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Evidences for the presence of chymotrypsin‐like activity in human spermatozoa with a role in the acrosome reaction
Author(s) -
Morales Patricio,
Socias Teresa,
Cortez Jacqueline,
Llanos Miguel N.
Publication year - 1994
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1080380214
Subject(s) - acrosome reaction , chymotrypsin , acrosome , sperm , biology , follicular fluid , benzamidine , biochemistry , enzyme , trypsin , oocyte , microbiology and biotechnology , embryo , botany
Abstract The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10 7 cells/ml, 37°C, and 5% CO 2 , After 4.5 hr, the chymotrypsin inhibitor TPCK (N‐Tosyl‐L‐Phenylalanine‐Chloromethyl Ketone) or the substrate ATEE (N‐Acetyl‐L‐Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome‐reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida‐induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N‐Benzoyl‐L‐Tyrosine Ethyl Ester), Succinyl‐Ala‐Ala‐Phe‐7‐Amido‐4‐Methyl‐Coumarin (Suc‐Ala‐Ala‐Phe‐AMC), and Succinyl‐Leu‐Leu‐Val‐Tyr‐7‐Amido‐4‐Methyl‐Coumarin (Suc‐Leu‐Leu‐Val‐Tyr‐AMC) inhibited the human follicular fluid‐induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc‐Ala‐Ala‐Phe‐AMC and Suc‐Leu‐Leu‐Val‐Tyr‐AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca 2+ , and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin‐like activity with a possible role in the acrosome reaction. © 1994 Wiley‐Liss, Inc.