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Regular Article |
a Center for Animal Biotechnology and Genomics,
b Department of Veterinary Anatomy & Public Health, College of Veterinary Medicine,
c Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.
pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Integrins are adhesion molecules that have been implicated in the porcine implantation cascade [7]. They comprise a large family of transmembrane heterodimeric glycoprotein receptors that are intimately associated with the cytoskeleton and signaling proteins [8]. Integrin receptors, composed of and ß subunits, participate in bidirectional signaling involving both "outside-in" (i.e., ligand activation at the cell surface to modify cytoskeletal organization, intracellular signaling, and gene expression) as well as "inside-out" (i.e., cytoplasmic domain transduction of intracellular signals to regulate ligand binding affinity) pathways [9]. At least seven integrin subunits are expressed by porcine endometrium and conceptus [7]. Interestingly, the subunits v, 4, 5, ß1, ß3, and ß5 (ß5, unpublished results) are expressed at the apical surface of both endometrial LE and conceptus trophectoderm (Tr). Moreover, LE expression of integrin subunits 4, 5, and ß1 increases during the period of maternal recognition of pregnancy, and 4, 5, v, ß1, and ß3 are localized to implantation sites [7]. These subunits potentially give rise to the integrin receptors vß1, vß3, vß5, 4ß1, and 5ß1 at the maternal-fetal interface during pregnancy and may function as part of an adhesion cascade that serves to generate both stable adhesion between apposing epithelial surfaces and activation of outside-in signal transduction [5].
Four of these integrin heterodimer receptors, vß1, vß3, vß5, and 4ß1, bind to the Arg-Gly-Asp (RGD) amino acid sequence that is found in osteopontin (OPN) [10–12]. OPN is a highly phosphorylated acidic glycoprotein that stimulates cell-cell adhesion, increases cell-extracellular matrix communication, and promotes cell migration (see reviews in [13–15]). Uterine, placental, and/or conceptus expression of OPN has been demonstrated in at least four species. OPN protein is expressed at high levels in the uterine glands and on the apical surface of LE during the midsecretory phase, in decidualizing stroma, and on invading cytotrophoblast in humans [16–18]. Similarly, OPN is found in the GE and decidualizing stroma of baboons [19]. In mice, OPN is produced by trophoblast, metrial gland cells of decidua, and placenta [20]. Recently, OPN expression has also been characterized in the ovine uterus [21–23].
Ovine uterine OPN is a 70-kDa protein that is cleaved by proteases at its Lys-Ser (KS) sequence to give rise to 45- and 24-kDa fragments [21, 22]. In sheep, OPN increases in uterine flushings from pregnant ewes between Days 11 and 17 [22]. Progesterone induces OPN mRNA expression in the endometrial GE of pregnant ewes [21, 23]; however, OPN protein is localized at the apical aspect of endometrial LE, GE, and conceptus Tr [22, 23]. These results suggest that OPN is secreted from the GE into the uterine lumen to bind integrin receptors expressed by LE and Tr. It has been hypothesized that OPN is a potential mediator of implantation in sheep that bridges integrin heterodimers expressed by Tr and the uterus to induce adhesion essential for conceptus attachment and placentation. However, OPN expression and function in the uterus of the pig, the only species that develops a true epitheliochorial placenta [24], have not been studied. Therefore, the objectives of this study were to 1) determine temporal and spatial expression of OPN in endometrium, uterine flushings, and conceptus/placental tissues during pregnancy in the pig and 2) perform functional analyses of potential OPN-integrin interactions at the apical surfaces of porcine conceptus Tr and endometrial epithelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experimental and surgical procedures involving animals were approved by the Agricultural Animal Care and Use Committee of Texas A&M University (Animal Use Protocol 7-127). Sexually mature crossbred gilts were observed daily for estrous behavior. Gilts exhibiting at least two estrous cycles of normal duration (18–21 days) were assigned randomly to cyclic or pregnant status. Forty-eight gilts were hysterectomized on either Days 9, 12, or 15 of the estrous cycle (Day 0 = estrus) or Days 9, 10, 12, 13, 14, 15, 20, 25, 30, 35, 40, 60, or 85 of pregnancy (n = 3 gilts