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Articles |
vß3 Integrin Expression in the Uterus and Conceptus During the Periimplantation Period1
a Departments of Animal Science, Veterinary Anatomy & Public Health,
b Center for Animal Biotechnology and Genomics,
c Institute of Biosciences and Technology, Texas A&M University System Health Science Center, College Station, Texas 77843-2471
d Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, Texas 77446
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The v and ß3 integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for vß3 integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is heavily phosphorylated, primarily on serine residues [2]. OPN binds primarily to vß3 integrin heterodimer on tissues via its Arg-Gly-Asp (RGD) sequence to promote cell-cell attachment and cell spreading via changes in cytoskeleton [3–7]. Upon freezing and thawing or treatment with proteases, the 70-kDa protein gives rise to 24-kDa and 45-kDa fragments that bind antigen and nonspecifically suppress T-helper lymphocytes, respectively [8, 9]. OPN has an Arg-Ser (RS) thrombin cleavage site; cleavage of the 301 amino acid OPN molecule at this site results in peptides of 137 and 164 amino acids, which may be further cleaved to the 24-kDa form [10]. OPN undergoes extensive posttranslational modification, especially in the glycosylation and phosphorylation of serine and threonine residues [2]. Increases in transcription of the OPN gene may be induced by interleukin-1 and -1ß, transforming growth factor ß1 (TGF-ß1), fibroblast growth factor (FGF), tumor necrosis factor (TNF), interferon gamma (IFN
), estrogen, progesterone, and 1,25-dihydroxyvitamin D3 [11–13]. Glucocorticoids may either inhibit (epithelial cells, bone, and kidney) or stimulate (cardiac muscle) expression of OPN [12, 14].
Originally isolated from bone [15], OPN has been found in epithelial cells and in secretions of the gastrointestinal tract, kidneys, thyroid, breast, and testes [10, 16–18], as well as in leukocytes, smooth muscle cells, and some tumor cells [19]. In the reproductive tract, OPN expression by secretory phase endometrial cells, invading trophoblast, decidual metrial glands, and placenta is temporally correlated with blastocyst invasion and placentation [17, 20].
The reported effects of OPN are to 1) stimulate cell-cell adhesion; 2) increase cell-extracellular matrix communication; 3) promote migration of immune cells, osteocytes, and tumor cells; 4) decrease cell death by reducing reactive oxygen species and nitric oxide production by injured tissues; 5) stimulate B cells to produce immunoglobulin; 6) induce changes in the phosphorylation state of focal adhesion kinase and paxillin; 7) stimulate phosphatidylinositol 3'-kinase activity; 8) alter intracellular calcium levels; and 9) affect tissue mineralization and promote calcium phosphate deposition in bone [3, 19, 21–28].
Expression of OPN mRNA increases in the uterine glands of pregnant ewes beginning at Day 13 [29], and protein is present in uterine flushings from Day 15 pregnant ewes [29]. The objectives of the present experiments were to 1) determine temporal and spatial expression of OPN protein in endometrium and uterine flushings during the ovine periimplantation period, 2) determine if OPN is expressed by trophectoderm, and 3) determine if its vß3 integrin receptor is present on both endometrium and trophectoderm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature western-range ewes of primarily Rambouillet breeding were observed daily for estrous behavior using vasectomized rams. After experiencing at least 2 estrous cycles of normal duration (16–18 days), ewes were assigned randomly on Day 0 (estrus/mating) to cyclic or pregnant status. Ewes assigned to pregnant status were mated to intact rams 3 times at 12-h intervals beginning at onset of estrus. 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 AG-239AG).
Experiment 1 Thirty-two ewes were ovariohysterectomized (n = 4 ewes/day) on Day 11, 13, or 15 of the estrous cycle or Day 11, 13, 15, 17, or 19 of gestation. At hysterectomy, uteri were flushed with 20 ml 0.9% NaCl, and flushes were frozen at -80°C. Pregnancy was verified by recovery of an apparently normal conceptus in uterine flushes. Several sections (1–1.5 cm) of uterine wall from the middle of each uterine horn were snap-frozen in Tissue-Tek Optimal Cutting Temperature compound (OCT; Miles Inc., Oneonta, NY), cooled in liquid nitrogen vapor, and stored at -80°C. The remaining endometrium was dissected from myometrium, frozen in liquid nitrogen, and stored at -80°C.
