Biomedical subjects

N M Delgado

Publications and source records attributed to N M Delgado.

At least 55 records · Page 3Linked to original sources

PubMedDate not supplied

Heparin and glutathione II: correlation between decondensation of bull sperm cells and its nucleons.

The correlation between the kinetics of bull sperm nuclear and nucleon decondensation induced by the action of physiological concentrations of heparin/GSH was studied. Sperm and nucleon suspensions were incubated at 37 degrees C in salt medium, at a constant concentration of either heparin or GSH and increasing concentrations of the other reagent. Even though nucleons are pretreated with DTT/CTAB, when they are incubated alone with GSH for 96 h, they remain intact, no matter which concentration is employed, and it was impossible to observe the slightest sign of nuclei decondensation. Therefore, rupture of disulfide bridges is not the main mechanism to induce nuclei decondensation and perhaps the GSH role resides in potentate the heparin effect by increasing its negative charge. Nevertheless, nucleons reach 95% of chromatin decondensation in the presence of heparin plus GSH or heparin alone. The fact that the correlation between heparin and GSH concentrations needed to induce sperm nuclei decondensation was 3- to 4-fold greater that in nucleons might be due to the complete lack of nucleon membranes. Heparin/GSH seem to induce nuclei decondensation by an ionic chromatin charge neutralization mechanism.

Animals↗

PubMedDate not supplied

Heparin-glutathione III: study with fluorescent probes as indicators of membrane status of bull sperm.

Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.

Acrosome↗

PubMedDate not supplied

Male contraception, IV: hypotonic-like effect from Echeveria gibbiflora on human sperm.

At present there is no accepted method for the regulation of male fertility. The most appropriate form of contraception would undoubtedly be to develop from traditional plant-derived folk drugs a contraceptive male method. Hypoosmotic shock is used for validate plasma membrane function and its fertilizing capacity of human sperm. Such effect is induced in human sperm in the presence of a purified fraction from Echeveria gibbiflora (PFEG) aqueous crude extract. The hypotonic-like effect included a distension of the plasma membrane over the acrosome region and in some occasions around the sperm middle piece. An enhanced activity of the immobilizing and agglutination effects was induced instantaneously after the addition of PEFG versus aqueous crude extract activity (OBACE). Using electron microscopy it was possible to observe a deposit of a "sticky" dense material intercalated along the plasma membrane. The membrane was sealed making it impossible to measure the viability or metabolic activity of the treated sperm by the fluorescence (FDA-IP) technique. It was also possible to corroborate the dispersion of the acrosomal content and the disappearance of the acrosomal and nuclear membrane. Results that makes PFEG hypotonic-like effect a serious candidate to conduct a study to determine the predicting capacity of this compound in human infertility and suggest that the plant may yield a compound suitable for use as male contraceptive agent.

Contraception↗

PubMedDate not supplied

Detection and bioimaging of nitric oxide in bovine oocytes and sperm cells.

Mammalian gametes contain constitutive nitric oxide synthases (NOS) to synthesize nitric oxide (NO). The detection and bioimaging of NO in bovine gametes is important to determine the regulatory roles of NO during the different events of fertilization. Diaminofluoresceins, new fluorescence indicators for NO, were applied to detect the release of NO from bovine gametes. These compounds yield green fluorescent triazolofluoresceins, which provide the advantages of high specificity, sensitivity, and simplicity for the detection of NO. In this study, we mapped the expression of NOS in the bovine sperm and ova. NOS activity in sperm first appeared in the acrosome, then 60 min later in the head, middle piece, cytoplasmic droplet, and tail. Cow ova had high NO activity in the cytoplasm and in the surrounding corona cells, but not in the zona pellucida. These results show that for bovine gametes, the synthesis NO by the NOS system presents clear patterns of time and spatial distribution that may be important for the different events of fertilization.

Acrosome↗

PubMedDate not supplied

Fluorescent berberine binding as a marker of internal glycosaminoglycans sulfate in bovine oocytes and sperm cells.

The use of berberine as a biological marker of glycosamineglycans sulfate was employed to corroborate the presence of heparin in mammalian oocytes and sperm and its distribution in all the structures, or only in some specialized zones, of the male and female gametes. Oocytes and sperms were treated with 1.8 mM berberine for the presence of heparin and examined 10, 30, 60, and 120 minutes later. We have found that heparin is homogeneously distributed in all the zones of bovine oocytes and in sperm cells. When sperm cells are first treated with 80 microM of heparin and then berberine, 40% of them display in their post acrosomal region an intense yellow fluorescence. This may be in relation to the high amount of heparin binding sites due to the presence of the reticular membranous like system in this sperm region and in its possible role whereby gametes recognize and adhere to one another. Therefore, the use of berberine as a fluorescent marker of heparin represents clear proof of the presence of GAGs and their binding sites in the outside and inside of mammalian gametes, reinforcing the importance they play in the events of the process of fertilization.

Animals↗

PubMedDate not supplied

Arrangement of beta tubulin in bull spermatozoa during nuclei decondensation.

