Role for the endometrial epithelial protein MFG-E8 and its receptor integrin αvβ3 in human implantation: results of an in vitro trophoblast attachment.

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Presentation transcript:

Role for the endometrial epithelial protein MFG-E8 and its receptor integrin αvβ3 in human implantation: results of an in vitro trophoblast attachment study using established human cell lines Carla Schmitz, M.D., Liang Yu, Ph.D., Silvina Bocca, M.D., Ph.D., Sandra Anderson, B.S., João Sabino Cunha-Filho, M.D., Bhaskara S. Rhavi, Ph.D., Sergio Oehninger, M.D., Ph.D. Fertility and Sterility Volume 101, Issue 3, Pages 874-882 (March 2014) DOI: 10.1016/j.fertnstert.2013.12.015 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunohistochemistry of Ishikawa and Jar cells (original magnification, ×200). (A) Ishikawa cells stained with anti-MFG-E8 antibodies. (B) Ishikawa cells stained with anti-integrin αvβ3 antibodies. (C) Jar cells stained with anti-MFG-E8 antibodies. (D) Jar cells stained with anti-integrin αvβ3 antibodies. Insets: NC = respective negative controls. Fertility and Sterility 2014 101, 874-882DOI: (10.1016/j.fertnstert.2013.12.015) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

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 αvβ3 (A) and with anti-MFG-E8 (B). (C, D) Jar cells stained with anti-integrin αvβ3 antibody (C) and with anti-MFG-E8 antibody (D). Note predominantly membrane immunolocation of staining. Fertility and Sterility 2014 101, 874-882DOI: (10.1016/j.fertnstert.2013.12.015) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A, B) Immunofluorescent images of live, nonfixed Jar cells (FITC, green; DAPI-counterstained nuclei, blue; merged, original magnification, ×400). (A) Jar cells stained with anti-integrin αvβ3. (B) Jar cells stained with anti-MFG-E8. Inset: NC = negative control. (C) Multicellular Jar spheroid attached to the epithelial monolayer of Ishikawa cells in the in vitro attachment assay (inverted microscopy, original magnification, ×200). (D) Immunoblotting of MFG-E8 in Ishikawa cells and Jar cells. MW = molecular weight. Note presence of single band at the expected 46-kDa location for both cell types. Fertility and Sterility 2014 101, 874-882DOI: (10.1016/j.fertnstert.2013.12.015) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Results of the attachment assay after antibody treatment, expressed as number of attached spheroids (mean ± SD). (A) For pretreatment of Ishikawa 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 αvβ3 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 significant effect. (D) On the other hand, pretreatment of Jar spheroids with anti-integrin αvβ3 antibodies resulted in a dose-dependent inhibition of attachment (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. Fertility and Sterility 2014 101, 874-882DOI: (10.1016/j.fertnstert.2013.12.015) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions