervals (0700 and 1900 h). Group 1 ewes received intrauterine infusions of 0.1 M potassium bicarbonate (2 ml/uterus/12 h) as vehicle. Group 2 ewes received intrauterine infusions of DIDS (10 mg/uterus/12 h) in 2-ml vehicle. Group 3 ewes received intrauterine infusions of DIDS (100 mg/uterus/12 h) in 2-ml vehicle. Ewes were observed daily for estrus in the presence of vasectomized rams. Blood samples were obtained from the jugular vein from d 11 to the day of onset estrus, and plasma was harvested to determine concentrations of progesterone by RIA (30). Numerical data are expressed as mean ± SEM.

Study 4: effects of inhibition of PGT on pulsatile release of PGF.
2{alpha}. Ewes (Suffolk crossbred) (n = 12) were checked daily for estrus (d 0) using a vasectomized ram. On d 6 of the estrous cycle, uterine horns were catheterized as described previously (27, 28, 29). On d 15 of the estrous cycle, ewes were assigned randomly into three groups (n = 4 ewes per group). Group 1 ewes received intrauterine infusions of vehicle, 0.1 M potassium bicarbonate (2 ml). Group 2 ewes received intrauterine infusions of DIDS 10 mg/2 ml. Group 3 ewes received intrauterine infusions of DIDS 100 mg/2 ml at 0 and 2 h to prevent PGT-mediated transport of PGF2{alpha}. Each ewe was then challenged with 30 IU oxytocin iv to induce a PGF2{alpha} pulse between time of injection of oxytocin and 3 h (31). Blood samples were obtained from the jugular vein (3 ml) at –60, –30, –15, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min after the oxytocin injection. Plasma was harvested, and concentrations of PGFM (a stable metabolite of PGF2{alpha}) were determined using an ELISA (32). Numerical data are expressed as mean ± SEM.

Study 5: cellular transport of PGF2{alpha} in ovine endometrial epithelial cells in vitro
Experiment 1.
Ovine immortalized endometrial epithelial cells (33) were cultured in DMEM/F12 medium containing 5% dextran charcoal-treated fetal bovine serum (DC-FBS) and 100 U penicillin/ml, 100 µg streptomycin/ml, and 2.5 µg amphotericin B/ml in humidified 5% CO2 and 95% air at 37 C. The cells were cultured in 100-mm tissue culture dishes. At 70% confluency, the cells were serum starved for 24 h and then treated with or without DIDS (500 µM) for 2 h. The DIDS medium was then replaced with DMEM/F12 medium. At the end of the 24-h experiment, total RNA and protein were isolated, and PGT mRNA and protein were analyzed by RT-PCR/Southern blot and immunoblotting, respectively. Cytotoxicity of DIDS, based on results of the trypan blue exclusion test, was not detected at 500 µM, a concentration chosen from dose-response experiments (data not shown) as we reported for HeLa cells (16).

Experiment 2.
The influx of PGF2{alpha} was determined as described previously (16, 18, 19) using cells cultured in 24-well plates and serum starved as described for experiment 1. The cells were pretreated with or without DIDS (500 µM) for 2 h and then medium was replaced, and cells were incubated with [3H]PGF2{alpha} [1.0 nM, well below the maximum affinity (Michaelis-Menten constant, Km)] for 0, 5, 10, 15, 20, or 40 min. At each time point, the cells were washed with ice-cold Hank’s Balanced Salt Solution, harvested using trypsin-EDTA, and [3H]PGF2{alpha} uptake was determined using a β-scintillation counter (Beckman Coulter Inc., Fullerton, CA). Influx rates were calculated as femtomole/milligram of protein/nanomolar concentration of radiolabeled PG added, and data are expressed as mean ± SEM.

Experiment 3.
The efflux of PGF2{alpha} was determined as described previously (16, 18, 19) using ovine endometrial epithelial cells cultured in 24-well plates, serum starved, and incubated with [3H]PGF2{alpha} (1.0 nM, well below the Km) for 10 min (the time point when maximum influx was found in experiment 2, the influx experiment). Medium was then replaced with fresh medium with or without DIDS (500 µM) (time zero), and cells were harvested at 0, 5, 10, 15, 20, or 40 min. The radioactivity remaining in the cells was counted using a β-scintillation counter. Efflux rates were calculated as percentages (%) based on the amount of radiolabeled PGF2{alpha} effluxed from the cells. The radioactivity remaining in the cells at time "0" was considered 100%. Data are expressed as mean ± SEM.

Experiment 4.
The effects of DIDS on PGT protein expression and its association with PGF2{alpha} transport in ovine endometrial epithelial cells were determined using ovine endometrial epithelial cells. The cells were cultured in 100-mm tissue culture dishes, serum starved, and treated with DIDS (500 µM) for 0, 5, 10, 15, 20, or 40 min. Total protein was isolated, and regulation of PGT protein expression was determined by immunoblotting.

