Intrauterine Inhibition of Prostaglandin Transporter Protein Blocks ...

BIOLOGY OF REPRODUCTION (2013) 89(2):27, 1–9 Published online before print 12 June 2013. DOI 10.1095/biolreprod.112.106427

Intrauterine Inhibition of Prostaglandin Transporter Protein Blocks Release of Luteolytic PGF2alpha Pulses Without Suppressing Endometrial Expression of Estradiol or Oxytocin Receptor in Ruminants1 JeHoon Lee,3 John A. McCracken,4 Sakhila K. Banu,3 and Joe A. Arosh2,3 3Reproductive

Endocrinology and Cell Signaling Laboratory, Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 4Department of Animal Science, University of Connecticut, Storrs, Connecticut

In ruminants, prostaglandin F2 alpha (PGF2alpha) is synthesized and released in a pulsatile pattern from the endometria luminal epithelial (LE) cells during the process of luteolysis. Prostaglandin transporter (PGT) is a 12-transmembrane solute carrier organic anion transporter protein that facilitates transport of PGF2alpha. The present study determined the effects of inhibition of PGT protein on pulsatile release of luteolytic PGF2alpha and the underlined cell-signaling mechanisms. The results indicate that intrauterine inhibition of the PGT protein inhibits the pulsatile release of PGF2alpha from the endometrium and maintains a functional corpus luteum. Surprisingly, inhibition of PGT-mediated luteolytic pulses is not associated with spatial regulation of estrogen and oxytocin receptors in the LE of the endometrium and is also not accompanied by decreased biosynthesis of PGF2alpha or increased catabolism of PGF2alpha by the endometrium. Importantly, PGT inhibitor increases expression of pERK1/2 proteins in the LE of the endometrium. Knock down of ERK1/2 genes in LE cells reverses the inhibitory effects of PGT inhibitor on release of PGF2alpha. In conclusion, intrauterine inhibition of PGT inhibits the pulsatile release of PGF2alpha from the endometrium without modulating spatial expressions of estrogen and oxytocin receptor proteins and metabolism of PGF2alpha at the time of luteolysis. Activation of ERK1/2 pathways and interactions between ERK1/2 and PGT protein appear to be important cell-signaling mechanisms that control PGT-mediated efflux transport function. PGT emerges as an important final component in the luteolytic machinery that controls the release of luteolytic pulses of PGF2alpha from the endometrium in sheep. endometrium, luteolysis, PGF2a, prostaglandin transporter, ruminants

INTRODUCTION In ruminants, prostaglandin F2a (PGF2a) is the luteolytic hormone [1]. During the process of luteolysis, PGF2a is 1Supported by Agriculture and Food Research Initiative Competitive Grant no. 2008-35203-19101 from the USDA National Institute of Food and Agriculture to J.A.A. and in part by USDA grant no. 2004-352014176 to J.A.M. 2Correspondence: Joe A. Arosh, Department of Veterinary Integrative Biosciences, College of Veterinary Medicine & Biomedical Sciences, Mail Stop: TAMU 4458, Texas A&M University, College Station, Texas 77843. E-mail: [email protected]