per day). Uterine flushings from Days 9, 12, or 15 of the estrous cycle and Days 9, 10, 12, 13, 14, or 15 of pregnancy were obtained by introducing and recovering 40 ml sterile Hanks balanced salt solution (HBSS; Sigma Chemical Co., St. Louis, MO) at hysterectomy, cleared of cellular debris by centrifugation (3000 x g for 10 min at 4°C), and frozen at -80°C until analyzed. Pregnancy was confirmed by the presence of conceptuses of normal morphology in the uterine flushings on Days 9, 10, 12, 13, 14, and 15 or by visual observation of conceptus tissues undergoing attachment and placentation from Days 20 through 85. Conceptuses from Days 9, 10, 12, 13, 14, and 15 were embedded in Tissue-Tek Optimal Cutting Temperature (OCT) Compound (Miles, Oneonta, NY) in concentric circles to maximize the amount of tissue available in cross-sections, snap-frozen in liquid nitrogen, and stored at -80°C. At hysterectomy, several sections (
0.5 cm) from the middle portion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2) for 24 h and then embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). Several sections were also embedded in OCT and handled as described for conceptus tissues. The remaining endometrium was dissected from myometrium, frozen in liquid nitrogen, and stored at -80°C for RNA and protein extraction. Porcine Tr and uterine epithelial cells were isolated from Day 12 pregnant gilts as previously described [25, 26].
RNA Isolation and Analyses
RNA isolation Total cellular RNA was isolated from endometrial tissue samples using TRIzol reagent (Gibco-BRL, Grand Island, NY) according to the manufacturer's recommendations.
Northern and slot blot analyses Northern blot hybridization analysis was performed as described previously [27] to determine the size of OPN transcripts. Twenty micrograms of total cellular RNA were hybridized with a 32P-labeled antisense complementary RNA (cRNA) probe generated against a linearized full-length porcine OPN cDNA [28]. Steady-state levels of OPN mRNA were assessed in endometrial total RNA samples by slot blot hybridization analysis as described previously [27] using 20 µg of endometrial total RNA and a 32P-labeled antisense OPN cRNA probe. The radioactivity in each slot was quantitated using an Instant Imager (Packard Instruments, Meridian, CT) and expressed as total counts.
In situ hybridization analysis The location of OPN mRNA expression in uterine and conceptus tissues was determined by in situ hybridization analysis as previously described [21]. Deparaffinized, rehydrated, and deproteinated uterine cross-sections (5 µm) were hybridized with 35S-radiolabeled antisense or sense cRNA probes for porcine OPN. Autoradiography was performed using Kodak NTB-2 liquid photographic emulsion (Kodak, Rochester, NY). Slides were exposed at 4°C for 1 wk, developed in Kodak D-19 developer, and counterstained with Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ).
Protein Isolation and Analyses
Protein isolation Endometrium was thawed and immediately homogenized in immunoprecipitation buffer (1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.1 mM EGTA, 0.2 mM Na3VO4, 0.2 mM PMSF, 50 mM NaF, 30 mM Na4P2O7, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) at a ratio of 1 g tissue per 5 ml buffer. Homogenates were passed through a 26-gauge needle, and cellular debris was cleared by centrifugation (16 000 x g, 15 min, 4°C). Uterine flushings were thawed and concentrated by ultrafiltration (2000 x g, 1 h) over 3000-molecular weight cutoff membranes (Amicon, Danvers, MA). Concentrations of protein in endometrial extracts and uterine flushings were determined using a Bradford protein assay (Bio-Rad Laboratories, Richmond, CA) with BSA as the standard.
Western blot analysis Proteins in endometrial extracts (30 µg per lane) or uterine flushings (20 µg per lane) were denatured in Laemmli buffer, separated on 10% SDS-PAGE gels, and transferred to nitrocellulose. Blots were then blocked, incubated with a cocktail containing rabbit polyclonal antibodies against recombinant human OPN (anti-hOPN; LF-123 and LF-124; 5 µg/ml; [29]) or normal rabbit serum (5 µg/ml), followed by incubation with goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugate (1:15 000 dilution of 1 mg/ml stock; KPL, Bethesda, MD). Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Piscataway, NJ) as previously described [22], and blots were quantitated by scanning densitometry using a GS-690 Imaging Densitometer and multianalyst software (Bio-Rad, Hercules, CA).
