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Research Article |
Department of Animal Science,3 Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078
Department of Animal Science,4 College of Agriculture and Life Sciences,
and Department of Veterinary Integrative Biosciences,5 College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843
ABSTRACT
Porcine trophoblast attachment to the uterine surface is associated with increased conceptus and endometrial production of prostaglandins. Conceptus secretion of estrogen on Day 12 of gestation is important for establishment of pregnancy; however, early (Days 9 and 10) exposure to exogenous estrogens results in embryonic mortality. Present studies established the temporal and spatial pattern of endometrial PTGS1 (prostaglandin-endoperoxide synthase 1) and PTGS2 expression during the estrous cycle and early pregnancy and determined the effect of early estrogen treatment on endometrial PTGS expression in pregnant gilts. Endometrial PTGS1 mRNA expression increased 2- to 3-fold after Day 10 of the estrous cycle and pregnancy, whereas PTGS2 mRNA expression increased 76-fold between Days 5 and 15 of the estrous cycle and pregnancy. Increased expression of the PTGS2 transcript was detected in the lumenal epithelium after Day 10 in both cyclic and pregnant gilts. There was a 10- and 20-fold increase in endometrial PTGS2 protein expression between Days 5 and 18 of the estrous cycle and pregnancy respectively. Administration of estrogen on Days 9 and 10 of gestation increased endometrial PTGS2 mRNA and protein on Day 10, but decreased PTGS2 mRNA and protein in lumenal epithelium (LE) on Day 12 of gestation compared to vehicle-treated gilts. The present study demonstrates that an increase in uterine epithelial PTGS2 expression occurs after Day 10 of the estrous cycle and early pregnancy in the pig. The conceptus-independent increase in the uterine LE indicates that a novel pathway exists for endometrial induction PTGS2 expression before conceptus elongation and attachment to the uterine surface. Epithelial expression of PTGS2 may serve as one of the signals for placental attachment and embryo survival in the pig. Early administration of estrogen on Days 9 and 10 of pregnancy alters endometrial PTGS2 mRNA and protein expression, which may, at least in part, represent a mechanism by which endocrine disruption of pregnancy causes total embryonic loss during implantation in the pig.
female reproductive tract, implantation, pregnancy, uterus
INTRODUCTION
The noninvasive attachment of the porcine conceptus forms an epitheliochorial type of placentation through its adhesion to the extracellular glycocalyx present on apical microvilli of the uterine lumenal epithelium (LE) [1]. Estrogen coordinates a complex program of events activating implantation within the progesterone-stimulated uterine environment of mice and rats [2, 3]. Developing pig conceptuses undergo rapid trophoblast elongation and secrete estrogen on Days 11 to 12 of pregnancy [4]. Release of estrogen by the elongating conceptuses serves as the signal from the conceptus for maternal recognition of pregnancy in the pig [5, 6]. The transient acute increase in estrogen release during rapid trophoblast elongation closely follows conceptus release of the proinflammatory cytokine, interleukin 1 beta (IL1B), which has been proposed to serve as the initial stimulus for conceptus elongation and attachment to the surface of uterine LE in the pig [7].
Implantation in the estrogen primed uteri of mice and rats requires endometrial prostaglandin (PG) synthesis [3]. The importance of endometrial PTGS (prostaglandin-endoperoxide synthase, also known as prostaglandin G/H synthase and cyclooxygenase) mRNA expression during implantation and decidualization was established using Ptgs2-null mice, which have an altered inflammatory response that results in reproductive defects in ovulation, fertilization, and implantation [8, 9]. Similar reproductive defects are not observed in Ptgs1-deficient mice, which are fertile and exhibit defects only with parturition [10]. During apposition of the mouse blastocyst to the uterine LE on Day 4.5 of pregnancy, ovarian estrogen increases synthesis of PGE 2 at the site of implantation [11], which is essential for implantation to occur. A dramatic increase of endometrial IL1B mRNA expression correlates with the rise in PGE 2, and an increase in endometrial IL1B secretion may stimulate PGE 2 production by elevating PTGS2 expression in the uterine epithelium [12, 13].
