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C Mendoza

Publications and source records attributed to C Mendoza.

At least 73 records · Page 4Linked to original sources
PubMed1996

Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

Acrosin↗
PubMed1996

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↗
PubMed1996

Single cell analysis of tyrosine kinase dependent and independent Ca2+ fluxes in progesterone induced acrosome reaction.

In this study we developed a single cell analysis protocol with which protein tyrosine kinase (PTK)-dependent and independent Ca2+ fluxes occurring in human spermatozoa in response to progesterone were evaluated. By recording the fluorescence emitted by fluo-3-loaded spermatozoa using a confocal laser scanning microscopy system it was possible not only to monitor relative changes in the intracellular free Ca2+ concentration ([Ca2+]i) but also to determine the time at which the acrosomal exocytosis began. The addition of progesterone produced a rapid transient [Ca2+]i increase in 35% of spermatozoa. In approximately 10% of spermatozoa, this initial [Ca2+]i increase was followed by a secondary [Ca2+]i increase beginning 2-10 min after the progesterone addition and leading to the acrosomal exocytosis in most of these spermatozoa. On the other hand, a rapid triggering of exocytosis during the initial [Ca2+]i increase was a relatively infrequent observation. The inhibition of PTK with genistein or herbimycin A did not influence the initial progesterone-induced [Ca2+]i increase but inhibited the secondary [Ca2+]i increase and the ensuing acrosomal exocytosis. The initial PTK-independent Ca2+ response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the ability to generate the secondary, PTK-dependent response developed during in-vitro capacitation.

Acrosome↗
PubMed1996

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↗
PubMed1996

Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development.

Spermatid injection into the oocyte cytoplasm has been shown recently to yield viable human embryos developing to term after transfer to the mother. This study provides details of the laboratory techniques related to round spermatid injection (ROSI) and elongated spermatid injection (ELSI) and focuses on some special features of zygote development associated with the use of these types of sperm precursor cells for fertilization. A spermatid-enriched fraction was obtained by centrifugation of cells from azoospermic ejaculates through a discontinuous Percoll gradient column. Individual round or elongated spermatids were identified in this fraction and injected deep into oocytes. Oocyte activation was boosted by a vigorous aspiration of the ooplasm at the time of injection. The fertilization rates after ROSI and ELSI were 45 and 44% respectively. A single large syngamy nucleus was detected in 36% of the zygotes that previously showed two normal-sized pronuclei. This condition did not appear to delay the first cleavage division. These observations underscore the importance of distinguishing the syngamy nucleus of diploid zygotes from the female pronucleus of haploid, parthenogenetically activated eggs.

Cell Separation↗
PubMed1995

Nongenomic steroid action: independent targeting of a plasma membrane calcium channel and a tyrosine kinase.

It is known that steroids can produce nongenomic effects on cells, such as opening of ionic channels, membrane receptor aggregation or changes in protein phosphorylation status. However, it is not known whether these different effects, when occurring concomitantly, are linked by a causal relationship or whether they are triggered independently by the steroid ligand. Here we show that progesterone opens a plasma membrane Ca2+ channel and activates a protein tyrosine kinase of human spermatozoa independently of each other because, on the one hand, tyrosine kinase inhibition does not affect the progesterone-induced Ca2+ influx and, on the other hand, the absence of extracellular Ca2+ does not preclude tyrosine-kinase-mediated progesterone-induced acrosomal exocytosis. These data suggest that steroids interact with multireceptor systems on the surface of responsive cells.

Calcium Channels↗
PubMed1995

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↗
PubMed1995

Alleviation of acrosome reaction prematurity by sperm treatment with egg yolk.

OBJECTIVE: To examine the effects of egg yolk on the spontaneous and ionophore-induced acrosome reaction (AR) and sperm-zona pellucida (ZP) binding in spermatozoa from patients with AR prematurity. DESIGN: Prospective analysis. SETTING: Private hospital, public research center, and a university-based laboratory. PATIENTS: In vitro fertilization patients selected on the basis of previous AR testing. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Frequency of the AR, number of ZP-bound spermatozoa. RESULTS: The pretreatment of spermatozoa with egg yolk decreased the frequency of AR occurring spontaneously during subsequent sperm incubation in capacitating medium but did not modify the sperm response to ionophore. The egg yolk-treated spermatozoa also showed a greater capacity of binding the ZP than their untreated counterparts. CONCLUSIONS: The pretreatment of spermatozoa with egg yolk alleviates the sperm-ZP binding deficiency resulting from premature initiation of the AR and may be helpful in IVF with patients showing this anomaly.

