Browsing by Author "BARROS, C"

Now showing 1 - 20 of 20
  • No Thumbnail Available
    Item
    CHANGES AT THE HAMSTER OOCYTE SURFACE FROM THE GERMINAL VESICLE STAGE TO OVULATION
    (1984) EBENSPERGER, C; BARROS, C
    Immature and ovulated hamster oocytes were studied with the scanning electron microscope. Immature oocytes at the germinal vesicle stage have their surface uniformly covered by microvilli. When meiosis has progressed to the 1st meiotic metaphase the overlying surface shows the differentiation of a circular area 19 .mu.m in diameter with a low density of microvilli. Later, from this region the 1st polar body emerges, and the oocyte surface at the point from which it was extruded shows a cluster of cytoplasmic, conical projections. When the zona-free oocytes are cultured at 37.degree. C for 5 min these projections disappear and the oocyte surface and that point becomes smooth. When the oocytes remain in the oviduct for several hours after ovulatioin these projections remain unchanged. The in vitro interactions of capacitated hamster sperm with the immature oocyte was always seen at microvillus surfaces and never associated with the differentiated regions.
  • No Thumbnail Available
    Item
    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.
  • No Thumbnail Available
    Item
    FINE-STRUCTURE OF THE MATURE SPERMATOZOON OF RHYNCHOCINETES-TYPUS CRUSTACEA DECAPODA
    (1983) DUPRE, E; BARROS, C
    R. typus spermatozoa obtained from the vas deferens have the shape of a round-headed nail. The head measures 30 .mu.m in diameter and 14 .mu.m of height. At the center of the flat face of the head emerges a single rigid spike of 53 .mu.m in length. Cross sections of this spike show that it has a wall of 0.4 .mu.m in thickness and a core of 0.6-0.8 .mu.m. The outer surface of the spike has a longitudinal striation. When the spermatozoa are placed in sea water it is possible to observe the unfolding of rays. The number of rays in different spermatozoa of the same individual varies from 9-13. Each ray is formed by a channel-like sheath that contains a rigid rod that occupies .apprx. 1/3 the length of the ray. This rod has a transverse striation with a periodicity of 185A. The rays are bound among them by a thin membranous sheet that is highly folded in vas deferens spermatozoa. At the distal end of each ray there is a rigid spine of 50 .mu.m in length. The nucleus is coplanar to the radial plane and it extends through the rays. The structure and ultrastructure of R. typus spermatozoa depart from that reported for spermatozoa of other Caridea species.
  • No Thumbnail Available
    Item
    HAMSTER OOCYTE FERTILIZABILITY AFTER 4-DEGREES-C STORAGE
    (1986) BARROS, C; HERRERA, E; FUENZALIDA, I; ARGUELLO, B
    The purpose of the present work was to study the feasibility of using hamster oocytes stored at 4.degree. C in M-2 culture medium for 24 and 48 hours in the evaluation of human sperm fertilizability. A total of 1,394 oocytes were stored for 24 hours and 1,234 were stored for 48 hours. After the storage period all the oocytes were stained with fluorescein diacetate, proving the physical integrity of the egg plasma membrane. Twenty-five and 22 semen samples were used to compare their ability to penetrate freshly ovulated and 24- and 48-hour-stored hamster oocytes. Freshly ovulated and 24-hour-stored oocytes were penetrated at percentages that in more than 95% of the cases showed no significant differences. The same experiments carried out with oocytes stored for 48 hours showed that in 75% of the cases no significant differences were found. The use of oocytes preserved at 4.degree. C when large numbers of semen samples are to be tested for fertilizability is recommended.
  • No Thumbnail Available
    Item
    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.