Immunocytochemical analysis OPN protein was localized in frozen uterine and conceptus tissue sections (4–8 µm) by immunofluorescence staining as previously described [22]. Frozen sections were fixed in -20°C methanol, permeabilized with 0.3% Tween 20 in 0.02 M PBS, blocked in 5% normal goat serum, and incubated overnight at 4°C with 20 µg/ml rabbit anti-hOPN (LF-123 and LF-124) or rabbit IgG as a control. Immunoreactive protein was then detected using a fluorescein-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA). Coverslips were placed over a layer of Prolong antifade mounting reagent (Molecular Probes, Eugene, OR).
Functional Analysis of OPN-Induced Integrin Activation
Polystyrene beads (6.0 µm; Polysciences Inc., Warrington, PA) were rinsed in high-phosphate PBS (hPBS; 0.1 M NaCl, 2.7 mM KCl, 5 mM Na2PO4, 0.85 mM KH2PO4, and 50 mM NaH2PO4; pH 7.4) and centrifuged (5 centrifugations at 7000 x g for 5 min each). Beads were coated with poly-L-lysine, RGD-containing recombinant rat OPN, or mutated OPN in which the RGD sequence was replaced with RGE or RAD sequences (100 µg/ml in hPBS [30]) and incubated at room temperature for 24 h under constant agitation. The beads were rinsed five times in hPBS as described previously, then incubated overnight with sterile BSA (1 mg/ml in hPBS) under constant agitation, and stored at 4°C [31]).
Porcine Tr [25] and endometrial LE cells [26] were seeded onto two-well Lab-Tek coverglass chamber slides (Nunc, Naperville, IL) and cultured for 48 h. Cells were then washed once in Dulbecco modified Eagle medium with F-12 salts (DMEM-F12; Sigma) plus 1 mg/ml BSA.
OPN-coated beads were diluted in DMEM-F12 + 1 mg/ml BSA to a final concentration of 2 x 106 beads per 2 ml per chamber, and incubated with cells at 37°C for 1 h in a humidified, 5% CO2/air incubator. Cells were then fixed with 4% paraformaldehyde in PBS (10.14 mM Na2PO4, 1.76 mM KH2PO4, 136.9 mM NaCl, 2.68 mM KCl) at room temperature for 10 min and rinsed twice with PBS, followed by a single rinse with PBS containing 2% BSA. Cells were incubated at room temperature for 30 min in 10% normal goat serum, followed by an overnight incubation at 4°C with mouse anti-talin clone 8d4 (7-3287; Sigma), after which cells were rinsed once in PBS/BSA and three times in PBS for 5 min each. Fluorescein-conjugated goat anti-mouse IgG (65-6411; Zymed) was diluted 1:200 and incubated with cells at room temperature for 1 h in the dark. Cells were rinsed twice with PBS and then twice with Hepes buffer (10 mM Hepes, 150 mM NaCl, and 0.08% NaN3). Mounting medium (0.2 M PBS, 2 mg/ml p-phenylenediamine and 90% glycerol; 400 µl per chamber) was then added, and slides were stored at 4°C in the dark before immunofluorescence analysis.
Photomicrography
Photomicrographs of representative fields of in situ hybridization and immunofluorescence preparations were evaluated with a Zeiss Axioplan2 microscope (Carl Zeiss, Thornwood, NY) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu Corporation, Bridgewater, NJ). Digital images were captured using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA) and MacIntosh PowerMac G3 computer (Apple Computer, Cupertino, CA). Black-and-white prints were electronically printed using a Kodak DS8650 color printer (Eastman Kodak Co., Rochester, NY).
Fluorescence imaging of ligand-coated beads was performed with a digital fluorescence imaging system consisting of a CCD camera and image-capture software (CELLscan; Scanalytics Inc., Bedford, MA) integrated with a Zeiss Axiovert inverted fluorescence microscope (Zeiss). Sequential optical slices from the basal-to-apical surface of the cells were collected with a high-numerical aperture objective lens. Image collection was initiated approximately 1 µm below the basal surface of the cell, and optical slices were collected at 0.5-µm steps up through the apical cell surface and attached beads. The percentage of OPN-coated beads with apical surface induced focal adhesions was determined by combined phase contrast-fluorescence imaging. Phase contrast microscopy was used to locate cells and count adherent beads. Fluorescence microscopy was then used to identify transmembrane accumulation of immunoreactive talin below the OPN-coated beads.
