Role for the endometrial epithelialprotein MFG-E8 and its receptorintegrin avb3 in human implantation:results of an in vitro trophoblastattachment study using establishedhuman cell lines

Carla Schmitz, M.D.,a Liang Yu, Ph.D.,a Silvina Bocca, M.D., Ph.D.,a Sandra Anderson, B.S.,a

Jo~ao Sabino Cunha-Filho, M.D.,a Bhaskara S. Rhavi, Ph.D.,b and Sergio Oehninger, M.D., Ph.D.a

a Department of Obstetrics and Gynecology, Eastern Virginia Medical School, The Jones Institute for ReproductiveMedicine, Norfolk, Virginia; and b Department of Mathematics and Statistics, Old Dominion University, Norfolk, Virginia

Objective: To investigate the role of MFG-E8 and its receptor integrin avb3 in the attachment of trophoblast cells to the endometrialepithelium.Design: Experimental in vitro study.Setting: Academic center.Patient(s): None.Intervention(s): By using a well-differentiated endometrial adenocarcinoma cell line (Ishikawa cells) and choriocarcinoma humantrophoblast cells (Jar cells), an in vitro assay mimicking human implantation was established. To investigate the impact of blockingMFG-E8 and integrin avb3, we pretreated the cell lines with antibodies against those proteins at different concentrations before theattachment assay.Main Outcome Measure(s): Attachment rate of Jar spheroids to the epithelial cell monolayer.Result(s): Pretreatment of Ishikawa cells with anti-MFG-E8 antibody caused a dose-dependent and significant inhibition ofattachment. On the other hand, pretreatment of Jar spheroids did not result in a significant effect on the attachment rate.Pretreatment of Ishikawa cells as well as Jar spheroids with anti-integrin avb3 antibodies resulted in a dose-dependent, significantinhibition of attachment.Conclusion(s): This study showed that blocking MFG-E8 and its receptor integrin avb3 in Ishikawa cells diminishes Jar spheroid

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attachment. Moreover, blocking integrin avb3 in the trophoblastic cells also diminished theirattachment to the Ishikawa monolayer. (Fertil Steril� 2014;101:874–82. �2014 by AmericanSociety for Reproductive Medicine.)Key Words: Attachment, integrin avb3, implantation, human endometrium, Jar spheroids,MFG-E8

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Received March 29, 2013; revised November 29, 2013; accepted December 5, 2013; published onlineJanuary 11, 2014.

C.S. has nothing to disclose. L.Y. has nothing to disclose. S.B. has nothing to disclose. S.A. hasnothing to disclose. J.S.C.-F. has nothing to disclose. B.S.R. has nothing to disclose. S.O. hasnothing to disclose.

C.S.'s present address is: Postgraduate Program, Medical Clinics of the Universidade Federal do RioGrande do Sul, Porto Alegre, Brazil.

J.S.C.-F.'s present address is: Department of Obstetrics and Gynecology, Universidade Federal do RioGrande do Sul, Porto Alegre, Brazil.

C.S. and L.Y. contributed equally to this work.Reprint requests: Sergio Oehninger, M.D., Ph.D., The Jones Institute for Reproductive Medicine, 601

Colley Ave., Norfolk, Virginia 23507 (E-mail: Oehninsc@evms.edu).

Fertility and Sterility® Vol. 101, No. 3, March 2014 0015-0282/$36.00Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc.http://dx.doi.org/10.1016/j.fertnstert.2013.12.015

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I t is well known that implantationrequires a receptive endometrium, anormal and functional embryo at

the blastocyst stage of development,and a synchronized dialogue betweenthem (1). Nevertheless, there are manyunknown factors that play an importantrole in this process (2, 3). Because humanimplantation studies in vivo are verylimited (mainly owing to ethicalreasons), have poor reproducibility, and

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lack capacity formanipulation/intervention, in vitro studies arethe most common ones performed (4–6).

Integrins are a diverse family of glycoprotein receptorsexpressed on the cell membrane as heterodimeric a and b sub-units involved in both cell–cell and cell–substratum adhesion(7, 8). One particular integrin, the avb3, has been studied inmany physiologic/pathologic processes, and it has beensuggested to be involved in implantation across manymammalian species, including mouse, rat, pig, rabbit, andhuman (8–12). In women, an adequate integrin avb3 receptorexpression seems to be necessary for a receptive endometrium(7, 13–15). Integrin's hallmark is the ability of individualfamily members to recognize multiple extracellular matrixligands, such as bone matrix proteins, collagens, fibronectins,fibrinogen, laminins, thrombospondins, vitronectin, and vonWillebrand factor (16, 17). Despite the structural diversity oftheir ligands, integrins recognize a core ligand motif centeredon an acidic residue (18).

