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

Sperm-egg fusion is believed to be mediated via specific molecular interactions. Integrin alpha6beta1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin alpha6beta1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin alpha6beta1 in sperm-egg 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 condition, solubilized proteins from eggs inhibited sperm-egg fusion. Western blot analysis under reducing conditions indicated that a major-labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti-integrin alpha6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin alpha6 subunit. To investigate the potential involvement of integrin alpha6beta1 in sperm-egg fusion, we next examined the localization of integrin alpha6 and beta1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm-egg fusion, the integrin alpha6 and beta1 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 accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin alpha6beta1 is involved in sperm-egg binding leading to fusion via direct association of the integrin alpha6 with sperm.