Acrosome↗
PubMed1995

Nongenomic effects of 17 beta-estradiol on maturing human oocytes: relationship to oocyte developmental potential.

There is increasing evidence for nongenomic steroid effects in various cell types. This study is the first to demonstrate that 17 beta-estradiol (E2) exerts a direct nongenomic effect on maturing human oocytes by inducing a series of transient increases in the intracellular free calcium concentration ([Ca2+]i). This effect of E2 appeared to be specific and was mediated by steroid action on the cell surface. The first of the E2-induced [Ca2+]i increases was induced by an influx of extracellular Ca2+ ions. However, the release of Ca2+ from intracellular stores contributed more significantly to [Ca2+]i waves characterizing the subsequent series of secondary [Ca2+]i increases, although the presence of extracellular Ca2+ still was an absolute requirement for these [Ca2+]i oscillations. The addition of E2 to oocyte maturation medium did not produce any apparent effects on either germinal vesicle breakdown or further progression of meiosis, but it did increase the fertilization and cleavage rates of the in vitro matured oocytes. These findings show that E2 can directly influence the quality of maturing oocytes. This effect is due to steroid action on the cell surface, implies Ca2+ as a second messenger, and contributes to oocyte capacitation for fertilization and early postfertilization development.

Calcium↗
PubMed1995

Patients with TSH-secreting pituitary tumor possess different TSH molecular isoforms.

The objective of the study was to demonstrate if TSH secreted by a pituitary tumor correlates with particular molecular forms of TSH. Aliquots of sera from two hyperthyroid patients with TSH-secreting pituitary tumor were subjected to gel filtration chromatography to be assayed for TSH. The assay was repeated in one case after transsphenoidal adenomectomy. Sera from two additional patients with long-lasting primary hypothyroidism, and from two subjects with normal thyroid function were also chromatographed. Molecular weight (mol wt) of isoforms was calculated on a calibration curve obtained with molecular markers. The molecular variants were characterized on the basis of elution volume, mol wt, and partition coefficient. Chromatographic profile of the two cases with TSH-secreting pituitary tumor exhibited mainly two peaks of TSH immunoreactivity. The major component of immunoreactivity eluted as a peak corresponding with the alpha-subunit of the glycoprotein. This pattern was different from that obtained in sera from two women with hypothyroidism, which was heterogeneous and presented a predominance of molecular variants corresponding with mol wt of 22 and 29 kD, and no alpha-subunit was present. In cases of normal thyroid function the chromatographic profile showed that the major proportion of TSH eluted simultaneously with the labeled standard and 22 kD marker. These results demonstrate that TSH secreted by pituitary tumors is composed of various molecular forms and the alpha-subunit is the major component. This chromatographic profile is different from that observed in other cases with elevated concentrations of TSH as it occurs in primary hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Most living acrosome-reacted spermatozoa do not fuse with the oocyte when inserted into the perivitelline space.

OBJECTIVE: To evaluate the frequency with which acrosome-reacted spermatozoa fuse with the oocyte after subzonal insemination (SUZI). DESIGN: The acrosome reaction frequency determined in sperm suspensions used for SUZI was related with the frequency of sperm-oocyte fusion after the microinsemination procedure. In some experiments, the acrosome reaction frequency was compared in sperm suspensions aspirated into the microinjection needle and in those leaving the needle. SETTING: Private hospital, public research center, and university-based laboratory. PATIENTS, PARTICIPANTS: Patients involved in a program of micromanipulation-assisted fertilization and healthy sperm donors. INTERVENTIONS: IVF-ET. MAIN OUTCOME MEASURES: Frequency of the acrosome reaction, frequency of sperm-oocyte fusion. RESULTS: The procedure of sperm aspiration to the microinjection needle followed by active swim-out of motile spermatozoa did not change the overall percentage of acrosome-reacted spermatozoa even though the subpopulation of acrosome-reacted spermatozoa leaving the needle was enriched in recently reacted cells. The frequency of sperm-oocyte fusion was considerably lower than the frequency of the acrosome reaction in both patient and donor sperm samples, irrespective of the number of spermatozoa inserted per egg. CONCLUSIONS: A spontaneous acrosome reaction does not necessarily predispose a spermatozoon to fuse with the oocyte after SUZI. It remains to be elucidated whether this relative inefficiency of acrosome-reacted spermatozoa is related to technical aspects of SUZI or whether, in a more general sense, the acrosomal exocytosis and the development of sperm-oocyte fusion capacity are distinctly regulated, though closely related, phenomena.

