N. M. Delgado

A kinetic study of the participation of zinc in human spermatozoa metabolism.

Abstract Incubation of washed human spermatozoa in the presence of 6 mM concentrations of EDTA, histidine and cysteine induces a release of about 75% of the zinc bound to the cells. No zinc is released by human spermatozoa when incubation is done in the absence of the mentioned reagents. No detectable amounts of calcium or magnesium were found to be released by the sperm cells under any of the experimental conditions tested. Zinc release induced by the presence of EDTA, histidine and cysteine is accompanied: 1) by a significant increase in oxygen uptake, both under basal conditions and in the presence of some substrates (glucose, pyrubate and succinate) and 2) by a significant increase in motility. This increase was greater with cysteine than with histidine, and greater with the latter than with EDTA. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and is related to the regulation of energy metabolism and probably to sperm capacitation.

Heparin Binding Sites in the Human Spermatozoa Membrane

The existence in the human spermatozoa membrane of receptorlike functional group for heparin was studied. Incubation of whole spermatozoa with tritiated heparin induced the specific binding of 745 +/- 112 pmol of heparin per 5 x 10(7) sperm cells with an intrinsic association constant KD = 3.6 x 10(-6) M. The specificity of binding was shown by the lack of competence in the binding process of some other glycosaminoglycans used at concentrations 20 x higher than heparin. However, dextran sulfate was a very efficient competitive agent. Autoradiography experiments showed that labeling was almost completely restricted to sperm cells in the process of nuclear decondensation. This technique showed the presence of a high amount of radioactive heparin in the isolated sperm membranes even after several washings. Heparin may participate both in the final part of the capacitation process (acrosome reaction) and in the decondensation of sperm nuclei.

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.

Correlation Between Sperm Membrane Destabilization by Heparin and Aniline Blue Staining as Membrane Integrity Index

Acidic aniline blue stain (AAB) was studied in relation to sperm membrane destabilization and nuclei decondensation by heparin. Untreated spermatozoa smears stained with AAB or vital stain shows 28.4% of stained and 71.6% of unstained nuclei. This behavior was also observed when incubation was done in the presence of 5 mM glutathione (GSH) used alone. In the presence of 21.6 microM heparin, staining of sperm cells commenced 10 min after heparin addition and was dependent on the incubation time. During the experiment 12.3% of the total cholesterol content and 20 micrograms protein/10(8) sperm cells were released. In the presence of 21.6 microM heparin-5 mM/GSH, swelling of sperm nuclei reach 95% after 150 min incubation. When this experiment was run along with AAB, the same average (45%) was seen in the first 30 min, which gives plenty of time to trigger the nucleis decondensation mechanism. The percentage of stained cells was of 71%, indicating that the histone is not completely replaced, and insuring a positive reaction with AAB stain. It would appear that AAB stain can be used as a membrane integrity index to confirm the destabilization effect of heparin on the sperm membrane.

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.

Exchange of Lipids Between Spermatozoa and Seminal Plasma in Normal and Pathological Human Semen

Normal and pathological semen were studied with regard to cholesterol and phospholipid content of sperm cells and seminal plasma. Spermatozoa from pathologic semen have similar concentrations of phospholipid-phosphorous and significantly higher cholesterol concentration than spermatozoa from normal semen. However, only oligoasthenospermic spermatozoa showed a significantly higher cholesterol/phospholipid ratio. Azoospermic seminal plasma showed the lowest values of both cholesterol and phospholipids, but the ratio of cholesterol to phospholipids was equal to that in normal spermatozoa. No significant difference was found in the cholesterol concentration of seminal plasma from oligoasthenospermic, asthenospermic, and normospermic subjects and only asthenospermic plasma showed a significantly lower concentration of this compound. Cholesterol and phospholipid exchange between sperm cells and seminal plasma was shown by the striking correlation between the lipid composition of seminal plasma with that of sperm cells.

Modification of human sperm metabolism by the induced release of intracellular zinc

Incubation of washed human spermatozoa in the presence of 6 mM histidine induces a release of about 75% of the zinc bound to the cells. Zinc release induced by the presence of histidine was accompanied by: 1) a significant increase in the utilization of exogenous 14C-labelled glucose, which is reflected in an increase in the production of 14CO2, 2) a small but significant decrease (−14%) in the utilization of fructose when this sugar is added as exogenous substrate, and 3) a highly significant decrease in the endogenous sperm phospholipids (>30%), an effect which is not inhibited by the addition of exogenous substrates. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and seems to be related to the regulation of energy metabolism and probably to sperm capacitation.

Cyclic-amp receptors in the human spermatozoa membrane

Abstract The binding properties of cyclic-AMP to human spermatozoa were studied. Incubation of whole human spermatozoa and of head and tail fractions with 14 C-cyclic AMP induced the binding of 7.8 ± 0.86 pmoles of the nucleotide per 5 × 10 7 sperm cells showing an intrinsic association constant of 15 × 10 −8 M. No significant inhibition of cyclic-AMP binding was caused by the addition of one hundred fold excess of AMP. However, when the excess AMP reached a thousand fold a slight but significant reduction (10%) was observed. These values were not modified by using sperm homogenates instead of intact sperm cells, which suggested that cyclic-AMP binding is to surface membranes and not to protein released from broken or damaged sperm. Binding was found to be unrelated with pH, between 6 to 8, but depended on the temperature of the incubation medium, showing a maximum at 20°C. The blocking of membrane sulfhydryl groups significantly inhibited (48%) cyclic-AMP binding to sperm cells.

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.

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. Copyright