Experiment 2 Conceptuses were recovered from the uterine lumen of Day 17 pregnant ewes (n = 2) by flushing with 20 ml (37°C) DMEM/F12 culture medium (Sigma, St. Louis, MO) containing penicillin G (100 IU/ml), streptomycin (0.1 mg/ml), and amphotericin B (0.25 µg/ml; Gibco-BRL, Grand Island, NY). Endometrium was minced with scalpel blades into small pieces (2–3 cubic mm). Aliquots (50 mg) of minced endometrium were placed into culture dishes (100 x 15 mm) with 5 ml culture medium and cultured for 24 h with rocking (6 cycles/min) under an atmosphere of 45% nitrogen:5% carbon dioxide:50% oxygen. Intact conceptuses were washed several times in culture medium and then incubated in 10 ml culture medium under conditions identical to those for the endometrium. The culture medium was then harvested and centrifuged (3000 x g for 10 min at 4°C), and both conceptuses and culture medium were stored at -80°C.
Stainsall Detection of OPN Protein
Conceptus tissue was thawed and immediately homogenized in extraction buffer (10 mM Tris [pH 7], 1 mM EDTA, 1 mM dithiothreitol [DTT], 100 µg/ml PMSF) at a ratio of 1 g tissue per 5 ml buffer. Homogenates were sonicated for 30 sec with a Mini Ultrasonic Cell Disrupter (Sonics & Materials, Inc., Danbury, CT) and clarified by centrifugation (10 000 x g for 15 min at 4°C). Concentrations of protein in conceptus extracts were determined using a Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard. Protein (120 µg) in endometrial extracts was denatured in Laemmli buffer and separated in 10% gels by one-dimensional (1D) SDS-PAGE [30]. After electrophoresis, gels were fixed (10% acetic acid:45% methanol:45% H2O) for 1 h at room temperature, and then soaked in 50% methanol:50% H2O for 4 h with two changes of this solution to remove acetic acid. Phosphoproteins were detected by staining overnight at room temperature (in the dark) with Stainsall [31]: 5 mg Stainsall (1-ethyl-2-[3-(1-ethylnaphtho[1,2-D]thiazolin-2-ylidene)-2-methylpropenyl]naphtho-[1,2-D]thiazolium bromide; Sigma) in 5 ml formamide, 25 ml isopropanol, 0.5 ml 3 M Tris-HCl (pH 8.8), and 69.5 ml H2O. Gels were then soaked in 40% methanol:60% H2O, placed on a gel light-box, and photographed through transmitted white light.
Western Blot Analysis
Endometrium or conceptus tissue was thawed and homogenized, and the concentration of protein was determined as described above. Uterine flushings and explant culture medium were dialyzed against 3 liters of cold dialysis buffer (10 mM Tris, pH 8.2) in Spectrapor 3 dialysis membrane (cutoff, Mr 3500; Spectrum, Houston, TX) for 24 h with stirring at 4°C. Dialysis buffer was changed 3 times. Samples were then concentrated by snap-freezing in liquid nitrogen followed by lyophilization. Proteins were resuspended in 10 mM Tris (pH 8.2), and protein concentrations were determined as described above. Proteins in endometrial extracts (120 µg), uterine flushings (8 µg), or explant culture medium (20 µg) were denatured in Laemmli buffer, separated using 10% (total monomer) 1D-SDS-PAGE, and transferred to nitrocellulose. Blots were blocked overnight in TBST (20 mM Tris [pH 7.5], 137 mM NaCl, 0.05% Tween-20) containing 5% dried milk. Blots were washed 3 times for 5 min each in TBST and then incubated overnight, rocking at 4°C in a cocktail containing rabbit polyclonal antibodies against recombinant human OPN (LF-123 and LF-124; 5 µg/ml) [32] or normal rabbit serum (5 µg/ml) in TBST containing 2% dried milk. Blots were then washed 3 times for 10 min each in TBST and placed in goat anti-rabbit IgG-horseradish peroxidase conjugate (1:15 000 dilution of 1 mg/ml stock; KPL, Bethesda, MD) for 1 h at room temperature while rocking. Blots were washed 3 times for 10 min each in TBST, and immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, Rochester, NY).