Received: 21 November 2012. First decision: 19 December 2012. Accepted: 15 May 2013. Ó 2013 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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synthesized and released from the endometrial luminal epithelial (LE) cells and superficial glandular epithelial cells (sGLE) in a pulsatile pattern that causes luteolysis. In sheep, continuous exposure of endometrium to progesterone (P4) for 8–10 days down-regulates expression of nuclear P4 receptor (PGR) in LE and sGLE cells between Days 11 and 13, thereby allowing a rapid increase in expression of nuclear estrogen receptor a (ESR-1) after Day 13, followed by an increase in expression of membrane oxytocin receptor (OXTR) after Day 14 of the estrous cycle [1, 2]. Pulsatile releases of oxytocin from the posterior pituitary and corpus luteum (CL) after Days 13–14 of the estrous cycle acts via oxytocin receptor to induce release of luteolytic pulses of PGF2a from LE between Days 14 and 16 of the estrous cycle [1]. Approximately five pulses of endometrial PGF2a secreted over a period of 48 h are required for complete CL regression in sheep [3]. Endometrial PGF2a is transported into each adjacent uterine vein, which joins the adjacent ovarian vein to form the uteroovarian vein. Luteolytic PGF2a pulses are transported from the uteroovarian vein into the ovarian artery locally through a unique vascular structure called the uteroovarian plexus (UOP) [4]. Arachidonic acid (AA) is the primary precursor for the synthesis of prostaglandins (PGs). Cytosolic phospholipase A2 liberates AA from phospholipids. Cyclooxygenases COX-1 and COX-2 convert AA into PGH2 [5]. PGH2 is then converted by specific enzymes to selective PGs, including PGF2a, PGE2, PGD2, PGI2, and TXA2 [6–10]. Prostaglandin F synthases PGFS-AKR1B1 (earlier known as AKR1B5) and PGFSAKR1C3 convert PGH2 into PGF2a. PGF2a exerts its biological effects via the seven-transmembrane G-protein coupled receptor FP [6]. Activation of FP in turn activates PKC and Ca2 þ cell-signaling pathways [6]. Catabolism of PGs is controlled initially by prostaglandin 15-dehydrogenase (PGDH), which catabolizes PGF2a into its inactive metabolite, 13,14-dihydro-15-keto PGF2a (PGFM) [11]. The transport of PGs through plasma membranes is poorly understood. Proposed mechanisms include simple diffusion, passive transport, active transport, countercurrent transport, and carrier-mediated transport. PGs predominantly exist as charged anions and diffuse poorly through plasma membranes in spite of their amphiphylic nature [7–9]. Although PGs cross cell membranes by simple diffusion, the estimated diffusion rate is probably too low to support a biological function [7–9]. Prostaglandin transporter (PGT) is a member of the 12transmembrane solute carrier organic anion transporter (OATP) 2A1 (SLCO2A1) [8, 9] that mediates both efflux and influx of PGF2a [8–10]. PGT recognizes PG substrates as anions. PGT transports PGF2a, PGE2, PGD2, and TxA2 in a competitive manner with different affinities for each of the PGs [8, 9]. The results from cysteine-scanning mutagenesis experiments of PGT indicate that substrate binding by PGT is within its

ABSTRACT

LEE ET AL.

transmembrane domains [8, 9]. 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 1position of carboxylate PG anions appears to be an important mechanism involved in PGT-mediated transport [8, 9, 11, 12]. In addition, PGT-mediated transport varies with the rate of cellular glycolysis as well as variations in intracellular and extracellular concentrations of PGs and cell membrane potential [8, 9]. The PGT inhibitor DIDS (4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonate), also known as anion transport inhibitor [8, 9] or PGT inhibitor [13], inhibits PGT-mediated transport of PGs [8, 10, 14]. We have cloned bovine and ovine PGTs and characterized their functional aspects. Our results demonstrate that PGT protein is abundantly expressed in the endometrium and UOP at the time of luteolysis in sheep and cows [10, 14]. We found that 1) the PGT inhibitor DIDS inhibited PGT-mediated transport of PGF2a from endometrial cells in vitro in a dosedependent manner [10, 11, 15]; 2) intrauterine infusion of DIDS (100 mg/uterus/12 h) from Days 11 to 15 of the estrous cycle maintained a functional CL and its P4 secretion and extended the interestrus interval for up to 35 days in sheep; 3) an acute intrauterine infusion of DIDS (100 mg/uterus) at 0000 and 0200 h on Day 15 of the estrous cycle in sheep inhibited an oxytocin-induced ectopic luteolytic PGF2a pulse, as measured by PGFM in the jugular venous plasma; and 4) a continuous infusion of DIDS (1.0 mg/0.1 ml/min) for 4 h into a uteroovarian vein inhibited the transport of [3H]-PGF2a as well as an oxytocin-induced ectopic luteolytic PGF2a pulse from the uterine vein to the ovarian artery through UOP on Day 15 of the estrous cycle in sheep. Based on our previous findings, we hypothesize that inhibition of PGT protein function in the endometrium inhibits pulsatile release of luteolytic PGF2a and thus maintains a functional CL and its secretion of P4 in sheep. Our objectives were to 1) determine whether pharmacological inhibition of the PGT protein blocks pulsatile release of PGF2a from the endometrium; 2) evaluate whether inhibition of PGT-mediated luteolytic pulses is associated with regulation of ESR-1 or OXTR in the endometrial LE; 3) identify whether inhibition of PGT-mediated luteolytic pulses is associated with decreased biosynthesis of PGF2a and/or increased catabolism of PGF2a in the endometrium; and 4) investigate the cell-signaling mechanisms that regulate PGT function in endometrial LE cells.

