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M Sousa

Publications and source records attributed to M Sousa.

At least 55 records · Page 3Linked to original sources

Developmental changes in calcium content of ultrastructurally distinct subcellular compartments of preimplantation human embryos.

The ultrastructural localization of mobilizable Ca2+ in different subcellular compartments of human oocytes and preimplantation embryos was studied using the potassium-pyroantimonate technique and transmission electron microscopy; the specificity was confirmed by chelation experiments and X-ray microanalysis. In unfertilized oocytes, Ca2+ was detected in small vesicles beneath the plasma membrane as well as in other forms of smooth endoplasmic reticulum (SER) and in mitochondria but not in cortical granules. In pronuclear zygotes and blastomeres of cleaving embryos, Ca(2+)-rich vesicles were no longer present close to the plasma membrane, and the entire periphery was poor in Ca(2+)-containing organelles which, however, were abundant in the perinuclear region. The uneven Ca2+ loading of SER and mitochondria from the pronuclear stage onwards suggests that Ca2+ release from both these types of organelle contributes to the embryonic Ca2+ signals. During mitosis, less Ca2+ was detected with organelles, but the antimonate reaction product was more abundant in the cytosol. These data suggest that, in addition to different forms of SER, mitochondria also act as a source of mobilizable Ca2+ in preimplantation human embryos. The previously described developmental and cell cycle related changes in the characteristics of Ca2+ signals are associated with the redistribution and structural reorganization of these organelles.

Antimony↗

Calcium responses of human oocytes after intracytoplasmic injection of leukocytes, spermatocytes and round spermatids.

Oocyte activation in mammals involves the action of a soluble sperm factor (SSF) that enables oocytes to develop a characteristic series of Ca2+ spikes (Ca2+ oscillations). SSF is also likely to be responsible for the Ca2+ oscillations driving oocyte activation after intracytoplasmic sperm injection [ICSI]. With an appropriate injection technique, Ca2+ oscillations do not develop spontaneously after ICSI but can be triggered by subsequent treatment of sperm-injected oocytes with Ca2+ ionophore. Here we show that Ca2+ oscillations, quite similar to those developing after ICSI, can be triggered by the ionophore treatment in human oocytes previously injected with human round spermatids. In contrast, oocytes injected with earlier spermatogenic cell (primary and secondary spermatocytes) and with non-germ cells (polymorphonuclear leukocytes) did not develop Ca2+ oscillations after the ionophore challenge although the subsequent injection of SSF did induce typical Ca2+ oscillations in these oocytes. Disintegration of the plasma membrane of the injected cells was detected in all cases by transmission electron microscopy. Thus, the absence of the typical oscillatory Ca2+ response in spermatocyte-injected oocytes was due to the actual deficiency of SSF in the spermatocytes rather than to a defective responsiveness of the injected oocytes or to the failure of SSF release into the oocyte cytoplasm. The ability of human round spermatids to induce a response to calcium in oocytes that is similar to that induced by mature spermatozoa may be important for normal embryonic development after spermatid conception.

Calcium↗

Developmental changes in calcium dynamics, protein kinase C distribution and endoplasmic reticulum organization in human preimplantation embryos.

Developmental changes in the Ca2+ dynamics of human zygotes and preimplantation embryos were related to changes in the distribution of endoplasmic reticulum (ER) and protein kinase C (PKC). The fertilization-induced Ca2+ oscillations were typically observed over > 5 h, were ryanodine-sensitive and showed a periphery-to-centre propagation of Ca2+ waves. At the same time, ER and PKC were accumulated in the cell periphery. After the appearance of pronuclei, ryanodine-sensitive Ca2+ oscillations of lower amplitude and frequency were observed until the pronuclear breakdown. However, Ca2+ waves then began in the perinuclear region, in the area of ER and PKC accumulation and spread towards the cell periphery. During the second to fourth cell cycle, small sinusoidal Ca2+ fluctuations were observed; sparse higher-amplitude Ca2+ spikes, superimposed on these basal fluctuations, appeared shortly before cell division. The sinusoidal Ca2+ fluctuations were asynchronous in individual blastomeres and disappeared progressively in arrested embryos. The direction of Ca2+ wave propagation and the distribution of ER and PKC were similar to the situation observed in pronuclear zygotes. In contrast to the zygotes, ryanodine did not arrest the Ca2+ oscillations but augmented their amplitude and frequency. These data suggest that human pre-embryos use different mechanisms of Ca2+ signalling in the early post-fertilization period, during the pronuclear development and during cleavage.