The increase in porcine conceptus IL1B expression occurs concomitantly with the morphological transformation of the conceptuses during trophoblast elongation [7]. Pig conceptuses and endometrium actively secrete PGs [14, 15], and conceptus expression of the PTGS2 gene increases immediately following trophoblast elongation and continues during conceptus attachment to the LE [16]. Pharmacological inhibition of PG synthesis does not affect trophoblast elongation in pigs [17]. However, inhibition of PG synthesis during the period of trophoblast attachment (Days 13 to 18) results in embryonic mortality [18]. These studies suggest that the acute release of conceptus IL1B during trophoblast elongation may have an obligatory role of enhancing PTGS2 synthesis for endometrial release of prostaglandins during the establishment of pregnancy in the pig. Although endometrial synthesis of prostaglandins has been well established, the patterns of PTGS1 and PTGS2 expression during the estrous cycle and early pregnancy have not been reported.
Although conceptus secretion of estrogen into the uterine lumen serves as one of the factors involved with establishment of pregnancy in the pig, the uterine endometrium is sensitive to the timing of estrogen exposure. Exposure of pregnant gilts to exogenous estrogens 48 h before the normal secretion by conceptuses on Day 12 results in total embryonic mortality before Day 30 of gestation [19]. Our laboratory previously demonstrated that conceptuses degenerate between Days 15 to 18 of gestation following administration of estrogen on Days 9 and 10 of pregnancy [20, 21]. Although conceptus degeneration is correlated with the spatiotemporal loss of the microvilli glycocalyx on the endometrial LE [22], changes in uterine gene expression that may adversely affect conceptus survival are unknown. We hypothesize that conceptus IL1B secretion stimulates induction of endometrial PTGS2, which may function in attachment of the conceptus to the uterine surface, and that the early administration of estrogen to pregnant gilts disrupts induction of uterine PTGS2 by conceptus IL1B, resulting in defective conceptus development and/or endometrial receptivity for placental attachment to the luminal surface.
Because endometrial PTGS1 and PTGS2 gene and protein expression have not been previously established during the porcine estrous cycle and early pregnancy, our first objective profiles endometrial expression of PTGS1 and PTGS2 during the estrous cycle and early pregnancy. The second objective evaluates endometrial PTGS1 and PTGS2 expression following treatment of gilts with estrogen on Days 9 and 10 of gestation.
MATERIALS AND METHODS
Animals
Research was conducted in accordance with the Guiding Principles for Care and Use of Animals promoted by the Society for the Study of Reproduction and approved by the Oklahoma State Institutional Care and Use Committee. Crossbred cycling gilts of similar age (8–10 mo) and weight (100–130 kg) were checked daily for estrous behavior with intact males. Gilts assigned to be bred were naturally mated with fertile crossbred boars at first detection of estrus, and subsequently at 12 and 24 h after detection of estrus.
Experiment I: Endometrial PTGS1 and PTGS2 Expression in Cyclic and Pregnant Gilts
Gilts were hysterectomized (n = 4 gilts/day) through midventral laparotomy on either Day 0, 5, 10, 12, 15, or 18 of the estrous cycle or Day 10, 12, 15, or 18 of pregnancy as previously described by Gries et al. [21]. Following induction of anesthesia with 1.8 ml i.m. administration of a cocktail consisting of 2.5 ml xylazine (100 mg/ml; Miles Inc.) and 2.5 ml Vetamine (ketamine HCl, 100 mg/ml; Molli Krodt Veterinary) in 500 mg of Telazol (tiletamine HCl and zolazepum HCl; Fort Dodge), anesthesia was maintained with a closed-circuit system of halothane (5%) and oxygen (1.0 L/min). The uterus was exposed via midventral laparotomy and the uterus and ovaries excised. Each uterine horn was injected with 20 ml PBS (pH 7.4) via the isthmus, and flushings were recovered in a petri dish. Conceptuses were removed from flushings of pregnant gilts, snap-frozen in liquid nitrogen, and stored at –80°C. Uterine flushings were cleared of cellular debris by centrifugation (1000 x g, 10 min, 4°C), supernatant collected, and uterine flushings stored at –20°C. Endometrial tissue was removed from the antimesometrial side of the middle portion of each uterine horn, immediately snap-frozen in liquid nitrogen, and stored at –80°C until used for extraction of protein and RNA.
Experiment II: Endometrial PTGS1 and PTGS2 Gene and Protein Expression Following Early Exposure of Pregnant Gilts to Estrogen
Bred gilts were randomly assigned to one of the following treatment groups: 1) vehicle: i.m. injection of corn oil (2.5 ml) on Days 9 and 10 of gestation; or 2) estrogen: 5 mg i.m. injection of estradiol cypionate (A.J. Legere) on Days 9 and 10 of gestation. Gilts were hysterectomized (n = 4 gilts/day) on Day 10, 12, 13, 15, or 17 of gestation as described in Experiment 1. Uterine horns were flushed with 20 ml PBS; conceptuses were removed, flushings were centrifuged to remove cellular debris, and supernatant was stored at –20°C. Endometrial tissue was harvested from the uterine horn and either fixed for in situ hybridization or snap-frozen as previously described in Experiment 1.