Recently, our group reported for the first time on a novelendometrial protein, milk fat globule-epidermal growthfactor (MFG-E8). Initially it was found that MFG-E8 was up-regulated in the human endometrium during the window ofimplantation in a microarray study comparing the geneexpression profile during the mid- vs. early luteal phase (19).We showed that MFG-E8 protein is mostly expressed in theendometrial epithelial cells and during the window of implan-tation in healthy ovulatory women, both in luminal and glan-dular epithelial cells, and with intense staining at both apicaland basal cellular compartments (20). The MFG-E8 receptor,integrin anb3, was present in the epithelial and stromal com-partments, with cycle-dependent and coincident peak expres-sions at mid-secretory phase, as previously described (20, 21).

The secreted glycoprotein MFG-E8 (also known as lactad-herin [SED1] and breast antigen 46 [BA46]) was initiallydescribed as a component of the milk fat globule membrane,which is mainly secreted in micro-vesicles (22). It has alsobeen suggested as a tumor marker in breast carcinomas (23,24). Human MFG-E8 is a 46-kDa glycoprotein peripherallyassociated with the cell membrane having two N-terminalepidermal growth factor-like repeats, one of which includesan arginine-glycine-aspartic acid motif that serves as theligand to its anb3 integrin receptor, facilitating cell adhesionand signal transduction. The two C-terminal discoidin C1–C2domains are homologous to coagulation factors V and VIIIand are responsible for binding to cell membrane phospho-lipids or cell surface carbohydrate moieties as well as to theextracellular matrix (25, 26).

MFG-E8 has a variety of functions in many extrauterinetissues related to apoptosis, cell adhesion and remodeling,neovascularization, and immunomodulation. In particular,it promotes phagocytosis of apoptotic cells by anb3/b5integrin-expressing phagocytes (27) as well as endothelialand epithelial cells (28, 29). In systemic endothelial cells,MFG-E8 binding to anb3/b5 integrins promotes vascularendothelial growth factor–induced survival and proliferation,leading to angiogenesis (30). It stimulates cell–cell andcell–extracellular matrix adhesion during sperm–oocyteinteraction (25, 31). In intestinal, mammary gland, andepididyimal epithelium, MFG-E8 regulates migration of

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epithelial cells (25, 32, 33). It also protects againstsymptomatic rotavirus infection (34).

On the basis of recent work from our laboratories, wepostulated MFG-E8 as a novel glycoprotein with key roles inthe regulation of endometrial function (20). We showed thatMFG-E8 protein was up-regulated by prolactin in primaryendometrial epithelial cell cultures. This finding supports amodulatory role for prolactin as a stromal–epithelial paracrinefactor controlling MFG-E8 production. We also demonstratedthat in epithelial Ishikawa cells, MFG-E8 protein secretion isup-regulated by hCG and secreted via microparticles (35).We also confirmed that MFG-E8 has proapoptotic activity inhuman endometrial stromal cells, suggesting that this mole-cule acts as a paracrine factor (36). Furthermore, we reportedthat MFG-E8 protein is highly expressed in human chorionicvilli at all trimesters of gestation (in both cytotrophoblasts andsyncytiotrophoblasts) and in murine implantation sites (37).Finally, we presented new evidence that recombinantMFG-E8 modulates endometrial endothelial cell proliferationand adhesion under in vitro conditions (38).

In the present experiments we investigated the role ofMFG-E8 and integrin avb3 in the attachment of trophoblastcells to the endometrial epithelium, to further define its func-tional roles during the early implantation events. We used anin vitro model with Jar spheroids and Ishikawa cells. TheIshikawa cell line is a human, well-differentiated endometrialadenocarcinoma cell line recognized as a well-characterizedmodel for studying endometrial function as a surrogate forepithelial endometrial cells (39, 40, 41). Jar cells are derivedfrom a human choriocarcinoma, tend to grow as multicellularspheroids when cultured under appropriate conditions, andare largely used in blastocyst/trophoblast attachment studies(1, 42–45). Ishikawa cells/Jar spheroids interactions havebeen demonstrated to represent an adequate model forstudying early implantation events (1, 40, 41).