Calcium↗

The role of ryanodine-sensitive Ca2+ stores in the Ca2+ oscillation machine of human oocytes.

This study was undertaken to localize ryanodine-sensitive Ca2+ stores in human oocytes and to evaluate their role in the Ca2+ oscillations responsible for oocyte activation at fertilization. The addition of ryanodine provoked a Ca2+ discharge from stores localized throughout the ooplasm with the exception of the cortical and subcortical peripheral regions. The ryanodine-induced discharge was typically followed by a short series of Ca2+ oscillations that only involved the cytoplasmic region populated by the ryanodine-sensitive stores. In contrast, the Ca2+ oscillations induced by the thiol reagent thimerosal or by spermatozoa at fertilization were of a much longer duration and also involved ryanodine-insensitive stores. Presumably, these ryanodine-insensitive stores are sensitive to inositol 1,4,5-trisphosphate (InsP3). The addition of ryanodine to oocytes during ongoing thimerosal- or sperm-induced Ca2+ oscillations inhibited the oscillations. These data suggest a co-operation between the ryanodine-sensitive and ryanodine-insensitive stores in maintaining the sperm-induced Ca2+ oscillations. In this two-store oscillation model, each periodic [Ca2+]i increase is triggered by a Ca2+ discharge from the peripheral, InsP3-sensitive stores inducing Ca(2+)-induced Ca2+ release from the ryanodine-sensitive stores. However, the pacemaker frequency of the Ca2+ discharges from the InsP3-sensitive stores is conditioned by the actual physiological state of the ryanodine-sensitive stores.

Calcium↗

Effects of protein kinase C activation and inhibition on sperm-, thimerosal-, and ryanodine-induced calcium responses of human oocytes.

Previous data have shown that protein kinase C (PKC) participates in the mechanism of sperm-induced calcium oscillations in mammalian oocytes, but the actual role of this enzyme in the oscillation mechanism is still unknown. In this study we show that drugs modulating PKC activity disturb the oscillations induced by spermatozoa, thimerosal and ryanodine, but in a different way for each of the three oscillogenic agents. Moreover, PKC inhibition interferes with the return of the intracellular free calcium concentration to basal values during the sperm- and ryanodine-induced calcium oscillations, but not during the thimerosal-induced calcium oscillations. When the PKC-modulating drugs were applied before any of the three oscillogens, the subsequent calcium oscillations were also disturbed. However, the first calcium spike induced by spermatozoa and thimerosal was little influenced by PKC activation or inhibition. On the other hand, ryanodine failed to produce any calcium response when the PKC activity was clamped to a high level. These data suggest that sustained high PKC activities impede calcium oscillations by interfering with the opening of the ryanodine-sensitive calcium release channel, whereas sustained low activities of the enzyme paralyse the channel in the open state.