Experiment 5.
The PGT gene was silenced to confirm its role in PGF2{alpha} transport in ovine endometrial epithelial cells. The cells were cultured in antibiotic-free DMEM/F12 medium with 10% DC-FBS in 24-well tissue culture plates. At 70–80% confluency, cells were used for PGT knockdown experiments using PGT siRNA duplex delivered by DharmaFect-1, per manufactures’ instructions. As internal controls, siGLO RISC-Free siRNA or mock siRNA was used. Briefly, siRNA duplexes (100 nM/well) and DharmaFect-1 (1 µl/well) were diluted in 50 µl antibiotic and serum-free DMEM/F12 medium separately, mixed gently, and incubated for 5 min at room temperature. Afterwards, PGT siRNA and DharmaFect-1 were mixed (total volume 100 µl) and incubated at room temperature for 20 min. Then, 100 µl siRNA:DharmaFect-1 complex was added to each well in a total volume of 400 µl/well antibiotic free DMEM/F12 medium with 10% DC-FBS. After 24 h, the medium was replaced with fresh DMEM/F12 with 10% DC-FBS and incubated for a second 24-h period. After 48 h transfection, influx experiments were performed using [3H]PGF2{alpha} as described previously. For efflux experiments, the culture media from normal and PGT knockdown cells were collected, and concentrations of effluxed PGF2{alpha} were measured by ELISA (32). Fluorescence-labeled siGLORISC-free siRNA was transfected separately, and transfection efficiency was estimated using a fluorescence microscope. A transfection efficiency greater than 80% was considered ideal for these experiments. The PGT gene was silenced up to 80–85% after 48–72 h based on RT-PCR analysis. PGT knockdown greater than 80% was considered ideal for the present experiments.

Study 6: regulation of transport of PGF2{alpha} in ovine endometrial epithelial cells in vitro.
The presence of multiple sites for several protein serine/threonine kinases and protein tyrosine kinases in extracellular loops, intracellular loops, and transmembrane domains of ovine PGT suggests a role(s) for these kinases in PGT-mediated cellular transport of PGs. Ovine endometrial epithelial cells were cultured in 24-well plates, and influx and efflux experiments were performed as described previously. For the influx study, the cells were pretreated with or without inhibitors of PKA (H-89, 50 nM), PKC (GF109203, 10 µM), EGFR (AG1478, 15 µM), ERK1/2 (U0126, 10 µM), P38MAPK (SB203580, 10 µM), JNK/SAPK (SP600125, 10 µM), or PI3K (LY294002, 50 µM) for 1 h, followed by treatment with DIDS (500 µM) for 2 h. The cells were then incubated with 1 nM [3H]PGF2{alpha} for 0, 5, 10, 15, 20, or 40 min, and influx of [3H]PGF2{alpha} was determined as described previously. For the efflux experiments, cells were incubated with 1 nM [3H]PGF2{alpha} for 10 min (time for maximum uptake), then treated with DIDS in the presence or absence of inhibitors of PKA, PKC, EGFR, ERK1/2, P38MAPK, JNK/SAPK, PI3K for 0, 5, 10, 15, 20, or 40 min, and efflux of [3H]PGF2{alpha} was calculated as described previously. Potential pathways identified on efflux of PGF2{alpha} were confirmed by determining phosphorylation of the respective proteins by Western blotting using the same experimental design and methods described previously.

Protein extraction and immunoblotting
Total protein was isolated from tissues and cells, and immunoblotting/Western blotting was performed as previously described (25, 26, 34). Briefly, tissues were homogenized in buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 1 mM diethyldithiocarbamic (DEDTC)] containing 0.1% Tween 20 and centrifuged at 30,000 x g for 1 h at 4 C. The homogenized tissue pellets (membranes, nuclei, mitochondria) were sonicated in Tris, EDTA, dithiothreitol sonication buffer [20 mM Tris (pH 8.0), 50 mM EDTA, 0.1 mM DEDTC and protease inhibitor cocktail 1 tab/50 ml] containing 1.0% Tween 20. The homogenates were centrifuged at 15,000 x g for 15 min at 4 C, and the supernatant (total protein) was stored at –80 C until analyzed. Proteins were also extracted from cells using this procedure except that the homogenization step was deleted. Protein concentration was determined using the Bradford method (35) and a Bio-Rad Protein Assay kit. Protein samples (50 µg) were resolved using 10% SDS-PAGE. Antihuman polyclonal PGT (1 µg/ml) or β-actin (1:5000) was used as the primary antibody. Goat antirabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (1:10,000). Chemiluminescent substrate was applied according to the manufacturer’s instructions. The blots were exposed to BioMax film, and densitometry of autoradiograms was performed using an Alpha Imager (Alpha Innotech Corp., San Leandro, CA).

Immunohistochemistry
Tissue sections were fixed in 4% buffered paraformaldehyde saline for 4 h at 4 C and processed using standard procedures (25, 26, 34). Paraffin sections (5 µm) were used for immunohistochemical localization of PGT protein using a Vectastain Elite ABC kit according to the manufacturer’s protocols. 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. Incubation with the primary antibody (antihuman polyclonal PGT, 1 µg/ml) was performed overnight at 4 C. The tissue sections were further incubated with the secondary antibody (goat antirabbit IgG biotinylated, 1:200) for 30 min at room temperature. For the negative control, PGT blocking peptide (2 µg/ml) was used according to our results (Fig. 2BGo) and manufacturer’s instructions. Between each step, tissue sections were washed in PBS. Photographs were captured using digital imaging and an image analysis workstation consisting of a Zeiss Axioplan 2 Research Microscope interfaced with a Zeiss Axiocam HR high-resolution color CCD camera with Zeiss Axiovision (Carl Zeiss, Thornwood, NY).


Figure 2
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  		FIG. 2. A, RT-PCR/Southern blot quantitation of ovine PGT. RT-PCR was performed using the one-step RT-PCR kit. Southern blotting was performed using [{alpha}32P]-dCTP labeled oPGT cDNA probe. Densitometry was performed using phosphoimager. Optimal PCR cycle was determined by plotting an amplification curve. B, Validation of human PGT antibody using ovine endometrial cells and tissues. Western blotting was performed using 50 µg protein per lane on 10% SDS-PAGE. a, Immunoblot using antihuman PGT antibody detected ovine PGT. b, PGT antibody preadsorbed with the synthetic peptide did not detect ovine PGT. As an internal control, β-actin was measured. Please see Materials and Methods for more details. IDV, Integrated density value.

