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Pregnancy |
Center for Animal Biotechnology and Genomics,3
Department of Veterinary Anatomy and Public Health,4 College of Veterinary Medicine,
Department of Animal Science,5 College of Agriculture and Life Sciences, Texas A&M University, College Station, Texas 77843
Department of Veterinary Anatomy, Histology and Embryology,6 Justus-Liebig-University, 35392 Giessen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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placenta, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Osteopontin (OPN) is a component of histotroph [5, 6] that is hypothesized to be involved in ovine implantation [7, 8]. This highly phosphorylated and glycosylated, secreted protein is present in various biological fluids [9]. It also has been found on epithelial cells of the gallbladder, pancreas, lung, sweat ducts, placenta, gastrointestinal, urinary, mammary, and reproductive tracts [10–14]. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments [15]. Secreted OPN functions as an extracellular matrix (ECM) protein that regulates the activity of a variety of cells by signaling through integrin or CD44 receptors [16, 17]. Reported effects of OPN include mediation of cell-cell adhesion, cell-ECM communication, and cell migration [16, 18].
Uterine OPN and its integrin receptors have become the focus of studies concerning conceptus adhesion for implantation in a number of species, including humans, in which OPN expression is increased by progesterone during the menstrual cycle, starting on Days 16–17, and secretory glandular epithelium (GE) appears to be a source of OPN localized on the apical LE surface [19]. In pregnant sheep, OPN protein is a component of histotroph that binds to the apical surface of LE and GE where several integrins are apically expressed and could form known OPN receptors, including 
vß3, vß1, 4ß5, 4ß1, and 5ß1 [6, 7]. The form of OPN protein that increases in uterine flushings from pregnant ewes between Days 11 and 17 is predominantly a 45-kDa cleavage fragment that binds with higher affinity to integrins and has greater biological activity than the native 70-kDa OPN [20]. The OPN protein is also present on trophectoderm of Day 14–19 ovine conceptuses despite the absence of conceptus OPN mRNA [6]. The OPN mRNA is expressed in the GE of pregnant ewes and is induced when ovariectomized ewes are given exogenous progesterone [5, 21]. Indeed, uterine gland knockout ewes lack uterine glands and exhibit a peri-implantation defect that is associated with the absence of OPN in uterine secretions [4, 8]. Therefore, OPN is proposed to be a progesterone-induced secretory product of the GE that binds to uterine LE and conceptus trophectoderm to mediate conceptus elongation and attachment, which is essential to maintain pregnancy through the peri-implantation period in ewes.
Although cell-cell adhesion, cell-ECM communication, and cell migration undoubtedly continue to be critical at the interface between uterus and placenta throughout pregnancy, to our knowledge endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in ewes. Our working hypothesis is that OPN is a component of histotroph that is secreted by the endometrial GE throughout pregnancy in the ewe and contributes to an ECM that supports maternal-conceptus adhesion and communication. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus and placenta between Days 25 and 120 of pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental and surgical procedures complied with the Guide for the Care and Use of Agricultural Animals and were approved by the Texas A&M University Institutional Agricultural Animal Care and Use Committee.
Mature, western-range ewes were observed daily for estrous behavior. After experiencing at least two estrous cycles of normal duration (16–18 days), ewes were assigned randomly on Day 0 (estrus/mating) to cyclic or pregnant status. Fifty-two ewes were ovariohysterectomized (n = 4 ewes/day) on Days 14, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, or 120 of pregnancy. At hysterectomy, uteri from Day 14 pregnant ewes were flushed with 0.9% NaCl to verify pregnancy by recovery of an apparently normal conceptus. Pregnancy was verified on all later days by visual observation of conceptus tissues undergoing attachment and placentation. Several sections (thickness, 
1–1.5 cm) from the middle 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 Tissue-Tek Optimal Cutting Temperature Compound (Miles, Oneonta, NY), snap-frozen in liquid nitrogen, and stored at -80°C. The remaining endometrium was dissected from the myometrium, frozen in liquid nitrogen, and stored at -80°C for RNA extraction.
Histological Analysis
Ovine uteri were sectioned (thickness, 5 µm), deparaffinized in CitraSolv (Fisher Scientific; Pittsburgh, PA), and rehydrated through a graded alcohol series to distilled water. Tissues were then exposed to a Masson trichrome staining procedure as previously described [8]. This staining procedure results in black nuclei, red cytoplasm and muscle fibers, and blue ECM components.
