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

Abstract

Sperm‐egg fusion is believed to be mediated via specific molecular interactions. Integrin α6β1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin α6β1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin α6β1 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 α6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin α6 subunit. To investigate the potential involvement of integrin α6β1 in sperm‐egg fusion, we next examined the localization of integrin α6 and β1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm‐egg fusion, the integrin α6 and β1 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 α6β1 is involved in sperm‐egg binding leading to fusion via direct association of the integrin α6 with sperm. Mol. Reprod. Dev. 56:412–423, 2000. © 2000 Wiley‐Liss, Inc.

CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion

“…In the mouse (using ZP-free eggs), clustering of integrins at sperm contact and fusion occurred concomitantly, and the percentage of eggs in which the integrin ␣6 and ␤1 subunits appeared clustered at a sperm-binding site was closely correlated to the percentage of eggs with fused sperm. This led Takahashi et al to suggest that sperm-egg fusion occurs at sites where the ␣6␤1 integrin is clustered (Takahashi et al, 2000). In our study, we observed, before fusion, a strong labelling of CD9 and to a lower extent of integrin ␣6␤1 at the site of interaction of the sperm with the oocyte.…”

“…At latter time, the sperm entry point could not be detected based on CD9 and integrin labelling, suggesting that these molecules diffuse rapidly in the plasma membrane after fusion. This result is consistent with the study of Takahashi et al who observed that the ␣6␤1 integrin disappeared from the site of sperm penetration (Takahashi et al, 2000). These data further suggest a role for integrin ␣6␤1 patches during sperm-egg fusion.…”

“…4A). This is similar to the previously reported repartition of the integrin ␣6␤1 on ZP-free eggs (Tarone et al, 1993;Takahashi et al, 2000). In wild-type oocytes, antibodymediated aggregation of ␣6 integrins resulted in the formation of small, regularly arranged patches.…”

The mechanism of sperm–oocyte fusion in mammals

“…The first member of the integrin family shown to be active in this regard was a6b1. GoH3, a functionblocking mAb against the a6 integrin subunit, inhibited both sperm -oocyte binding and fusion, and peptides derived from a sequence of a part or all of the fertilin b disintegrin domain bound to a6b1 integrin (Almeida et al 1995, Chen & Sampson 1999, Takahashi et al 2000. However, the contribution of a6b1 integrin to the spermoocyte interaction remains questionable because some inhibition studies using GoH3 mAb under different conditions did not show blocking of the interaction (Evans et al 1997a, Miller et al 2000.…”

Cyclic QDE peptide increases fertilization rates and provides healthy pups in mouse

“…The first member of the integrin family shown to be active in sperm-egg fusion was the a6b1 integrin. Hence, GoH3, a function-blocking mAb directed against the a6 integrin subunit, inhibits sperm-oocyte binding and consequently fusion (5,8,9). Recently we showed that a6b1 integrin is also present in mouse sperm and involved in the fertilizing process (10).…”