 
Immunofluorescence
Ovine endometrial epithelial cells were cultured in four-well Lab-Teck chamber glass slides (Nalge Nunc Intl., Rochester, NY). At 70% confluency the cells were treated or not treated with DIDS (500 µM) for 40 min. The cells were fixed in methanol for 10 min. Nonspecific bindings were blocked by incubating the cells in 10% goat serum for 1 h at room temperature. Incubation with the primary antibody (antihuman polyclonal PGT, 1 µg/ml) was performed overnight at 4 C. The cells were further incubated with secondary antibody, horse radish peroxidase-conjugated Alexa fluor 594 goat antirabbit IgG (1:100) (Molecular Probes, Invitrogen) for 45 min at room temperature. Nuclei were stained with DAPI (ProLong Gold Antifade; Molecular Probes, Invitrogen). For the negative control, PGT blocking peptide (2 µg/ml) was used based on results shown in Fig. 1BGo and manufacturer’s instructions. Photographs were captured using digital imaging and an image analysis workstation as described previously.


Figure 1
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  		FIG. 1. Proposed transmembrane model of ovine PGT based on structural and hydropathy analysis of amino acid sequences (58 59 ). I–XII indicate the 12 transmembrane domains (TMDs). Both amino and carboxyl termini are located intracellularly. Amino acid residues associated with structural and functional integrity of ovine PGT protein are indicated and highlighted. Arg561 and Lys614 are located in TMDs XI and XII, respectively. Eight Cys-Cys Zinc-finger motifs exist in the extracellular loop. Val611 and Ala 612 in transmembrane XII domain are ovine specific. Four N-linked glycosylation sites are present in the extracellular loop. Multiple serine/threonine/tyrosine phosphorylation sites for various protein kinases are predicted in the extracellular and intracellular loops and TMDs. The kinases and the potential sites of action are indicated.

 
ELISA
Concentrations of the stable metabolite of PGF2{alpha}, PGFM, were measured in plasma using a commercially available PGFM assay kit (catalog no. 404610; Neogen Corp., Lexington, KY) according to the manufacturer’s instructions. The sensitivity or minimal detection limit of this assay was 20 pg/ml.

Progesterone assay
Concentrations of progesterone in plasma were determined using DSL-3900 ACTIVE Progesterone Coated-Tube RIA Kit according to the manufacturer’s instructions (Diagnostic Systems Laboratories, Webster, TX). The RIA used rabbit anti-progesterone immunoglobulin-coated tubes and iodinated progesterone. The primary antiserum cross-reacts 6.0, 2.5, 1.2, 0.8, 0.48, and 0.1% with 5{alpha}-pregnane-3,20-dione, 11-deoxycorticosterone, 17{alpha}-hydroxyprogesterone, 5β-pregnane-3,20-dione, 11-deoxycortisol, and 20β-dihydroprogesterone, respectively. The progesterone standard curve (0–10.57 ng/ml) was provided in the assay kit. The sensitivity or minimum detection limit of this assay is approximately 0.12 ng/ml. The intraassay variation was 8.8%.

Statistical analyses
Statistical analyses were performed using general linear models of Statistical Analysis System (SAS Institute Inc., Cary, NC). In study 2, effects of day on PGT mRNA and protein expression were determined by least squares regression analysis using β-actin data as the covariate. Effects of treatment on concentrations of progesterone on different days (study 3) and PGFM (study 4) at different time points and day x time x treatment interactions were analyzed using repeated measures multivariate ANOVA (MANOVA). In study 5, effects of DIDS (experiment 1) on PGT mRNA and protein were analyzed by one-way ANOVA. Effects of treatment with DIDS were determined by analyzing for treatment, time, and treatment x time interactions (experiments 2–4) on PGF2{alpha} transport using MANOVA. Effects of PGT siRNA (experiment 5) on PGF2{alpha} transport were studied by one-way ANOVA. Effects of DIDS and different inhibitors, time and treatment x time interactions on PGF2{alpha} transport (study 6) were analyzed by repeated measures MANOVA. Numerical data are expressed as mean ± SEM. Statistical significance was considered as P < 0.05. The statistical model accounted for sources of variation, including treatments, replicates, and ewes, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Molecular characterization of ovine PGT
The ovine PGT cDNA (GenBank/European Bioinformatics Institute accession no. DQ026455) open reading frame consisted of 1935 nucleotides, encoding 644 amino acids. Hydropathy analysis revealed the presence of 12 hydrophobic domains characteristic of other members of the Solute Carrier Organic Anion Transporter Family Member 2A1 (14, 15). Both amino and carboxyterminal domains were located intracellularly. The predicted amino acid sequence of ovine PGT shared 92, 85, 83, 81, and 79% identity with bovine, canine, human, mouse, and rat homologs, respectively. For ovine PGT, 29 (4.5%) amino acids (Asp and Glu) were negatively charged, 55 (8.5%) amino acids (Arg and Lys) were positively charged, and the remaining 560 (87%) amino acids were neutral. Thus, the net positive charges make ovine PGT cationic. Hydropathy and structural analyses of ovine PGT suggested that arginine (R561) and lysine (K614) in 11th and 12th transmembrane domains, respectively, are involved in ligand binding (14, 15). Multiple serine, threonine, and tyrosine phosphorylation sites for various kinases, including PKA, PKC, CaM II/IV, GRK, GSK, IKK, JAK, SRC, MAPKKK, MAPK, AKT, mTOR, EGFR, PDGFR, IGFR, FGFR, and VEGFR, were predicted in the extracellular and intracellular loops and transmembrane domains of ovine PGT (Fig. 1Go).

