Immunohistochemical localization of integrin alpha V beta 3 and osteopontin suggests that they do not interact during embryo implantation in ruminants
© Kimmins et al; licensee BioMed Central Ltd. 2004
Received: 20 December 2003
Accepted: 28 April 2004
Published: 28 April 2004
It has been suggested that trophoblast attachment requires co-expression of integrin alpha V beta 3 and its ligand osteopontin at the fetal-maternal interface. Until now the expression patterns of integrin alpha V beta 3 and osteopontin in the pregnant bovine uterus were unknown. The objectives of this study were to localize integrin alpha V beta 3 and osteopontin in bovine and sheep endometrium during the periimplantation period and to compare the distribution patterns using antibodies that had not yet been tested in sheep.
Cell compartments within endometrial tissue sections were scored for immunohistochemical staining intensity and data were analyzed to determine the effects of day of pregnancy or cycle.
In pregnant bovine endometrium, integrin alpha V beta 3 was detected in luminal epithelium, stroma, myometrium and smooth muscle. A strong band of immunoreactivity was observed in the subepithelial stroma of intercaruncular regions, but there was reduced reactivity in the caruncles and glands. Bovine trophoblast did not express integrin alpha V beta 3 at any stage of pregnancy. In ovine endometrium a different pattern of staining for integrin alpha V beta 3 was observed. Reactivity was not present in the luminal epithelium or trophoblast. There was strong staining of the deep glands and no reactivity in the superficial glands. Osteopontin distribution was similar for sheep and cattle. For both species, apical staining was present on the luminal epithelium and glands and on embryonic tissues.
In ruminants, integrin alpha V beta 3 and osteopontin do not co-localize at the fetal-maternal interface indicating that these proteins could not interact to facilitate embryo attachment as has been proposed in other species.
Comparative studies of placentation in domestic animals show that implantation is similar among these species [1–4]. However differences in the molecular properties of receptive endometrium are just now being explored. Placentation in ruminants is categorized as non-invasive as chorionic cell migration into maternal tissue is restricted to the luminal epithelium [5, 6]. The preimplantation period in cattle and sheep is longer than in rodents, carnivores and primates. This period coincides with secretion of pregnancy supporting proteins from the glandular epithelium and trophoblast production of the pregnancy recognition factor, interferon-tau (IFN-τ) . Unique to ruminants are caruncles. These dense aglandular connective tissue regions are covered by columnar epithelium and their location defines where the finite placentae will form [1, 8]. The molecular determinants which prevent villus development in the intercaruncular endometrial tissue, or promote development in the caruncles, are not yet identified.
Integrins are heterodimeric glycoproteins that facilitate cell-cell and cell-extracellular matrix attachment and are key facilitators of cellular processes involved in tissue remodeling such as cell migration and de-adhesion [9–11]. In many species endometrial expression of integrins and their extracellular matrix (ECM) ligands is correlated with embryo attachment, and uterine differentiation during the receptive phase and pregnancy [12–17]. The most promiscuous integrin in terms of ligand interactions is integrin αvβ3, which binds Arg-Gly-Asp (RGD) motifs in fibronectin, osteopontin and laminin, among others [18, 19]. The affinity of integrins for their ligands is dependent on cell type, and a change in the activation state that is mediated by phosphorylation of the cytoplasmic domain (inside out signaling), and ligand availability (outside in signaling) [20, 21]. Integrin activity is augmented in the presence of growth factors such as the increased binding of integrin αvβ3 observed in smooth muscle cells in the presence of insulin-like growth factor I (IGF-1) .
Osteopontin was first described as a secreted 60-kDa phosphoprotein associated with bone ECM and as a lymphokine expressed by activated lymphocytes and macrophages . Expression has since been identified on the epithelium of many tissues such as kidney, breast, and the reproductive tract . Osteopontin expression and distribution differs between cycling and pregnant sheep; little secretion occurs in the glands of cycling animals while secretion increases from glandular epithelium during the periimplantation period . As a secreted protein of the ruminant uterus regulated by progesterone, osteopontin has been proposed to support conceptus growth and act as an adhesive between trophoblast and luminal epithelium via integrin αvβ3 [25, 26].