Animals All the 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 were housed in Texas A&M University Physiology Field Laboratory/Research Barn, Department of Animal Science. Suffolk crossbred ewes that had exhibited at least two estrous cycles of normal duration (17–18 days) were used in this study. Estrus (Day 0) was detected using vasectomized rams. On Day 6 of the estrous cycle, a catheter was inserted into the lumen of each uterine horn. On Day 11 of the estrous cycle, ewes were assigned randomly to the following treatments groups. Ewes in the control group (n ¼ 5) received intrauterine infusions of 0.1 M potassium bicarbonate (2 ml/uterus/12 h) as vehicle from Days 11 to 15 at 12-h intervals (0700 and 1900 h). Ewes in the treatment group (n ¼ 5) received 10 mg/uterus/12 h intrauterine infusions of DIDS, a PGT inhibitor, in 2 ml of vehicle from Days 11 to 15 at 12-h intervals (0700 and 1900 h). Jugular venous blood samples were collected daily from Days 11 to 16 and assayed for P4. Blood samples were collected hourly from 0700 h on Day 15 to 0700 h on Day 16 and assayed for PGFM. On Day 16, the experimental ewes were ovariohysterectomized, and endometria and corpora lutea were harvested.

Protein Extraction and Western Blot Analysis

Immunohistochemistry Tissue sections were fixed in 4% Tris-buffered paraformaldehyde saline (pH 7.4) and processed using standard procedures. Paraffin sections (5 lm) were cut, and immunohistochemistry was performed 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. The tissue sections were blocked in 10% goat serum for 1 h at room temperature and incubated overnight at 48C with primary antibodies for ESR1 (1:200), OXTR (1:50), ERK1/2 (1:100), PGT (1:250) and COX-2 (1:250), and PGDH (1:500) at the concentrations recommended by the manufacturers. Then, tissue sections were further incubated with the secondary antibody (biotinylated IgG) for 45 min at room temperature. For the negative control, serum or IgG from respective species with reference to the primary antibody at the respective dilution was used. Between each step, the tissue sections were washed in PBS. Digital images were captured using a Zeiss Axioplan 2 Research Microscope (Carl Zeiss, Thornwood, NY) with an AxioCam HR digital color camera. The intensity of staining for each protein was quantified using ImagePro Plus 6.3 image processing and analysis software according to the manufacturer’s instructions (Media Cybernetics, Inc., Bethesda, MD). The detailed methods for quantification are given in the instruction guide. In brief, six images of endometrial LE, at 3400 magnification, were captured randomly without hot-spot bias in each tissue section per animal. Using the selection tools in the Image-Pro Plus software, LE was demarcated in the given sections and the integrated optical intensity (IOD) of immunostaining was quantified under the RGB mode. Numerical data were expressed as least square mean 6 SEM. This technique is more quantitative than conventional blind scoring systems, and the validity of the quantification was reported previously by our group [4, 14, 17, 18].

MATERIALS AND METHODS 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 and PhosStop (Roche Applied Biosciences, Indianapolis, IN); DIDS and anti-human mouse b-actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO); antihuman rabbit PGT,COX-2, PGDH polyclonal antibody, and PGFM ELISA kits (Cayman Chemical, Ann Arbor, MI); ESR-1 (Santa Cruz Biotechnology, Santa Cruz, CA); OXTR (LifeSpan BioSciences Inc., Seattle, WA) ERK1/2 (Cell Signaling Technology, Danvers, MA); goat anti-rabbit or anti-mouse immunoglobulin G (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); P4 radioimmunoassay (RIA) kits (Diagnostic Systems Laboratories, Webster, TX); and ERK1/2 siRNA (small interfering RNA), siGLO RISC-free siRNA, and Dharmafect-1 (Dharmacon Inc., Lafayette, CO). The other chemicals used were molecular biological grade from Fisher (Pittsburgh, PA) or Sigma-Aldrich.

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Total protein was isolated from the endometrial tissue as previously described [14, 16]. Briefly, tissues were homogenized in TED buffer (50 mM Tris, pH 8.0, 10 mM ethylenediaminetetraacetic acid [EDTA], and 1 mM diethyldithiocarbamic acid [DE-DTC]) containing 0.1% Tween-20 and centrifuged at 30 000 3 g for 1 h at 48C. The homogenized tissue pellets (which consisted of membranes, nuclei, and mitochondria) were sonicated in TED sonication buffer, which consisted of 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 100 lM DE-DTC, 1% Tween-20, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail tablets: complete EDTA-free (1 tablet/50 ml) and PhosStop (1 tablet/10 ml). Sonication was performed using a Microson ultrasonic cell disruptor (Microsonix, Inc., Farmingdale, NY). Sonicated tissues were centrifuged at 15 000 3 g for 15min at 48C, and the supernatants (total proteins) were stored at 808C until analyzed. Protein concentration was determined using the Bradford method and a Bio-Rad protein assay kit. Protein samples (75 lg) were resolved using 10% SDSPAGE. Anti-human polyclonal PGT (1:500) or b-actin (1:5000) was used as the primary antibody. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (1:10 000). Chemiluminescent substrate was applied according to the manufacturer’s instructions (Pierce Biotechnology). The blots were exposed to Blue x-ray film, and densitometry of autoradiograms was performed using an Alpha Imager (Alpha Innotech Corp., San Leandro, CA).