The number of PCR cycles was optimized for the PGT gene to fall within the linear range of PCR amplification. Results showed that the optimal number of PCR cycles was 27 for detection of PGT gene in ovine endometrial tissues and cells (Fig. 2AGo). Similarly, results showed that the optimal number of PCR cycles was 16 for detection of the β-actin gene in ovine endometrial tissues and cells (data not shown). Specificity of the human PGT antibody for use with ovine endometrium was confirmed by using a PGT blocking peptide along with an antibody to PGT. Different concentrations (0.5 µg, 1 µg, and 2 µg/ml) of PGT antibody were tested, and 1 µg/ml was chosen based on a strong immunoreactive protein band at 45 kDa. The PGT blocking peptide was tested at different concentrations (1, 2, 3, and 4 µg/ml), and 2 µg/ml or more effectively blocked the immunoreaction (Fig. 2BGo). These results confirmed specificity and validity of the commercial human PGT polyclonal antibody for use with ovine endometria.

Expression of PGT in ovine endometrium
Results (Fig. 3Go, A–D) demonstrated that PGT mRNA and protein were abundantly expressed in ovine endometria, and expression increased (P < 0.05) from d 3 and 6 to d 14–16 of the estrous cycle. During the period of luteolysis, i.e. d 14–16 of the estrous cycle, expression of PGT protein was more abundant in endometrial luminal and glandular epithelia than in stromal cells (Fig. 3EGo).


Figure 3
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  		FIG. 3. Expression of PGT in ovine endometria during the estrous cycle. A, RT-PCR/Southern blot analysis of PGT mRNA expression. As an internal standard, β-actin mRNA was measured. B, Densitometry of PGT:β-actin mRNA ratio. Expression of PGT mRNA was affected by the day of the estrous cycle (P < 0.05). C, Western blot analysis of PGT protein expression. As an internal standard, β-actin protein was measured. D, Densitometry of PGT:β-actin protein ratio. Expression of PGT protein was affected by the day of the estrous cycle (P < 0.05). Representative blots were shown. E, Immunohistochemical localization of PGT protein in ovine endometria. PGT protein was expressed in luminal epithelium (LE), glandular epithelium (GL), and stratum compactum on d 14 and 16 of the estrous cycle. Pictures are at x400 magnification. Immunohistochemistry was performed using the Vectastain Elite ABC kit as described in Materials and Methods. IDV, Integrated density value; STR, stroma.

 
Effects of inhibition of PGT on luteolysis in sheep
We examined whether inhibition of PGT with DIDS, the PGT inhibitor, from d 11–15 of the estrous cycle affected interestrus intervals of cyclic ewes. Control ewes and ewes treated with 10 mg DIDS had normal interestrus intervals of 17–18 d. However, ewes that received 100 mg DIDS had prolonged (P < 0.05) interestrus intervals of 35–52 d (Fig. 4AGo). Accordingly, concentrations of progesterone decreased (P < 0.05) on d 16 to basal level at estrus in control ewes and in ewes treated with 10 mg DIDS, whereas the progesterone concentrations were higher (P < 0.05) on d 16–18 and remained above 3 ng/ml for ewes treated with 100 mg DIDS until the ewes returned to estrus (Fig. 4BGo). These results indicated that inhibition of PGT-mediated transport of PGF2{alpha} from the uterus prevented luteolysis to extend lifespan of corpora lutea and their secretion of progesterone, and length of interestrus intervals. Furthermore, these results indicated that the doses of DIDS used were not cytotoxic to the ovine endometrium. If the luteolytic mechanism is blocked by the temporary presence of the conceptus or intrauterine infusions of interferon {tau}, ewes have prolonged interestrus intervals that are highly variable among ewes and experiments (36). The period of time required for eventual regression of the corpus luteum is assumed to be related to the time required for the endometrium to reestablish the luteolytic mechanism.


Figure 4
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  		FIG. 4. A, Effects of DIDS on interestrus intervals in ewes. a, Effects of DIDS 100 mg vs. CONTROL or DIDS 10 mg on interestrus intervals (P < 0.05). B, Effects of DIDS on concentrations of progesterone in plasma in control (CONT) ewes, and in ewes treated with DIDS 10 or 100 mg. The temporal changes in progesterone concentrations on d 16–18 were different (P < 0.05) for DIDS 100 mg treated ewes compared with control and DIDS 10 mg treated ewes. C, Effects of DIDS on oxytocin-induced changes in concentrations of PGFM. Temporal changes in concentrations of PGFM between 0 and 50 min were not different between control (CONT) and DIDS 10 mg treated ewes but were lower (P < 0.05) in ewes treated with 100 mg DIDS. More details on treatment regimen are given in Materials and Methods. OT, Oxytocin.

 
Next, we determined whether inhibition of PGT by DIDS affected oxytocin-induced pulsatile release of endometrial PGF2{alpha} in ewes on d 15 of the estrous cycle. Results (Fig. 4CGo) indicated that oxytocin induced a PGF2{alpha} pulse that peaked (P < 0.05) at 10 min and returned to basal levels at 50 min in control ewes and in ewes treated with 10 mg DIDS. In contrast, 100 mg DIDS inhibited (P < 0.05) oxytocin-induced release of PGF2{alpha} from the endometria in ewes. The mean concentrations of PGFM were lower (P < 0.05) between 10 and 30 min in ewes treated with 100 mg DIDS than for control ewes and ewes treated with 10 mg DIDS. These results indicated that inhibition of PGT reduced the ability of the endometrium to release PGF2{alpha} in response to oxytocin.

Cellular transport of PGF2{alpha} in ovine endometrial epithelial cells
We used immortalized ovine endometrial epithelial cells as model to determine mechanisms involved in PGT-mediated transport of PGF2{alpha}. First, it was determined that PGT mRNA and protein were expressed abundantly, and that DIDS decreased (P < 0.05) expression of PGT mRNA and protein in ovine endometrial epithelial cells (Fig. 5Go).


