Browsing by Author "VIGIL, P"

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    ALKYLATING-AGENTS AND MOUSE SPERMATOGENESIS - EFFECTS OF A SINGLE DOSE OF CYCLOPHOSPHAMIDE
    (1985) VIGIL, P; BUSTOSOBREGON, E
    Cyclophosphamide (CFA) is one of the alkylating agents which has now been used with some success in the treatment of human neoplasias and renal disease. To evaluate how this drug could affect the seminiferous epithelium, a single dose of CFA (200 mg/kg per wt) was given to 20 adult mice (strain A/Sw). The effect of the drug was compared with a control group to which physiological saline solution was injected. Mice were sacrificed by cervical dislocation at 4 different time intervals after the drug administration to evaluate the action of the drug in different stages of spermatogenesis. The effect of the drug was appreciated as soon as 4 days after its administration. The initial damage to the epithelium was characterized by vacuolization of the Sertoli cells. Of all germ cells, primary spermatocytes showed the highest sensitivity to the drug. A high percent of teratozoospermia in the experimental group, when compared to the control group, was observed at all time intervals. The way CFA affects the morphology of mammalian spermatozoa and the usefulness of the animal model presented are discussed.
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    GAMETE MEMBRANE-FUSION IN HAMSTER SPERMATOZOA WITH REACTED EQUATORIAL SEGMENT
    (1989) VIGIL, P
    Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies showed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.
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    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.
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    HUMAN SPERM-CERVICAL MUCUS INTERACTION AND THE ABILITY OF SPERMATOZOA TO FUSE WITH ZONA-FREE HAMSTER OOCYTES
    (1988) BARROS, C; JEDLICKI, A; FUENZALIDA, I; HERRERA, E; ARGUELLO, B; VIGIL, P; VILLASECA, P; LEONTIC, E
    Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4.degree.C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband''s semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P < 0.05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.
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    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.
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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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    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.
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    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.
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    ROUND-HEADED SPERMATOZOA - A MODEL TO STUDY THE ROLE OF THE ACROSOME IN EARLY EVENTS OF GAMETE INTERACTION
    (WILEY, 1990) VONBERNHARDI, R; DEIOANNES, AE; BLANCO, LP; HERRERA, E; BUSTOSOBREGON, E; VIGIL, P
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    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.
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    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.
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    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.
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