  • 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
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    IS ACTIVATED SPERMATOZOON REALLY CAPACITATED
    (1977) BARROS, C; BERRIOS, M
    A culture medium (7N) consisting only of salts and devoid of any macromolecule was prepared to test its effect upon survival, activation and acrosome reaction of golden hamster spermatozoa. Incubation of spermatozoa in medium 7N allowed a temporal separation of activation and acrosome reaction. More than 70% of spermatozoa were activated after 4 h at 37.degree. C while the acrosome reaction remained very low (8%). Activated spermatozoa with intact acrosomes were incubated with zona-free hamster oocytes at 37.degree. C for 1-2 h. When these oocytes were examined by EM it was found that the spermatozoa were closely bound to the egg plasma membrane, mainly by the plasma membrane overlying the acrosome, but in no instance had the gamete membranes fused. Prolonged incubation resulted in an engulfment of the sperm by the oocyte in a phagocytic manner. An activated spermatozoon is not capacitated since it cannot fertilize oocytes.
  • No Thumbnail Available
    Item
    LOCALIZATION OF MICROFILAMENTS AND A TUBULIN-LIKE PROTEIN IN CRUSTACEAN (RHYNCHOCINETES-TYPUS) SPERMATOZOON
    (1991) PEREZ, C; ROCO, M; CASTRO, A; DUPRE, E; SCHATTEN, G; BARROS, C
    Sperm from the decapod crustacean Rhynchocinetes typus undergo dramatic shape changes as they pass from the vas deferens to seawater and interact with the oocyte envelopes. Using FITC-phalloidin and antitubulin antibodies, we were able to localize microfilaments and a tubulin-like protein in R. typus spermatozoon. Microfilaments and the tubulin-like protein were associated with the sperm rays and spines, but were absent at the spike and at its base. Folded and unfolded spermatozoa display similar fluorescence patterns. SDS-PAGE of whole spermatozoa and electrotransfer to nitrocellulose confirmed the presence of actin and two proteins at 97 kd and 120 kd that bind to tubulin antibodies (tubulin-like proteins). These results demonstrate the presence of actin, but not tubulin, and localize microfilaments in these sperm. It is proposed that this cytoskeletal component is active in sperm during crustacean fertilization.
  • No Thumbnail Available
    Item
    PRESERVATION OF HAMSTER OOCYTES TO ASSAY THE FERTILIZING-CAPACITY OF HUMAN-SPERMATOZOA
    (1982) QUINN, P; BARROS, C; WHITTINGHAM, DG
    Between 70 and 80% of zona-intact hamster ova survived freezing after slow cooling (.apprx. 0.3.degree. C/min) to -80.degree. C in Medium PB1 containing 1.5 or 2.0 M-DMSO before transfer to -196.degree. C. After slow warming (.apprx. 8.degree. C/min), there was no difference in survival if the DMSO [dimethyl sulfoxide] was diluted out by a slow stepwise or a rapid single addition of medium. When slow cooling was terminated -40.degree. C by direct transfer to -196.degree. C, up to 75% of the ova survived rapid warming (.apprx. 500.degree. C/min) and rapid dilution if the medium contained 2.0 M-DMSO. The survival rates were calculated on the basis of the number of thawed ova which retained their normal morphological appearance after a 1 h incubation before removal of the zona pellucida with trypsin. All of these ova were penetrated after incubation with mouse spermatozoa, indicating that the freezing procedure per se does not adversely affect the penetration of frozen-thawed hamster ova by heterologous spermatozoa. There was no difference in the penetration rate of human spermatozoa into frozen (34%) or fresh (42%) oocytes when a Hepes[N-2-hydroxyethylpiperazine-N''-2-ethanesulfonic-acid]-buffered Tyrode solution, containing 30 mg BSA[bovine serum albumin]/ml and 2.0 M-DMSO, was used as the freezing medium. However, fewer ova frozen in Medium PB1, containing 4 mg BSA/ml and 2.0 M-DMSO, were penetrated by human spermatozoa (18%) compared with freshly collected ova (38%). Zona-free ova did not survive the freezing procedure as well as zona-intact ova. The survival of hamster oocytes stored at -196.degree. C offers a convenient means of supplying and transporting these ova for the assessment of the fertilizing capacity of human and other heterologous spermatozoa.
