Biomedical subjects

N M Delgado

Publications and source records attributed to N M Delgado.

At least 19 recordsLinked to original sources

PubMed1999

Effects of a purified fraction from Echeveria gibbiflora aqueous crude extract on guinea-pig spermatozoa.

Guinea-pig spermatozoa in the presence of a purified fraction from Echeveria gibbiflora aqueous crude extract suffer a hypotonic-like effect. The phenomena exhibited included a distension of the plasma membrane over the acrosome region, inducing the formation of a huge 'head-bubble'. The agglutination effect was so enhanced that instead of inducing sperm clusters, it produced cane-like 'stalk' structures. The immobilizing activity was induced instantaneously after the addition of the purified fraction. At electron microscope level it was possible to observe a heavy amount of electron dense material of the purified fraction embedded or intercalated along the plasma membrane. It was also possible to corroborate the dispersion of the acrosomal content and the disappearance of the external acrosome membrane. The purified fraction induced loosening of the plasma membrane all along the sperm cell, however, the distension of the membrane was only produced in the apical portion of the sperm head and not in the post equatorial region. The results suggest that the plant may yield a compound suitable for use as a vaginal barrier or male contraceptive agent.

Acrosome↗

PubMed1992

RNA metabolism during the sexual differentiation of rat hypothalamus.

Modifications in the basal molecular biology parameters (total concentrations of RNA, DNA, proteins, rRNA, tRNa, free and polysomal bound poly-A+ mRNA) have been determined daily in the growing hypothalamus of male and female rats from day 1 to day 8 after birth. Changes observed in the parameters studied in this work occurred mainly in the first 48 h after birth. In males tRNA and free mRNA (f-mRNA) contents decreased from day 1 to day 2 and then their concentrations remained more or less constant. Total mRNA significantly decreased from day 1 to day 3 and showed a further significant decrease from day 6 to day 8. Polysomal bound-mRNA (b-mRNA) decreased from day 1 to day 3, then increased to day 6, and finally decreased once more from day 6 to day 8. The b-mRNA/f-mRNA, mRNA/rRNA and mRNA/total RNA ratios showed a bimodal behavior with a first peak on day 2, and a second, smaller peak, on days 6-7. The changes observed in the females on the first 2-3 days of life were the inverse of those observed in the males, most of the parameters studied showed a sharp increase from day 1 to day 2 or to day 3 and then a drastic decrease. The only exception to this behavior was the b-mRNA/f-mRNA ratio which showed a small decrease from day 1 to day 2, followed by a continuous increase from day 2 to day 8. b-mRNA concentrations, after the sharp decrease from day 2 to day 3 of life, increased from day 3 to day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Differences in lipoprotein composition between heads and tails of human sperm: an infrared spectroscopy study.

Human spermatozoa and their fractions (heads and tails) have been studied by infrared spectroscopy. Protein conformation in isolated human spermatozoa heads, although predominantly of the alpha helix or random coil type, has a significant proportion of antiparallel B structure. Spectra of isolated spermatozoa tails show that proteins exist in this fraction preponderantly in pleated-sheet conformation (parallel and antiparallel). The quantity and type of lipids seem to be drastically different between heads and tails of spermatozoa. Head lipids are scarce and difficult to extract, and they are apparently tightly bound to proteins, highly unsaturated, and rich in free hydroxyl and carboxyl groups. Tail lipids are more abundant and more easily extractable. Head phospholipids are probably phosphatidylcholine, cephalins, and inositols, and tail phospholipids are preponderantly plasmalogen-type lecithins and sphingomyelins. The presence of specific infrared bands points to the existence in tails of important amounts of sulfur compounds, probably sulfolipids or sulfoglycolipids.

Humans↗

PubMed1990

Glycosaminoglycan-sulfate as plasma membrane component of pig spermatozoa.

The effect of specific glycosaminoglycan-hydrolyzing enzymes on the ruthenium red staining of pig spermatozoa was studied. Washed spermatozoa were incubated at 35 degrees C in buffer or with neuraminidase 0.5 units/ml, heparinase 0.2 mg/ml, or chondroitinase ABC 2.0 units/ml. After incubation sperm cells were washed, stained with ruthenium red and studied under the electron microscope. Anionic sites in the surface of untreated spermatozoa follow regularly the plasma membrane, but present are numerous processes constituting what has been defined as the glycocalyx. Neuraminidase did not affect the distribution of ruthenium red on the surface of the spermatozoa, but eliminated almost completely the processes of the glycocalyx. Heparinase caused loss of the ruthenium red-stained sites on the membrane surface of pig spermatozoa with less influence on the dense processes of the glycocalyx. A similar loss of ruthenium red-stained sites was observed with nitrous acid treatment. A striking effect of treatment with chondroitinase ABC was the production of a typical acrosome reaction.