Acrosome↗
PubMed1993

A plasma-membrane progesterone receptor in human sperm is switched on by increasing intracellular free calcium.

Recent studies have revealed the presence of an atypical progesterone receptor on the plasma membrane of a relatively small subpopulation of human sperm cells. In this study, we show that the receptor is present in many cells in a latent form and that the receptor function in these cells is switched on asynchronously by increasing intracellular free Ca2+ levels. Under physiological conditions, this process occurs during sperm capacitation, but it can be mimicked by a short sperm exposure to the Ca(2+)-mobilizing agent thapsigargin. These observations suggest a role of Ca2+ as a modulator of nongenomic steroid effects on the cell surface.

Biological Transport↗
PubMed1993

Progesterone action on the human sperm surface is potentiated by an egg-associated acrosin activator.

The progesterone-induced Ca2+ influx and acrosomal exocytosis in human sperm are recently described examples of non-genomic steroid action on the cell surface. These progesterone effects are known to be inhibited by synthetic protease inhibitors. In this study we tested a hypothesis that a physiological activator of the sperm protease acrosin modulates the sperm response to progesterone. It was found that the activator augments the amplitude of the progesterone-induced Ca2+ transient and accelerates the progesterone-induced acrosomal exocytosis. These observations suggest a physiological significance of the modulation of nongenomic steroid effects by protease regulators.

Acrosin↗
PubMed1993

Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin.

Pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) have previously been shown to mediate the zone pellucida-induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona-induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction-promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 microgram/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction.

Acrosome↗
PubMed1993

Insights into the function of a sperm-surface progesterone receptor: evidence of ligand-induced receptor aggregation and the implication of proteolysis.

Human spermatozoa have recently been introduced as a model for the study of rapid, nongenomic effects of steroids on the cell. Indirect evidence suggests the presence of a novel type of progesterone receptor on the cell surface; some cellular responses mediated by the receptor have been shown to be sensitive to protease inhibitors, but the molecular identity and the mode of function of this receptor are not known. Recent biochemical evidence showed that Ca2+ influx and a Ca(2+)-dependent exocytotic event (the acrosome reaction) can be induced in human sperm by antibody-mediated aggregation of the cell-surface progesterone receptor. These data suggested that progesterone receptor aggregation, occurring after ligand binding, may represent an early reaction in the signal transduction pathway. In this study we used cytological methods to examine ligand-induced changes in the distribution of the progesterone receptor in the sperm plasma membrane. We also examined the effects of trypsin and of trypsin inhibitors on the function of the receptor. Under the conditions of this study, neither trypsin nor trypsin inhibitors affected sperm viability, motility, or the acrosome reaction. However, the trypsin treatment completely abolished the ligand-binding activity of the sperm progesterone receptor. On the other hand, trypsin inhibitors did not influence the ligand binding despite their inhibitory effect on the ligand-induced exocytosis. The treatment with trypsin inhibitors was thus used to prevent the exocytotic reaction and so to preserve the plasma membrane for the study of ligand-induced receptor migration. The distribution of ligand-receptor complexes in the sperm acrosomal region remained homogeneous during incubation at 4 degrees C, but warming to 37 degrees C entailed a rapid formation of patches, followed by migration of the complexes towards the sperm equatorial region and ending by a virtually complete disappearance of the complexes from the anterior acrosomal region. It is concluded that aggregation is an early response of the sperm-surface progesterone receptor to ligand binding, that trypsin inhibitors block the function of the receptor downstream of the aggregation, and that some mechanism must exist in the plasma membrane to protect the ligand-binding site from digestion while permitting the protease action in the signal transduction mechanism.

Acrosome↗