Immunocytochemical Analysis
Frozen sections (4–8 µm) of endometrial and conceptus tissues were cut with a cryotome (Lipshaw Manufacturing Co., Detroit, MI) and mounted on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Conceptuses were present in the uterine lumen of sections from Day 19 pregnant ewes because these uteri were not flushed prior to uterine tissue collection. Sections were fixed in -20°C methanol for 10 min, permeabilized with 0.3% Tween-20 in 0.02 M PBS, and then blocked in antibody dilution buffer (2 parts 0.02 M PBS, 1.0% BSA, 0.3% Tween-20 [pH 8.0] and one part glycerol) containing 5% normal goat serum for 1 h at room temperature. Sections were rinsed in PBS and incubated overnight at 4°C with 20 µg/ml rabbit IgG against either human OPN [32], v integrin (Chemicon, Temecula, CA), or ß3 integrin (Chemicon). Following 3 rinses in PBS for 10 min each, sections were incubated with fluorescein-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA) for 1 h at room temperature and again washed in PBS 3 times for 10 min each. Sections were then overlaid with a coverslip and Prolong antifade mounting reagent (Molecular Probes, Eugene, OR), viewed with a Zeiss Photomicroscope III (Carl Zeiss Inc., Thornwood, NY) equipped with a fluorescein filter, and photographed on T-MAX 3200 film (Kodak).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoreactive 70-, 45-, and 24-kDa OPN forms were detected in endometrial extracts from cyclic and pregnant ewes with no apparent effect of day or pregnancy status (Fig. 2). Note that at least 3 immunoreactive bands > 80 kDa were increased in extracts from pregnant over cyclic ewes. However, the 45-kDa form of OPN increased in uterine flushings from pregnant ewes after Day 13, and the 70-kDa form increased after Day 15.
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Immunoreactive OPN was localized predominantly on the apical surface of the glandular epithelium (GE) and luminal epithelium (LE) in endometrium of Day 15 cyclic and pregnant ewes (Fig. 3, A–D). Immunoreactive OPN was present in endometrium from both cyclic (Day 11, 13, or 15) and pregnant (Day 11, 13, 15, 17, or 19) ewes. However, OPN appeared to be present in secretory granules in the uterine glands of pregnant ewes beginning at Day 15. The OPN protein was also detectable on Day 19 trophectoderm (Fig. 3E).
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Endometrial and conceptus explants from Day 17 pregnant ewes were cultured, and the medium was analyzed by Western blot using rabbit anti-rhOPN antiserum (Fig. 4). The 70-, 45-, and 24-kDa forms of OPN were present in culture medium from endometrial explants, while only 70- and 45-kDa bands were detected in culture medium from conceptus explants. These immunoreactive proteins were similar in size to proteins detected in ovine endometrial extracts and uterine flushings. Interestingly, the 45-kDa OPN form released from endometrium migrated more rapidly on SDS-PAGE than did OPN released by conceptus. Posttranslational modification of OPN phosphorylation in ROS 17/2.8 osteoblast-like cells by 1,25-(OH)2 D3 treatment has previously been reported [35]. Also, in rat long bone, 2 phosphorylation variants of OPN occur. The more phosphorylated form migrates more slowly on SDS-PAGE [35].