Uterine Tissue Fixation Procedures
Endometrial tissue sections (
1.0 cm) were excised from the bottom 40 cm of the uterine horn, which was not flushed with PBS. Tissue sections were placed in freshly prepared 4% paraformaldehyde in PBS (pH 7.2) and gently agitated at room temperature for 24 h. Solution was replaced with 70% EtOH (v/v in H2O) and gently agitated for an additional 24 h. Fixed endometrial tissue was dehydrated in a series of graded ethanol changes followed by xylene, and was then embedded in Paraplast-Plus (Oxford Labware).
RNA Extraction
Total RNA was extracted from endometrial tissue using RNAwiz reagent (Ambion, Inc.). Approximately 0.5 g of endometrial tissue was homogenized in 5.0 ml of RNAwiz using a Virtishear homogenizer (Virtis Company Inc.). Total RNA was resuspended in 500 µl of diethyl pyrocarbonate-treated water and further purified by phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. Samples were treated with DNase I (Invitrogen) according to manufacturer protocol to eliminate possible DNA contamination. RNA was stored at –80°C. Total RNA was quantified with a spectrophotometer at an absorbance of 260 nm, and purity was verified using the 260/280 ratio.
Quantitative One-Step RT-PCR
Quantitative analysis of endometrial PTGS1 and PTGS2 mRNA was performed using quantitative RT-PCR (qRT-PCR) as previously described in our laboratory [23]. The PCR amplification was performed in a reaction volume of 15 µl using an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The transcripts were evaluated using dual labeled probes with 6-Fam (5' reporter dye), and TAMRA (3' quenching dye). Primer and probe sequences for the amplification of PTGS1 and PTGS2 gene expression (Table 1) were generated from porcine sequences available in the NCBI database. Total RNA (100 ng) was assayed in duplicate using thermocycling conditions for one-step cDNA synthesis for 30 min at 50°C and for 15 min at 95°C, followed by 45 repetitive cycles for 15 sec at 95°C and for 1 min at 60°C. To determine possible genomic contamination, samples were assayed in the absence of reverse transcriptase. Samples either did not amplify or the amplification curve was greater than 8 cycles beyond the target amplification for mRNA expression. Ribosomal 18S RNA was assayed in each sample to normalize RNA loading, as previously described by our laboratory [7].
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Template amplification was quantified by determining the threshold cycle (CT) based on the fluorescence detected within the geometric region of the semilog plot. Theoretically, in the geometric region one cycle is equivalent to the doubling of the PCR target template. Sample dilutions were made for each assay to determine the actual PCR efficiency, which was 1.91 and 1.82 for PTGS1 and PTGS2, respectively. Using the comparative CT method [23], relative quantification of mRNA abundance was determined for the endometrial samples. Sample expressions of PTGS1 and PTGS2 mRNA were normalized with ribosomal 18S expression to adjust for sample loading. Relative units of target mRNA expression were calculated by arbitrarily setting the lowest sample measurement (CT) as one relative unit, which was used as the baseline standard to compare mRNA abundance for all other samples. Relative mRNA abundance was calculated using the PCR efficiencies for PTGS1 and PTGS2 in the formula 1.91–CT and 1.82–CT.