MATERIALS AND METHODSCell Lines and Culture Conditions

Current work in our laboratories using endometrial tissueand cells has been approved by the Eastern Virginia MedicalSchool institutional review board (#04-10-FB-0279 and#09-07-EX-0153, active projects). In this particular studyconsent was not required because the use of established cellslines is considered exempt. The human endometrial adeno-carcinoma cell line Ishikawa was generously provided byC. Lockwood, M.D. from Yale University. Cells used in theseexperiments were from passage 10 and were characterized byimmunostaining as cytokeratin-18þ and vimentin�, andwere also estrogen receptor aþ and bþ, as previously re-ported by us (data not shown) (4, 35). Ishikawa cells werecultured in Dulbecco's modified essential medium/F12containing 100 U/mL penicillin and 100 mg/mLstreptomycin (Gibco) and supplemented with 5% fetalbovine serum (Atlanta Biological) in 75-mL flasks at 37�Cin a humid atmosphere with 5% CO2. The cells were thenplated in four- well plates (Thermo Fisher Scientific) untilreaching 100% confluence and used in subsequentexperiments.

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The human choriocarcinoma Jar cell line was purchasedfrom ATCC. Cells used in these experiments were frompassage 6 and were characterized by immunostaining ascytokeratin-7þ and hCGþ, as previously reported by us (datanot shown) (4, 46). The Jar cells were cultured in RoswellPark Memorial Institute 1640 medium (Gibco) with 5% fetalbovine serum (Atlanta Biological) and 100 U/mL penicillinand 100 mg/mL streptomycin (Gibco). After reaching 80%confluence, Jar cells were detached with 0.05% trypsin(Gibco). A single-cell suspension was transferred into anagarose-coated Petri dish and cultured for 2 days to formspheroids. All cultures were conducted in an incubator with5% CO2 at 37�C. The diameter of Jar spheroids as measuredby inverted microscopy was between 150 and 200 mm (4, 46).

Immunocytochemistry

The Dako EnVisionþ System-HRP kit (DAB, Dako) was usedto perform immunocytochemistry. Ishikawa cells weregrown on glass coverslips and fixed with methanol at�20�C for 10 minutes. After washing with phosphate-buffered saline, endogenous peroxidase activity wasremoved with 3% hydrogen peroxide for 10 minutes.Nonspecific binding sites were blocked with 2% normalgoat serum for 30 minutes at room temperature. Cells wereincubated overnight at 4�C with the following mouse mono-clonal primary antibodies prepared in 1% normal goat

FIGURE 1

Immunohistochemistry of Ishikawa and Jar cells (original magnification,�20cells stained with anti-integrin avb3 antibodies. (C) Jar cells stained withantibodies. Insets: NC ¼ respective negative controls.Schmitz. MFG-E8 and integrin avb3 implantation roles. Fertil Steril 2014.

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serum: anti-MFG-E8 (Abcam, catalog no. ab17787) at a1:1,000 dilution, and anti-anb3 integrin (Abcam, catalogno. ab78289) at a 1:10 dilution. The cells were then incubatedwith a goat anti-mouse secondary antibody labeled withhorseradish peroxidase (Dako) for 30 minutes, then with3,3-diaminobenzidine chromogen solution and substratebuffer for 5–10 minutes at room temperature. Slides werecounterstained with Mayer's hematoxylin (Sigma) thendehydrated with ethanol, cleared in xylene, and mountedwith mounting media. Negative controls included sectionsthat were treated with a nonimmune IgG control antibody(normal mouse ascites, clone NS-1; Sigma) at a similar con-centration as primary antibodies. Representative imageswere photographed with an Olympus microscope using anOlympus Q-color 3 camera (20).