In mice, humans, baboons and sheep, it has been postulated that co-expression of integrin αvβ3 and its ligand osteopontin on uterine epithelium is required for trophoblast attachment and/or invasion; blocking integrin αvβ3 in mice prevents embryo implantation [15, 16, 25, 27]. Potential osteopontin receptors include CD44 and integrins αvβ3, αvβ1, α9β1, αvβ5 and α5β1 . In sheep, several of these integrin subunits are expressed on the apical surface of uterine epithelium and on conceptus trophoblast, although no change in their expression patterns has been observed between cyclic and pregnant ewes [16, 29]. Despite the similarity in placental anatomy of sheep and cattle this widespread distribution pattern of αvβ3 is not observed in cycling cattle. In cycling heifers, integrin αvβ3 is most strongly expressed in intercaruncular subepithelial stroma, and its expression is temporally regulated with the estrous cycle at least partially under the influence of the reproductive steroids [14, 30].
Until now the distributions of integrin αvβ3 and its ligand osteopontin have not been studied in the pregnant cow. The distribution of these proteins has been studied in cycling and pregnant sheep, however, the integrin αvβ3 results were unlike what we observed in cycling cattle. This is surprising since histologically placentation in sheep and cows is similar. The objectives of the present study were to localize the expression of integrin αvβ3 in bovine and sheep endometrium during the periimplantation period and to compare the distribution patterns using two monoclonal antibodies that had not been tested in sheep. In addition, the expression of osteopontin during the estrous cycle and periimplantation period in bovine and ovine endometrium was examined using an antibody to bovine osteopontin.
Materials and methods
All procedures performed were in accordance with the guidelines of the Canadian Council on Animal Care and were reviewed and approved by the Nova Scotia Agricultural College Animal Care and Use Committee. Sexually mature heifers of mixed beef breeds were synchronized to estrus using Estrumate® (500 μg cloprostenol, Schering Canada Inc.; QUE, Canada). Animals were observed for estrous behavior three times a day and had cycle lengths of 19 to 20 days. Uteri were collected at slaughter on days 0 (estrus), 1, 3, 6, 10, 14, 15, 16, 17, 18, 19 and 20 of the estrous cycle, or days 16, 18, 21, 24 or 30 of pregnancy with at least three animals at each day. To confirm estrous cycle stage, blood samples taken at heat and slaughter were assayed for progesterone by radioimmunoassay (RIA) using a Coat-A-Count kit (Diagnostic Products Corporation, Los Angeles CA).
Mature Rideau Arcott ewes were synchronized to estrus using intravaginal progesterone pessaries for 14 days followed by observation for estrous behavior in the presence of an intact ram. Ewes were randomly assigned to either cyclic or pregnant status, and mating to rams occurred over a 2-day period. Reproductive tracts were collected on days 13 and 17 of the cycle, and on day 13 of pregnancy (n = 5).
A single sample of mammary gland parenchyma was excised from a randomly selected lactating cow at a provincial abattoir to serve as a positive control tissue for osteopontin antibody reactivity.
Ovarian dating was performed at collection according to the criteria of Ireland  and pregnancy was established by the presence of an embryo or trophoblast in the uterine lumen. The ipsilateral horn was dissected into 1 cm3 cross-sectioned blocks and frozen in liquid nitrogen (-196°C) at the collection site before transfer to an ultra low temperature freezer (-80°C) for storage.
Two murine monoclonal antibodies specific to human integrin αvβ3 were used for immunohistochemical localization. Clone LM609 (Chemicon, Temecula, CA) targeted the functional binding region of the integrin αvβ3 complex of several species, including cattle , and clone BV4 (Abcam product number ab7167, Novus Biologicals, Littleton, CO) was directed against the full length integrin αvβ3 of endothelial cells. Osteopontin was localized using an anti-bovine milk osteopontin polyclonal antibody  kindly provided by Dr. G. Killian (Pennsylvania State University, Almiquist Research Centre, University Park, PA). Horseradish peroxidase (HRP) conjugated donkey-anti-rabbit IgG (Pierce, Rockford, IL) was used for detection of osteopontin. Negative controls were rabbit IgG (ICN Biomedical Inc., Aurora, ON, Canada) or purified mouse IgG (Chemicon) and were used at the same concentration as primary antibodies. Optimal dilutions of primary antibodies were empirically determined, and fixation and detection methods were selected based on the conditions that allowed for maximum specific immunostaining.
