Browsing by Author "LLANOS, MN"

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    EVIDENCES FOR THE PRESENCE OF CHYMOTRYPSIN-LIKE ACTIVITY IN HUMAN SPERMATOZOA WITH A ROLE IN THE ACROSOME REACTION
    (1994) MORALES, P; SOCIAS, T; CORTEZ, J; LLANOS, MN
    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 x 10(7) cells/ml, 37 degrees C, and 5% CO2. 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 Ca2+, 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. (C) 1994 Wiley-Liss, Inc.
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    STUDIES OF LYSOPHOSPHOLIPIDS RELATED TO THE HAMSTER SPERM ACROSOME REACTION IN-VITRO
    (1993) LLANOS, MN; MORALES, P; RIFFO, MS
    Phospholipase A2 and lysophospholipids have been implicated in the mammalian sperm acrosome reaction. In this study we further investigated the role of this enzyme and lysophospholipids on the acrosome reaction of hamster spermatozoa. Hamster epididymal spermatozoa were incubated under capacitation and acrosome reaction-inducing conditions. After 3.0 and 3.5 h, the spermatozoa were treated with different doses of lysophosphatidylcholine for 12 min. Then the percentage of motility, hyperactivation, and acrosome reaction was evaluated by light microscopy. Lysophosphatidylcholine, 10 mug/ml, was the highest acrosome reaction-inducing dose without an effect on sperm motility. Lysophosphatidylcholine induced the acrosome reaction only when added to spermatozoa capacitated for a minimum of 2 h. This effect was apparent after 1 min of its addition and reached a plateau after 5 min. Lysophosphatidylethanolamine and lysophosphatidylinositol were also effective in inducing the acrosome reaction. Lysophosphatidylserine did not have any effect on the reaction, but caused an increase in sperm hyperactivation. Sperm treated with the phospholipase A2 inhibitors quinacrine dihydrochloride and p-bromophenacyl-bromide showed an inhibition of the spontaneous occurrence of the acrosome reaction. These inhibitors, however, did not block the acrosome reaction induced by lysophosphatidylcholine. The time course of the lysophosphatidylcholine-induced acrosome reaction was the same whether control or inhibitor treated spermatozoa were used. These results suggest that the membrane events of the acrosome reaction initiate with the activation of the phospholipase A2, thus producing the fusogen agents necessary for this exocytotic event. (C) 1993 Wiley-Liss, Inc.