Figure 5
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  		FIG. 5. Effect of DIDS on PGT expression in ovine endometrial cells. A, DIDS (500 µM) decreased (P < 0.05) PGT mRNA expression. The internal standard was β-actin mRNA. B, Densitometry of PGT:β actin mRNA ratio indicated that DIDS inhibited (P < 0.05) PGT mRNA expression. C, DIDS decreased (P < 0.05) PGT protein expression. The internal standard was β-actin protein. D, Densitometry of PGT:β actin protein ratio indicated that DIDS inhibited (P < 0.05) PGT protein expression. Representative blots are shown. More details are given in Materials and Methods. CONT, Control; IDV, Integrated density value.

 
Second, PGT-mediated influx and efflux of PGF2{alpha} in ovine endometrial cells in the presence or absence of DIDS was studied. Results indicated that influx rates of [3H]PGF2{alpha} peaked (P < 0.05) at 10 min and then decreased to basal levels by 15–20 min (Fig. 6AGo). Efflux of [3H]PGF2{alpha} occurred rapidly (P < 0.05) during the first 5–10 min, leaving less than 20% of [3H]PGF2{alpha} inside the cells, and values reached basal levels within 40 min (Fig. 6BGo). DIDS inhibited (P < 0.05) both influx and efflux of [3H]PGF2{alpha} from ovine endometrial epithelial cells, which was determined by Western blotting (Fig. 6Go, C and D), likely resulted from inhibition (P < 0.05) of expression of PGT protein in a time-dependent manner by DIDS. Immunofluorescence analysis (Fig. 6EGo) also revealed that DIDS decreased PGT protein within 40 min in ovine endometrial epithelial cells.


Figure 6
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  		FIG. 6. Cellular transport of PGF2{alpha} in ovine endometrial epithelial cells was assessed using influx and efflux experiments in the presence or absence of the PGT inhibitor DIDS, as described in Materials and Methods. A, Influx of PGF2{alpha} by endometrial epithelial cells was inhibited (P < 0.05) by DIDS. B, Efflux of PGF2{alpha} by endometrial epithelial cells was inhibited (P < 0.05) by DIDS. C, Western blot analyses of PGT protein and β-actin protein, as the internal control (CONT). D, Densitometry of PGT:β-actin protein ratio indicated that DIDS inhibited (P < 0.05) expression of PGT protein between 10 and 40 min. Representative blots are shown. E, Immunofluorescence analysis of PGT protein expression and its cellular localization in normal and DIDS treated cells. DIDS decreased the abundance of PGT protein at 40 min. Numerical values are means ± SEM for three experiments. IDV, Integrated density value.

 
Because PGT is abundantly expressed in ovine endometrial epithelial cells, siRNA was used to silence the PGT gene and confirm its role in the transport of PGF2{alpha}. Silencing the PGT gene prevented (P < 0.05) influx of PGF2{alpha} up to 95%, and changes in the influx pattern (Fig. 7AGo) were similar to those resulting from treatment with DIDS to inhibit PGT protein in ovine endometrial epithelial cells. Because PGT knockdown prevented influx of [3H]PGF2{alpha}, it was not possible to determine PGT-mediated efflux of PGF2{alpha} by incubating [3H]PGF2{alpha}. To confirm the role of PGT in release of PGF2{alpha} in this cell line, we compared release of PGF2{alpha} from normal and PGT knockdown cells. The results (Fig. 7BGo) indicated that silencing of PGT inhibited (P < 0.05) 75–80% release of PGF2{alpha} from ovine endometrial epithelial cells.


Figure 7
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  		FIG. 7. Effects of PGT gene knockdown on influx and efflux of PGF2{alpha} in endometrial epithelial cells. A, PGT siRNA inhibited (P < 0.05) influx of PGF2{alpha} compared with control (CONT) and treatments with siGLO RISC-Free siRNA and Mock siRNA. B, PGT siRNA inhibited (P < 0.05) efflux of PGF2{alpha} compared with control and treatments with siGLO RISC-Free siRNA or Mock siRNA. Numerical values are presented as means ± SEM for three experiments.

 
Regulation of cellular transport of PGF2{alpha} in ovine endometrial epithelial cells
Multiple sites for various protein kinases and protein tyrosine kinases in the extracellular loops, intracellular loops, and transmembrane domains are present in ovine PGT. Therefore, we evaluated roles of PKA, PKC, EGFR, and PI3K pathways in PGT-mediated transport of PGF2{alpha} in ovine endometrial epithelial cells. The results indicated that inhibition of PKA (Fig. 8Go, A and B) and PKC (Fig. 8Go, C and D) pathways prevented (P < 0.05) effects of DIDS on PGT-mediated influx, but not efflux of PGF2{alpha}. Interestingly, inhibition of EGFR pathways (Fig. 8Go, E and F) inhibited (P < 0.05) effects of DIDS on PGT-mediated influx and efflux of PGF2{alpha}. Inhibition of PI3K (Fig. 8Go, G and H) did not reverse effects of DIDS on PGT-mediated influx and efflux of PGF2{alpha}. Inhibition of PKA, PKC, and EGFR had no effect on PGT-mediated transport of PGF2{alpha} in the absence of DIDS.