Western Blot Analysis of PTGS1 and PTGS2
Cytoplasmic protein was extracted from endometrial tissue using T-PER reagent (Pierce Inc.) to which a protease inhibitor cocktail, HALT (Pierce Inc.), was added. A total of 250 mg of endometrial tissue was homogenized in 5 ml T-PER reagent on ice using a Virtishear homogenizer (Virtis Company Inc.). Following homogenization, samples were centrifuged at 10 000 x g for 5 min, and supernatant was collected and stored at –20°C until it was used for Western blot analysis. Cytoplasmic protein concentration in the supernatant was determined with the Bio-Rad Protein Assay Kit II (Bio-Rad). Samples (30 µg protein) were placed in a 95°C water bath for 90 sec in equal volumes of sample denature buffer (0.125 M Tris-HCl [pH 6.8], 205 mg glycerol, 4% SDS, 10% ß-mercaptoethanol, and 0.0025% bromophenol blue) and protein separated in a 10% SDS polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp.) using a semidry immunoblot apparatus (MilliBlot-SDS System) at 350 V for 3 h. Membranes were washed with Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl) containing 0.05% Tween 20 (TTBS) and then blocked with 5% nonfat dried milk for 1 h. After being washed 3 times with TTBS for 10 min, the membrane was incubated for 2 h with rabbit anti-human PTGS2 polyclonal antibody (1:2000), or sheep anti-human PTGS1 polyclonal antibody (1:2000) (Calbio-Chem Inc.). Following incubation with primary antibody, membranes were washed 3 times with TTBS for 10 min each, and the membrane was incubated with goat anti-rabbit secondary antibody (PTGS2; 1:1000), or mouse anti-sheep secondary antibody (PTGS1; 1:1000) (Bio-Rad) for 1 h. Membranes were washed with TTBS 3 times for 10 min, followed by addition of color development solution (Immuno-Blot kit; Bio-Rad). Densities of the resultant stained bands were measured with a Densitometer 2100 (Bio-Rad).
In Situ Hybridization
Expression of PTGS2 mRNA was localized in paraffin-embedded porcine uterine tissue by in situ hybridization using methods previously described by Johnson et al. [24]. Briefly, deparaffinized, rehydrated, and deproteinated uterine cross sections (5 µm) were hybridized with radiolabeled antisense or sense porcine PTGS2 cRNA probes synthesized by in vitro transcription with [
-35S] uridine 5-triphosphate (PerkinElmer Life Sciences). After hybridization, washes, and RNase A digestion, autoradiography was performed using NTB-2 liquid photographic emulsion (Eastman Kodak). Slides were exposed at 4°C for 6 days, developed in Kodak D-19 developer, counterstained with Harris modified hematoxylin (Fisher Scientific), dehydrated, and protected with coverslips.
Enzyme-Linked Competitive Binding Assays
The IL1B protein content in uterine luminal flushings collected in Experiment 2 was quantified using a commercial ELISA (R&D Systems) as previously described in our laboratory [7]. All samples, standards, and controls were assayed in duplicate. The intra-assay coefficient of variation of the IL1B ELISA was 4.9%. Total contents of PGE 2 and PGF2 in the uterine flushings collected in Experiment 2 were quantified using a commercial ELISA (R&D Systems) validated for uterine flushing in our laboratory in accordance with the manufacturer's specifications. Samples were analyzed in duplicate with a single assay. The intra-assay coefficients of variation for the PGE 2 and PGF2 assays were 5.1% and 4.9%, respectively.
Microscopy and Digital Imaging
Photomicrographs of representative brightfield and darkfield images of in situ hybridization were evaluated with a Zeiss Axioplan2 microscope (Carl Zeiss) interfaced with an Axioplan HR digital camera and Axiovision 3.0 software. Digital images were captured and photographic plates assembled using Adobe Photoshop 6.0 (Adobe Systems Inc.)
Statistical Analysis
Data were analyzed by least-squares ANOVA using the Proc Mixed model of the Statistical Analysis System [25]. The statistical model used to analyze cyclic and pregnant endometrial PTGS1 and PTGS2 gene and protein expression in Experiment 1 included the effects of day, reproductive status (cyclic or pregnant), and day x reproductive status interaction. The statistical model used to analyze endometrial PTGS1 and PTGS2 gene expression and uterine flushing PG content in Experiment 2 included the effects of day, treatment, and day x treatment interaction. Because of unequal variances of PGE 2 and PGF2 content in the uterine flushings, data were transformed using log and square root transformations for the statistical analysis.
RESULTS
Experiment I
Endometrial PTGS1 and PTGS2 mRNA expression. Endometrial PTGS1 mRNA expression (Fig. 1A) was affected by day (P < 0.001), but not pregnancy status. Endometrial expression of PTGS1 increased approximately 3-fold after Day 10 of the estrous cycle and pregnancy. PTGS1 mRNA expression remained elevated to Day 18 of the estrous cycle and pregnancy.
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Endometrial PTGS2 mRNA expression (Fig. 1B) was affected by day (P < 0.0001), but not pregnancy status. Endometrial PTGS2 mRNA expression was lowest on Day 5 of the estrous cycle. Expression of PTGS2 mRNA increased approximately 76-fold on Day 12 of the estrous cycle and pregnancy. Endometrial PTGS2 expression remained elevated on Day 18 in both cyclic and pregnancy gilts.
