Calcium (hydrogen-1-malate) hexahydrate on Echeveria gibbiflora leaves and its effect on sperm cells.

Echeveria gibbiflora is a plant widely used for its contraceptive activity in traditional Mexican medicine. Data on calcium crystals in plants are not outstanding. In the case of the Echeveria gibbiflora leaves, however, its quality, quantity, and salt type are quite surprising; one striking result of its X-ray crystallographic data shows the presence of calcium bis (hydrogen-1-malate) hexahydrate [2(C4H5O(5)1), Ca(1)2+, 6(H2O1)]. This highly soluble compound might explain the rapid shape changes of calcium crystals. Because SEM-EDS analysis shows that calcium malate crystals were obtained in a highly pure state and the immobilization and agglutination pattern that OBACE show on human and bull spermatozoa are not found even when high concentrations of calcium bis (hydrogen-1-malate) hexahydrate salt are present it is not feasible to involucrate molecules as calcium malate as part of the OBACE contraceptive activity.

Nucleons, I: A model for studying the mechanism of sperm nucleus swelling in vitro.

The nucleon, a highly organized chromatin structure, was studied to learn if its swelling takes place by the action of heparin/GSH, without the participation of any mechanism provided by sperm membranes, subcellular organelles, or other proteins foreign to the sperm nucleus. Sperm suspensions of guinea pigs and rats were incubated with 9 mM DTT and 1% CTAB. The nucleons obtained from washed epididymal spermatozoa appear under a phase-contrast microscope to preserve their original nucleus shape and to completely lack the acrosome, middle piece, and tail. In an electron microscope, nucleon thin sections show a slight nuclear chromatin decompressed from the periphery toward the center. An outstanding result was that the nucleon swelling pattern by heparin/GSH showed the same classic organization into hub-like nuclear bodies joined by a network of chromatin fibers ranging in thickness from 25 to 1.5 nm. Under the conditions of this study there was no need of any membrane or subcellular structure. At stage IV, all the thick fibers disappear, leaving only thin bead fibers on a string. With respect to nuclear swelling there is no doubt that the sperm chromatin is organized in a special form that decides a specific required pattern of unpacking.

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.

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 nuclei's 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.

DNA unpacking in guinea pig sperm chromatin by heparin and reduced glutathione.

The kinetics of nuclear decondensation and DNA unpacking induced by the action of a physiological concentration of heparin and glutathione of guinea pig spermatozoa was studied. Sperm (acrosomeless) suspensions were incubated at several different temperatures (37, 40, 43, and 46 degrees C), with a constant concentration of either heparin (50 microM) or reduced glutathione (12.5 mM) and increasing concentrations of the other reagent. Nuclei spermatozoa remained highly condensed when incubated in the medium alone or in either GSH or heparin alone for up to 72 h. Swelling of nuclei spermatozoa was initially observed during the first 20 min of incubation. The sperm nuclei initiate decompaction at the central part of the nuclear structure while at the periphery there remain numerous residues of densely packed chromatin. The swollen chromatin pattern presents the characteristic organization into "hub-like" nuclear bodies that measured 10-100 nm diameter joined by a network of chromatin fibers. At full nuclei decondensation chromatin end fibers are loose, probably meaning that DNA is not organized into loop domains. DNA presence was verified by the use of ethidium bromide and acridine orange.

Effect of heparin-reduced glutathione on hamster sperm DNA unpacking and nuclear swelling.

This study examined the kinetics of sperm nuclear decondensation induced by the action of physiological concentrations of heparin and glutathione in hamster sperm nuclei as a chromatin model that contains protamine P1 and P2. Sperm suspension was incubated at different temperatures (37, 40, 43, and 46 degrees C) in media, keeping constant the concentration of either heparin or GSH and increasing concentrations of the other reagent. Spermatozoa nuclei without any treatment, incubated for 72 h, appear densely condensed. Swelling of hamster spermatozoa nuclei was observed after 30 min of incubation in the presence of efficient concentrations of heparin-GSH. The extent of this time lag was significantly reduced at higher temperatures. DNA presence was verified by the use of ethidium bromide, acridine orange, and Feulgen stain. Phase-contrast microscopy shows that nuclear decondensation begins at the equatorial levels, with DNA highly condensed at the acrosome pole, and the basal pole as the DNA attachment point. Electron microscopy observations showed that hamster sperm nuclei initiates its decompaction at the peripheral regions and this behavior remains until late stages of decondensation, nevertheless, the chromatin is organized into "hub-like" nuclear bodies that measured 10-100 nm in diameter, joined by a network of chromatin fibers with apparent reduction in number. At the decondensation full stage, the network seems to be wide open with a reduced number of hub-like nuclear bodies present in the interlace. DNA is not organized into topologically constrained loop domains and is attached to the basal plate instead of to the nuclear matrix or any other structure.

Differential decondensation of class I (rat) and class II (mouse) spermatozoa nuclei by physiological concentrations of heparin and glutathione.

The kinetics of sperm nuclear decondensation induced by the action of physiological concentrations of heparin and glutathione was studied by comparing two rodents: the rat, with very stable protamine P1 containing chromatin (class I nuclei), and the mouse, with protamine P1 and protamine P2 (class II nuclei). Sperm suspensions were incubated at different temperatures (37, 40, 43, and 46 degrees C) in media while keeping a constant concentration of either heparin or GSH and increasing concentrations of the other reagent. Spermatozoa nuclei without any treatment incubated for 72 h appear densely condensed. Swelling of mouse spermatozoa nuclei was observed after 30 min of incubation in the presence of efficient concentrations of heparin-GSH. The extent of this time lag was significantly reduced at higher temperatures. This behavior was also observable in the rat, but required time lags of 3-4 h. Electron microscopy observations showed that the pattern of nuclear decondensation was different in both animal species. Mice sperm nuclei initiates its decompaction by the peripheral regions and this behavior remains until late stages of decondensation. On the contrary, rat spermatozoa nuclei decondense initially at the central part of the nuclei while the periphery remains condensed, showing numerous residues of densely packed chromatin. In both cases, the chromatin is organized into "hub-like" nuclear bodies joined by a network of chromatin fibers.

Energy metabolism of spermatozoa during pronucleus formation induced in vitro by heparin-reduced glutathione. I. Glucose uptake.

Glycolitic metabolism under basal conditions and its modifications by the combined action of heparin and GSH were studied in human sperm. Respirometric data indicated that the amount of U. L. [14C]-glucose converted to 14CO2 increased with the incubation time, being almost linear for up to 60 min and then leveling off at 120 and 150 min (594 and 620 nmol of [14C]-glucose/10(8) spermatozoa, respectively). When spermatozoa were incubated in the presence of heparin-GSH such behavior completely changed, showing a decrease (approximately 50%) in glucose metabolism with values of 254 and 366 nmol of [14C]-glucose/10(8) spermatozoa at the same incubation times as the basal consumption. When these results were compared with the kinetic of the swollen nuclei it was seen that at 30 min 44% of the spermatozoa have its nuclei swollen with a glucose uptake value of 91 nmol/10(8) spermatozoa, and at 150 min when nearly all the spermatozoa nuclei are swollen (95%) the glucose uptake increases fourfold more than the initial rate at 30 min. Therefore, it is possible to suggest the existence of an energy contribution by the sperm to the male pronuclei formation mechanism.