To determine reactivity of bovine anti-milk osteopontin antibodies with bovine and ovine endometrium, 0.3 g samples of endometrial and mammary gland tissues were thawed and homogenized in 2 ml extraction buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 10% v/v glycerol, 1% v/v NP-40, 0.5 mM PMSF and 10 μg/mL aprotinin). Concentrations of protein were determined using a Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard. Following denaturation in Laemmli buffer, proteins (200 μg) were separated using 10% SDS-PAGE and blotted to PVDF membrane. Blots were blocked in PBS containing 0.05% (v/v) Tween-20 (PBS-T) and 2% (w/v) BSA for 60 min at RT. Blots were washed 3 times for 5 min each in PBS-T, then incubated for 2 h in PBS-T containing 1% BSA and 2.5 μg/ml anti-bovine osteopontin or rabbit IgG (negative control). Blots were washed 3 times for 5 min each, then incubated for 1 h in PBS-T containing 1% BSA and donkey anti-rabbit IgG conjugated to horseradish peroxidase. Following 3 washes for 5 min each in PBS-T, and one wash in PBS, immunoreactive proteins were detected using a chemiluminescent substrate (ECL®, Amersham Biosciences Inc., Baie d'Urfe, Quebec, Canada).
Immunoperoxidase staining for integrins was performed on 5 to 8 μm cryostat cross-sections of endometrium. Serial sections were mounted on 3-aminopropyl-triethoxysilane- coated-Superfrost Plus® slides (Fisher Scientific, Whitby, ON, Canada), then fixed in acetone for 10 min and air dried overnight. Cut sections were stored at -80°C until use. Sections were blocked with 2% w/v BSA in PBS (pH 7.2 to 7.4) for 30 min. Primary antibodies were diluted in 2% w/v BSA in PBS and applied at concentrations of 10 μg/ml for anti-osteopontin, 0.6 μg/ml for anti-αvβ3 clone LM609 (Chemicon), and 0.2 μg/ml for anti-αvβ3 clone BV4 (Novus Biologicals). All primary antibodies were allowed to bind for 2 h, then the slides were washed in PBS 3 times for 3 min each, and secondary antibody was applied and allowed to react for 45 min followed by washing in PBS. For amplification of integrin αvβ3 signal, the Vectastain Elite ABC kit® (Vector laboratories, Burlingame, CA) was used. To prevent non-specific staining, avidin (reagent A) was used at half the recommended concentration. Immunostaining was visualized using the chromogen metal enhanced diamino benzidine (integrin αvβ3; Pierce) or 3-amino-9-ethylcarbazole (osteopontin; Sigma, Saint Louis, MO). Slides were counterstained with Mayers Hematoxylin (Electron Microscopy Sciences, Fort Washington, PA) and coated with Aquaperm® mounting media (Fisher). Slides were viewed on a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany) and photographed on Ektachrome 160T tungsten film (Eastman Kodak, Rochester, NY). Slides were scanned and images assembled in Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA). All experiments were replicated at least three times for each animal on different days.
Cell compartments within sections were scored by two independent observers on a six point scale (0-negative, 1-very weak, 2-weak but clearly positive, 3-intermediate, 4- high, and 5- very high) for staining intensity. Data were checked for normalcy using the univariate procedure in SAS™ (Statistical Analysis Software Version 8, SAS Institute Inc. Cary NC) and log transformed if transformation corrected to a normal distribution. Normal data were analyzed using a general linear models procedure to examine the effect of day on staining intensity, and the Duncan's multiple range test was used to compare population means. Nonparametric statistical comparisons between day of cycle, day of pregnancy or cycle stage (stage1-metestrus, stage 2-early diestrus; stage 3-late diestrus; stage 4-proestrus and estrus) were made by the Kruskal-Wallis test of population medians . When significant effects were observed, Mann-Whitney's two sample rank test  was used to test the equality of population medians. The selected alpha level was 0.05.