In situ hybridization analysis. In situ hybridization revealed that uterine LE and glandular epithelium of the endometrium were devoid of PTGS2 mRNA on Days 5 to 10 of the estrous cycle (Fig. 2). However, PTGS2 mRNA expression within LE of the endometrium was abundant by Day 15 of the estrous cycle. In pregnant gilts (Fig. 2), endometrial PTGS2 mRNA cellular localization was localized in the LE beginning on Day 12 of gestation. Expression of PTGS2 mRNA remained abundant in the LE on Day 15 of gestation.
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Western blot analysis. Western blot analysis of the PTGS1 detected a single 72-kDa protein that remained relatively constant through Day 10 of the estrous cycle and pregnancy (Fig. 3A). On Day 12 of the estrous cycle and pregnancy, there was an increase in intensity of the 72-kDa band for PTGS1 that continued through Day 18.
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Western blot analysis of endometrial protein detected a single 72-kDa immunoreactive product to the PTGS2 antiserum across all days of the estrous cycle and early pregnancy (Fig. 3B). Endometrial expression of the 72-kDa product (Fig. 3C) was affected by day (P < 0.04) and status (P < 0.03). PTGS2 expression was apparent on Day 0 but decreased on Day 5 of the estrous cycle, with levels of PTGS2 protein increasing 20-fold from Days 5 to 15 of the estrous cycle (Fig. 3C). PTGS2 protein increased 10-fold between Days 10 and 15 of pregnancy. Overall endometrial content of PTGS2 between Days 10 and 18 was approximately 3-fold greater in cyclic compared to pregnant gilts.
Experiment 2
Conceptus development, viability, and IL1B secretion. Normal spherical conceptuses were recovered from the flushing media of vehicle- and estrogen-treated gilts on Day 10 of pregnancy. On Day 12 of pregnancy, a few pregnancies contained spherical conceptuses, but filamentous conceptuses were recovered in the majority of the gilts. Viable peri-attachment conceptuses were recovered in both vehicle- and estrogen-treated gilts on Day 13 of pregnancy, but conceptuses were fragmented and degenerating in estrogen-treated gilts harvested on Days 15 and 17 of pregnancy. Content of IL1B in uterine flushings was not different (P < 0.46) between vehicle- and estrogen-treated gilts. A day effect (P < 0.01) was detected for the uterine lumenal content of IL1B, which increased with conceptus elongation on Days 12 and 13 of pregnancy (Fig. 4).
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Endometrial PTGS1 and PTGS2 mRNA expression. Endometrial PTGS1 mRNA expression was affected by day (P < 0.01), but not estrogen treatment (P > 0.10). Expression of PTGS1 mRNA increased 2-fold in the endometrium between Days 10 and 15 of gestation (Fig. 5A).
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A day x treatment interaction (P < 0.01) was detected for endometrial PTGS2 mRNA expression (Fig. 5B). In vehicle-treated gilts, mRNA expression increased 5-fold from Day 10 to Day 12 of pregnancy and remained elevated to Day 18. However, estrogen treatment altered the normal expression pattern compared to that seen in vehicle-treated gilts. Endometrial PTGS2 mRNA expression in estrogen-treated gilts was 2.9-fold greater on Day 10 of pregnancy compared to vehicle-treated gilts. However, PTGS2 mRNA expression was 5.1-fold greater in vehicle- compared to estrogen-treated gilts on Day 12 of pregnancy. Endometrial expression of PTGS2 decreased from Day 10 to Day 12 in estrogen-treated gilts. Endometrial PTGS2 mRNA expression was similar between treatment groups on Days 13, 15, and 17 of gestation.
In situ hybridization analysis Endometrial PTGS2 gene expression was localized in the LE on Day 12 of gestation in vehicle-treated gilts (Fig. 6) as previously described in Experiment 1. Expression of PTGS2 remained in the LE from Day 12 to Day 17 of gestation. Treatment of gilts with estrogen on Days 9 and 10 altered the temporal pattern of PTGS2 expression within the endometrium. Expression of PTGS2 in the LE of estrogen-treated gilts was greatly attenuated on Days 12 and 13 of gestation compared to vehicle-treated gilts.
