Browsing by Author "SALGADO, AM"

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    EFFECT OF INVIVO OOCYTE AGING ON SPERM CHROMATIN DECONDENSATION IN THE GOLDEN-HAMSTER
    (1986) JEDLICKI, A; BARROS, C; SALGADO, AM; HERRERA, E
    Aged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase-contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase-contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.
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    INHIBITION OF THE ACROSOME REACTION BY TRYPSIN-INHIBITORS AND PREVENTION OF PENETRATION OF SPERMATOZOA THROUGH THE HUMAN ZONA-PELLUCIDA
    (1993) LLANOS, M; VIGIL, P; SALGADO, AM; MORALES, P
    In this study we evaluated the effect of several trypsin inhibitors (p-aminobenzamidine: pAB; N-alpha-p-tosyl-L-lysine-chloromethyl-ketone: TLCK and p-nitrophenyl-p'-guanidino-benzoate: NPGB) on sperm binding and penetration of the human zona pellucida. Motile spermatozoa, selected by a two-step Percoll gradient, were incubated at 1 X 10(7) cells ml-1 at 37-degrees-C and in 5% CO2 for 4.5 h. This was followed by the addition of 1 mmol pAB l-1 or phosphate-buffered saline (control) for 30 min. Three to four non-viable human oocytes were then added to each sperm suspension and incubated for 3 h. The numbers of spermatozoa bound to the human zona pellucida and in the perivitelline space were determined by phase contrast microscopy. The results showed that pAB significantly inhibited zona penetration by spermatozoa (56 +/- 8% oocytes penetrated, control versus 0 +/- 0% oocytes penetrated, pAB, mean +/- SEM), without modifying spermatozoa-zona pellucida binding. The inhibition of zona penetration was due to a block of the acrosome reaction normally induced by the human zona pellucida. In separate experiments, sperm suspensions pretreated with 1 mmol pAB l-1 or 10 mumol NPGB l-1 exhibited a marked decrease in the percentage of acrosome reactions on the zona surface (85 +/- 4% and 76 +/- 3% inhibition, respectively). In addition, the inhibitors prevented the acrosome reaction induced by human follicular fluid (percentage of acrosome-reacted spermatozoa: control 8 +/- 2; follicular fluid 25 +/- 3; pAB 6 +/- 2; NPGB 8 +/- 1; TLCK 12 +/- 2). Electron microscope studies suggested a significant inhibition of the membrane fusion events of the acrosome reaction in the inhibitor-treated spermatozoa. These results are the first to show that trypsin inhibitors block sperm penetration of the human zona pellucida owing to an inhibition of the acrosome reaction. In addition, they suggest a role for a trypsin-like enzyme during the acrosome reaction of human spermatozoa.
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