J.D. Bleil and P.M. Wassarman. 1986. Journal of Cell Biology. 102:1363-1371.
Sixteen years ago, ZP3, one of three glycoproteins found in the extracellular coat, zona pellucida, of mammalian eggs was determined by Bleil and Wassarman to be a sperm receptor. Six years later, they reported the results from binding of radioiodinated egg ZP3 from mice to acrosome-intact and acrosome-reacted sperm . As expected of a mouse egg's sperm receptor, visual evidence of ZP3 binding to the heads of acrosome-intact sperm was obtained. Not surprisingly, ZP3 failed to bind acrosome-reacted sperm. Additional information was obtained with labeled ZP2, the second of the zona glycoproteins. An unexpected ability of this glycoprotein to bind to acrosome-reacted sperm heads was revealed. It would thus appear that both ZP3 and ZP2 play key roles in mammalian sperm binding during fertilization.
Under fertilizing conditions in vitro, sperm attach loosely and without specificity to the zona pellucida outside mammalian eggs . Species-specific binding between the sperm plasma membrane and receptors in the zona pellucida follows. This triggers a cascade of events in which the plasma membrane at the anterior end of the sperm head fuses with the outer acrosomal membrane. Both are shed, resulting in the exposure of the inner acrosomal membrane and subsequent release of acrosomal enzymes. This is what is commonly referred to as the acrosome reaction.
The fate of the acrosome-reacted sperm is what was of interest to Bleil and Wassarman. These sperm remain attached to the egg and subsequently penetrate the zona pellucida with the aid of an acrosomal protease. The three glycoproteins of the zona are appropriately named ZP1, ZP2, and ZP3. While the function of ZP1 at the time of Bleil and Wassarman's research was unknown, ZP3 had already been determined to act as a sperm receptor . The investigators set out to further define sperm binding to ZP3 and, in the process, revealed a possible role for ZP2 in fertilization.
Bleil and Wassarman looked for binding of ZP3 to acrosome-reacted and acrosome-intact sperm. Using EGTA to prevent the acrosome reaction and the ionophore A23187 to induce the event, acrosome-reacted and acrosome-intact sperm were obtained from mice. These could be distinguished under Nomarski Differential Microscopy by the presence of a thick ridge on the sperm heads of acrosome-intact sperm. This ridge was absent in acrosome-reacted sperm.
The glycoproteins isolated from the zona pellucida were radiolabeled with radioactive iodine. The investigators reported radioiodonated-ZP3 binding to only acrosome-intact sperm. Acrosome-reacted sperm were devoid of the labeled glycoprotein. To further characterize this finding, a competition assay was performed in which the binding of a fixed amount of radioiodonated-ZP3 to acrosome-intact sperm was measured in the presence of unlabeled solubilized egg zonae pellucidae (ZP1, ZP2, and ZP3). Increasing concentrations of the unlabeled zonae inhibited ZP3 binding. Bleil and Wassarman were thus able to conclude that there are a limited number of binding sites on the sperm head, for which the radiolabeled and unlabeld ZP3 compete.
The role of ZP3 was thus becoming a little clearer, but what about ZP2? Preliminary studies had suggested that ZP2 in any form lacked sperm receptor or acrosome-reaction inducing activities . It was therefore somewhat surprising that Bleil and Wassarman achieved minimal, yet significant, binding of radioiodinated ZP2 to acrosome-intact sperm and an impressively high level of binding to acrosome-reacted sperm (as much as 50 times the level of ZP3). These observations suggest that ZP2 binds very well to the inner acrosomal membrane, which is exposed following the acrosome reaction.
The preferential binding of ZP3 to acrosome-intact sperm came as no surprise. The two investigators showed that ZP3 prevents binding of sperm to unfertilized eggs under conditions that support fertilization in vitro. Neither ZP1 nor ZP2 had any effect on binding, suggesting that ZP3 is the sperm receptor . Only acrosome-intact sperm bind to eggs . It thus follows that the inhibition of binding due to ZP3 is attributable to an interaction with the plasma membrane overlying the heads of acrosome-intact sperm. Once the acrosome reaction has taken place resulting in the loss of the plasma membrane at the anterior portion of the sperm head, binding of ZP3 can no longer occur as observed with the acrosome-intact sperm.
How do the reported findings on ZP3 and ZP2 relate to one another? The researchers proposed that ZP2 may act as a secondary receptor, interacting with the inner acrosomal membrane after the binding and release of ZP3. In this way its function would be to retain binding between the sperm and egg following ZP3 dissociation. This would support the finding that once bound to the zona pellucida, sperm that have undergone the acrosome reaction remain bound . Free-swimming, acrosome-reacted sperm, however, can not bind to zona . If ZP2 is in fact a secondary receptor, the question arises as to why free-swimming acrosome-reacted sperm fail in this regard. Bleil and Wassarman suggested that the interaction between ZP2 and the acrosome-reacted sperm is too weak to bring about binding to the egg yet sufficient in strength to maintain prior binding initiated by ZP3. However, this is subject to experimental verification.
The big surprise is the apparent binding of ZP2 to acrosome-intact sperm at a low, but significant, level. The prevailing view is that this binding may actually reflect binding of ZP2 to the inner acrosomal membrane of sperm heads in the process of undergoing the acrosome reaction. The methods used by Bleil and Wassarman were unable to detect partially reacted acrosomes.
Moving on from his collaborative discovery concerning the role of ZP3 in fertilization, Wassarman later showed the need for a ZP3 in the initiation of the acrosome reaction , thus establishing a second function for this glycoprotein. Further research is required to identify the role of the ZP1 in the fertilization process as well as to explain the unexpected ZP2 binding results reported by Bleil and Wassarman in 1986.
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Copyright © Tim Ayas, Patrick Mitchell, Makela Nkendirim and Marco Perizzolo. 1996 This material may be reproduced for educational purposes only provided credit is given to the original source.
September 27, 1996