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Immunoreactive v and ß3 integrin subunits were localized to endometrial LE and GE from Day 19 pregnant ewes (Fig. 5). Immunoreactive v and ß3 integrin subunits were also detected in the trophectoderm of Day 19 ovine conceptuses (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 has been established in other species. OPN is expressed at high levels in decidual cells and endometrial GE of secretory-phase nonpregnant human uterus [16, 17], and cytotrophoblast of the chorionic villus is also immunoreactive for OPN [36, 37]. Although v and ß3 integrins are expressed constitutively by human endometrial epithelium, they increase during the periimplantation period [38], and epithelial ß3 expression is reduced in infertile women [39]. OPN has not been localized to the preimplantation human embryo; however, v and ß3 integrins are present on oocyte and early blastomere surfaces [40]. Similarly, OPN is co-expressed with vß3 integrins in invading cytotrophoblast, GE, and decidualizing stromal cells of the baboon [41]. In mice, injection of RGD peptides into the uterine lumen reduces implantation rate [42], probably through interference of ligand interaction with vß3 integrins. OPN is produced by mouse trophoblast, metrial gland cells of decidua, and placenta [20], whereas v and ß3 integrins are present in mouse endometrial epithelium [43]. Alpha v and ß3 integrin subunits have also been detected in porcine uterine epithelia and conceptuses [44].
Implantation is a complex and progressive process involving local tissue remodeling for which cytokines and adhesion molecules play essential roles in preparing the endometrium and trophectoderm. OPN can act as both a cytokine and an adhesion molecule. The presence of the GRGDS sequence in OPN suggests interaction with cell surface integrins [45]. OPN promotes attachment and spreading of cells in vitro. Rat osteosarcoma cells attach and spread on surfaces coated with OPN [46], and cultured human smooth muscle cells are capable both of adhering and migrating to OPN in a gradient-dependent manner [24]. It has been suggested that OPN and vß3 facilitate angiogenesis and vascular remodeling because these proteins are co-localized in smooth muscle cells following balloon angioplasty [47]. OPN binding to vß3 integrins also results in the rapid generation of intracellular signals. Phosphoinositide turnover and phosphatidylinositol trisphosphate production occur in response to osteoclast interaction with OPN [26]. Intracellular calcium is also effected by OPN, although cytosolic calcium may either increase [48] or decrease [3] with osteoclast exposure to OPN. OPN is also a cytokine of the immune system. It is secreted by activated T-lymphocytes [22], expressed by monocytes and macrophages during inflammation after tissue injury [49], and is present in granulated metrial gland cells, which are differentiated natural killer cells [50]. OPN binds the cell surface proteoglycan CD44 [9]. As such, OPN serves as a pro-adhesive cytokine immobilized on the luminal surface of endothelium to recruit leukocytes to the endothelial surface and trigger conversion of integrin molecule expression to an active adherence configuration [51]. Binding of OPN to vß3 integrins, expressed by endometrial cells and trophectoderm, may trigger a similar conversion.
Our working hypothesis is that the conceptus enhances expression, secretion, and/or proteolytic cleavage of OPN by uterine epithelium. This OPN then binds vß3 integrin heterodimer expressed by trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for attachment and superficial implantation. A model summarizing the present working hypothesis for the role of ovine uterine OPN in superficial implantation is presented in Figure 6. This model is supported by present results indicating both day and pregnancy-status effects on amounts of OPN mRNA in GE [29] and OPN protein in uterine flushings. The 45-kDa form of OPN increased in uterine flushings from pregnant ewes. This suggests differential post-translational processing of OPN in the pregnant uterus, perhaps due to effects of pregnancy on expression of uterine thrombin, which can cleave the 70-kDa form [9]. The 45-kDa form of OPN has greater binding affinity for ligand than the native 70-kDa form [52], thus adhesive interactions of OPN may be regulated by thrombin cleavage [53]. Prothrombin and thrombin are secreted by the rat uterus [54], but there are no reports of thrombin expression in the ruminant uterus. OPN protein is found on the apical surface of endometrial LE, is present in endometrial GE, and can be detected on trophectoderm. OPN mRNA, however, is present only in the GE of pregnant ewes, but not LE or trophectoderm [29]. Therefore, OPN is not synthesized by LE and trophectoderm, but presumably binds to these surfaces. This binding may be through RGD interaction with v and ß3 integrins that are present on ovine conceptus trophectoderm and maternal endometrial epithelium, or it may be to other components of the extracellular matrix besides integrins through sites other than the RGD. The immunoreactive OPN found in conceptus conditioned medium is likely the result of release of OPN that was bound to cell surface receptors on trophectoderm in utero.
