The first contact of mammalian gametes is the binding of the spermatozoon to the zona pellucida of the egg. Previous work has shown that binding of the spermatozoon to the
zona in the mouse occurs prior to the acrosome reaction and that
trypsin inhibitors block this initial binding. This suggests that the sperm surface contains a trypsinlike binding site that functions by an active site mechanism to effect initial
zona binding. When
suspensions of twice-washed spermatozoa were incubated with the
serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (
MUGB), the titrant was hydrolyzed at a rate of 8 pmoles/min-10(6) cells.
MUGB was found to inhibit the binding of spermatozoa to the zona pellucida. The degree of inhibition and the rate of hydrolysis of
MUGB by washed spermatozoa depend on the concentration of titrant, with half maximal effects at 13 microM and a linear correlation with r = 0.99. The analogous lysyl and arginyl
trypsin substrates containing
7-amino-4-methylcoumarin as the fluorogenic leaving group were not hydrolyzed under the same conditions and did not inhibit
zona binding. Both binding of sperm to
zona-intact eggs and the hydrolysis of
MUGB by sperm are inhibited by p-nitrophenyl guanidinobenzoate, soybean
trypsin inhibitor, and
acid-solubilized zonae. The linear correlation coefficients of the inhibition of sperm binding and
MUGB hydrolysis by these three substances are greater than 0.92. This "trypsinlike" sperm site is essential for sperm binding to the
zona: its stereospecificity is unique in that it reacts with
trypsin inhibitors but not with
trypsin substrates.