Analysis of the role of egg integrins in sperm-egg binding and fusion

Sperm-egg fusion is believed to be mediated via specific molecular interact ions. Integrin alpha 6 beta 1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin alpha 6 beta 1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin alpha 6 beta 1 in sperm-eg g interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin-labeled egg surface proteins. Under this condit ion, solubilized proteins from eggs inhibited sperm-egg fusion. Western blo t analysis under reducing conditions indicated that a major-labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti-integ rin alpha 6 antibody GoH3 indicated that the 135 kDa egg surface molecule t hat bound to sperm was the integrin alpha 6 subunit. To investigate the pot ential involvement of integrin alpha 6 beta 1 in sperm-egg fusion, we next examined the localization of integrin a6 and beta 1 subunits before and aft er fertilization by confocal laser microscopy. At an early stage of sperm-e gg fusion, the integrin alpha 6 and beta 1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm-egg fusion, indicating that integrin receptors are accumu lated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin alpha 6 beta 1 is involved in sperm-egg binding l eading to fusion via direct association of the integrin alpha 6 with sperm. Mol. Reprod. Dev. 56:412-423, 2000. (C) 2000 Wiley-Liss, Inc.