Statistical Analyses
Slot blot hybridization data and integrated optical density measurements from Western blots were subjected to least-squares ANOVA using the General Linear Models (GLM) procedures of the Statistical Analysis version 8.1 for Windows (SAS Institute, Cary, NC). Total counts were adjusted for differences in sample loading using the 18S rRNA data as a covariate for slot blot data analysis. The initial measurement of band optical density on Day 9 was used as a covariate for Western blot data analysis. All tests of statistical significance were performed using the appropriate error terms according to the expectation of mean squares. The percentage of OPN-coated beads in contact with cells that exhibited apical focal adhesions was obtained by multiplying the ratio of focal adhesion-positive beads by the total number of beads in contact with the cell by 100. Three to five separate experiments on different days were performed for each of the ligands tested. Data collected from each experiment were subjected to arcsine transformation and analyzed by the Tukey multiple comparison test using GLM. Data are presented as mean percentage of beads inducing apical focal adhesions ± SD. A P value of less than 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot analysis indicated that two OPN mRNA transcripts of 1.5 and 3.8 kilobase (kb) were present in endometrial extracts from pregnant pigs (Fig. 1). The 1.5-kb transcript was evident by Day 25, and the 3.8-kb transcript increased after Day 45 (Fig. 1). Steady-state levels of total OPN mRNA were not different between Days 9 and 15 of the estrous cycle and pregnancy (Day x status, P > 0.05) (Fig. 2). However, OPN mRNA expression increased after Day 25 of pregnancy (cubic, P < 0.01).
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In situ hybridization analysis revealed OPN mRNA expression in three distinct cell types (Fig. 3). On Day 9 of the estrous cycle and pregnancy, OPN transcripts were detected in a small percentage of cells in the subepithelial stratum compactum of the endometrial stroma (Fig. 3). In pregnant gilts, OPN mRNA was present in the LE and GE. Expression along the LE was barely detectable on Day 12 but dramatically increased in discrete regions of the LE by Day 15. It is noteworthy that the OPN expression was greatest in LE in close proximity to conceptus tissue and very weak or absent in LE without adjacent conceptus Tr (Fig. 3). However, all LE expressed OPN mRNA from Days 20 through 85 of pregnancy. Expression of OPN mRNA in GE was first detected on Day 35 and increased markedly through Day 85 of pregnancy. Day 15–25 conceptuses did not express OPN mRNA (Fig. 3).
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Expression of OPN Protein in Porcine Endometrial Extracts, Uterine Flushings, and Endometrial/Conceptus Frozen Sections
Immunoreactive 70- and 45-kDa OPN forms were detected in all endometrial tissues from cyclic and pregnant gilts (Fig. 4A). A third 25-kDa OPN form was occasionally detected (data not shown). Expression of the 70-kDa OPN did not change during pregnancy (P > 0.25). However, abundance of the 45-kDa form was affected by day of pregnancy (cubic, P < 0.03) with higher levels on Day 9 and between Days 35 and 85 of pregnancy. Only the 70-kDa OPN form was detected in uterine flushes from both cyclic and pregnant gilts (Fig. 4B).
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OPN protein was detectable on endometrial LE and GE on Day 12 of pregnancy, but expression increased in LE and GE on Day 30 and was present along the entire uterine-placental interface by Day 45 (Fig. 5A). Immunoreactive OPN was also detected in a subpopulation of cells dispersed within the stratum compactum of the stroma on Day 9 (Fig. 5A). In situ hybridization indicated the absence of OPN mRNA expression in conceptus Tr; however, OPN protein was detected on conceptus Tr on Days 12 and 13 of pregnancy (Fig. 5B).
