A complex series of events results in sperm-egg
fusion. These events include sperm binding to the zona pellucida,
acrosome reaction of the sperm, and sperm binding to the egg plasma
membrane. Many proteins are involved in the latter step that immediately
precedes fusion, and we are studying this step. One of the best
characterized proteins is fertilinb that is an integral membrane
protein on the surface of the acrosome-reacted sperm. A second
sperm protein, cyritestin, has also been implicated in sperm-egg
binding. Fertilinb and cyritestin are members of the ADAM (A Disintegrin
and Metalloprotease) family of proteins (1,2). The disintegrin
domain is the moiety of the ADAM involved in sperm-egg binding.
Previous work in our laboratory, as well as others, identified
the three amino acid sequence, glutamate-cysteine-aspartate, ECD,
of the fertilinb disintegrin domain as the minimum sequence required
for inhibition of sperm-egg binding (3-6). Many different linear
peptides containing the ECD sequence have been synthesized, and
the IC50 for inhibition of sperm-egg binding is typically 500
µM. These peptides have been tested in a variety of species,
e.g., guinea pig, mouse, monkey and human, and inhibit fertilization
in all of them. Thus, ECD is a promising pharmacophore for development
of inhibitors of fertilization. Although not as extensive, the
existing mutagenesis studies on cyritestin suggest that the glutamine
of the analogous sequence in cyritestin, QCD, is minimally required
for inhibition in mouse (4,7).
Recent work from several laboratories has identified integrin
a6b1 as the egg plasma membrane integrin receptor for fertilinb.
A combination of photoaffinity labeling experiments (11) and cell-binding
assays with recombinant wild-type and mutant proteins (5,8) suggest
that fertilinb binds directly to integrin a6b1. This interaction
may be inhibited with small peptides that contain the binding
sequence of fertilinb or recombinant protein fragments that correspond
to the disintegrin domain of fertilinb (3,4,11-15). On the basis
of these experiments, it has been proposed that fertilinb-a6b1
binding is a precursor to sperm-egg fusion. However, integrin
gene knockout experiments have further highlighted the complexity
of this system. Female mice with a conditional knockout of integrin
b1 in their oocytes are fertile in vivo and in vitro (16). This
type of discrepancy between blocking and knockout experiments
has been observed with other integrins, e.g., those involved in
angiogenesis (17,18). There may be redundancy of ligand-receptor
pairs in wild-type fertilization that makes the a6b1-fertilinb
binding interaction one of several required for wild-type fertilization.
The biology of sperm ligand-egg receptor interactions is quite
complex. Regardless of this complexity, it is clear that peptides
derived from the fertilinb and cyritestin disintegrin binding
loops inhibit sperm-egg binding in vitro. Thus, mimics of these
peptides may be used to probe biological function. For the probes
to be useful, they must be receptor selective, and the design
of more potent and receptor-specific inhibitors requires a detailed
understanding of the structural requirements for binding. Moreover,
the mechanism of action of our polyvalent inhibitors is unclear.
Are they simply competitive inhibitors or are they activating
an egg signalling pathway that blocks further sperm adhesion and
fusion? Finally, selective, potent inhibitors may be developed
into compounds suitable as in vivo probes of activity that allow
temporal control of function blocking.
