L. Huacuja

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.

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.

Male pronuclei formation release of phosphorylation of histone H-3 during decondensation of human sperm nuclei activated in vitro by heparin.

The release and phosphorylation/dephosphorylation mechanisms of human spermatozoa histone during nuclei in vitro decondensation by heparin was studied. Washed sperm cells were incubated in the presence of 32P and in the absence or presence of heparin. The results showed an increase in the incorporation of 32P of 20 times greater in the presence of heparin than in the absence of heparin (the control sample). In some cases the incorporation of 32P into histones was confirmed by its isolation. To validate these results a phosphorylation kinetic of isolated sperm histone, used as a substrate, was performed. The amount of 32P was not a linear function of time, and maximal phosphorylation was reached in 60 min. A measurement of 32P incorporated as a function of the amount of histone, shows a linear relationship of up to 50 micrograms of protein, with a rapid saturation thereafter with the incorporation of 220 nm and with a KD = 442 x 10(-6) mol/L. 32P incorporation, independent of exogenous cAMP, was related to alkaline pH but was totally dependent on temperature--with a maximum of 37 degrees C. The only histone released was histone H-3. Phosphorylation/dephosphorylation is involved during male pronuclei formation.

Elemental composition of subcellular structures of human spermatozoa. A study by energy dispersive analysis of X-ray.

Abstract The elemental chemical composition of selected intracellular structures (membrane, mitochondria and nucleus) of the human spermatozoa were studied by the combined use of energy dispersive analyses of X-rays and electron microscopy and the results compared with those obtained by atomic absorption spectrometry of isolated subcellular structures (heads and tails) of the same type of cells. In nuclei EDAX studies showed the following relative concentrations P>S>Mg, K, Zn, Si>Ca, Fe. The mitochondrial spectrum showed the presence of important concentrations of Ca>Fe, K, P>Mg, S>Mn. Human spermatozoa membrane was found to be particularly rich in Ca, S and Z n. By atomic absorption it was found that K was the most concentrated element in both isolated fractions (heads and tails). Sperm heads were found richer than tails in Na, Cu and Zn, while sperm tails had higher concentrations of Ca. The zinc concentration of human sperm cells and their subfractions was considerably higher than the reported Zn concentration in any other human cells or their subfractions.

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.

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.