Immunofluorescence Detection of MFG-E8 andIntegrin anb3

Cells were cultured on glass coverslips and fixed with coldmethanol, rinsed in phosphate-buffered saline, and blockedwith normal goat serum at room temperature. Antibodiesused for immunofluorescence were primary anti-MFG-E8(1:1,000) and anti-integrin avb3 (1:50) antibodies as shownabove, with a fluorescein isothiocyanate (FITC)-conjugatedsecondary goat anti-mouse antibody (Santa Cruz Biotech-nology). Coverslips were mounted onto glass slides using

0). (A) Ishikawa cells stained with anti-MFG-E8 antibodies. (B) Ishikawaanti-MFG-E8 antibodies. (D) Jar cells stained with anti-integrin avb3

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FIGURE 2

Immunofluorescence of Ishikawa and Jar cells (FITC, green; DAPI-counterstained nuclei, blue; merged, original magnification, �630). (A, B)Ishikawa cells stained with anti-integrin avb3 (A) and with anti-MFG-E8 (B). (C, D) Jar cells stained with anti-integrin avb3 antibody (C) andwith anti-MFG-E8 antibody (D). Note predominantly membrane immunolocation of staining.Schmitz. MFG-E8 and integrin avb3 implantation roles. Fertil Steril 2014.

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mounting medium and were visualized using a fluorescencemicroscope, Olympus BX50, equipped with a DP-70 Olympuscamera. Staining with 40,6-diamidino-2-phenylindole (DAPI)was performed to visualize nucleus of all cells (VECTASHIELDmounting medium with DAPI, Vector Laboratories).

In addition to standard cell fixation, we also performedstaining of live, nonfixed cells following techniques describedpreviously (47). Jar cells were placed and cultured in eight-well chamber slides until 70%–80% confluence, then wereincubated with primary antibodies diluted in complete growthmedium in the incubator for 2 hours, followed by washingthree times. An FITC-conjugated secondary goat anti-mouseantibody was incubated with cells for 2 hours at room temper-ature. A nonimmune IgG control antibody (normal mouseascites; clone NS-1, Sigma) was used as negative control.Mounting and analysis were the same as above.

Protein Extraction and Immunoblotting

For total lysate protein extraction, Ishikawa and Jar cellsgrown in T-75 flasks were harvested in radio immunoprecipi-tation assay lysis buffer supplemented with Halt protease in-hibitor cocktail (Pierce Thermo Scientific). Samples weresonicated, followed by centrifugation at 10,000 rpm for 10 mi-nutes at 4�C. After centrifugation, supernatants were collectedfor total protein quantification by bicinchoninic acid protein

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assay using bovine serum albumin as the standard (Sigma-Al-drich). The protein extracts were loaded and were separated byelectrophoresis on 10% sodium dodecyl sulfate–polyacryl-amide gel, followed by electrotransfer onto a polyvinylidenefluoride membrane. Immunoblotting was performed usingstandard techniques as previously reported by us (20, 35–38).The antibody used was anti-MFG-E8 (Abcam). Protein bandswere detected by exposure to a chemiluminescent substrate.

Jar Spheroids–Ishikawa Cells Attachment Assay

The coculture system was set up into wells in four-well plates(Nunc). Ishikawa cells were plated as a monolayer; in parallel,20 Jar spheroids were placed in a Petri dish and carefullytransferred into each well with a pipette tip and visualizedunder the microscope. Depending on the experiment, eitherthe Ishikawa cells or the Jar spheroids were subjected to anti-body pretreatment as presented below, and results comparedwith untreated controls. After 30 minutes of coculture of Jarspheroids and Ishikawa cell monolayer, the four-well platewas transferred into a bucket of a centrifuge (the top facingthe bottom of the swinging bucket) and then centrifuged at1,000 rpm for 5 minutes to remove unbound and any looselyattached spheroids. The attached Jar spheroids were countedunder regular optic microscopy (�40) (4, 46). The number ofseeded spheroids was constant (n ¼ 20) per tested antibody

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dose (and control). There were 10 replicates per investigatedcondition (antibody dose and control) that were performedon three different experimental days. Additionalexperiments were performed in a similar fashion using anegative control with pretreatment with a nonimmune IgGcontrol antibody (normal mouse ascites; clone NS-1 Sigma)at equal concentration as primary antibodies and comparedwith nontreated (non-IgG) conditions.

Treatment Conditions

First, Ishikawa cells in the four-well plates were pretreatedwith the anti-MFG-E8 antibodies at concentrations of 1 mg/mL and 2 mg/mL or with the anti-integrin avb3 antibodiesat concentrations of 1 mg/mL, 10 mg/mL, and 50 mg/ml for2 hours, after which Jar spheroids were seeded on top of themonolayer for the attachment assay. When Ishikawa cellswere treated, no treatment was added to Jar spheroids for30 minutes.