Eukaryotic cells have internal scaffolding of microtubules cytoskeleton that gives them their characteristic shapes. We analyzed by immuno-fluorescence the shift and distribution of tubuline during in vitro bull sperm nuclei swelling by the action of heparin-reduced glutathione physiological decondensing agents. Sperm tubulin display a pattern that shows tubulin fluorescence all over the head, leaving the acrosome tip devoid of tubulin. In the second stage we can observe that the acrosomal zone is practically devoid of fluorescence and a net of fluorescent microtubules that seems to be anchored in the basal plate in the postacrosomal region. It is also possible to observe green spots of tubulin fluorescence in the nucleus periphery, that might represent clusters of chromatin hub-like bodies and/or the microtubule organizing center (MTOC). In the third stage, practically all tubulin moves backwards to the basal plate in the neck region of the sperm nuclei remaining in only the green fluorescence spots in the periphery of the swollen sperm nuclei. The results allow us to assume that tubulin mechanism rearrangement is considered to be necessary for the normal fertilization process.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

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.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMedDate not supplied

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.

Cell Nucleus↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

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.

Aniline Compounds↗

PubMedDate not supplied

Human urinary glycosaminoglycans as accurate method for ovulation detection.

Urinary glycosaminoglycans (GAGs) content showed a characteristic pattern of fluctuation during normal and hormonally induced cycles with a distinct peak at ovulation. This peak of maximal GAGs concentration (106.7 +/- 46.2 micrograms/mL in urine) was centered according to the day of the midcycle LH surge, which serves as the reference point, designated day 0. All cycles are presumed to be ovulatory on the basis of biphasic BBT nadir, ultrasonographic studies, and progesterone assays (greater than 5 ng/mL of serum) during the secretory phase. In hormonally induced menstrual cycles, a noticeable increase in GAGs concentration (70%) was observed. This indirect evidence suggests a possible correlation between GAGs content and hormonal effect. All types of GAGs, with the exception of heparin, have been found in urine, chondroitin sulfate C being the major component at time of ovulation. These results strongly suggest that urinary GAGs determination is a precise method for ovulation detection.

Adult↗

PubMedDate not supplied

A rapid method for ovulation detection.

Uronic acid (UA) content in whole urine showed a characteristic pattern of fluctuations during menstrual cycle (24 cycles) with a distinct peak on the day of ovulation (midcycle peak 1043 + 68 micrograms/ml urine. This peak of maximal UA concentration was validated by ultrasonography (abdominal and/or vaginal scanner and basal body temperature. In hormonal induced menstrual cycle (5 cycles) a significant decrease in Ua concentrations were observed during the follicular phase (20%) and in the midcycle peak (11%) when they were compared with the same values in the normal cycle. Nevertheless, two days after the ovulation peak occurs a second peak can be observed (1344 + 799 micrograms UA/ml urine). On the other hand in the anovulatory cycles (5 cycles) the same diminishing behavior in the UA concentrations were observed in both phases of the cycle (50-60%) with the expected lack of the midcycle peak. Results that clearly show that UA excretion in urine reflect a excellent correlation with the menstrual cycle allowing us to propose this technique as an accurate and reliable method to detect ovulation.

Adult↗

PubMedDate not supplied

[Follicular maturation in the swine. Changes in the protein secretion pattern during follicular growth].

The formation of fluid, maturation and follicular differentiation are physiological processes unknown to this date. Therefore, changes in the electrophoretic pattern of proteins and glycoproteins in different states of maturation were studied, as well as the volume and protein concentration. Follicular fluid was collected from 43 follicles with a diameter of 2 mm., 45 follicles with a diameter of 5 mm. and 49 of 9 mm. The average of the concentration of protein for the 2 mm. follicles was 106 +/- 56.3 mg/ml of follicular fluid, for the 5 mm. ones 68.3 +/- 37.1 mg/ml and the 9 mm. ones had 52.9 +/- 21.3 mg/ml. In the protein and glycoproteins characterized by electrophoresis, a consistent pattern of 3 bands was found, and none were PAS positive in the 2 mm. follicles. In the 5 mm. follicles a 10 band pattern was found, of which 1 was PAS positive. In the last plane, the 9 mm. pattern was 12 and 2 respectively. The average value of follicular fluid was 5.3 +/- 1.8 microliters, for the 5 and 9 mm. it was 57 mu 25 microliters and 190 +/- 77 microliters respectively. Statistical analysis between volume and size show a highly significant correlation, thus suggesting the use of one of them to extrapolate the other.

Animals↗

PubMedDate not supplied

[Agglutinating and immobilizing activity of an ethanol extract of Bursera fagaroides on human and other mammalian spermatozoa].

Human spermatozoa and those obtained from mouse epididymis incubated in ethanolic from mouse epididymis incubated in ethanolic extracts of the cortex of Bursera fagaroides became agglutinated and immobilized in 100% with 30% of viability decrease. This double effect occurred in 50% of pig'ssperm and 50% of rabbit sperm when the concentration was increased tenfold. The pattern of progressive motility was affected 80% of rat's spermatocytes without agglutination. The values remained the same when the extracts were partially purified with salts of Zn++ and Ba++ or by filtration in a sephadex g-10 column. Analysis with thin layer chromatography of both purified extracts produced 3 compounds in comparison with the ethanolic unpurified extract. The extract of the plant Bursera fagaroides contains 3 compounds, which apparently are glycosides with a potent activity upon agglutination -immobilization and a low effect upon spermatocytes viability, which might be used as contraceptives .

Animals↗