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Functional Activation of Integrin Receptor Signaling by OPN in Porcine Endometrial LE and Conceptus Tr Cells
The capacity for OPN to function as a ligand for integrins present at the apical surfaces of porcine LE and Tr cells was evaluated using an in vitro bead assay adapted from Miyamoto et al. [32] to measure integrin-mediated "outside-in" signaling. Figure 6 illustrates focal adhesions resulting from reorganization of cytoskeletal elements at the cytoplasmic side of the interface between OPN-coated beads and the apical cell surface of porcine Tr cells. These focal adhesions, indicated by immunocytochemical localization of the cytoskeletal protein talin, are the result of integrin activation in response to OPN binding and aggregation of cytoskeletal proteins. Approximately 20% of the beads in contact with LE and 42% in contact with Tr exhibited accumulation of talin at the bead-cell interface (Table 1). Similar aggregates were detected in Tr cells stained with antibody directed against -actinin (data not shown). No focal adhesions were induced by poly-L-lysine-coated beads or beads coated with mutant recombinant OPN in which the RGD sequence was replaced with RAD or RGE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two OPN transcripts were detected in endometrium from pregnant pigs. The 1.5-kb transcript is similar in size to that described in porcine bone [33] and ovine uterine endometrium [21]. The 3.8-kb transcript may be an alternative splice variant reported for mouse kidney cells [34] and human bone and decidual cells [16], or it may be a product of a second promoter that is present in the porcine OPN gene and silent in bone cells [33]. The identity of the 3.8-kb OPN transcript in the porcine uterus remains to be determined.
In pigs, there is a complex temporal and spatial pattern of OPN mRNA expression that involves at least three separate sites within the uterus. These include 1) a small number of cells present in subepithelial stratum compactum of the stroma on Day 9 of the estrous cycle and pregnancy, 2) discrete regions of the uterine LE adjacent to conceptus tissue beginning on Day 12 and throughout the LE surface by Day 20 of pregnancy, and 3) the endometrial GE beginning on about Day 35 and increasing to Day 85 of pregnancy.
Although some aspects of the complex pattern of endometrial OPN mRNA expression in pigs are similar to those described for sheep [21], expression of OPN mRNA in the uterine LE of pregnant gilts is novel. Typically, epithelial expression of OPN mRNA is limited to the uterine GE of species that have invasive implantation, including primates [17, 35] and mice [20]. Similarly, in sheep, which have central and noninvasive implantation, OPN mRNA is limited to GE of pregnant or progesterone-treated ovariectomized ewes [21, 23] and is not detected in LE. Patterns of OPN expression in pregnant pigs, sheep, mice, and primates may relate to differences in type of placentation. Primates and rodents have hemochorial placentation with initial attachment of Tr to LE followed by rapid degeneration of LE and stromal decidualization [36, 37]. Sheep have syndesmochorial placentation in which the endometrial LE undergoes transformation and regional disintegration, resulting in areas of attenuated LE during pregnancy [38]. In contrast, the pig is the only species that has true epitheliochorial placentation in which the LE remains intact throughout pregnancy [24]. It follows that if epithelial-derived OPN is critical to pregnancy, the pig uterine LE synthesizes OPN throughout pregnancy, whereas other species must use OPN from GE to ensure uninterrupted expression. It is noteworthy that the fibroblast growth factor 7 (FGF-7), normally produced in stroma, is uniquely expressed in porcine LE prior to Day 35 of pregnancy, and in both LE and GE between Days 35 and 85 of pregnancy with its receptor expressed by LE, GE, and Tr [39]. These similar results for OPN and FGF-7 emphasize that functional properties of LE and interactions between LE and Tr are critical to the processes of central implantation and true epitheliochorial placentation in pigs.
Expression of OPN mRNA in pig uterus is barely detectable in LE on Day 12 of pregnancy, increases in discrete regions of the LE by Day 15, and is expressed maximally in all LE by Day 20 of pregnancy (Fig. 3). Interestingly, this spatial pattern of OPN expression is temporally coordinate with the morphological and biological changes that conceptuses undergo between Days 10 and 24 of gestation and corresponds to the periods for pregnancy recognition and early adhesion between Tr and uterine LE. During this period, conceptus Tr produces estrogen for pregnancy recognition and achieves intimate contact with uterine LE for noninvasive implantation [2, 4]. Although speculative, a paracrine factor from the conceptus may be responsible for local increases in OPN mRNA in LE. This putative conceptus factor(s) remains to be determined; however, the increase in OPN coincides with the decline of progesterone receptor (PR) in LE [40] and down-regulation of PR by progesterone may be required for up-regulation of OPN expression [22, 41].