RNA Isolation and Analyses
RNA isolation Total cellular RNA was isolated from endometrial tissue samples using TRIzol reagent (Invitrogen, Carlesbad, CA) according to the manufacturer's recommendations.
Slot-blot analysis Steady-state levels of OPN mRNA were assessed in endometrial total RNA samples by slot-blot hybridization analysis as described previously [22] using 20 µg of endometrial total RNA and 32P-labeled antisense ovine OPN cRNA probe [5]. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with 32P-labeled antisense 18S rRNA cDNA (pT718S; Ambion, Austin, TX). The radioactivity in each slot was quantified by a Typhoon 8600 Imager (Molecular Dynamics, Piscataway, NJ) and expressed as total counts.
In situ hybridization analysis Osteopontin mRNA expression in ovine uterine tissues was localized by in situ hybridization as previously described [5]. Deparaffinized, rehydrated, and deproteinated uterine cross-sections (thickness, 5 µm) were hybridized with 35S-radiolabeled antisense or sense cRNA probes for ovine OPN [5]. Following washes and RNase A digestion, slides were dipped in Kodak NTB-2 liquid photographic emulsion (Kodak, Rochester, NY), stored at 4°C for 5 days, developed in Kodak D-19 developer, counterstained with Harris modified hematoxylin (Fisher Scientific), dehydrated, and protected with coverslips.
Immunofluorescence Analyses
Antibodies used for immunofluorescence staining included rabbit anti-OPN amino terminal (LF-124) and carboxyl terminal (LF-123) [23] and a fluorescein-conjugated goat antibody against rabbit immunoglobulin G (Chemicon International, Temecula, CA). Proteins were localized in frozen ovine uterine cross-sections as previously described [6]. Briefly, tissues were fixed in -20°C methanol, permeabilized with 0.3% Tween 20 in PBS, blocked in 10% normal goat serum, incubated overnight at 4°C with 2 µg/ml of primary antibody, and detected with fluorescein-conjugated secondary antibody. Slides were overlaid with a coverglass and Prolong antifade mounting reagent (Molecular Probes, Eugene, OR).
Photomicrography
Photomicrographs of representative fields of in situ hybridization, Masson trichrome staining, and immunofluorescence staining 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 an Apple PowerMac G3 computer (Apple Computer, Cupertino, CA).
Statistical Analysis
Data from slot-blot hybridization analyses were subjected to least-squares ANOVA using the general linear models procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Effects of day on steady-state levels of endometrial OPN mRNA were examined by regression analysis. Hybridization data (relative units) were normalized for differences in sample loading using the 18S rRNA data as a covariate in ANOVA. Data are presented as least-square-means relative units with overall SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In support of our working hypothesis that OPN is a component of histotroph contributing to an ECM that supports maternal-conceptus adhesion and communication throughout pregnancy, Masson trichrome staining of paraffin-embedded, Day 35 cross-sections revealed the deposition of ECM (blue staining) along the interface between uterine LE and placental tissue (Fig. 1). The following results indicate that OPN is an important constituent of this ECM.
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Expression of OPN mRNA in Total Endometrium
Temporal changes in steady-state levels of OPN mRNA in total ovine endometrium from Days 25 through 120 of pregnancy were quantified by slot-blot hybridization (Fig. 2). The OPN mRNA remained constant through Day 40. Steady-state levels of endometrial OPN mRNA were affected (P < 0.001) by day of pregnancy. Overall, OPN mRNA levels were lowest on Days 20–40, increased 39-fold from Days 40 to 100, and remained maximal thereafter (cubic effect of day, P < 0.0001, r2 = 0.94) (Fig. 2).
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Localization of OPN mRNA Within Endometrial Glands
In situ hybridization analysis of endometrial cross-sections revealed expression of OPN mRNA in the middle to deep GE from Days 20 through 120 of pregnancy (Fig. 3). An increase in total hybridization was evident concomitant with the hypertrophy in endometrial glands that occurs between Days 50 and 60 of pregnancy (Fig. 3). As previously reported, OPN mRNA was also present in endometrial stratum compactum stroma between Days 25 and 120 of pregnancy [24]. However, hybridization was much less in these cells than in GE and is not readily apparent in Figure 3 because of the relative intensity of the GE signal.
