Browsing by Author "MORALES, P"

Now showing 1 - 10 of 10
  • No Thumbnail Available
    Item
    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.
  • No Thumbnail Available
    Item
    HUMAN CERVICAL-MUCUS - RELATIONSHIP BETWEEN BIOCHEMICAL CHARACTERISTICS AND ABILITY TO ALLOW MIGRATION OF SPERMATOZOA
    (1993) MORALES, P; ROCO, M; VIGIL, P
    Cervical mucus is produced throughout the menstrual cycle. Sperm migration, however, is possible only during the periovulatory period of the cycle. Cervical mucus is also produced during the amenorrhoeic post-partum period. Post-partum mucus is very similar to luteal phase mucus except that it can allow sperm migration. In this study, mucus samples obtained from all these periods were classified according to their capacity to allow sperm migration. The biochemical characteristics of mucus samples that did (peri-ovulatory and 40% of post-partum samples) and did not (luteal and 60% of post-partum samples) allow sperm migration were then compared. Mucus samples with positive sperm migration showed the highest percentage of water and lowest protein and glycoprotein concentration (per ml of mucus). In addition, post-partum mucus samples with positive sperm migration showed lower concentrations of proteins and glycoproteins than post-partum mucus samples that did not allow sperm migration. However, the amount of glycoproteins per mg of protein was similar between post-partum samples that were positive and negative for sperm migration. These data suggest that the carbohydrate composition of the glycoproteins is playing a key role in the ability of cervical mucus to accept spermatozoa.
  • No Thumbnail Available
    Item
    HUMAN SPERMATOZOA SELECTED BY PERCOLL GRADIENT OR SWIM-UP ARE EQUALLY CAPABLE OF BINDING TO THE HUMAN ZONA-PELLUCIDA AND UNDERGOING THE ACROSOME REACTION
    (1991) MORALES, P; VANTMAN, D; BARROS, C; VIGIL, P
    Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37-degrees-C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P < 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.
  • No Thumbnail Available
    Item
    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.
  • No Thumbnail Available
    Item
    PENTOXIFYLLINE INCREASES SPERM PENETRATION INTO ZONA-FREE HAMSTER OOCYTES WITHOUT INCREASING THE ACROSOME REACTION
    (1993) MORALES, P; LLANOS, M; YOVICH, JL; CUMMINS, JM; VIGIL, P
    Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml-1 of pentoxifylline at 37-degrees-C, 5% CO2 for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 X 10(6) cells ml-1. One hundred microlitres of each sperm suspension was then deposited under oil and 30-40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.
  • No Thumbnail Available
    Item
    POSTPARTUM CERVICAL-MUCUS - BIOLOGICAL AND RHEOLOGICAL PROPERTIES
    (1991) VIGIL, P; PEREZ, A; NEIRA, J; MORALES, P
    In this study we have evaluated the score, sperm migration and ultrastructural characteristics of cervical mucus present in amenorrhoeic women under exclusive breastfeeding at 30, 60, 90, 120, 150 and 180 days post-partum. Periovulatory mucus samples from seven normally cycling women were used as a control. The average scores of post-partum and periovulatory mucus were 4.6 +/- 0.4 and 14.1 +/- 0.5 respectively. Twenty-one (39%) of the 54 post-partum cervical mucus samples and all (100%) periovulatory mucus samples allowed sperm migration. Positive sperm migration into post-partum mucus was observed at all time intervals studied. The only parameter that correlated with sperm migration into post-partum mucus was ferning formation. Sperm migration was obtained in all post-partum mucus samples with a score > 8, but samples with scores between 2 and 7 also showed sperm penetration. Scanning electron microscopic studies showed the characteristic spongy appearance of periovulatory mucus. Post-partum mucus was formed by a dense mesh (rocky appearance), when samples were generally unable to sustain sperm migration, but samples where sperm migration occurred showed small areas of spongy mucus mixed with areas in which a dense mesh and high cellularity was observed.