PGF2ALPHA TRANSPORT IN OVINE ENDOMETRIUM


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ELISA Concentrations of PGF2a and the stable metabolite of PGF2a, PGFM, were measured in plasma using a commercially available enzyme immunoassay kits (Cayman Chemical) according to the manufacturer’s instructions. The intraassay coefficient of variation (CV) was determined at multiple time points on the standard curve as described by the manufacturer in each assay and compared between assays. The sensitivity, or minimal detection limit, of the PGF2a assay was 3.9 pg/ml, and the intra- and interassay CVs were 9.4% and 12.5%, respectively [14]. The sensitivity, or minimal detection limit, of the PGFM assay was 23 pg/ml, and intra- and interassay CVs were 6.8 % and 8.8 %, respectively. The mean concentration of PGFM refers to the average value of all the blood samples taken in 24h. The basal concentration of PGFM refers to the average value of blood samples taken in 24 h by eliminating the three peak values of each pulse.

Progesterone Assay

FIG. 2. Intrauterine infusion of DIDS decreased PGFM pulse amplitude and mean and basal concentrations of PGFM on Days 15–16 of the estrous cycle from the ovine endometrium. Ewes received intrauterine infusions of vehicle (CONT; n ¼ 5) or DIDS (n ¼ 5) from Days 11 to 15 at 12-h intervals. Jugular venous blood samples were collected at 1-h intervals from 0700 h on Day 15 to 0700 h on Day 16, and concentrations of plasma PGFM were determined using ELISA. The mean concentration of PGFM refers to the average value of all the blood samples taken in 24 h. The basal concentration of PGFM refers to the average value of blood samples taken in 24 h by eliminating the three peak values of each pulse. Data are expressed in mean 6 SEM (*P , 0.05). More details are given in Materials and Methods.

ERK1/2 Gene Knockdown The ERK1/2 gene was knocked down to confirm its role in PGT-mediated release of PGF2a in LE cells. The endometrial LE cells were cultured in antibiotic-free Dulbecco modified Eagle medium/F12 (DMEM/F12) with 10% dextran/charcoal-treated fetal bovine serum (DC-FBS) in 24-well tissue culture plates. At 70%–80% confluency, cells were used for the knockdown experiments. ERK1/2 gene was silenced using SMARTpool siRNA duplex delivered by DharmaFect-1 per the manufacturer’s instructions. As internal controls, siGLO RISC (RNA-inducing silencing complex)-free siRNA or mock siRNA were used. According to the manufacturer’s instructions, SMARTpool siRNA consisted of at least four individual siRNA duplexes targeted against a specific gene and designed using a bioinformatics technology known as SMARTselection. This resulted in the generation of siRNAs more than 97% of the time, and the targeted message level was reduced by more than 70% within 24 h after transfection. We preferred SMARTpool siRNA in order to use more than one siRNA duplex from different regions of the gene of interest to avoid nonspecific indirect effects of single siRNA duplex to a single region of the target gene as we described before [17]. Briefly, siRNA duplexes (100 nM/ well) and DharmaFect-1 (1 ll/well) were diluted in 50 ll antibiotic and serumfree DMEM/F12 medium separately and incubated for 5 min at room temperature. Afterwards, ERK1/2 siRNA and DharmaFect-1 were mixed (total volume of 100 ll) and incubated at room temperature for 20 min. Then 100 ll siRNA:DharmaFect-1 complex were added to each well in a total volume of 400 ll/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 72-h posttransfection, PGF2a release/ efflux experiments were performed using [3H]-PGF2a as described below. Fluorescence-labeled siGLO RISC-free siRNA was transfected separately, and the transfection efficiency was estimated using a fluorescence microscope. A transfection efficiency of greater than 80% was considered ideal for these experiments. Knockdown of ERK1/2 or EGR-1 resulted in 80% decrease at protein level after 96 h based on Western blot analysis as we described before.