Expression of OPN mRNA in the endometrial GE was detected on Day 35 and increased markedly between Days 40 and 85 of pregnancy (Fig. 3). These are the first results showing uterine expression of OPN during later stages of pregnancy in any species. In sheep, OPN mRNA in GE increases in response to sequential treatment of ovariectomized ewes with progesterone and placental lactogen [22, 41]. Progesterone may also be responsible for OPN expression in uterine GE of pigs. Progesterone regulates endometrial secretory activity in the pig, and secretory activity increases rapidly after Day 35 of pregnancy, reaching maximal levels between Days 60 and 75 [42]—a temporal pattern that mirrors transcriptional activity of the OPN gene in GE. The observation that the 70-kDa form of OPN can be detected in uterine flushings while the 45-kDa form increases in endometrial extracts coincident with increasing secretory activity of GE between Days 25 and 80 (Fig. 4) suggests that OPN may be a secretory protein in porcine histotroph. The 45-kDa OPN has greater binding affinity for integrin receptors than does the native 70-kDa form [43], and OPN protein is prevalent on conceptus Tr and at the uterine-placental interface that potentially express the OPN integrin receptors vß1, vß3, vß5, and 4ß1 [5, 7].
The morphology and distribution of OPN mRNA- and protein-positive cells in the stratum compactum of the stroma on Day 9 of the estrous cycle and pregnancy suggest that these are immune cells. OPN, also called early T-cell activation factor-1 (Eta-1), is an established component of the immune system that is secreted by activated T lymphocytes [44], induced in monocytes and macrophages by tissue injury [45], and present in CD8- CD4- granulated metrial gland cells [46]. Recently, OPN was shown to be a key component of Type-1 (Th-1) immunity that up-regulates interleukin 12 (IL-12) and down-regulates IL-10 expression by macrophages [47]. Macrophage production of IL-12 promotes a cell-mediated or Th-1 immune response that effectively protects against the growth of infectious viral and bacterial pathogens, whereas IL-10 inhibits this response and favors humoral or Th-2 immunity (see reviews [48, 49]). Thus OPN, a protein critical for immune cell recruitment and cytokine production in Th-1-mediated immunity, is expressed in the uterus of Day 9 pregnant pigs, whereas the maternal immune response in primates and rodents is biased toward humoral immunity and away from cell-mediated immunity [50]. The significance of OPN expression by this population of "immune-like" cells in the compact stroma of porcine endometrium requires further study. But one may speculate that because insemination in pigs is intrauterine, OPN expressing immune cells promote a Th-1 cytokine profile within the uterus to protect against infectious pathogens introduced during mating.
The present study also indicates that OPN protein is associated with the apical surface of porcine conceptus Tr and uterine LE, while previous studies have identified potential OPN integrin receptors including vß1, vß3, vß5, 4ß1, and 5ß1 at the apical surfaces of these cell types in vivo [5, 7]. Moreover, results of the present study confirm that OPN binds and activates integrins on porcine uterine LE and conceptus Tr cells in vitro. In this model system [32], integrin receptor binding to RGD sequences immobilized on OPN-coated polystyrene beads triggered the aggregation of integrins and subsequent transmembrane accumulation of the cytoskeletal proteins talin and -actinin to the ß integrin subunit cytoplasmic domain in porcine cells. These aggregates composed of extracellular matrix (ECM) proteins, integrins, and cytoskeletal proteins are known as focal adhesions [8], and resulted from RGD-integrin interactions because cytoskeletal assembly was not observed when the RGD sequence of OPN was mutated to RAD or RGE. The identity of activated integrins in porcine cells remains unknown; however, v, 4, 5ß1, ß3, and ß5 integrin subunits are present both in vivo and in vitro [5, 7, 26]. It is reasonable to predict, therefore, that uterine-derived OPN binds integrins within the uterus and conceptus to initiate functional intracellular signals that influence conceptus development, adhesion, and placentation in the pig.