Figure 8
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  		FIG. 8. Effects of PKA, PKC, EGFR, and PI3K pathways on influx and efflux of PGF2{alpha} in ovine endometrial epithelial cells. Influx and efflux experiments were performed in the presence or absence of PGT inhibitor DIDS, as described in Materials and Methods. PKA (H-89, 50 nM), PKC (GF109203, 10 µM), EGFR (AG1478, 15 µM), and PI3K (LY294002, 50 µM) were indicated by PKA-I, PKC-I, EGFR-I, and PI3K-I, respectively. In the control (CONT) group, cells received no treatment. In the DIDS group, cells were treated with DIDS. In the PKA-I or PKC-I or EGFR-I or PI3K-I + DIDS group, cells treated with respective inhibitor and DIDS. In the PKA-I or PKC-I or EGFR-I or PI3K-I group, cells were treated with only respective inhibitor. A, DIDS inhibited (P < 0.05) the influx of PGF2{alpha}, which was prevented (P < 0.05) by PKA-I, but PKA-I did not inhibit normal influx of PGF2{alpha}. B, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was not prevented by PKA-I, and PKA-I did not inhibit normal efflux of PGF2{alpha}. C, DIDS inhibited (P < 0.05) the influx of PGF2{alpha}, which was partially prevented (P < 0.05) by PKC-I, whereas PKC-I did not inhibit normal influx of PGF2{alpha}. D, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was not prevented by PKC-I, and PKC-I did not inhibit normal efflux of PGF2{alpha}. E, DIDS inhibited (P < 0.05) the influx of PGF2{alpha}, which was prevented by the EGFR-I, and EGFR-I did not inhibit normal influx of PGF2{alpha}. F, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was prevented (P < 0.05) by EGFR-I, and EGFR-I did not inhibit normal efflux of PGF2{alpha}. G, DIDS inhibited (P < 0.05) the influx of PGF2{alpha}, which was not prevented by the PI3K-I, and PI3K-I did not inhibit normal influx of PGF2{alpha}. H, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was not prevented by PI3K-I, and PI3K-I did not inhibit normal efflux of PGF2{alpha}.The numerical values presented are means ± SEM from three experiments.

 
Determination of EGFR downstream pathways on PGT-mediated influx and efflux of PGF2{alpha} revealed that inhibition of ERK1/2 pathways (Fig. 9Go, A and B) reversed (P < 0.05) effects of DIDS on PGT-mediated efflux, but not influx of PGF2{alpha}. In contrast, inhibition of JNK/SAPK pathways (Fig. 9Go, C and D) prevented (P < 0.05) effects of DIDS on PGT-mediated efflux and influx of PGF2{alpha}. Inhibition of P38 MAPK (Fig. 9Go, E and F) did not modulate effects of DIDS on either efflux or influx of PGF2{alpha}. Inhibition of ERK1/2 and JNK/SAPK had no effect on PGT-mediated transport in the absence of DIDS.


Figure 9
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  		FIG. 9. Effects of ERK1/2, JNK/SAPK, and P38MAPK pathways on influx and efflux of PGF2{alpha} in ovine endometrial epithelial cells. Influx and efflux experiments were performed in the presence or absence of PGT inhibitor DIDS, as described in Materials and Methods. ERK1/2 (U0126, 10 µM), JNK/SAPK (SP600125, 10 µM), and P38MAPK (SB203580, 10 µM), were indicated by ERK-I, JNK-I, and P38-I, respectively. In the control (CONT) group, cells received no treatment. In the DIDS group, cells were treated with DIDS. In the ERK-I or JNK-I, or P38-I + DIDS group, cells treated with respective inhibitor and DIDS. In the ERK-I or JNK-I or P38-I group, cells were treated with only respective inhibitor. A, DIDS inhibited (P < 0.05) the influx of PGF2{alpha}, which was not prevented by ERK-I, and ERK-I did not inhibit normal influx of PGF2{alpha}. B, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was prevented (P < 0.05) by ERK-I, but ERK-I did not inhibit normal efflux of PGF2{alpha}. C, DIDS inhibited (P < 0.05) influx of PGF2{alpha}, which was prevented (P < 0.05) by JNK-I, whereas JNK-I did not inhibit normal influx of PGF2{alpha}. D, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was prevented (P < 0.05) by JNK-I, but JNK-I did not inhibit normal efflux of PGF2{alpha}. E, DIDS inhibited (P < 0.05) influx of PGF2{alpha}, which was not prevented by P38-I, and P38-I did not inhibit normal influx of PGF2{alpha}. F, DIDS inhibited (P < 0.05) efflux of PGF2{alpha}, which was not prevented by P38-I, and P38-I did not inhibit normal efflux of PGF2{alpha}. The numerical values presented are means ± SEM from three experiments.

 
We determined effects of DIDS on phosphorylation of ERK1/2 and JNK/SAPK proteins and found that DIDS phosphorylated ERK1/2 proteins (Fig. 10Go, A and B) between 15 and 40 min (P < 0.05), and JNK/SAPK proteins (Fig. 10Go, C and D) between 5 and 20 min (P < 0.05). This time-dependent phosphorylation of ERK1/2 proteins was associated with efflux of PGF2{alpha}, and phosphorylation of JNK/SAPK proteins was associated with both efflux and influx of PGF2{alpha} in ovine endometrial epithelial cells. Collectively, these results indicated that phosphorylation of ERK1/2 and JNK/SAPK impaired PGT-mediated efflux of PGF2{alpha} in ovine endometrial epithelial cells.