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Western blot analysis: PTGS1 and PTGS2. Western blot analysis did not detect any differences in endometrial PTGS1 protein expression between estrogen-and vehicle-treated gilts (Fig. 7). The expression pattern of endometrial PTGS2 protein was different between vehicle- and estrogen-treated gilts (Fig. 8A). A day x treatment effect (P < 0.001) was detected for endometrial PTGS2 protein expression (Fig. 8B). In vehicle-treated gilts, PTGS2 protein expression was faint on Day 10 and increased 5-fold on Day 12. Day 13 endometrial content of PTGS2 was low, but PTGS2 was abundant on Days 15 and 17 of pregnancy. Estrogen-treated gilts showed a shift in PTGS2 expression: PTGS2 was abundant on Day 10, but this was followed by a dramatic decline of PTGS2 expression on Day 12 (Fig. 8B). Endometrial content of PTGS2 protein increased from Day 13 to Day 17 in the estrogen-treated gilts.
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ELISA assay of prostaglandins in uterine flushings.
Estrogen treatment did not alter the total uterine content of PGE 2 or PGF2 in uterine flushings of pregnant gilts. Total lumenal content of PGF2 increased across the days of pregnancy (P < 0.001). Uterine lumenal content of PGF2 was 4.1, 0.11, 0.69, 8.43 and 9.18 ng on Days 10, 12, 13, 15, and 17 of gestation, respectively. Content of PGF2 in the uterine flushings increased 80-fold from Day 12 to Day 17 of gestation. Content of PGE 2 in the uterine flushings also increased across the days of gestation (P < 0.001). Uterine lumenal content of PGE 2 was 1.0, 52.5, 223.7, 1008.5 and 1348.7 ng on Days 10, 12, 13, 15, and 17 of gestation, respectively. Following conceptus elongation on Day 12, there was a 20-fold increase of PGE 2 in the uterine flushings on Day 15. Uterine content of PGE 2 continued to increase during the period of placental attachment to the uterine surface.
DISCUSSION
Endometrial synthesis of prostaglandins is essential for establishment of pregnancy in species with both invasive [9, 13, 26] and noninvasive forms of implantation [18]. Implantation in the pig occurs through noninvasive attachment between the trophoblast and uterine surface epithelial microvilli [27]. Endometrial expression of PTGS1 increased only slightly during late diestrus and early pregnancy in the pig; however, significant changes in endometrial PTGS2 expression were observed over the same period. Prostaglandins are involved with many changes in tissue vascularity and proinflammatory responses [28]. The large increase in endometrial PTGS2 expression after Day 10 of pregnancy coincides with the time of rapid conceptus development, conceptus secretion of estrogen and IL1B, and trophoblast attachment to the endometrial LE in the pig [4]. A number of growth factors and cytokines stimulate prostaglandin synthesis through induction PTGS2 mRNA expression [28, 29]. IL1B induces PTGS2 expression through activation of the transcription factor nuclear factor kappa B (NF-
B) [30]. Expression of IL1B increases before initiation of blastocyst implantation in the mouse [31–33], and it may be an initiator of conceptus-uterine interaction during pregnancy in women [34]. Expressions of IL1 receptor types I and II (IL1R1 and IL1R2) in the endometrium are both molecular markers for uterine receptivity in mice [35]. During rapid trophoblastic elongation on Day 12 of pregnancy, pig conceptuses secrete large amounts of IL1B, and there is an increase in endometrial IL1R1 mRNA expression [7].
Our working hypothesis was that conceptus expression of IL1B triggers transcription of inflammatory cytokines and factors involved with endometrial receptivity to implantation in the pig. The increase in endometrial PTGS2 mRNA and protein expression on Day 12 of pregnancy correlates with peak conceptus secretion of IL1B during trophoblast elongation [7]. However, this increase is not pregnancy-specific; a similar increase in endometrial PTGS2 mRNA and protein expression also occurs in cyclic gilts. Therefore, the increase in endometrial PTGS2 mRNA expression is not the direct result of conceptus IL1B secretion, but is rather part of a complex and sequential uterine program to accomplish placental attachment in the pig. The significant increase in endometrial PTGS2 mRNA occurs in the endometrial LE from Day 10 to Day 18 of the estrous cycle and early pregnancy, where it is optimally placed to possibly mediate placentation in pigs [18]. Thus, our research has established that PTGS2 increases in the endometrial LE during the period of porcine conceptus attachment to the uterine surface, which is independent of conceptus IL1B secretion. Results indicate a uterine mechanism exists for inducing PTGS2 mRNA expression specifically in endometrial LE during the estrous cycle.