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    RELATIONSHIP BETWEEN THE LENGTH OF SPERM PREINCUBATION AND ZONA PENETRATION IN THE GOLDEN-HAMSTER - A SCANNING ELECTRON-MICROSCOPY STUDY
    (1984) BARROS, C; JEDLICKI, A; BIZE, I; AGUIRRE, E
    Hamster spermatozoa are able to fertilize a high percentage of zona-intact hamster oocytes when they are preincubated for 2 h in a chemically defined medium. From this time on, the longer the preincubation time the lower the percentage penetration. Spermatozoa preincubated for > 6 h are unable to cross the zona pellucida, retaining their ability to fuse with zona-free hamster oocytes. Zona-intact hamster oocytes were observed with the scaning electron microscope. When the oocytes were inseminated with spermatozoa preincubated for 1-5 h the outer surface of the zona showed the penetrating spermatozoa and the sperm tracks made by those that failed to cross it. With longer preincubation times no penetrating spermatozoa were observed, and very few sperm tacks were present on the outer surface of the zona. Control experiments showed that neither eggs, spermatozoa, nor fertilization were affected by the medium recovered after long preincubations. Care should be taken regarding the preincubation time when using the in=vitro fertilization technique.
  • No Thumbnail Available
    Item
    SCANNING ELECTRON-MICROSCOPE STUDY OF INVITRO PREPENETRATION GAMETE INTERACTIONS
    (1985) JEDLICKI, A; BARROS, C
    Hamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. Sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin-treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatoza. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona-reacted and in trypsin-treated hamster eggs inseminated in vitro with homologous spermatozoa.
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    SPERM-EGG INTERACTIONS IN THE SHRIMP RHYNCHOCINETES-TYPUS
    (1986) BARROS, C; DUPRE, E; VIVEROS, L
    Sperm-egg interaction in Rhynchocinetes typus was studied with the phase-contrast and scanning electron microscopes. R. typus spermatozoa present in the vas deferens have the shape of a round-headed nail. After contact with seawater it is possible to observe the unfolding of the rays of stellate arms, giving the spermatozoon the appearance of an inverted umbrella. From the center of the flat face of the umbrella emerges a spike with longitudinal striations. Ovarian eggs and spermatozoa were mixed in vitro by agitating them for two minutes in Millipore-filtered seawater. The first gamete contact was established by the spermatozoa through the tip of the spike, which exerted a lytic action on the egg envelopes. After the rigid spike was completely inside the egg, the rays became aligned parallel to each other and began to enter the eggs. Toward the final stages of ray entry, it was possible to observe fusion of the ray membranes with one another, and later the fusion process continued toward the tip of the radial spines. Concomitantly, the egg surface that surrounds the sperm swelled in a circular fashion and formed a fertilization cone. After the spermatozoa entry was complete, a scarlike mark appeared at the place on the egg surface through which penetration occurred. The whole penetration process was completed within 45-60 minutes.
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    ULTRASTRUCTURAL OBSERVATIONS OF INCORPORATION OF GUINEA-PIG SPERMATOZOA INTO ZONA-FREE HAMSTER OOCYTES
    (1977) BARROS, C; HERRERA, E
    Zona-free hamster oocytes inseminated in vitro with acrosome-reacted guinea-pig spermatozoa were examined with the EM. Guinea-pig spermatozoa, in the vicinity of the oocytes, consistently lacked the whole acrosome including the equatorial segment region. In cross fertilization, sperm-egg membrane fusion does not differ significantly from that of normal fertilization. It was sometimes possible to observe protrusions of oocyte cytoplasm containing sperm chromatin in the process of dispersion.
  • No Thumbnail Available
    Item
    ULTRASTRUCTURE OF THE HEAD OF CTENOMYS MAULINUS SPERMATOZOON WITH SPECIAL REFERENCE TO THE NUCLEUS
    (1982) FEITO, R; BARROS, C
    The spermatozoa of the neotropical hystricomorph rodent C. maulinus [tuco tuco] were examined cytochemically and under the transmission electron microscope. The head is flattened dorsoventrally. At the caudal end of the head there is a process oriented parallel to the tail. This process corresponds to a cylindrical extension of the nucleus, which constitutes a unique feature among mammals.
  • No Thumbnail Available
    Item