well plates, and the ERK1/2 gene was knocked down as described above. After 72-h posttransfection, the cells were incubated with [3H]-PGF2a (1.0 nM, which is well below the Michaelis-Menten kinetics constant [Km]) for 10 min as described before. Then, the medium was replaced; this point was considered as zero, and the cells were then harvested at 0, 5, 10, 15, 20, and 40 min. At each point, the cells were washed with ice-cold Hank balanced salt solution, harvested using trypsin-EDTA, and [3H]-PGF2a uptake was determined using a beta scintillation counter (Beckman Coulter Inc., Fullerton, CA). Efflux rates were calculated in percentage (%) as the amount of [3H]-PGF2a effluxed from the cells. The radioactivity remaining in the cells at time zero was considered as 100%. Data were expressed as the mean 6 SEM of three experiments.

Statistical Analyses Statistical analyses were performed using general linear models of Statistical Analysis System (SAS Institute Inc., Cary, NC). The effects of treatment (control vs. DIDS), time, and treatment 3 time interactions on PGF2a transport was analyzed by repeated measures using multivariate ANOVA. Comparison of means was tested by Wilks lambda or orthogonal contrast tests. The effects of DIDS on basal and mean concentration of PGF, on the mean concentration of P4, and mean weight of corpus luteum on Day 16 were analyzed by one-way ANOVA. Numerical data are expressed as mean 6 SEM. Simple linear correlation was used to determine association between P4 and weight of CL. Statistical significance was considered with P , 0.05. The statistical model accounted for sources of variation including treatments, replicates, and ewes as appropriate.

Efflux/Release Experiments PGF2a efflux experiments in LE cells were performed as described previously [10, 14] with little modifications. The LE cells were cultured in 24-

3 FIG. 1. Intrauterine infusion of DIDS inhibited pulsatile release of PGF2a from the ovine endometrium on Days 15–16 of the estrous cycle. A–D) Control group. E–F) DIDS-treated group. Ewes received intrauterine infusions of vehicle (n ¼ 5) or DIDS (n ¼ 5) from Days 11 to 15 at 12-h intervals. Jugular venous blood samples were collected at 1-h intervals from 0700 h on Day 15 to 0700 h on Day 16 and assayed for plasma PGFM using ELISA. Data are expressed for four individual animals and compared between the groups (*P , 0.05). More details are given in Materials and Methods.

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Concentrations of P4 in plasma were determined using the DSL-3900 ACTIVE Progesterone Coated-Tube RIA Kit according to the manufacturer’s instructions (Diagnostic Systems Laboratories). The RIA used rabbit anti-P4 immunoglobulin-coated tubes and iodinated P4. The primary antiserum crossreacts 6.0%, 2.5%, 1.2%, 0.8%, 0.48%, and 0.1% with 5a-pregnane-3,20dione, 11-deoxycorticosterone, 17a-hydroxyprogesterone, 5b-pregnane-3,20dione, 11-deoxycortisol, and 20b-dihydroprogesterone, respectively. The P4 standard curve (0–10.57 ng/ml) was provided in the assay kit. The sensitivity, or minimum detection limit, of this assay is ;0.12 ng/ml, and the intraassay CV was 8.8%.


FIG. 3. Intrauterine infusion of DIDS maintained concentrations of progesterone (P4) (A) and luteal (CL) weight (B) on Day 16 of the estrous cycle in sheep. Ewes received intrauterine infusions of vehicle (CONT; n ¼ 5) or DIDS (n ¼ 5) from Days 11 to 15 at 12-h intervals. Jugular venous blood samples were collected on Days 11–16 of the estrous cycle for P4 assay, and corpus lutea were collected on Day 16 at necropsy. Data are expressed in mean 6 SEM (*P , 0.05). More details are given in Materials and Methods.