Alkaloids↗

The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

The UDP-Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein-linked Man7-9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a unique property: it glucosylates misfolded, but not native, glycoproteins. It has been proposed that the glucosyltransferase participates, together with calnexin, in the control mechanism by which only properly folded glycoproteins can exit from the endoplasmic reticulum. In this paper it is demonstrated that the glucosyltransferase recognizes two elements in the acceptor substrates: the innermost N-acetylglucosamine unit of the oligosaccharide and protein domains exposed in denatured, but not in native, conformations. Both determinants have to be covalently linked. In many cases the first element is not accessible to macromolecular probes in native conformations. Concerning the protein domains, it is demonstrated here that the glucosyltransferase interacts with hydrophobic amino acids exposed in denatured conformations. More disordered conformations, i.e. those exposing more hydrophobic amino acids, were found to be those having higher glucose acceptor capacity. It is suggested that both accessibility of the innermost N-acetylglucosamine unit and binding to hydrophobic patches determine the exclusive glucosylation of misfolded conformations by the glucosyltransferase.

Acetylglucosamine↗

Sperm-induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery.

Temporal and spatial characteristics of explosive periodic increases (spikes) of intracellular free Ca2+ concentration ([Ca2+]i) induced by sperm in human oocytes (Ca2+ oscillations) were analyzed by confocal laser scanning microscopy and compared to Ca2+ oscillations induced in oocytes by the thiol reagent thimerosal. During the steady-state period of sperm-induced Ca2+ oscillations, each individual [Ca2+]i spike invariably began from a focus in oocyte periphery and spread throughout the entire peripheral region before propagating to the central ooplasm. This peripheral Ca2+ wave was immediately followed by an explosive [Ca2+]i increase in the central ooplasm. However, this central [Ca2+]i rise only peaked when [Ca2+]i in the peripheral ooplasm was already on the decline. Moreover, the peak [Ca2+]i values were always considerably higher in the oocyte center than in the periphery. In contrast, thimerosal-induced Ca2+ oscillations did not show this particular form of propagation. These data show that sperm-induced Ca2+ oscillations have a unique pattern of spatial dynamics and suggest that the bulk of Ca2+ mobilized during each spike is released from stores that have a relatively high threshold for Ca(2+)-induced Ca2+ release (CICR). These stores are poorly developed, if not absent, in the oocyte cortex, and CICR from them is triggered by previous CICR from another type of store with a lower threshold that are preferentially located in the oocyte cortex and act as a detonator.

Calcium↗

More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore.

OBJECTIVE: To examine whether fertilization rates after intracytoplasmic sperm injection can be increased by artificial oocyte activation. DESIGN: Oocytes that failed to fertilize spontaneously by 24 hours after intracytoplasmic sperm injection were treated either with calcium ionophore to induce activation or with solvent only to serve as control. The ability of ionophore-treated and control oocytes to achieve delayed fertilization was compared. SETTING: Private hospital and public research center. PATIENTS: Infertile couples treated by intracytoplasmic sperm injection. INTERVENTIONS: Intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Fertilization and cleavage rates. RESULTS: The mean rate of spontaneous fertilization after intracytoplasmic sperm injection was 32%, but 88% of the oocytes that failed to fertilize spontaneously did so after subsequent exposure to calcium ionophore. Most of these oocytes underwent at least one apparently normal cleavage division. In contrast, delayed fertilization of oocytes not treated with ionophore was an exceptional finding. If only oocytes remaining intact after intracytoplasmic sperm injection are taken into account, the mean global fertilization rate of ionophore-enhanced intracytoplasmic sperm injection was 91%. CONCLUSIONS: These results show that the failure of oocyte activation is the main cause of fertilization failure after intracytoplasmic sperm injection. If an appropriate, clinically applicable treatment is found to overcome this problem, intracytoplasmic sperm injection can be expected to yield fertilization rates far exceeding those of standard IVF with normal spermatozoa.

Adult↗

Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca2+ fluxes and oocyte cytoplasmic dislocation.

