Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.
Copyright 2000 Academic Press.