RESULTS Inhibition of PGT Protein Inhibited the Pulsatile Release of PGF2a from the Endometrium and Maintained Functional CL Experimental sheep were treated with intrauterine infusions of either vehicle or the PGT inhibitor DIDS from Days 11 to 15 of the estrous cycle. Beginning of Day 15, jugular blood samples were collected at 1-h interval for 24 h, and PGFM was measured in the plasma. In control sheep, three PGFM pulses were detected at ;8-h intervals between 0700 h on Day 15 and 0700 h on Day 16 (Fig. 1). The mean amplitude of the three pulses was ;1000–1400 pg/ml. However, such PGFM pulses were absent in DIDS-treated sheep. In control sheep, the mean concentration of PGFM was ;750–800 pg/ml while the basal concentration of PGFM was ;200–250 pg/ml. By contrast in DIDS-treated sheep, the mean concentration of PGFM decreased (P , 0.05) to ;210–250 pg/ml, and the basal concentration of PGFM decreased (P , 0.05) to 180–200 pg/ ml (Fig. 2). In control sheep, the concentration of P4 in the jugular venous plasma was 4.5 ng/ml on Day 11 and decreased to 1.3 ng/ml on Day 16 of the estrous cycle. The mean luteal weight of ;200–250 mg indicated that luteal regression had begun. In DIDS-treated sheep, the concentration of P4 in the jugular venous plasma was maintained (P , 0.05) at ;4.4 ng/ ml between Days 11 and 16 of the estrous cycle (Fig. 3). It was clearly apparent, based on the mean luteal weight ;600–750 mg (P , 0.05), that the CL had not entered into the regression process (Fig. 3). DIDS Inhibited Function but Not Expression of PGT Protein in the Ovine Endometrium We determined whether PGT inhibitor DIDS regulates expression of PGT protein in the endometrium. The Western blot results showed that DIDS did not decrease the expression of PGT protein in the endometrium on Day 16 of the estrous cycle (Fig. 4). In addition, immunohistochemistry results indicated that DIDS did not decrease the spatial expression of PGT protein in LE and sGLE cells on Day 16 of the estrous cycle (Fig. 5, A and B). These results together indicate that DIDS inhibits PGT-mediated release of PGF2a pulses from the endometrium by inhibiting PGT function.

FIG. 4. Intrauterine infusion of DIDS did not decrease expression of prostaglandin transporter (PGT) protein on Day 16 of the estrous cycle in sheep. A) Western blot analysis of PGT. b-Actin protein was measured as an internal control. B) Densitometry was performed using Alpha Imager. Ewes received intrauterine infusions of vehicle (CONT; n ¼ 5) or DIDS (n ¼ 5) from Days 11 to 15 at 12-h intervals. Data are expressed in mean 6 SEM. Densitometry was performed using Alpha Imager. More details are given in Materials and Methods.

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Downloaded from www.biolreprod.org. FIG. 5. Intrauterine infusion of DIDS increased expression of pERK1/2 in LE of the endometrium on Days 16 of the estrous cycle in sheep. Ewes received intrauterine infusions of vehicle (CONT; n ¼ 5) or DIDS (n ¼ 5) from Days 11 to 15 at 12-h intervals. Immunohistochemical localization of proteins was performed using Vectastain Elite ABC kit as described in Materials and Methods. A–B) Estrogen receptor alpha (ESR-1). C–D) Oxytocin receptor (OXTR). E– F) Cyclooxygenase 2 (COX-2). G–H) Prostaglandin transporter (PGT). I–J) Prostaglandin dehydrogenase (PGDH). K–L) Phosphorylated extracellular signal regulated kinases 1/2 (pERK1/2). M–N) Control IgG. GL, glandular epithelium; IOD, integrative optical density; LE, luminal epithelium; STR, stroma. Original magnification 3400. O) Relative quantification of pERK1/2 expression in LE using Image-Pro Plus. P) Western blot analysis of pERK1/2 in total endometrial cell lysates. Data are expressed in mean 6 SEM (*P , 0.05).

Inhibition of PGT-Mediated Luteolytic Pulses of PGF2a Was Not Associated with a Change in the Expression of ESR-1 or OXTR in the Endometrium

Inhibition of PGT-Mediated Luteolytic Pulses of PGF2a Was Not Associated with Changes in PGF2a Biosynthesis or Catabolism in the Endometrium

As we detailed, spatial and temporal expression of ESR-1 and OXTR in LE are required for synthesis of PGF2a by the endometrium. Our results (Fig. 5, C–F) indicated that ESR-1 and OXTR proteins were abundantly expressed in LE of the endometrium on Day 16 of the estrous cycle and DIDS did not suppress their expression. These results indicate that DIDS inhibits PGT-mediated release of PGF2a pulses from the LE of the endometrium through novel a mechanism that is not controlled by spatial regulation of ESR-1 and OXTR proteins.