OBJECTIVE: To analyze the mechanism by which modifications of the intracytoplasmic sperm injection (ICSI) technique influence success rates. DESIGN: Prospective clinical study supplemented with an experimental analysis of Ca2+ fluxes provoked by the injection procedure. SETTING: Private hospital and public research center. PATIENTS: Patients treated by IVF and ICSI. INTERVENTIONS: Intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates and intracellular free Ca2+ concentration. RESULTS: The inclusion of vigorous aspiration of oocyte cytoplasm improved outcomes of ICSI. In a series of 100 consecutive cases treated with this technique, the fertilization and pregnancy rates were 87% of total metaphase II oocytes injected and 52% of total treatment cycles, respectively. Enhanced Ca2+ influx into the injected oocytes and dislocation of the oocyte cytoplasm, including the development of a focus of persistent Ca2+ discharge around the injected sperm head, were the main characteristics of this highly successful technique. CONCLUSIONS: Vigorous aspiration of oocyte cytoplasm may facilitate fertilization after ICSI by increasing the oocyte Ca2+ load at the time of injection, by establishing a more intimate contact of the injected sperm head with oocyte intracellular Ca2+ stores, or by a conjunction of these mechanisms.

Aniline Compounds↗

A cytochemical study of the nucleolus and nucleolus-related structures during human spermatogenesis.

Besides the components of typical nucleoli, three other nuclear structures were identified in spermatogonia by positive silver staining: dense centers, fibrillar regions, and dense granules. There was a close relationship between the dense centers and fibrillar regions in spermatogonia and spermatocytes, whereas the dense granules, which appeared dispersed throughout the nucleoplasm of spermatogonia, became localized at the periphery of chromosomes in spermatocytes. From the beginning of spermiogenesis, these three structures then appeared in direct relationship with the dense fibrillar component of the nucleolus. At the Golgi phase, a new nuclear structure also appeared in close relationship with the dense fibrillar component. As it was morphologically similar to the fibrillar region, but not silver stained, it was designated fibrillar structure. With further spermatid development, as the dense fibrillar component gradually disappeared, a sharp increase in the number of the associated dense granules was observed; these granules then disappeared as well. In spermatids at cap phase, each fibrillar region appeared intimately associated with several fibrillar structures. In maturing spermatids, silver staining became confined to the fibrils that appeared located inside the nuclear vacuole. The nuclear vacuole also contained a dense fibrillar structure in intimate relationship with these fibrils and the peripheral condensed chromatin. Ethidium bromide-PTA, Na-tungstate and EDTA regressive staining suggest the presence of RNP in the fibrillar regions, dense granules and intravacuolar fibrils, and the presence of DNP in the fibrillar structures.

Cell Nucleolus↗

Comparison of Ca2+ responses in human oocytes fertilized by subzonal insemination and by intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the consequences of bypassing the normal interaction between the sperm and oocyte surfaces for the form of Ca2+ responses developing in oocytes at fertilization. DESIGN: Oocytes were fertilized by subzonal insemination (SUZI) (maintaining the normal interaction between cell surfaces of both gametes) or by direct intracytoplasmic sperm injection, and changes in intracellular free Ca2+ concentration were evaluated by confocal laser scanning microscopy after loading oocytes with a fluorescent Ca2+ indicator. SETTING: Private hospital, public research center, and university-based laboratory. PATIENTS, PARTICIPANTS: Patients participating in an assisted reproduction program. INTERVENTIONS: In vitro fertilization, SUZI, intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Changes in intracellular free Ca2+ concentration. RESULTS: All oocytes fertilized after SUZI showed an oscillatory Ca2+ response introduced by a short initial phase with faster Ca2+ oscillations. In contrast, oocytes fertilized after intracytoplasmic sperm injection did not show a similar change in the oscillation rhythm. In both cases, Ca2+ increases were propagated throughout the ooplasm in a wave-like manner. CONCLUSIONS: The results show that there is a relationship between gamete surface contact and the form of Ca2+ fluxes accompanying fertilization. When the contact between gamete surfaces is skipped by direct sperm injection to the ooplasm, a delayed, truncated Ca2+ response is produced which, however, can maintain the form of Ca2+ waves and Ca2+ oscillations typical of normal fertilization.