Increased expression of PTGS2 in the uterine LE is temporally associated with the loss of the progesterone receptor (PGR) from the uterine LE [36]. It is possible that PTGS2 expression in LE may be induced through activation of the NF-B pathway (see [37]), which has been detected in the human endometrium [38]. Inhibition of NF-B activation blocks induction of PTGS2 mRNA expression in trophoblast cells [39]. Further, expression of another NF-B regulated cytokine, fibroblast growth factor 7 (keratinocyte growth factor), is increased in porcine endometrial LE following loss of epithelial PGR during the estrous cycle and early pregnancy [40]. Indeed, a mutual negative interaction between NF-B (subunit P-65) and PGR has been suggested [41], because loss of the PGR in the endometrial epithelia is associated with increased activation of NF-B in women [42], and NF-B activation has been implicated in opening the window of implantation in the mouse uterus [43]. Interestingly, the NF-B system is involved with many inflammatory events and with the release of cytokines [44], events that occur during early conceptus development and attachment to the uterine surface [4]. One uterine pathway to activate NF-B independently of conceptus IL1B secretion may be progesterone-stimulated endometrial expression of receptor activator of nuclear factor B ligand (RANKL). In the developing mammary gland, progesterone stimulates RANKL expression to activate NF-B for development of the lobuloalveolar buds [45]. Gene expression of RANKL increases in the porcine endometrium on Day 10 of the estrous cycle (unpublished results), which could activate NF-B to increase PTGS2 mRNA expression as observed in our study. We propose the hypothesis that changes in endometrial PTGS2 gene expression may be regulated through progesterone-induced RANKL expressions causing cell-specific changes in PGR and NF-B, which could play an important role in regulating conceptus development and timing of implantation in the pig. Further studies are needed to investigate the possible interaction of RANKL, PGR, and NF-B in the porcine uterus during the establishment of pregnancy.
Pope et al. [19] were the first to demonstrate the detrimental effects of early estrogen exposure on porcine embryo survival. Although slight reductions in the uterine surface glycocalyx occur during placentation in the pig [28], early estrogen exposure results in uterine surface microvilli devoid of glycocalyx by Day 16 of gestation [22]. Breakdown of the glycocalyx is not an immediate response, but rather occurs 5 days after early estrogen exposure. The present study provides evidence that exogenous exposure of gilts to estrogen on Days 9 and 10 of pregnancy does not affect conceptus IL1B secretion during elongation but does alter mRNA and protein expression of the inducible PTGS system on Days 12 and 13 of gestation. Inappropriate early exposure of the uterus to estrogen advances the normal induction of PTGS2 in the LE. Although uterine PG content in uterine flushing was not different between vehicle- and estrogen-treated gilts, the majority of the high lumenal content of PGs on Days 12–15 of pregnancy originates from the developing conceptuses [1, 4]. In a previous study, Morgan et al. [20] did not detect any difference in uterine lumenal PGF2 content between vehicle- and estrogen-treated gilts. It is difficult to determine endometrial production of PGs from uterine flushings, because porcine conceptuses are the major contributors of PGs in the lumen during this day of gestation [4] and cannot be separated from the smaller contribution of the endometrium. In cyclic gilts administered estrogen on Day 11, the lumenal content of PGs does not increase until Day 15 of the induced pseudopregnancy [46]. Thus, the presence of elevated endometrial PTGS2 mRNA does not necessarily correspond to changes to PGs in the uterine lumen until Day 15. The premature increase of PTGS2 on Day 10 provides evidence that the administration of estrogen on Days 9 and 10 of gestation stimulates an early increase in the uterine LE before Day 12. However, there is a dramatic decline in PTGS2 expression in the uterine LE on Day 12, when the conceptuses would undergo trophoblast elongation and initiate attachment to the LE. Alteration in PTGS2 mRNA and protein expression in the uterine LE is specific to Days 10 to 13, because there is no difference in vehicle- and estrogen-treated gilts on Days 15 and 17. Although the present study is correlative and does not provide definitive evidence that short-term alteration of endometrial PTGS2 expression causes embryonic loss during the period of placental attachment, Ptgs2-null mice have an altered inflammatory response, resulting in defects causing implantation failure [9]. Induction of PTGS2 expression promotes PGE synthesis [47], and it is known that endometrial secretion of PGE increases during peri-implantation in pigs [4], perhaps to stabilize the extracellular matrix, inflammation, and immune functions.