  • No Thumbnail Available
    Item
    SPERM BINDING TO THE HUMAN ZONA-PELLUCIDA AFTER MIGRATION THROUGH HUMAN CERVICAL-MUCUS
    (1995) VIGIL, P; RIQUELME, R; MORALES, P
    During lactational amenorrhea a special type of cervical mucus, similar to that found during the luteal phase, is produced. This mucus, however, is able to support sperm migration. In the study described, the ability of spermatozoa to bind to the human zona pellucida (hZP) after migration through periovulatory and post-partum mucus was studied. Mucus was obtained from exclusively breastfeeding women in amenorrhea at 30, 60, 120 and 180 days post-partum. Periovulatory mucus samples from normally cycling women were used as a control. Flat capillary tubes were filled with BWW culture medium at the top and cervical mucus at the bottom. The tubes were immersed in a semen reservoir and the spermatozoa allowed to migrate through the mucus for 3 h into the culture media. Then the spermatozoa were coincubated with 3-4 hZP for 30 min and the number of bound spermatozoa per zona was counted. Periovulatory cervical mucus had an average Insler score of 14 +/- 0.5 as compared to 4.6 +/- 0.4 for post-partum mucus. Spermatozoa recovered from periovulatory mucus were always able to bind to the hZP. Spermatozoa recovered from post-partum mucus, however, were able to bind to the hZP in only 68 +/- 7% of the cases. Moreover, spermatozoa recovered from post-partum mucus bound to the ZP in lower numbers than did spermatozoa recovered from periovulatory mucus (p<0.03). These results suggest a greater ability of sperm-hZP binding after migration through periovulatory mucus and they also indicate that sperm binding to the ZP is possible even after sperm migration through a low quality mucus.
  • No Thumbnail Available
    Item
    SPERM OOCYTE INTERACTION - STUDIES ON THE KINETICS OF ZONA-PELLUCIDA BINDING AND ACROSOME REACTION OF HUMAN SPERMATOZOA
    (1994) MORALES, P; VIGIL, P; FRANKEN, DR; KASKAR, K; COETZEE, K; KRUGER, TF
    Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10(7) cells ml-1 after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h under-went the acrosome reaction (range 9-43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.
  • No Thumbnail Available
    Item
    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.
  • No Thumbnail Available
    Item
    THE ACROSOME REACTION-INDUCING ACTIVITY OF INDIVIDUAL HUMAN FOLLICULAR-FLUID SAMPLES IS HIGHLY VARIABLE AND IS RELATED TO THE STEROID CONTENT
    (1992) MORALES, P; LLANOS, M; GUTIERREZ, G; KOHEN, P; VIGIL, P; VANTMAN, D
    In this study, we have evaluated the relationship between the acrosome reaction-inducing activity of individual human follicular fluid samples and their steroid content. Eighteen samples of follicular fluid were obtained during egg retrieval in six patients undergoing assisted fertilization. Motile spermatozoa were incubated in modified Tyrode's medium (26 mg/ml bovine serum albumin) for 20 h at 1 X 10(7) cells/ml. In a single experiment, aliquots of a semen specimen were simultaneously treated with an aliquot of each follicular fluid sample. The percentage of acrosome reacted spermatozoa was determined using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC - PSA) lectin. The fluids were also analysed by radioinimunoassay to determine the levels of progesterone, 17-alpha-hydroxy-progesterone, testosterone and oestradiol. The results showed that there was a positive, highly significant correlation between the acrosome reaction-inducing activity and the progesterone level of each follicular fluid sample (r = 0.72, P < 0.005). Additionally, treatment of the follicular fluid samples with charcoal-dextran caused both a decrease in progesterone concentration and the total loss of the acrosome reaction-inducing activity. The addition of progesterone restored the acrosome reaction-inducing ability in 88% of samples. These data support the idea that progesterone in follicular fluid is the molecule responsible for inducing the acrosome reaction in human spermatozoa.