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Localization of OPN Protein in the Uterus and Placenta
The location of OPN protein in pregnant ovine uterus and placenta was assessed by immunofluorescence staining of frozen cross-sections using the LF-123 rabbit anti-OPN antibody that detects both native and the carboxyl half of OPN starting at the Arg-Ser (RS) thrombin-cleavage site or the LF-124 antibody that detects both native and the amino half of OPN ending at the RS site (see Fig. 5A).
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OPN protein in endometrial glands In agreement with in situ hybridization results, immunoreactive OPN protein (LF-123 antibody) was present in the endometrial GE of all ewes between Days 20 and 120 of pregnancy (Fig. 4).
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OPN protein at the uterine-placental interface Immunolocalization of OPN protein by immunofluorescence staining of serial cross-sections from Days 14 through 120 of pregnancy with LF-123 and LF-124 antibodies is illustrated in Figure 5B. Previous studies have established that LF-124 antibody detects a 45-kDa OPN cleavage product that contains the Arg-Gly-Asp (RGD) integrin-binding sequence and is secreted into the uterine lumen of ewes during early pregnancy, whereas LF-123 antibody does not detect 45-kDa OPN [6]. Both antibodies immunoreact with native 70-kDa OPN in immunofluorescence staining. Importantly, however, clear differences exist in the endometrial distribution of native 70-kDa OPN and the secreted 45-kDa fragment, which is exclusively, continuously, and abundantly present along the apical surface of LE, on trophectoderm, and along the entire uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy (Fig. 5B). As previously reported [24], OPN protein is also expressed in uterine stroma between Days 30 and 120 of pregnancy (Fig. 5B). Stromal OPN is detectable with either the LF-123 or LF-124 antibody, indicating expression of native 70-kDa OPN.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Steady-state levels of total endometrial OPN mRNA increased approximately 30-fold between Days 40 and 80 of gestation. Although increases in total OPN mRNA in the sheep are confounded by a complex temporal and spatial pattern of expression that includes both an increase in OPN gene expression in stratum compactum stroma that begins between Days 20 and 25 of pregnancy [24] as well as constitutive expression within the GE, even a perfunctory examination of mRNA localization (Fig. 3) clearly indicates that the major source of OPN is GE. This is readily seen by comparing the relative magnitude of hybridization between stroma and GE on Days 80 and 100 of pregnancy.
Changes in OPN mRNA correlate with growth of endometrial glands that occurs as the uterus remodels to accommodate rapid conceptus development and growth during the last two-thirds of pregnancy [25]. During ovine gestation, GE undergoes hyperplasia through Day 50, followed by hypertrophy and maximal production of histotroph after Day 60 [26]. A similar correlation between GE development and gene expression during pregnancy has been shown for the well-defined secretory product of GE, uterine milk protein (UTMP) [26]. Endometrial gland morphogenesis and, by inference, increases in GE gene products, such as OPN and UTMP, likely are influenced by sequential uterine exposure to estrogen, progesterone, interferon-tau, placental lactogen, and placental growth hormone [27, 28]. Using an ovariectomized ewe model, administration of progesterone increased GE expression of OPN and UTMP [21–27], and injection of a progesterone-receptor antagonist prevented progesterone effects on OPN mRNA [21]. Furthermore, intrauterine infusion of placental lactogen to progesterone-treated ovariectomized ewes increased OPN and UTMP levels over those with progesterone alone [27, 28]. Interestingly, regulation of OPN and UTMP genes diverge in regard to exposure to growth hormone. Whereas infusion of growth hormone into progesterone-treated ovariectomized ewes increased GE expression of UTMP mRNA, OPN mRNA was not affected [27, 28]. It is important to note that sequential treatment of ovariectomized ewes with progesterone, interferon-tau, placental lactogen, and growth hormone resulted in GE development similar to that observed under the influence of placental lactogen from the placenta during pregnancy[28].