Second, Jar spheroids were pretreated with either anti-MFG-E8 antibodies at concentrations of 1 mg/mL and 2 mg/mL or with anti-integrin avb3 antibodies at a concentrationof 50 mg/mL, for 15 minutes, after which Jar spheroids wereseeded on top of the (untreated) Ishikawa cell monolayer forthe attachment assay.

The anti-MFG-E8 and anti-integrin avb3 antibody dosesused for treatment were chosen on the basis of an in vitro hu-man endometrial endothelial cell adhesion and proliferation

FIGURE 3

(A, B) Immunofluorescent images of live, nonfixed Jar cells (FITC, green; DA(A) Jar cells stained with anti-integrin avb3. (B) Jar cells stained with antiattached to the epithelial monolayer of Ishikawa cells in the in vitro attacImmunoblotting of MFG-E8 in Ishikawa cells and Jar cells. MW ¼ molelocation for both cell types.Schmitz. MFG-E8 and integrin avb3 implantation roles. Fertil Steril 2014.

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study previously performed, in which these concentrationsshowed stimulatory effects (38).

Statistical Analysis

Ten replicates were run in three experiments, and 20 spher-oids were seeded per replicate, for a total of 200 spheroidstested per investigated dose and for control. Overall effectsof antibody preincubation on attachment were analyzed byanalysis of variance, followed by a post hoc test (Tukey'stest as per equal sample sizes per group) as appropriate. AP value of < .05 was considered statistically significant.

RESULTSImmunohistochemical studies confirmed the presence of MFG-E8 and integrin avb3 in Ishikawa and also in Jar cells, as de-picted in Figure 1. Figure 2 shows immunofluorescence imagesof Ishikawa and Jar cells for integrin avb3 and MFG-E8, de-picting typically predominant membrane locations.

To address specificity of antibodies, two further experi-ments were performed. Figure 3A and B show immunofluo-rescence results of live, nonfixed Jar cells, confirmingspecific binding and showing similar localization of integrinavb3 and MFG-E8 on these cells as that observed in fixedslides. Figure 3C shows a representative photograph of a Jarspheroid attached to the Ishikawa cell monolayer during thein vitro attachment assay. In addition, Figure 3D presents re-sults of immunoblotting for MFG-E8 in Ishikawa and Jar

PI-counterstained nuclei, blue; merged, original magnification, �400).-MFG-E8. Inset: NC ¼ negative control. (C) Multicellular Jar spheroidhment assay (inverted microscopy, original magnification, �200). (D)cular weight. Note presence of single band at the expected 46-kDa

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FIGURE 4

Results of the attachment assay after antibody treatment, expressed as number of attached spheroids (mean � SD). (A) For pretreatment ofIshikawa cells with anti-MFG-E8 antibodies, a dose-dependent inhibition of attachment was evident (overall effect, P<.0003). *P<.0001 vs.control. (B) For pretreatment of Ishikawa cells with anti-integrin avb3 antibodies, a dose-dependent inhibition of attachment rate was evident(overall effect, P<.0001). *P<.0001 vs. control. (C) Pretreatment of Jar spheroids with anti-MFG-E8 antibodies did not result in a significanteffect. (D) On the other hand, pretreatment of Jar spheroids with anti-integrin avb3 antibodies resulted in a dose-dependent inhibition ofattachment (overall effect, P<.001). *P<.01 vs. control. (E, F) No impact of pretreatment of Ishikawa or Jar cells on attachment, respectively,with an irrelevant IgG antibody vs. control.Schmitz. MFG-E8 and integrin avb3 implantation roles. Fertil Steril 2014.

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cells, confirming the expression of the expected 46-kDaprotein in both cell types.

Figure 4 presents results of the attachment assays. For Ish-ikawa cells (upper panel), treatment with an anti-MFG-E8antibody at doses of 1 mg/mL and 2 mg/mL was performed

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before the attachment assay. A dose-dependent inhibition ofattachment was evidenced (overall effect, P< .0003), with anapproximate decrease of Jar spheroids attachment rate by49% for every one-unit (1 mg) increase in antibody dosage.Treatment with the highest antibody dose (2 mg) was

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significantly lower than controls (P< .0001), with a reductionof attachment by 76% (Fig. 4A). Treatment with anti-integrinavb3 antibodies at doses of 1 mg/mL, 10 mg/mL, and 50 mg/mLvs. controls was carried out before the attachment assay. Adose-dependent inhibition of attachment rate was observed(overall effect, P< .0001), and the attachment rate decreasedby approximately 2% per every one-unit (1 mg) increase inantibody dosage. The treatment with 4 mg was significantlylower than controls (P< .0001), with a reduction of attachmentby 81% (Fig. 4B).

