We are studying protein-protein interactions
that occur during fertilization. Two sperm proteins, fertilinb
and cyritestin, members of the still expanding family of ADAM
proteins (A Disintegrin and Metalloprotease), are involved in
sperm-egg binding. Biochemical and genetic experiments suggest
there are multiple ADAM receptors on the egg. Assays to examine
each step in the kinetic pathway of fertilization individually
do not exist. Specific reagents are necessary for blocking studies
that will allow temporal control of inhibition, be complementary
to genetic deletion experiments, and allow elucidation of the
role of individual receptors in fertilization. We have developed
several reagents containing the fertilinb or cyritestin binding
motifs presented in either monovalent or polyvalent format. These
reagents block fertilization, parthenogenically activate eggs
or do both, and the polyvalent inhibitors are significantly more
effective than their monovalent counterparts. We are assaying
these reagents to determine their specificity for implicated receptors,
as well as their competitive nature. We are determining whether
inhibition is due to direct competition of ADAM sperm protein
or to activation of the egg that results in blocking of sperm
fusion. We are examining whether inhibition with fertilinb and
cyritestin binding motifs has synergistic effects. We are using
mouse eggs lacking putative receptors to identify in which step
of the sperm-egg fusion pathway each receptor functions. Testing
of these molecules in knock out eggs will provide insight into
the receptor activation required for initiation of development
in the egg. Using conformationally constrained tripeptides to
mimic the disintegrin binding loops of fertilinb and cyritestin,
we are synthesizing fertilization inhibitors to have a higher
specificity and affinity for a single step.
Our goal is to develop specific tools that will allow dissection
of the role of various egg receptors in the sperm binding and
adhesion pathway. The ultimate goal is to use these inhibitors
in vivo in order to explore the correspondence between in vitro
results and in vivo function. This dissection will help us to
define a molecular pathway for sperm-egg adhesion and fusion.
We hypothesize that the correct density and ratio of ADAM ligands
presented to the egg will activate it and prevent fertilization
by sperm. Identification of the cellular receptors of these activating
agents will provide new leads for blocking fertilization in vivo.
