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Characterization of the mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors
Abstract:
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.
Insights
Spermatozoa utilize a unique trypsin-like binding site on their surface for initial egg zona pellucida binding. This site, essential for fertilization, is inhibited by specific protease inhibitors, not substrates.
Area of Science:
- Reproductive Biology
- Molecular Andrology
- Cellular Biochemistry
Background:
- Mammalian gamete interaction begins with sperm binding to the egg's zona pellucida.
- This initial binding precedes the acrosome reaction and is inhibited by trypsin inhibitors.
- A trypsin-like binding site on the sperm surface, functioning via an active site mechanism, is hypothesized.
Purpose of the Study:
- To investigate the enzymatic nature of the sperm surface binding site for the zona pellucida.
- To characterize the specificity and function of this putative trypsin-like site.
- To determine if this site is essential for sperm-zona pellucida binding.
Main Methods:
- Incubation of washed spermatozoa with the serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (MUGB).
- Measurement of MUGB hydrolysis rates by spermatozoa.
- Assessment of MUGB's effect on sperm-zona pellucida binding in the presence of inhibitors like p-nitrophenyl guanidinobenzoate and soybean trypsin inhibitor.
Main Results:
- Spermatozoa hydrolyzed MUGB at a specific rate (8 pmoles/min-10^6 cells), indicating a trypsin-like enzymatic activity.
- MUGB significantly inhibited sperm-zona pellucida binding, with concentration-dependent effects.
- Inhibition of MUGB hydrolysis and sperm binding showed strong positive correlations with various inhibitors, suggesting a shared active site.
Conclusions:
- A unique, trypsin-like sperm surface site, characterized by its active site mechanism, is crucial for initial binding to the zona pellucida.
- This site exhibits unique stereospecificity, reacting with trypsin inhibitors but not with typical trypsin substrates.
- The findings elucidate a key molecular interaction in mammalian fertilization.