Animals↗

PubMed1989

Heparin and glutathione: physiological decondensing agents of human sperm nuclei.

It has been proposed that reduced glutathione (GSH) or other thiol reagents may participate in the basic mechanism by which sperm-decondensing activity is accomplished. However, in vitro, these reagents seem to be inactive and require the presence of other chemicals, usually detergents. Heparin binds specifically to the sperm membrane and provokes the decondensation of human sperm and the activation of DNA transcription and synthesis. However, the concentrations at which these effects occur seem to be higher than those expected under physiological conditions. In the present study, thiol reagents at 10 mM concentration, either alone or combined, were completely ineffective in inducing any significant nuclear decondensation after prolonged exposures (24 hr) of incubation. Heparin, 153.8 microM, was capable of inducing only a small increase in nuclear swelling. However, GSH at concentrations as low as 0.1 mM in combination with heparin induces decondensation of human sperm nuclei in vitro. When GSH concentration was kept constant at 5 mM, nuclear decondensation was induced with heparin at concentrations as low as 11.6 microM, and a maximal decondensation (90%) was obtained with only 21.6 microM of heparin. The latter is more than ten times less than the minimal active concentration of heparin used alone.

Cell Nucleus↗

PubMed1988

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states.

Binding of N-formyl-methionyl-L-leucyl-[3H]phenylalanine (fML[3H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (Kd = 12.3 +/- 0.5 nM, n = 22,285 +/- 65,008 binding sites per sperm cell) and a second one (Kd = 700 +/- 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (Kd = 6.4 +/- 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.

Cell Membrane↗

PubMed1988

Size-uniform heparin fragments as nuclear decondensation and acrosome reaction inducers in human spermatozoa.

Using size- uniform mixtures of di-, tetra-, octa- and decasaccharides obtained from the depolymerization of heparin with heparinase, we have studied the activity that these low molecular weight heparin fragments may have on the acrosome reaction and sperm nuclei decondensation processess. Swelling of human spermatozoa nuclei was stimulated by heparin and their fragments and was dependent on the incubation time and directly correlated with the size of the fragment tested. Disaccharides were unable to increase the number of swollen nuclei. At short times (2-8 hrs) decasaccharides were the most active substances tested, including heparin. Only heparin and the tetra- and decasaccharides showed a significant increase in the number of acrosome-reacted spermatozoa, both fragments were more active than heparin at 2 hour incubation. Hexa- and octasaccharides induced a slight increase in the number of acrosome reacted spermatozoa and disaccharides were ineffective. The presence in animal systems of oligosaccharides derived from macromolecules and having specific biochemical properties, remembers the recent discovery of to those mediated by oligosaccharins in plants may exist in animals.

Acrosome↗

PubMed1988

Structure of human sperm chromatin: a study on the accessibility of DNA to macromolecules.

The structure of human sperm chromatin compared with somatic chromatin (liver) was studied by titration of the exposed DNA-phosphate groups with poly-1-lysine (3000 and 28,100 MW) and by their susceptibility to the hydrolytic action of micrococcal nuclease and DNase I. With both sizes of polylysine used, the binding values were significantly lower for sperm chromatin (0.31 +/- 0.05) than for liver chromatin (0.52 +/- 0.05), indicating the presence of about 30% and 52% of free phosphate groups, respectively. Interaction with liver chromatin left no polylysine molecules partially unbound ("wastage") even when 28,100 MW polylysine was used; on the contrary, sperm chromatin showed 26% of "wasted" polylysine even when the smaller polymer was used, indicating that in sperm chromatin the accessible DNA zones are usually no longer than 42 A, that is, 12 base pair. Sperm chromatin was notably more susceptible to both micrococcal nuclease and DNase I action than liver chromatin. However, in the presence of saturating concentrations of polylysine they were similarly protected. Micrococcal nuclease and DNase I hydrolysis products of sperm fractions when submitted to electrophoresis produced a polydisperse smearing pattern along the gel that was difficult to correlate with the presence of nucleosomal structure.

Chromatin↗

PubMed1987

Increased acrosome-reaction inducing activity of glycosaminoglycans by partial hydrolysis.