Conceptus elongation, implantation, and placentation are progressive events involving adhesion molecule-dependent remodeling of endometrium and Tr. Since OPN expression in the porcine uterus temporally and spatially corresponds to these events, OPN may mediate conceptus Tr attachment, growth, and spreading on uterine LE. Indeed, the RGD sequence of OPN mediates interactions with cell surface integrins in adhesion cascades as they bind their ECM ligand(s) to mediate adhesion (implantation), cause cytoskeletal reorganization to stabilize adhesion (elongation of conceptuses and placentation), and transduce cellular signals through numerous signaling intermediates [5]. There is considerable evidence that OPN promotes attachment and spreading of cells in vitro. Rat osteosarcoma cells attach to and spread on surfaces coated with OPN [51], and cultured human smooth muscle cells both adhere and migrate to OPN [52]. Recently, Fisher et al. [53] have shown that purified OPN is flexible along its entire length in solution, allowing for regions within the protein to use several interspersed binding sequences for interaction with different proteins. Proteins that lack structure in solution are therefore able to bridge between two or more proteins. It is noteworthy that OPN is believed to bridge cell surface integrins to hydroxyapatite during resorption of bone matrix [54], and complement factor H binds OPN that is already complexed to either vß3 integrin or CD44 to inhibit the alternative complement lysis pathway [55]. The presence of OPN protein at the apical surfaces of Tr and LE throughout porcine pregnancy may indicate a role for OPN as an adhesive between Tr and LE essential for attachment and epitheliochorial placentation.
Clearly, the role(s) of uterine OPN during porcine pregnancy remains to be determined. However, the complex and extensive pattern of OPN expression in the uterus throughout pregnancy strongly suggests physiological relevance. In sheep, OPN may interact with integrins to influence conceptus elongation, initial attachment to uterine LE, and implantation [21]. It is reasonable to speculate that OPN performs similar functions during porcine pregnancy and may also be involved in placentation as endometrial immune cells, LE, and GE sequentially express OPN. The regulation and immediate functional implications of these expression events may be independent but temporally and spatially orchestrated to maintain successful pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Research supported by USDA-NRICGP grant 95-35203-6337 to F.W.B. and R.C.B., USDA-NRICGP grant 95-35203-6223 to R.C.B., and NIH 1-F32-HD08501-01A1 to G.A.J. 
2 Correspondence: Fuller W. Bazer, Department of Animal Science and Center for Animal Biotechnology and Genomics, 442 Kleberg Center, Texas A&M University, College Station, TX 77843-2471. FAX: 979-862-2662; fbazer{at}cvm.tamu.edu
3 Current address: Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS 66160-7400
4 Current address: Department of Animal and Veterinary Science, Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-2330
5 Equal contribution by both authors
Accepted: October 18, 2001.
Received: July 26, 2001.
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L. A. Jaeger, A. K. Spiegel, N. H. Ing, G. A. Johnson, F. W. Bazer, and R. C. Burghardt Functional Effects of Transforming Growth Factor {beta} on Adhesive Properties of Porcine Trophectoderm Endocrinology, September 1, 2005; 146(9): 3933 - 3942. [Abstract] [Full Text] [PDF]
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T. E Spencer, G. A Johnson, F. W Bazer, and R. C Burghardt Implantation mechanisms: insights from the sheep Reproduction, December 1, 2004; 128(6): 657 - 668. [Abstract] [Full Text] [PDF]
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G. A. Johnson, R. C. Burghardt, F. W. Bazer, and T. E. Spencer Osteopontin: Roles in Implantation and Placentation Biol Reprod, November 1, 2003; 69(5): 1458 - 1471. [Abstract] [Full Text] [PDF]
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G. A. Johnson, R. C. Burghardt, M. M. Joyce, T. E. Spencer, F. W. Bazer, C. A. Gray, and C. Pfarrer Osteopontin Is Synthesized by Uterine Glands and a 45-kDa Cleavage Fragment Is Localized at the Uterine-Placental Interface Throughout Ovine Pregnancy Biol Reprod, July 1, 2003; 69(1): 92 - 98. [Abstract] [Full Text] [PDF]
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