Immunohistochemical localization of integrin αVβ3 in early pregnant bovine endometrium
Immunohistochemical localization of integrin αVβ3 in ovine endometrium
Osteopontin distribution in cyclic bovine endometrium
Osteopontin distribution in early pregnant bovine endometrium
The distribution of osteopontin was similar in cryosections prepared from pregnant animals to those prepared from cyclic animals, although reactivity scores for the apical surface of the luminal and glandular epithelium decreased with advancing pregnancy (Fig. 4D,4E,4F,4G). Staining was lowest in glandular epithelium in samples from day 30 of pregnancy in comparison to other pregnancy stages examined (P < 0.05;Fig. 4G and Fig. 5B). Trophoblast was weakly reactive, and in many sections from day 24 and day 30 pregnant animals, extraembryonic membrane luminal to trophoblast, probably allantois, expressed moderate to high levels of osteopontin (Fig. 4E,4F,4G).
Distribution of osteopontin in ovine endometrium
In pregnant bovine endometrium reactivity scores for integrin αVβ3 were always highest in the stroma underlying the luminal epithelium of intercaruncular tissues, and weak or no staining was detected in the caruncles or stroma underlying the glands. This concurs with our previous study of cycling cattle . In cows, integrin αVβ3 may be involved in constraining trophoblast and endometrium in the intercaruncular regions, since the growth of both tissues to form the chorionic villi and intervening maternal septae occurs only in the caruncles. We hypothesize that subepithelial stromal integrin αVβ3 is involved in regulating the behavior of the overlying epithelium and adjacent trophoblast. Such a signal from stroma underlying the uterine epithelium is logical, since the epithelium itself is modified rapidly after attachment by trophoblast binucleate cell migration and fusion with maternal epithelium to form giant cells . This migration is maximal about day 24, then subsides to the extent that trinucleate, rather than giant cells, form and there is some regeneration of the luminal epithelium . The observation in the current study that this is preceeded by increased expression of stromal integrin αVβ3 provides support for a role of this integrin in constraining invasion.
A comparison of the distribution of integrin αvβ3 and osteopontin in ovine and bovine endometrium.
Apical surface LE
In cattle, the transient disappearance of subepithelial integrin αVβ3 on day 16 of the cycle, but not day 16 of pregnancy, suggests a possible role for this integrin in the onset of luteolysis. Our more recent studies have shown that this temporary downregulation may be mediated by estrogen , a known stimulator of the luteolytic mechanism . Expression of IGF-1, like integrin αvβ3, is high at estrus and throughout the cycle is restricted to the subepithelial stroma . Furthermore, in tissue from day 16 cycling cows expression of IGF-1 mRNA in this region reached a cycle low which preceded the proestrous increase beginning on day 17 . Similar patterns of IGF-1 protein expression were observed by Ohtani and coworkers  in cyclic heifers, although the downregulation was observed on day 14–15, rather than day 16–17, consistent with the shorter estrous cycle generally observed in heifers compared to cows. Again, similar to what was observed for integrin αvβ3 in the current study, Robinson et al.  found that pregnant cows were more likely to express stromal IGF-1 mRNA on day 16 than nonpregnant animals, although the results were not conclusive. IGF-1 mRNA expression in sheep was evident in caruncular as well as intercaruncular stroma . In other cell systems IGF-1 and integrin αvβ3 are known to have cooperative signaling roles . Both proteins have been linked to prostaglandin production via cyclooxygenase 2 [40–43], which has been localized to the luminal epithelium of cows . Differences in expression patterns of IGF-1 and integrin αvβ3 may mark a subtle difference in the mechanisms regulating prostaglandin release between sheep and cows.
In this study integrin αVβ3 was absent from bovine trophoblast, but was expressed at moderate intensity in the glandular epithelium, and in the luminal epithelium until it was modified by binucleate cell migration. In ovine endometrium, glandular epithelium exhibited a strong pericellular expression but no reactivity was observed in the luminal epithelium (Table 1). The pattern of integrin αVβ3 expression we observed in ovine endometrium was not only different from cows, but also from what has previously been reported for the ewe . This difference in localization patterns could be attributable to the antibodies used. In this study we used two antibodies that targeted different epitopes of the integrin heterodimer αVβ3., whereas the earlier study used antibodies targeting the individual integrin subunits, αV and β3. Johnson et al.  reported antibody reactivity for these subunits on the apical surfaces of uterine epithelium and trophoblast. Monomeric integrin subunit protein is not normally expressed at the cell surface [9, 10], so it is possible that other integrin heterodimers, such as integrins αVβ1 or αVβ5, were being detected.