Calcium↗

Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection.

Human oocytes that failed to display signs of fertilization by 44 h after intracytoplasmic sperm injection (ICSI) were processed for electron microscopic analysis. All oocytes were arrested at metaphase II. The first polar body contained intact cortical granules and chromosome clumps, which were not surrounded by a nuclear envelope but still associated with microtubules. When a second globular body was present, it always showed the same ultrastructure, indicating that it had originated from fragmentation of the first polar body and not from the resumption of the second meiotic division. The most prominent organelles of the oocyte cytoplasm were the smooth endoplasmic reticulum and mitochondria. In the oocyte cortex, cortical granules were intact, with no signs of incipient or incomplete cortical reaction. Oocyte chromosomes were found in the oocyte periphery near the locality of the first polar body extrusion. They consisted of dense aggregates of chromatin associated with microtubules. The chromatin of the injected spermatozoon was demembranated and partially decondensed. In some cases, vesicular and tubular structures, apparently of oocyte origin, were associated with the periphery of the sperm chromatin mass but they never formed a continuous layer. These data suggest that fertilization failure after ICSI is basically a failure of oocyte activation.

Cell Nucleus↗

Human oocyte activation after intracytoplasmic sperm injection.

Oocyte activation is a series of events triggered by the fertilizing spermatozoon and necessary for the beginning of the embryonic development. Calcium plays a pivotal role in this process. Here we used confocal laser scanning microscopy to examine the changes in the concentration of intracellular free calcium ([Ca2+]i) in human oocytes after intracytoplasmic sperm injection (ICSI). The first considerable but short (< 2 min) increase in [Ca2+]i was detected immediately after the penetration of the microinjection needle into the ooplasm. This rise by itself did not provoke oocyte activation and was also obtained after the injection of medium without spermatozoa. After a lag period of 4-12 h, oocytes that were subsequently activated initiated a second period of [Ca2+]i changes. These changes were sperm-dependent and followed one of two alternative patterns, a non-oscillatory one and an oscillatory one. The non-oscillatory pattern resembled the changes described previously during parthenogenetic activation of mammalian oocytes. The oscillatory pattern was similar to the changes accompanying normal fertilization in different mammalian species. It is concluded that the initial [Ca2+]i rise provoked by the ICSI procedure is not responsible for oocyte activation, and that a release of a sperm factor(s) is required to initiate this process.

Calcium↗

Flocculation of Kluyveromyces marxianus is induced by a temperature upshift.

An upshift of the growth temperature from 26 to 40 degrees C in the presence of calcium leads to the aggregation of Kluyveromyces marxianus cells and to the formation of flocs. Analysis of cell wall proteins, either by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of extractable mannoproteins or by immunolocalization, revealed an accumulation of a protein with Mr 37 kDa(p37), upon flocculation. Immunological studies confirmed the homology of this protein with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When mRNA isolated from cells growing at 40 degrees C was translated in vitro, a 35 kDa newly labelled protein was synthesized and immunoprecipitation assays showed that this protein is recognized by p37-antiserum, suggesting that the 35 kDa polypeptide might be an unglycosylated precursor for of p37. The results indicated that the presence of this cell wall mannoprotein closely related to GAPDH is dependent on the growth temperature, suggesting its role as adhesin.

Calcium↗

Purification of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi by affinity chromatography.

The major cysteine proteinase from Trypanosoma cruzi, cruzipain, can be obtained essentially homogeneous, starting from crude epimastigote extracts, in one step, by affinity chromatography on Cystatin-Sepharose (specific for cysteine proteinases) or ConA-Sepharose (specific for high mannose N-linked glycoproteins). The methods offer considerable potential for enzyme purification from scarce sources, such as other parasite stages or radioactively labelled material with high specific radioactivity.

Animals↗

Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.

Acrosome↗