We investigated whether inhibition of PGT-mediated PGF2a pulses was associated with decreased PGF2a biosynthesis and/ or increased catabolism of PGF2a to PGFM by the endometrium. We measured PGF2a and PGFM in the uterine flushing on Day 16 in control and DIDS-treated sheep. The results indicated that DIDS did not decrease intrauterine concentration of PGF2a or increase the intrauterine concentration of PGFM compared to controls (Fig. 6). Moreover, COX-2 and PGDH proteins were abundantly expressed in LE of the endometrium in control and DIDS-treated groups (Fig. 5, G–J). These results 6

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FIG. 6. Intrauterine infusion of DIDS did not affect intrauterine concentrations of PGF2a (A) and PGFM (B) on Day 16 of the estrous cycle in sheep. Ewes received intrauterine infusions of vehicle (CONT; n ¼ 5) or DIDS (n ¼ 5) from Days 11 to 15 at 12-h intervals. Uterine flushings were collected on Day 16 of the estrous cycle, and PGF2a and PGFM concentrations were determined using ELISA. Data are expressed in mean 6 SEM. More details are given in Materials and Methods.

in total protein lysate (composed of epithelium and stroma) did not differ between control and DIDS groups. These results suggest that DIDS either increases expression of pERK1/2 protein in luminal epithelium or shifts cell-specific expression of pERK1/2 protein from luminal epithelium to stratum compactum of the stroma. In addition, DIDS did not affect the total expression of pERK1/2 protein in endometrial tissues, as shown by Western blot analysis. We then used oLE cells as a model system to knock down the ERK1/2 gene using siRNA and confirmed the role for ERK1/2 pathways in PGT-mediated release of PGF2a. The ERK1/2 siRNA approach resulted in the knockdown of total ERK1/2 protein up to 75%–80% at 72-h posttransfection (Fig 7A). The results (Fig. 7B) indicated that loss of function of ERK1/2 reversed (P , 0.05) the inhibitory effects of DIDS on PGT-mediated release of PGF2a by up to 80% in the oLE cells. Collectively, these results indicate that DIDS inhibits PGTmediated release of PGF2a through ERK1/2-signaling pathways in the ovine endometrium.

Role for ERK Pathways in PGT-Mediated Pulsatile Release of PGF2a in the Ovine Endometrium We reported a role for ERK1/2 pathways in PGT-mediated release of PGF2a in ovine luminal endometrial epithelial (oLE) cells in vitro [14, 17]. Therefore, we determined the effects of DIDS on the expression of pERK1/2 in the endometrium in vivo. The immunohistochemistry results indicated that pERK1/2 protein is highly expressed in stratum compactum of stroma compared to LE. DIDS increased (P , 0.05) or shifted expression of pERK1/2 protein in the LE cells compared to control (Fig. 5, K, L, and O). The Western blot results (Fig. 5P) results indicated that expression of pERK1/2

FIG. 7. Effects of ERK1/2 siRNA on the inhibitory effects of DIDS on PGT-mediated release/efflux of PGF2a in LE cells. A) Effects of siRNA on expression of total ERK1/2 protein at 72-h posttransfection. B) PGF2a efflux experiments were performed as described in the Materials and Methods. aCONT versus DIDS (P , 0.05). bDIDS versus DIDS þERK1/2-siRNA (P , 0.05). All the numerical values are expressed in mean 6 SEM of three (n ¼ 3) independent experiments.

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indicate that DIDS does not affect the expression of COX-2 and PGDH proteins in LE of the endometrium and the absence of PGF2a pulses during DIDS treatment is not due to either a decrease in the biosynthesis or an increase in catabolism of PGF2a by the endometrium.