The differential staining observed when serial cross-sections were immunolabeled with LF-123 and LF-124 antibodies provides significant insights regarding the mechanistic role of OPN during pregnancy. These antibodies were generated against the amino (LF-124) and carboxyl (LF-123) terminal fragments of recombinant human OPN that form when the native 70-kDa protein is cleaved by thrombin at its RS sequence [23]. Both immunoglobulins detect the native 70-kDa OPN, but only the LF-124 antibody recognizes the 45-kDa cleavage fragment that contains the integrin-binding RGD sequence and has increased cell attachment and spreading properties through increased accessibility of the RGD to integrin receptors [20]. Only the 45-kDa OPN supports 9ß1- and 4ß1-mediated cell migration and adhesion [29, 30] as well as attachment through several other non-RGD sites [31]. This fragment is the predominant form of OPN in uterine flushes from pregnant ewes [5, 6]. The 45-kDa OPN fragment is clearly present at the apical surface of uterine LE on Day 14 of pregnancy, on Day 20 conceptus trophectoderm, and along the entire uterine-placental interface of both placentomal and interplacentomal regions through Day 120 of gestation. Because OPN mRNA is synthesized by GE, but not by LE or conceptus, immunodetection of 45-kDa OPN protein at these locations confirms that OPN is secreted by GE and binds to all sites of direct attachment between conceptus and uterus. The source of OPN protein that localizes at the interface between caruncular epithelium and chorion likely is OPN secreted from neighboring GE in the intercaruncular endometrium. However, stromal and immune cells of placentomes express OPN, which may be transported to the interface between caruncular epithelium and chorion [24, 32].
Implantation/placentation is a progressive process involving adhesion molecule-dependent remodeling of endometrium and conceptus trophectoderm. Recently, OPN and its integrin receptors have become the focus of studies concerning adhesion for implantation. Lessey et al. [33] have established that transient endometrial expression of vß3 and 4ß1 integrins is cycle-dependent and defines the implantation window in women. Altered expression of these integrins is correlated with several causes of infertility [34]. Null mutations of v, 5, ß1, or ß5 integrin genes in mice lead to peri-implantation lethality and failure of chorion-allantois fusion [35], whereas functional blockade of v and ß3 integrins in mice reduces the number of implantation sites [36]. Endometrial integrin expression has also been reported in several species that exhibit noninvasive implantation, including the sheep, pig, goat, and cow [6, 7, 37–39]. Uterine, placental, and/or conceptus expression of OPN has been demonstrated in cows, pigs, mice, baboons, humans, and sheep. Preliminary studies in cows suggest that OPN protein is localized to the apical surface of GE, to stroma, and on trophectoderm cells of midpregnancy cows [40]. In pigs, OPN mRNA is synthesized in discrete regions of the uterine LE between Days 12 and 15 of pregnancy, is present throughout the entire LE thereafter, and appears in GE by Day 30 [41]. In the mouse, OPN is transcribed by trophectoderm, metrial gland cells of decidua, and placenta [42], whereas OPN protein is found in GE and decidualized stroma of baboons [43]. In women, high levels of OPN mRNA were first localized by Young et al. [10] to decidual stromal cells as well as endometrial glands of nonpregnant secretory uterus. The OPN transcript was later detected in hypersecretory endometrial GE of pregnant women [11, 44]. Omigbodun et al. [45] subsequently determined that progesterone increased OPN protein on cytotrophoblasts of the chorionic villus, but not syncytial trophoblasts, and concluded that OPN mediates adhesive and/or signaling events between these trophoblast cell types via v integrins. Importantly, endometrial expression of OPN is increased by progesterone during the menstrual cycle, starting on Days 16–17, and secretory GE appears to be the source of OPN protein that is localized on the apical LE [19, 44].
In sheep, OPN is a component of histotroph that increases in uterine flushings from pregnant ewes between Days 11 and 17 [6]. Exposure to progesterone induces OPN mRNA expression only in endometrial GE, but OPN protein is present on the apical aspect of the endometrial LE and trophectoderm [5, 6, 21]. Indeed, OPN has been proposed to bridge integrin receptors expressed by trophectoderm and uterus to induce adhesion between LE and trophectoderm essential for initial conceptus attachment in sheep and humans [6, 7, 44, 46]. Two recent reports using high-density microarray screening of human endometrium during the receptive phase for implantation have confirmed that OPN increases markedly during the window of implantation. A comparison of gene expression between endometrium from Days 8 to 10 of the cycle with Days 8–10 postmidluteal LH surge showed an 8.1-fold increase in OPN mRNA [47]. Similarly, microarray comparison of endometrium from Days 2 to 4 with Days 7 to 9 post-LH surge showed a 12.3-fold up-regulation [48]. Results from the present study support the results from studies of pigs [41] and cows [40] and firmly establish that glandular OPN continues to have a role in maternal-fetal interactions well beyond the receptive phase for implantation. The role of OPN at the uterine-placental interface is not clear, but this ECM protein has potential to mediate adhesion, remodeling, and cell-cell/cell-ECM communication at sites of contact between maternal- and fetal-derived tissues. The presence of the highly adhesive [20] and flexible [49] 45-kDa OPN cleavage fragment containing multiple binding sequences for interaction with different proteins [50] and capable of multimer formation [46] supports the hypothesis that OPN continues to serve as an adhesive ligand that bridges receptors on uterus and placenta throughout gestation. However, other roles for OPN are possible. For example, OPN fragments are known to initiate cell proliferation and migration [51] and may be involved in cytoskeletal reorganization for placentation. In addition, ECM proteins, such as OPN, can bind integrins to promote survival in cells that would otherwise undergo apoptosis [52], and OPN sequesters Factor-H, a plasma protein that binds and inactivates complement pathway components, to the cell surface and inhibits complement-mediated cell lysis [53]. Interestingly, both OPN and another endometrial secretory protein, decay-accelerating factor, which functions in a similar way to inhibit complement-mediated cell lysis, exhibit comparable temporal and spatial increases of expression in the peri-implantation human endometrium [54]. Therefore, OPN may serve to stabilize cells at the uterine-placental interface.