Moderate but consistent osteopontin expression was observed in uterine epithelium and trophoblast of sheep and cattle. It has been proposed that osteopontin or another matrix molecule could act as a "bridge" for integrin αVβ3, connecting trophoblast and uterine epithelium during embryo attachment [27, 45, 46]. Integrin αVβ3 is thought to be the functionally most important receptor for osteopontin in bone and vascular tissue [47, 48], and experiments with the Ishikawa endometrial cell line suggest that this is also true for human endometrial cells . Since in this study integrin αVβ3 was not detected on trophoblast or the apical surface of the luminal epithelium of either sheep or cattle, another integrin may be anchoring the bridge. The integrins αVβ1, αVβ5, α4β1, α8β1 and α9β1, as well as CD44, can bind osteopontin [28, 29]. Bovine trophoblast and uterine epithelium express β1 integrins [17, 49], but the alpha subunits associated with β1 in these tissues have not been fully characterized. We have not been able to detect the integrin α4 subunit at the fetomaternal interface , therefore it is unlikely that integrin α4β1 is the interaction partner for osteopontin in bovine uterine epithelium.
The pattern of expression of osteopontin was generally similar in bovine endometrium to what has been observed in other species, including ovine (Table 1) and human endometrium [13, 25, 51]. However, unlike what has been observed in those species, the scores for osteopontin reactivity in luminal epithelium decreased during the luteal phase in cyclic heifers, and strong apical reactivity was not observed on trophoblast, luminal or glandular epithelial cells during implantation as has been observed in sheep [current study, ]. Although the human and murine promoters for osteopontin possess progesterone response elements, the role of progesterone is complex in endometrium. The downregulation we observed occurred when progesterone levels are high but expression of progesterone receptors in epithelium is low [52, 53]. Sheep infused with progesterone showed increased osteopontin mRNA in the glands, however no progesterone receptors were expressed by these cells . These experiments suggest that progesterone may act in concert with local modulators of osteopontin expression, which include hepatocyte growth factor, transforming growth factor β 1 and epidermal growth factor [15, 47, 54], perhaps via a stromal mediated paracrine mechanism.
In human placenta, the chorionic villus cytotrophoblasts but not the overlying syncytiotrophoblast  produce osteopontin. It has been proposed that differentiated syncytiotrophoblasts control a paracrine communication loop with underlying cytotrophoblasts by releasing progesterone to stimulate secretion of osteopontin, which in turn binds and activates integrin αvβ3 on the adjacent syncytial trophoblast . Engagement of integrin αvβ3 may then signal increased adhesion, or other intracellular events that promote normal tissue function. In the cow, there is no syncytial trophoblast per se but there are syncytial plaques and trinucleate cells formed by migration and fusion of trophoblast binucleate cells with maternal luminal epithelium . These are exposed to progesterone from the trophoblast  and showed reactivity to osteopontin antibodies in the current study. However, integrin αvβ3 was not expressed on trophoblast and expression decreased in the fetomaternal hybrid epithelium as it formed from day 24 to 30. On the other hand the underlying stroma of the hybrid epithelium showed increased expression of integrin αvβ3 through this period. Whether this is related to a paracrine loop similar to what occurs in human placenta and/or a response to extracellular matrix changes occurring at this time  remains to be determined.
Integrin αvβ3 in the subepithelial stromal cells of bovine endometrium may have a role in the epithelial-stromal signaling events that regulate remodeling of pregnant epithelium and trophoblast during attachment in the cow. The endometrial distribution of integrin αvβ3 we observed in sheep endometrium differed from the distribution in cows. Apparently at the molecular level, the signaling mechanisms between the epithelium and stroma that lead to formation of the synepitheliochorial placenta of sheep are not the same as in cows. At the fetomaternal interface in both species, the distribution patterns of integrin αvβ3 and osteopontin did not overlap. This suggests that, unlike in primates and rodents, in ruminants these proteins could not act together to facilitate embryo attachment.
The authors thank Dr. Gary Killian, Pennsylvania State University, for his generous gift of antibody to bovine osteopontin and Dr. Brian K. Hall, Dalhousie University, for his helpful insights and guidance. We also appreciate the assistance of staff in the NSAC ruminant section, Brookside Abattoir, Westmorland Institute and HUB Meat Packers Inc. with the animals. This work was financed by an NSERC postgraduate scholarship to S. K. and grants to L.M. from the Dairy Farmers of Canada, the Nova Scotia Technology Development Program and NSERC.
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