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It should be noted that OPN mRNA is expressed by scattered immune cells localized beneath the LE within the stratum compactum on Days 1–9 of the estrous cycle [29] and that small amounts of OPN protein are present in uterine flushings of cyclic ewes (Fig. 2). Release of OPN by immune cells may explain the accumulation of OPN protein on LE of cyclic ewes, evident in Figure 3, and the consistent detection of OPN protein in uterine tissue extracts (Fig. 2). In a manner similar to immunoglobulins, OPN may be basally endocytosed by LE to eventually become exocytosed at the apical surface [55]. However, any number of proteins can pass through the pericellular space [56, 57]. In rats, estrogen acts on lateral junctions, causing an increase in the diameter of the pericellular spaces that results in fluid movement directly from the stromal compartment to the lumen. Because a wide array of epithelia express OPN [17], it is possible that OPN protein is present on the LE of cyclic ewes to stabilize some aspect of the conformation or maintenance of epithelial cells in general.
Temporal expression of OPN mRNA in GE is similar to that for progesterone-responsive uterine milk proteins [58] and the IFN
-responsive Mx protein [59]. The gene for OPN has a putative progesterone receptor response element in its promotor region, and progesterone up-regulates OPN mRNA in human cytotrophoblast [37]. The protein translated from this mRNA and secreted into the uterine lumen is presumably available as ligand for vß3 integrins. Although vß3 integrin heterodimer is present on Day 19 trophectoderm and endometrium, and is receptive to OPN binding, other OPN receptors may be involved. The integrin heterodimers vß1 and vß5 have affinities for the RGD motif of OPN that are similar to vß3 [60], and OPN can promote leukocyte adhesion through the 4ß1 integrin heterodimer [61]. Interestingly, 4 and ß1 integrins are expressed by porcine uterine LE during the period of maternal pregnancy recognition, and their expression increases in response to progesterone [62]. Also OPN contains a cryptic binding sequence recognized by the 9ß1 integrin heterodimer [53], and OPN binds the cell surface proteoglycan CD44 [23].
Collectively, results from rodent, human, and, now, ruminant models suggest that OPN secreted by epithelium binds integrins on luminal surfaces, and that this interaction is important for luminal epithelial communication with the external environment [17]. However, an essential role for OPN during implantation in mice has recently been discounted because OPN-null, mutant mice remain fertile [65], whereas inactivation of v integrin causes perinatal lethality [66]. Apparently no single ECM is essential to the implantation process in mice. Loss of one of these proteins can be compensated for by another ligand that binds a common receptor. Indeed redundancy in ECM/integrin expression and interaction may ensure the ability of the conceptus to attach to the uterine epithelium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Fuller W. Bazer, Department of Animal Science and Center for Animal Biotechnology and Genomics, 442D Kleberg Center, Texas A&M University, College Station, TX 77843-2471. FAX: 409 862 2662; fbazer{at}cvm.tamu.edu
3 Current address: Department of Animal and Veterinary Science, Agricultural Sciences Building, University of Idaho, Moscow, ID 83844-2330.
Accepted: May 24, 1999.
Received: February 19, 1999.