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DISCUSSION It is well accepted that the pulsatile release of endometrial PGF2a induces luteolysis in ruminants [1]. A minimum of five pulses of PGF2a given over 48 h are necessary to consistently cause luteolysis in sheep [3]. We have found that PGT protein is abundantly expressed in the endometrium at the time of luteolysis and that inhibition of PGT by DIDS from Days 11 to 16 of the estrous cycle extends interestrous intervals up to 35 days by maintaining a functional CL in sheep [14]. The present study investigated the underlying physiological and molecular control of PGT function at the time of luteolysis. The results indicate that intrauterine inhibition of the PGT protein by DIDS between Days 11 and 15 of the estrous cycle completely inhibits the pulsatile release but not the basal release of PGF2a of from the endometrium on Day 16 of the estrous cycle. These findings are in agreement with our previous report that 80% of PGF2a is transported through PGT-dependent mechanisms and the other 20% of PGF2a is mediated through PGT-independent mechanism in the ovine endometrium [4, 14, 17]. As detailed, increased expression of ESR-1 and OXTR in LE is one of the prerequisite for secretion of luteolytic PGF2a pulses from the endometrium [1]. Inhibition of expression of ESR-1 and OXTR proteins in the LE of the endometrium is required to prevent luteolytic PGF2a pulses during establishment of pregnancy [2, 19]. Our results indicated that DIDS did not decrease expression of ESR-1 and OXTR proteins in LE compared to control. This indicates that DIDS inhibits PGTmediated transport of PGF2a pulses without suppressing ESR-1 and OXTR in the LE. Although ESR-1 and OXTR are directly associated with the synthesis and secretion of PGF2a from LE [1], PGT protein is required to transport PGF2a pulses from the endometrium into the uterine venous system. These new findings suggest that PGT protein is a final component of the luteolytic machinery that controls the release of luteolytic PGF2a pulses in the ovine endometrium. Endometrial biosynthesis and catabolism of PGF2a control the net intrauterine production of PGF2a and eventually its pulsatile secretion [1]. In addition, recent studies have reported that PGT most likely functions as an influx transporter and facilitates catabolism of PGs in kidney and endothelial cells [20–22]. Therefore, we examined whether inhibition of PGT by DIDS decreased intrauterine biosynthesis or increased intrauterine catabolism of PGF2a. Our results indicated that inhibition of PGT by DIDS did not either decrease the biosynthesis or increase the catabolism of PGF2a by the endometrium. These results indicate that absence of PGTmediated PGF2a pulses in DIDS-treated sheep is not due to decreased intrauterine biosynthesis of PGF2a or increased intrauterine catabolism of PGF2a. These results further confirm that transport of PGF2a is an important mechanism that regulates the pulsatile release of PGF2a from the ovine endometrium. PGT belongs to the OATP family that shares common structural features that include: 12-transmembrane domains, multiple glycosylation sites, multiple phosphorylation sites in the intracellular loops, extracellular loops and transmembrane domains, presence of clusters of cysteine zinc-finger motifs in the extracellular loops, and the presence of predominantly positive charged amino acid residues [23]. Phosphorylated state of several serine, threonine, and tyrosine residues in response to PKC [24, 25], MAPK [26, 27], and AKT [28] regulate phosphorylation, cell surface expression, internalization, stability, maximum affinity (Km) and maximum transport velocity (Vmax) of OATPs [23, 29]. We have reported in our previous study that PKA and PKC pathways are involved

ACKNOWLEDGMENT We gratefully acknowledge Dr. Fuller W. Bazer, Department of Animal Sciences, for providing immortalized ovine endometrial luminal epithelial cells, and Drs. Bazer and Thomas E. Spencer, Department of Animal Sciences (current address: Department of Animal Sciences, Washington State University), for providing technical help in surgery and animal husbandry.

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in PGT-mediated influx/uptake transport, ERK1/2 pathways are involved in PGT-mediated efflux/release transport, and JNK pathways are involved in PGT-mediated efflux and influx in LE cells [14]. These results together suggest that the functional activity (release or uptake) of PGT can be modulated by its interaction with specific intracellular cell-signaling pathways [14, 20]. The results of the present study indicate that PGT and ERK1/2 proteins are colocalized in the LE of the endometrium on Day 16 of the estrous cycle. Intrauterine infusion of DIDS increases phosphorylation of ERK1/2 proteins in the LE of endometrium. In order to confirm role for ERK1/2 pathways in PGT-mediated release of PGF2a, we knocked down ERK1/2 genes using siRNA in oLE cells in vitro and found that loss of function of ERK1/2 reversed the inhibitory effects of DIDS on PGT-mediated release of PGF2a by up to 80%. Taken together, these results indicate that DIDS inhibits PGT-mediated release of PGF2a through ERK1/2-signaling pathways in the ovine endometrium. It appears that the interaction between ERK1/2 and PGT are the important cell-signaling cascades required to inhibit PGT-mediated pulsatile release of PGF2a from the ovine endometrium [14, 17]. At present, the specific molecular mechanisms through which ERK1/2 interact with PGT and control functional activity of PGT are not known. The results of the present study along with our previous reports in sheep [4, 14, 17] confirm that PGT is a functional efflux transporter that facilitates the release of PGF2a pulses from the endometrium at the time of luteolysis in sheep. Ongoing studies are aimed at knocking down the PGT gene in LE of the endometrium in vivo to further define the role of PGT in the release of luteolytic PGF2a pulses from the ovine endometrium. In conclusion, we have found that intrauterine inhibition of PGT inhibits the pulsatile release of PGF2a from the ovine endometrium. Activation of ERK1/2 pathways and interaction between ERK1/2 and PGT appears to be important cellsignaling mechanisms that control PGT-mediated efflux transport. PGT emerges as an important final component that controls the release of endometrial PGF2a pulses at the time of luteolysis in sheep.


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