The possibility that partial hydrolysis of glycosaminoglycan-sulfates (GAGs) such as occurs during the last phases of follicular maturation could play some role in the activity of follicular fluid as an inducer of the acrosome reaction was explored. Hydrolysis of follicular fluid GAGs (ff-GAGs) for 30 min with low-pH HNO2 substantially increased (more than 3 times) its capacity to induce the acrosome reaction. This increase was significantly reduced when the time of hydrolysis was either shorter (10 min) or longer (60 min). Partial hydrolysis of spermatozoa GAGs by direct incubation of sperm cells with chondroitinase ABC was also capable of inducing the acrosome reaction.

Acrosome↗

PubMed1986

Taurine content and amino acid composition of human acrosome.

The presence and concentration of taurine was determined by amino acid analysis in human spermatozoa acrosomes isolated by the method of Srivastava. Taurine is one of the four amino acids whose concentration is higher in the acrosomal extracts, being only lower than histidine, methionine and lysine. It is worth mentioning that these four amino acids constituted 50% the free amino acid concentration in this organelle. The role that this high concentration of taurine, and also the presence of considerable amounts of methyl histidine may have in the functioning of the acrosome, is discussed.

Acrosome↗

PubMed1986

Mode of entry of glycosaminoglycan sulfate during epididymal maturation of bull spermatozoa. I. Charge neutralization mechanism.

Heparin uptake by bull spermatozoa from each one of main epididymis regions was investigated to determine whether the heparin binding was modified during epididymal maturation, the heparin internalization differed during the maturation process, and the charge neutralization by histone or polylysine enhances heparin internalization. From our results we conclude that membrane binding of heparin is greater in the spermatozoa from the caput epididymis than in those of the cauda epididymis region (9.41 +/- 2.27 vs. 2.89 +/- 0.72 pmole of [3H]heparin) and that membrane binding of heparin occurs also at 4 degrees C (4.97 +/- 1.36 vs. 1.05 +/- 0.54 pmole of [3H]heparin). It was also clearly shown that heparin uptake requires active metabolism of the cell and that internalization decreases according to the progress of the epididymal maturation process [6.34 +/- 1.25 (caput) vs. 1.74 +/- 0.72 (cauda)]. Finally, charge neutralization of heparin by histone or polylysine did not enhance heparin internalization.

Animals↗

PubMed1985

Effect of divalent ions in acrosome reaction induced by glycosamineglycans in porcine spermatozoa.

Magnesium, calcium, and zinc at the concentration of 10 microM are capable of inducing a "true" acrosome reaction in the pig spermatozoa judged by the criteria of the fusion of the acrosome and the plasmatic membrane at the anterior region or the sperm nucleus. The optimal percent of acrosome reaction reached by any of the ions tested as a whole was 50%. When glycosamineglycan sulfate (GAGs) plus 10 microM of Mg++, Ca++, or Zn++ was added, they reach to 70-80% of acrosome reaction. At the electrom microscope, thin sections taken from pig spermatozoa treated with ions, GAGs, or ion + GAGs under optimal experimental conditions revealed the same pattern of acrosomal reaction. Results suggest the important role that divalent cations play in general in the induction of the acrosome reaction and question the so-called essential role of calcium ions.

Acrosome↗

PubMed1984

Glycosamineglycan sulfate as acrosomal reaction-inducing factor of follicular fluid.

Follicular fluid from different mammalian species possesses two factors responsible for the induction of capacitation: a sperm-stimulating factor and an acrosomal reaction-inducing factor. The glycosamineglycan-sulfate (GAGs) extracted from pig follicular fluid induce acrosome reaction in pig spermatozoa which exhibit no morphological difference between the GAGs-induced reaction and the natural one. Acrosomal reaction commenced 30 min after the addition of GAGs and depended on GAGs concentration reaching 80% of acrosomal reacted spermatozoa after 6 hr of incubation with 7 mg of GAGs/ml. Chemical composition differs with the chemical data that characterize them as proteoglycans since those we obtained were practically protein free (2%). Another difference resides in the uronic acid content, which is almost twofold higher (59%). Electron microscope observations of the acrosomal reacted spermatozoa revealed that the addition of 10 mg/ml of trypsin soybean inhibitor did not interfere with any of the acrosomal reaction steps. The active capacitating factors may also originate from the follicular fluid released into the genital tract during ovulation.

Acrosome↗

PubMed1984

Heparin-induced release of DNA template restrictions in human sperm zinc-depleted nuclei.