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vß3 integrin stimulates immediate cell signals in osteoclasts. J Biol Chem 1991; 266:20369–20374.vß3 potentiate bone resorption. J Biol Chem 1993; 263:19433–19436.vß3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest 1995; 95:713–724.vß3 by osteopontin stimulates phosphatidylinositol 3-hydroxy kinase activity. Endocrinology 1995; 136:2984–2992.[Abstract]v in human and mouse endometrium: expression in stromal and glandular cells. Mol Hum Reprod 1996; 2:527–534.vß3 integrin ligands increase intracellular [Ca++] in rat osteoclasts and mouse derives osteoclast-like cells. Am J Physiol 1994; 266:C376–381.9ß1 integrin. J Biol Chem 1996; 271:28485–28491.vß1 and vß5. J Biol Chem 1995; 270:26232–26238.4ß1 integrin. J Cell Sci 1998; 111:1165–1174.[Abstract]This article has been cited by other articles:
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  G. Song, M C. Satterfield, J. Kim, F. W Bazer, and T. E Spencer Progesterone and interferon tau regulate leukemia inhibitory factor receptor and IL6ST in the ovine uterus during early pregnancy Reproduction, March 1, 2009; 137(3): 553 - 565. [Abstract] [Full Text] [PDF]
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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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 K. A. Dunlap, D. W. Erikson, R. C. Burghardt, F. J. White, K. M. Reed, J. L. Farmer, T. E. Spencer, R. R. Magness, F. W. Bazer, K. J. Bayless, et al. Progesterone and Placentation Increase Secreted Phosphoprotein One (SPP1 or Osteopontin) in Uterine Glands and Stroma for Histotrophic and Hematotrophic Support of Ovine Pregnancy Biol Reprod, November 1, 2008; 79(5): 983 - 990. [Abstract] [Full Text] [PDF]
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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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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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M. C. Satterfield, K. A. Dunlap, K. Hayashi, R. C. Burghardt, T. E. Spencer, and F. W. Bazer Tight and Adherens Junctions in the Ovine Uterus: Differential Regulation by Pregnancy and Progesterone Endocrinology, August 1, 2007; 148(8): 3922 - 3931. [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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J. J Muniz, M. M Joyce, J. D Taylor II, J. R Burghardt, R. C Burghardt, and G. A Johnson Glycosylation dependent cell adhesion molecule 1-like protein and L-selectin expression in sheep interplacentomal and placentomal endometrium. Reproduction, April 1, 2006; 131(4): 751 - 761. [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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C A. Gray, K. A Dunlap, R. C Burghardt, and T. E Spencer Galectin-15 in ovine uteroplacental tissues Reproduction, August 1, 2005; 130(2): 231 - 240. [Abstract] [Full Text] [PDF]
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 S. Mirkin, M. Arslan, D. Churikov, A. Corica, J.I. Diaz, S. Williams, S. Bocca, and S. Oehninger In search of candidate genes critically expressed in the human endometrium during the window of implantation Hum. Reprod., August 1, 2005; 20(8): 2104 - 2117. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D Carpenter, T. H Welsh Jr, R. C Burghardt, L. J Spicer, and T. E Spencer The IGF system in the neonatal ovine uterus Reproduction, March 1, 2005; 129(3): 337 - 347. [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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M. H. Melner, N. A. Ducharme, A. R. Brash, V. P. Winfrey, and G. E. Olson Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes Biol Reprod, February 1, 2004; 70(2): 406 - 414. [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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 K. Nagaoka, H. Nojima, F. Watanabe, K.-T. Chang, R. K. Christenson, S. Sakai, and K. Imakawa Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon {gamma}-inducible Protein 10 kDa (IP-10), during Early Gestation J. Biol. Chem., August 1, 2003; 278(31): 29048 - 29056. [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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G. A. Johnson, R. C. Burghardt, M. M. Joyce, T. E. Spencer, F. W. Bazer, C. Pfarrer, and C. A. Gray Osteopontin Expression in Uterine Stroma Indicates a Decidualization-Like Differentiation During Ovine Pregnancy Biol Reprod, June 1, 2003; 68(6): 1951 - 1958. [Abstract] [Full Text] [PDF]