PTGS2 is not the only endometrial gene that is affected by endocrine disruption with early exposure of the uterus to estrogen in the pig. An alternative cause of embryonic death could be the result of changing the window of receptivity with early estrogen exposure. A recent study in our laboratory demonstrated that estrogen exposure on Days 9 and 10 of pregnancy resulted in a premature proteolysis of insulin-like growth factor (IGF)-binding proteins and a loss of IGF1 and IGF2 48 h before the normal decline in uterine luminal IGFs on Day 12 and 13 [48]. Thus, loss of pig conceptuses during the period of trophoblast attachment following early estrogen exposure may be caused by an advancement of uterine gene programming that disrupts the normal timing of conceptus attachment to the uterine surface resulting in later embryonic death. Microarray analysis revealed that endometrial expression for a number of genes is advanced on Day 12 of gestation by early estrogen exposure (unpublished results). Future studies on the role of NF-B in uterine and conceptus development are needed to establish the role of estrogen and IL1B in regulating the proinflammatory response that occurs during early pregnancy in the pig.
ACKNOWLEDGMENTS
The authors would like to thank Mr. Steve Welty for the care and feeding of the animals used in the study and the Oklahoma State University Recombinant DNA/Protein Resource Facility for the sequencing of cDNA.
FOOTNOTES
1 Supported by the National Research Initiative Competitive Grant 2002-35203-12262 from the USDA Cooperative State Research, Education, and Extension Service and Oklahoma Agriculture Experiment Station project H-02465. Manuscript approved for publication by the Director of the Oklahoma Agriculture Experiment Station. 
2 Correspondence: Rodney D. Geisert, Department of Animal Science, Oklahoma State University, Animal Science Building, Room 114, Stillwater, OK 74078. FAX: 405 744 7390; Rodney.Geisert{at}okstate.edu
Received: 15 August 2005.
First decision: 13 September 2005.
Accepted: 27 January 2006.
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 M. M. Joyce, J. R. Burghardt, R. C. Burghardt, R. N. Hooper, F. W. Bazer, and G. A. Johnson Uterine MHC Class I Molecules and {beta}2-Microglobulin Are Regulated by Progesterone and Conceptus Interferons during Pig Pregnancy J. Immunol., August 15, 2008; 181(4): 2494 - 2505. [Abstract] [Full Text] [PDF]
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J. W. Ross, M. D. Ashworth, F. J. White, G. A. Johnson, P. J. Ayoubi, U. DeSilva, K. M. Whitworth, R. S. Prather, and R. D. Geisert Premature Estrogen Exposure Alters Endometrial Gene Expression to Disrupt Pregnancy in the Pig Endocrinology, October 1, 2007; 148(10): 4761 - 4773. [Abstract] [Full Text] [PDF]
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 A. Waclawik and A. J Ziecik Differential expression of prostaglandin (PG) synthesis enzymes in conceptus during peri-implantation period and endometrial expression of carbonyl reductase/PG 9-ketoreductase in the pig J. Endocrinol., September 1, 2007; 194(3): 499 - 510. [Abstract] [Full Text] [PDF]
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M. M. Joyce, R. C. Burghardt, R. D. Geisert, J. R. Burghardt, R. N. Hooper, J. W. Ross, M. D. Ashworth, and G. A. Johnson Pig Conceptuses Secrete Estrogen and Interferons to Differentially Regulate Uterine STAT1 in a Temporal and Cell Type-Specific Manner Endocrinology, September 1, 2007; 148(9): 4420 - 4431. [Abstract] [Full Text] [PDF]
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 M. M. Joyce, J. R. Burghardt, R. C. Burghardt, R. N. Hooper, L. A. Jaeger, T. E. Spencer, F. W. Bazer, and G. A. Johnson Pig Conceptuses Increase Uterine Interferon-Regulatory Factor 1 (IRF1), but Restrict Expression to Stroma Through Estrogen-Induced IRF2 in Luminal Epithelium Biol Reprod, August 1, 2007; 77(2): 292 - 302. [Abstract] [Full Text] [PDF]
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H. Ka, S. Al-Ramadan, D. W. Erikson, G. A. Johnson, R. C. Burghardt, T. E. Spencer, L. A. Jaeger, and F. W. Bazer Regulation of Expression of Fibroblast Growth Factor 7 in the Pig Uterus by Progesterone and Estradiol Biol Reprod, July 1, 2007; 77(1): 172 - 180. [Abstract] [Full Text] [PDF]
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