The addition of heparin to human sperm zinc-depleted nuclei releases DNA template restrictions. Spermatozoa depleted of zinc were assayed for (3H-methyl), thymidine incorporation was observed (27,500 +/- 1,248 dpm of 3H methyl-thymidine). Sperm cells incubated in the presence of 10 mg/ml of soybean trypsin inhibitor shows no effect in sperm nuclear swelling or in the release of DNA template restrictions. This process runs in a parallel fashion to the nuclear swelling induced by heparin, suggesting that swollen nuclei might be the source of DNA template. This was confirmed by autoradiographic studies, since all the sperm cells whose nuclei were judged swollen by morphological criteria also appeared labeled. The fact that there was no need for ATP generating system or of exogenous DNA polymerase emphasized the control role that zinc plays in the physiology of the human spermatozoa.

Cell Nucleus↗

PubMed1984

Capacitation of human spermatozoa by heparin.

Zona-free hamster oocytes were incubated in vitro with human spermatozoa treated with heparin for 5, 10, 20, and 30 min. Sperm penetration was observed 2, 4, and 8 h after insemination. Sperm penetration was successful in 40%, 60%, and 69% of the oocytes treated for 10 min with heparin. Feminine and masculine pronuclei were clearly identifiable. In these samples, 80% of the sperm was alive and there was approximately 68% progress motility. Live sperm were observed for up to 24 h. Since glycosamineglycan sulfates are normally present in the ooplasm, zona pellucida, follicular and uterine fluids, this might suggest a direct participation of heparin in human sperm capacitation.

Animals↗

PubMed1983

Effect of steroid hormones on membrane sugar transport in human spermatozoa.

Using the model of exchange transport, we found that glucose transport through the human spermatozoa membrane (447 +/- 54 pmoles/min/10(8) cells) is probably the rate-limiting step in sugar utilization. Sugar transport was more efficient for glucose than for fructose (182 +/- 32 pmoles/min/10(8) cells) and depends on a highly asymmetric carrier with at least two transporting sites. Transport was drastically dependent on pH with an optimal pH of 7.4, showing a decrease of more than 60% with a change of 1 pH unit. Testosterone and 17-B estradiol increased the amount of transported sugar (619 +/- 73 and 922 +/- 110 pmoles/min/10(8) cells, respectively), while progesterone has no effect.

Biological Transport, Active↗

PubMed1983

Effect of zinc on decondensation of human spermatozoa nuclei by heparin.

Swelling sperm nuclei were assayed, exposing them to the combine action in increasing amounts of seminal plasma 0.1-1.0 ml (124 +/- 21 micrograms of Zn++/ml). An inhibition of almost 30% in the swollen spermatozoa nuclei was observed with 0.1 ml of seminal plasma (12 micrograms of Zn++/ml) reaching to 71% with 74-124 micrograms of Zn++/ml. Inactivated seminal plasma (boiling) induced the same percentage of inhibition (73%) than normal seminal plasma. Dialyzed seminal plasma (31 micrograms of Zn++/ml) produced an inhibition of 88% of swollen spermatozoa nuclei, the same percentage produced with 30-35 micrograms of zinc ions (ZnCl2). Previous release of sperm zinc by preincubation with EDTA 6 mM changed the decondensation kinetics, making sperm nuclei more susceptible to the action of the glycosaminoglycan. No effect was observed in the presence of calcium ions. Therefore, zinc, among its several physiological roles, may act as a nuclear chromatin stabilizer.

Cell Nucleus↗

PubMed1983

Heparin-induced nuclei decondensation of mammalian epididymal spermatozoa.

Decondensation of mammalian epididymal spermatozoa nuclei has been induced by exposure of intact spermatozoa to heparin, including those species in which ejaculated sperm were not susceptible to this treatment. This process occurred in the absence of any disulfide bond cleaving reactant. Swelling of caput epididymal spermatozoa nuclei commenced about 30 min after the addition of heparin, reaching 88% in rat, 33% in rabbit, 26% in pig, and 62% in bull of swelled nuclei after 6 hr of incubation at 37 degrees C with 5000 USP of heparin per ml. Corpus epididymal spermatozoa nuclei of rat and rabbit underwent decondensation at 50 degrees C reaching 24% and 22% of swelled nuclei, respectively, after 6 hr of incubation. The nuclei of the sperm cells of pig and bull from this epididymal region remained highly condensed as well as the nuclei of the cauda epididymal spermatozoa of all the species assayed. Electron microscope observations of the caput epididymal spermatozoa nuclei treated with heparin revealed that the chromatin is organized into nuclear bodies joined by a network of cross-linked and branched chromatin fibers in the species studied.

Animals↗