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A. H. King, Z. Jiang, J. P. Gibson, C. S. Haley, and A. L. Archibald Mapping Quantitative Trait Loci Affecting Female Reproductive Traits on Porcine Chromosome 8 Biol Reprod, June 1, 2003; 68(6): 2172 - 2179. [Abstract] [Full Text] [PDF]
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K.B.C. Apparao, M. J. Illera, S. A. Beyler, G. E. Olson, K. G. Osteen, M. H. Corjay, K. Boggess, and B. A. Lessey Regulated Expression of Osteopontin in the Peri-Implantation Rabbit Uterus Biol Reprod, May 1, 2003; 68(5): 1484 - 1490. [Abstract] [Full Text] [PDF]
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 J. M. Borthwick, D. S. Charnock-Jones, B. D. Tom, M. L. Hull, R. Teirney, S. C. Phillips, and S. K. Smith Determination of the transcript profile of human endometrium Mol. Hum. Reprod., January 1, 2003; 9(1): 19 - 33. [Abstract] [Full Text] [PDF]
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D. M. MacIntyre, H. C. Lim, K. Ryan, S. Kimmins, J. A. Small, and L. A. MacLaren Implantation-Associated Changes in Bovine Uterine Expression of Integrins and Extracellular Matrix Biol Reprod, May 1, 2002; 66(5): 1430 - 1436. [Abstract] [Full Text] [PDF]
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J. E. Garlow, H. Ka, G. A. Johnson, R. C. Burghardt, L. A. Jaeger, and F. W. Bazer Analysis of Osteopontin at the Maternal-Placental Interface in Pigs Biol Reprod, March 1, 2002; 66(3): 718 - 725. [Abstract] [Full Text] [PDF]
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 K. B. C. Apparao, M. J. Murray, M. A. Fritz, W. R. Meyer, A. F. Chambers, P. R. Truong, and B. A. Lessey Osteopontin and Its Receptor {alpha}v{beta}3 Integrin Are Coexpressed in the Human Endometrium during the Menstrual Cycle But Regulated Differentially J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4991 - 5000. [Abstract] [Full Text] [PDF]
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G. A. Johnson, F. W. Bazer, L. A. Jaeger, H. Ka, J. E. Garlow, C. Pfarrer, T. E. Spencer, and R. C. Burghardt Muc-1, Integrin, and Osteopontin Expression During the Implantation Cascade in Sheep Biol Reprod, September 1, 2001; 65(3): 820 - 828. [Abstract] [Full Text] [PDF]
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M. D. Stewart, G. A. Johnson, C. A. Gray, R. C. Burghardt, L. A. Schuler, M. M. Joyce, F. W. Bazer, and T. E. Spencer Prolactin Receptor and Uterine Milk Protein Expression in the Ovine Endometrium During the Estrous Cycle and Pregnancy Biol Reprod, June 1, 2000; 62(6): 1779 - 1789. [Abstract] [Full Text]
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M. J. Illera, E. Cullinan, Y. Gui, L. Yuan, S. A. Beyler, and B. A. Lessey Blockade of the {alpha}v{beta}3 Integrin Adversely Affects Implantation in the Mouse Biol Reprod, May 1, 2000; 62(5): 1285 - 1290. [Abstract] [Full Text]
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G. A. Johnson, T. E. Spencer, R. C. Burghardt, K. M. Taylor, C. A. Gray, and F. W. Bazer Progesterone Modulation of Osteopontin Gene Expression in the Ovine Uterus Biol Reprod, May 1, 2000; 62(5): 1315 - 1321. [Abstract] [Full Text]
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T. E. Spencer, A. Gray, G. A. Johnson, K. M. Taylor, A. Gertler, E. Gootwine, T. L. Ott, and F. W. Bazer Effects of Recombinant Ovine Interferon Tau, Placental Lactogen, and Growth Hormone on the Ovine Uterus Biol Reprod, December 1, 1999; 61(6): 1409 - 1418. [Abstract] [Full Text]
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G. A. Johnson, R. C. Burghardt, T. E. Spencer, G. R. Newton, T. L. Ott, and F. W. Bazer Ovine Osteopontin: II. Osteopontin and {alpha}v{beta}3 Integrin Expression in the Uterus and Conceptus During the Periimplantation Period Biol Reprod, October 1, 1999; 61(4): 892 - 899. [Abstract] [Full Text]
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