The role of the osteopontin-integrin αvβ3 interaction at implantation: functional analysis using three different in vitro models.
OBJECTIVE
Does the interaction between integrin and its ligand osteopontin (OPN) mediate embryonic attachment to endometrial epithelium at implantation?


CONCLUSIONS
OPN of epithelial origin binds the receptor integrin αvβ3 at the maternal surface to support adhesion during the early stages of implantation.


BACKGROUND
Integrin αvβ3 and OPN are both present in the endometrial luminal epithelium in the mid-secretory phase.


METHODS
Microscopy of attachment sites of blastocysts (mouse, n = 151, human, n = 8) and OPN- or BSA-coated beads (n = 488) interacting with Ishikawa cell monolayers at 24 and 48 h. Levels of epithelial OPN or integrin αvβ3 were altered by siRNA-mediated targeting and the results compared with non-targeting siRNA or mock-transfected controls.


METHODS
In vitro modelling of early implantation with human endometrial cells (Ishikawa) and mouse or human embryos or ligand-coated beads. Immunolocalization of antigen around attached embryos was measured by image analysis with multiple repeats (n > 3), allowing a gradient of relative intensity to be detected. Attachment was quantified using a stability scale and protein expression documented by indirect immunofluorescence. Protein associations were probed by pulldown assays.


RESULTS
Integrin and OPN levels were increased in epithelial cells near to attached embryos. The pulldown assay confirmed OPN-integrin αvβ3 binding (n > 3). Decreased attachment stability of mouse embryos observed after siRNA knock-down of integrin αvβ3 or OPN itself, or OPN-coated beads after knock-down of integrin αvβ3, was tested for significance using Kruskal-Wallis with Dunn's post hoc tests.


CONCLUSIONS
In vitro model. Attachment data using human embryos is limited by embryo availability. Mouse embryo attachment to human cells involves a species crossover so must be interpreted with caution. Ligand-coated beads allow specific molecular interactions mediating attachment to be probed, but obviously lack the adhesion and signaling repertoire of a live embryo.


CONCLUSIONS
Some of the literature identifies reduced integrin αvβ3 expression in infertile endometrium; these findings predict that embryo attachment stability will be reduced in vivo if integrin levels are low. We suggest that the robustness of the initial attachment of the embryo affects its ability to progress to the post-epithelial phase of implantation; some poorly attached embryos will be lost.


BACKGROUND
No external funds were used for this study, which was supported by funds from the Universities of Manchester and Oxford. None of the authors has any conflict of interest to declare.


BACKGROUND
N/A.
Maternal and Fetal Health Research Centre, Institute of Human Development, University of Manchester, St. Mary's Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Level 5-Research, Manchester, UK.
Youn-Jung
YJ
Kang
Karen
K
Forbes
Janet
J
Carver
John D
JD
Aplin
D000818:Animals; D001755:Blastocyst / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line; D010064:Embryo Implantation / Q000502:physiology*; D004717:Endometrium / Q000166:cytology* / Q000378:metabolism; D005260:Female; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D053495:Osteopontin / Q000378:metabolism* / Q000502:physiology; D034622:RNA Interference
D039302:Integrin alphaVbeta3; D053495:Osteopontin
endometrium; implantation; integrin αvβ3; osteopontin