Figure 10
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  		FIG. 10. Effects of DIDS on activation of ERK1/2 and JNK/SAPK pathways in ovine endometrial epithelial cells. A, Western blot analysis of phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) proteins. B, Densitometry of pERK1/2:tEPK1/2 protein ratio indicated that DIDS phosphorylated ERK1/2 protein and that levels of phosphorylation increased over time (P < 0.05) from 15–40 min. C, Western blot analysis of phosphorylated JNK/SAPK (pJNK/SAPK) and total JNK/SAPK (tJNK/SAPK) protein. D, Densitometry of pJNK/SAPK:tJNK/SAPK protein ratio. DIDS phosphorylated JNK/SAPK protein in a time-dependent manner (P < 0.05), with levels of phosphorylation increased from 0–5 min and then declined gradually. The numerical values presented are means ± SEM from three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well accepted that pulsatile release of endometrial PGF2{alpha} induces luteolysis in ruminants (20). The present study provides the first published results to indicate that the ability of ovine endometrial cells to release PGF2{alpha} as basal secretion or pulsatile secretion, such as that induced by oxytocin, is facilitated by a PGT-mediated mechanism(s) in vivo. Cell signaling pathways PKA, PKC, and MAPK regulate PGT-mediated transport of PGF2{alpha} in ovine endometrial cells in vitro. This is the first report to demonstrate a functional role(s) for PGT in endometrial release of PGF2{alpha} to affect luteolysis and to identify specific cell signaling pathways associated with regulation of PGT-mediated transport of PGF2{alpha} in the ovine endometrium. Our results add a new dimension to the existing paradigm regarding the luteolytic mechanism in ruminants and mechanisms regulating cellular transport of PGF2{alpha}.

Results of the present study indicated that expression of PGT mRNA and protein were regulated in a spatial and temporal pattern in the ovine endometrium during the estrous cycle, as described for bovine endometria (16). In the present study, inhibition of PGT, using DIDS, prevented oxytocin-induced release of luteolytic pulses of PGF2{alpha}, which prevented luteolysis and, therefore, extended interestrus intervals in ewes. These results strongly suggest that PGT facilitates release of luteolytic PGF2{alpha} pulses and that inappropriate function or expression of PGT would result in the failure of ewes to exhibit normal estrous cycles.

The present study validated the utility of an immortalized ovine endometrial epithelial cell line (33) as a model to evaluate and understand mechanisms associated with PGT-mediated transport of PGF2{alpha}. We demonstrated that these cells expressed abundant PGT mRNA and protein, and that DIDS inhibited PGT-mediated influx (>95%) and efflux (>80%) of PGF2{alpha} by inhibiting both PGT function and its expression in a time-dependent manner. Furthermore, silencing of PGT gene impaired 90–95% of influx and 75–80% of efflux of PGF2{alpha}. Our results, using pharmacological and genomic approaches to inhibit PGT, indicate that 80% of PGF2{alpha} release is mediated by a PGT-mediated mechanism and that only 20% of PGF2{alpha} release is due to simple diffusion in ovine endometrial epithelial cells (13, 14, 15).

The present study examined PKA, PKC, EGFR, and PI3K cell signaling pathways and PGT-mediated transport of PGF2{alpha}. The results indicated that: 1) inhibition of PKA and PKC pathways prevented effects of DIDS on influx, but not efflux of PGF2{alpha}; 2) inhibition of EGFR pathways prevented effects of DIDS on both influx and efflux of PGF2{alpha}; and 3) inhibition of PI3K had no effect on either influx or efflux of PGF2{alpha}. Thus, PKA and PKC pathways could affect influx of PGF2{alpha}, whereas EGFR pathways affect efflux of PGF2{alpha} in ovine endometrial epithelial cells. Our current interest is to determine mechanisms responsible for PGT-mediated release of PGF2{alpha} by ovine endometrial cells. Because EGFR pathways affect both influx and efflux of PGF2{alpha}, EGFR downstream pathways were examined. We found that inhibition of ERK1/2 pathways reversed effects of DIDS on efflux, but not influx of PGF2{alpha}, and that inhibition of JNK/SAPK pathways prevented effects of DIDS on both efflux and influx of PGF2{alpha}. Inhibition of P38 MAPK did not alter effects of DIDS on either influx or efflux of PGF2{alpha}. DIDS phosphorylated ERK1/2 and JNK/SAPK proteins in a distinct time-dependent manner that was associated with efflux and influx of PGF2{alpha}, respectively. These results suggest that ERK1/2 pathways affect efflux of PGF2{alpha}, and that JNK/SAPK pathways regulate both efflux and influx of PGF2{alpha} in ovine endometrial epithelial cells.

There is little information in the literature on regulation of expression of PGT mRNA and protein and PGT-mediated transport mechanisms (14, 15). PGT transports PGF2{alpha}, PGE2, PGD2, and TxA2 in a competitive manner with different affinities for each of the PGs. Results from cysteine-scanning mutagenesis experiments of PGT indicated that substrate binding by PGT is within its transmembrane domains (14, 15). Site-directed mutagenesis of arginine at 561 (R561) in the 11th transmembrane domain and lysine 614 (K614) in the 12th transmembrane domain of rat PGT suggests that these cationic amino acids are essential for binding and transport of PGs by PGT (14, 15, 18, 37). PGT-mediated transport appears to involve an electrogenic anion exchange mechanism. PGT is reported to recognize PG substrates as anions, and electrostatic attractions between cations of PGT and the 1-position of carboxylate PG anions appear important for mechanisms involved in PGT-mediated transport (14, 15, 18, 37). Furthermore, PGT-mediated transport varies with rates of cellular glycolysis, and variations in intracellular and extracellular concentrations of PGs and cell membrane potential (14, 15).

PGT belongs to the organic anion transporter (OAT) family that shares common structural features, including: 12-transmembrane domains; multiple glycosylation sites; multiple phosphorylation sites in the intracellular loops, extracellular loops, and transmembrane domains; presence of clustered cysteine residues in the extracellular loops; and presence of specific charged amino acid residues (38, 39). Several transport mechanisms have been proposed for OAT family members based on structure-function analyses (38, 39): 1) inappropriate glycosylation at specific sites impairs trafficking of OATs to the plasma membranes (40); 2) phosphorylation state of several serine, threonine, and tyrosine residues in response to PKC (39, 41, 42, 43, 44, 45), MAPK (46, 47), and AKT (48) regulate phosphorylation, cell surface expression, internalization, stability, Km, and maximum transport velocity of OATs (38, 49); 3) dysfunction of charged amino acid residues such as lysine and arginine regulate binding and transport capabilities of OATs (38, 49, 50, 51); and 4) impaired activities of clustered cysteine residues affect trafficking of OATs toward the cell membrane, and regulate conformation of OATs and interactions with substrates (38, 49, 52). Expression of OAT3 mRNA is regulated by cAMP responsive element binding protein-1 and activating transcription factor 1, which act via cAMP-response element (53). Progesterone inhibits OAT4 activity by causing redistribution of OATs from cell surface to intracellular compartments (54). Interestingly, expression and function of OATs depend on the individual OAT, cell type, and physiological state of cells (38, 49). Whether PGT shares the same regulatory pathways as other OATs is an important issue that requires further investigation.

Results of the present study indicated that DIDS decreased PGT mRNA and protein expression, and inhibited PGT-mediated transport of PGF2{alpha} in ovine endometrial cells in vitro, and PGT-mediated release of PGF2{alpha} from ovine endometria in vivo. Inhibition of various cell-signaling pathways selectively reversed actions of DIDS on PGT-mediated efflux, influx, or both. It is possible that DIDS also affects other OATs in the ovine endometria to cause PGT-independent effects. However, this report to characterize PGT-mediated transport of PGs in ruminant endometria (16, 24, 55) and to demonstrate that DIDS inhibited PGT-mediated transport of PGs (16, 18, 24, 55). DIDS treatment did not result in endometrial epithelial cell death in vitro, and DIDS treated ewes returned to estrus at specific time intervals, suggesting the absence of toxic effects of DIDS in ovine endometria at the doses used.

DIDS has two negative charges and two isothiocyano groups that covalently bind with amines (56, 57). DIDS inhibits the activity of OATs by either reversibly, by rapidly interacting with positively charged amino acid residues, or irreversibly, by slowly reacting covalently and preferentially with lysine residues of the transporter. However, similar mechanistic studies have not been done with PGT. Results from the present investigation indicate that DIDS activates JNK/SAPK and ERK cell signaling pathways for PGT-mediated transport of PGF2{alpha} in ovine endometrial epithelial cells. Ovine PGT has multiple serine, threonine, and tyrosine residues that can be affected by various kinases, including PKA, PKC, EGFR, MAPK, and AKT (Fig. 1Go). This suggests additional mechanisms by which DIDS could regulate expression and activity of PGT.

Based on the literature (38, 49, 56, 57) and results of the present study, we propose that the effects of DIDS on PGT-mediated transport of PGF2{alpha} in ovine endometrial cells might be mediated through one or more of the following mechanisms: 1) direct binding of DIDS with positively charged amino acid residues of PGT such as R561 and K614 in 11th and 12th transmembrane domains, respectively; 2) DIDS-induced multiple cell signaling cross talk may regulate phosphorylation/dephosphorylation, trafficking, surface expression, internalization, and conformation of PGT protein in a short time (5–40 min) to interfere with binding and transport; 3) DIDS may directly phosphorylate/dephosphorylate specific serine, threonine, and tyrosine residues in either intracellular loops, extracellular loops, or transmembrane domain of PGT protein to facilitate preferential efflux and influx; 4) DIDS may activate specific transcriptional factors and cis acting elements, and regulate PGT gene transcription long term (~24 h); and/or 5) DIDS may exert mechanisms explained previously for OATs. Further studies are required to unravel mechanisms for regulation of PGT expression and PGT-mediated influx and efflux of PGs in endometria.

We have reported that DIDS prevents PGT-mediated PGE2 transport in ovine endometrial epithelial cells in vitro (55). PGT siRNA not only inhibited PGF2{alpha} transport but also PGE2 (Banu, S. K., and J. A. Arosh, unpublished data). Expression of PGT mRNA and protein were lower in ovine endometria on d 14–16 of pregnancy compared with the estrous cycle (55). The present study focused on PGT-mediated transport of PGF2{alpha} from the endometria in association with luteolysis in ewes. Ongoing experiments will determine PGT-mediated competitive transport of PGF2{alpha} and PGE2 and associated cell signaling pathways in ovine endometrial epithelial cells in vitro, and the role of PGT in transport of PGF2{alpha} and PGE2 at the time of recognition of pregnancy in ewes in vivo.

In conclusion, PGT mRNA and proteins expressed in ovine endometrial epithelia in a spatial and temporal pattern influence the estrous cycle, particularly the release of luteolytic PGF2{alpha}. Inhibition of PGT prevents oxytocin-induced pulsatile release of PGF2{alpha}. The ERK1/2 and JNK/SAPK cell signaling pathways appear to be most important in regulation of PGT-mediated release of PGF2{alpha} by ovine endometrial epithelial cells.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online September 27, 2007

Abbreviations: DC-FBS, Dextran charcoal-treated fetal bovine serum; DIDS, 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonate; EGFR, epidermal growth factor receptor; JNK, Jun-amino-terminal kinase; Km, maximum affinity; MANOVA, multivariate ANOVA; OAT, organic anion transporter; PG, prostaglandin; PGF2{alpha}, prostaglandin F2{alpha}; PGFM, 13, 14-dihydro-15-keto prostaglandin F2{alpha}; PGT, prostaglandin transporter; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; SAPK, stress-activated protein kinase; siRNA, small interfering RNA.

Received August 6, 2007.

Accepted for publication September 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S E Ulbrich, K Schulke, A E Groebner, H D Reichenbach, C Angioni, G Geisslinger, and H H D Meyer
Quantitative characterization of prostaglandins in the uterus of early pregnant cattle
Reproduction, August 1, 2009; 138(2): 371 - 382.
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