Collectively, OPN mRNA and protein localization in the present study confirm that OPN is a component of ovine histotroph that binds to the uterine-placental interface, where it could have profound influence throughout gestation. Experiments that address physiological, cellular, and molecular mechanisms involved in OPN-mediated attachment, proliferation, migration, and signal transduction using in vitro, ex vivo, and in vivo models are warranted to define the role of OPN during pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Greg A. Johnson, Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4458. FAX: 979 845 7249; gjohnson{at}cvm.tamu.edu
Received: 20 November 2002.
First decision: 18 December 2002.
Accepted: 27 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrin expression in the uterus and conceptus during the peri-implantation period. Biol Reprod 1999 61:892-899vß3 integrin stimulates immediate cell signals in osteoclasts. J Biol Chem 1991 266:20369-20374vß3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol Metab 2001 86:4991-50004ß1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin. J Biol Chem 2001 276:13483-134899ß1-mediated adhesion and migration to thrombin-cleaved osteopontin. Exp Cell Res 1998 242:351-360[CrossRef][Medline]v integrins. J Cell Biochem 1996 62:123-131[CrossRef][Medline]vß3 integrin adversely affects implantation in the mouse. Biol Reprod 2000 62:1285-1290This article has been cited by other articles:
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  R. C Burghardt, J. R Burghardt, J. D Taylor II, A. T Reeder, B. T Nguen, T. E Spencer, K. J Bayless, and G. A Johnson Enhanced focal adhesion assembly reflects increased mechanosensation and mechanotransduction at maternal-conceptus interface and uterine wall during ovine pregnancy Reproduction, March 1, 2009; 137(3): 567 - 582. [Abstract] [Full Text] [PDF]
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G. Song, M. C. Satterfield, J. Kim, F. W. Bazer, and T. E. Spencer Gastrin-Releasing Peptide (GRP) in the Ovine Uterus: Regulation by Interferon Tau and Progesterone Biol Reprod, August 1, 2008; 79(2): 376 - 386. [Abstract] [Full Text] [PDF]
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F. J White, R. C Burghardt, J. Hu, M. M Joyce, T. E Spencer, and G. A Johnson Secreted phosphoprotein 1 (osteopontin) is expressed by stromal macrophages in cyclic and pregnant endometrium of mice, but is induced by estrogen in luminal epithelium during conceptus attachment for implantation. Reproduction, December 1, 2006; 132(6): 919 - 929. [Abstract] [Full Text] [PDF]
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G. Song, F. W. Bazer, G. F. Wagner, and T. E. Spencer Stanniocalcin (STC) in the Endometrial Glands of the Ovine Uterus: Regulation by Progesterone and Placental Hormones Biol Reprod, May 1, 2006; 74(5): 913 - 922. [Abstract] [Full Text] [PDF]
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F. J. White, J. W. Ross, M. M. Joyce, R. D. Geisert, R. C. Burghardt, and G. A. Johnson Steroid Regulation of Cell Specific Secreted Phosphoprotein 1 (Osteopontin) Expression in the Pregnant Porcine Uterus Biol Reprod, December 1, 2005; 73(6): 1294 - 1301. [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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T. E. Spencer, G. A. Johnson, R. C. Burghardt, and F. W. Bazer Progesterone and Placental Hormone Actions on the Uterus: Insights from Domestic Animals Biol Reprod, July 1, 2004; 71(1): 2 - 10. [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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