For Jar spheroids (Fig. 4, middle panel), pretreatment ofJar spheroids with anti-MFG-E8 antibody did not result in asignificant effect on attachment rate (Fig. 4C). Conversely,pretreatment of Jar spheroids with anti-integrin avb3antibodies showed a dose-dependent inhibition of attachment(overall effect, P< .001), and treatment with the highestantibody dose (2 mg) was significantly lower than controls(P< .01), with a reduction of attachment by 44% (Fig. 4D).

Pretreatment with IgG antibody (negative control)showed no difference of attachment compared with control,untreated conditions (P>.7) in either Ishikawa (Fig. 4E) orJar (Fig. 4F) cells.

DISCUSSIONPrevious studies have shown that integrin avb3 and MFG-E8are expressed in primary human epithelial cells, more inten-sively during the window of implantation (7, 19, 20). Ourstudy confirmed that both of them are also expressed inIshikawa cells (20, 35, 48). Furthermore, we demonstratedthat both proteins are present in trophoblast Jar cells byimmunochemistry and immunofluorescence. Integrin avb3has been previously found to be expressed in mouse andhuman embryos (49, 50), and MFG-E8 has been shown tobe expressed in human placental trophoblasts in all threetrimesters of pregnancy (36). Here we show for the first timethat Jar cells express MFG-E8. After these characterizationswe were able to proceed with our experiments to test ourhypothesis that blocking MFG-E8 and integrin avb3 by spe-cific antibodies might impair the attachment of Jar spheroids(mimicking the trophoblast/embryo) to the Ishikawa cellmonolayer (simulating the endometrial epithelium).

Integrins divalent-cation-dependent binding to theirligands regulates many cellular processes, such as remodelingof bone, homeostasis, fertilization, and implantation (15, 18).Integrin avb3 and its ligands' roles in implantation have beensubject to extensive investigation in the last few years. It wasshown that blocking integrin avb3 and Lewis Y, adifucosylated oligosaccharide carried by this integrin, inepithelial RL95-2 cells diminishes Jar spheroids attachment(5). Another study performed in Ishikawa cells knocked downintegrin avb3 and demonstrated diminished attachment of ratblastocysts to those cells (51). In agreement with these results,we found diminished attachment of Jar's spheroids to Ishikawacells when this integrin was blocked. Additionally, we alsoshowed that blocking integrin avb3 present in the trophoblastcells decreases attachment, an effect not reported before.

MFG-E8 is a glycoprotein that contains two epidermalgrowth factor domains, a proline/threonine-rich domain,

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and two factor-VIII-homologous domains (25, 26). It has anessential role in an adequate functioning immune system,promoting the engulfment of apoptotic cells bymacrophages and dendritic cells (52). MFG-E8 dysfunctionseems to be implicated in some pathologic processes, suchas tumor progression and systemic lupus erythematosus (53,54). Furthermore, MFG-E8 promotes angiogenesis and is acrucial mediator of vascular endothelial growth factor proan-giogenic effect, and its therapeutic potential has been pointedout for future disease treatment, such as sepsis and certaintypes of cancers (55–57).

Although its function in mediating phagocytic engulf-ment seems to be the most investigated one, MFG-E8's rolein the implantation process has also received some attentionrecently. It has been shown that there is an increase in itsendometrial expression during the window of implantationin human (19, 20). Here we demonstrated that blockingMFG-E8 in Ishikawa cells significantly diminishes Jarspheroid attachment, although blocking the protein in theJar spheroids did not have the same effect. To the best ofour knowledge this is the first evidence of MFG-E8 adhesionproperties in a trophoblast/endometrial epithelial cell modelthat simulates human implantation.

In conclusion, results demonstrated that independentblockage ofMFG-E8and its receptor integrinavb3 in Ishikawacells significantly diminished Jar spheroid attachment. More-over, we showed that blockage of integrin avb3 in the tropho-blastic tissue (Jar cells) also diminished their attachment to theIshikawa monolayer. Further characterization of MFG-E8 andintegrin avb3 intracellular pathways involved in this processis required for a better understanding of the roles of theseproteins in normal and pathologic human implantation.

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