Biomedical subjects

E Tosti

Publications and source records attributed to E Tosti.

At least 19 recordsLinked to original sources

PubMed2002

Fertilization and activation currents in bovine oocytes.

One of the first events that occurs at fertilization is a transient modification of the electrical properties of the oocyte plasma membrane. The whole-cell voltage clamp technique was used to demonstrate an outward ion current and a hyperpolarization of the plasma membrane after fertilization in bovine oocytes. These electrical events, together with measurement of internal calcium concentrations, were also recorded after injection with sperm factor and exposure to parthenogenetic activators, such as Ca(2+) ionophore, ethanol and thapsigargin. Experiments were carried out simultaneously in immature and in vitro matured oocytes. Significant differences were recorded in the activation current and hyperpolarization among oocyte activators and between immature and matured oocytes. However, outward ion current and Ca(2+) release showed similar dynamics. The injection of the calcium chelator EGTA completely abolished both ion current and hyperpolarization, indicating that these electrical events are calcium dependent. Addition of specific calcium releasers, such as 1,4,5-inositol trisphosphate (IP(3)) and caffeine, triggered ion activation current and hyperpolarization indicating that IP(3) and ryanodine receptors are active in both immature and matured oocytes. Different ion channel inhibitors were used to characterize the channels underlying outward currents. Only addition of rIberiotoxin caused a complete inhibition of the current, indicating the involvement of high conductance Ca(2+)-activated K(+) channels in generating activation current. In conclusion, these findings provide evidence that bovine oocyte activation is associated with Ca(2+)-dependent electrical events. Oocytes have the potential to react to different activators even when immature; however, oocyte maturation seems to increase sensitivity to physiological activators, such as spermatozoa and sperm factor, and chemicals, such as ethanol.

Animals↗

PubMed2001

Progesterone induces activation in Octopus vulgaris spermatozoa.

The purpose of the present study was to determine whether Octopus vulgaris spermatozoa are activated by progesterone stimulation. Spermatozoa were collected from the spermatophores in the Needham's sac of the male (MS) and from the spermathecae of oviducal glands of the female (FS). We used transmission (TEM) and scanning (SEM) electron microscopy to study the morphology of untreated, Ca2+ ionophore A23187 and progesterone-treated MS spermatozoa, and untreated FS spermatozoa. We showed that ionophore and progesterone stimulation of MS spermatozoa induce breakdown of the membranes overlapping the acrosomal region, exposing the spiralized acrosome. These modifications resemble the acrosome reaction observed in other species. FS stored in the spermathecae did not show the membranes covering the acrosomal region present in the MS spermatozoa. When ionophore and progesterone treatments were performed in Ca2+-free artificial sea water, no changes were observed, suggesting the role of external calcium in modifying membrane morphology. Lectin studies showed a different fluorescence distribution and membrane arrangement of FS-untreated spermatozoa with respect to the MS, suggesting that spermatozoa transferred in the female genital tract after mating, are stored in a pre-activated state. The plasma membrane of the untreated MS and FS spermatozoa was labelled with Progesterone-BSA-FITC, indicating the presence of plasma membrane progesterone receptor. Taken together these data suggest that progesterone induces an acrosome- like reaction in MS spermatozoa similar to that induced by calcium elevation. In addition progesterone may play a role in the pre-activation of spermatozoa stored in the female tract, further supporting the hypothesized parallelism between cephalopods and vertebrates.

Acrosome Reaction↗

PubMed2001

[Serotoninergic processes in cells of early embryos of the sea urchin Paracentrotus lividus].

Agonists of serotonin receptors generate specific inward currents in the cells of early embryos of sea urchin Paracentrotus lividus. Dose-dependent current generated by 5-HT3-agonist 5-HTQ shows complex volt-amperic characteristics with reversal potential -25 and +15 mV. The 5-HTQ effect seems to be due to the activity of channels of mixed conductivity. The 5-HTQ effect is more obvious during cleavage furrow formation. The findings suggest presence of serotonin receptors in the surface membrane of blastomers and their activity play a certain role in regulation of cellular events during cleavage division in the early sea urchin embryo.

Animals↗

PubMed2000

Ca(2+) current activity decreases during meiotic progression in bovine oocytes.

By using the whole cell voltage-clamp technique, we studied changes in plasma membrane permeability at different meiotic stages of bovine oocytes. Follicular oocytes were matured in vitro and activated by Ca(2+) ionophore. Oocytes at germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), and meiosis exit were used for electrophysiological recording. By clamping the oocytes at -30 mV, we found that the L-type voltage-dependent Ca(2+) channels were active at the GV stage and that their activity decreased after the GVBD stage. Furthermore, the resting potential decreased from the GV to the MI stage and increased again at MII. A significant decrease of the steady-state conductance occurred from the GV to the MI stage, followed by a sharp increase at the MII stage. With the addition of organic L-type Ca(2+) channel blockers (nifedipine and verapamil), we inhibited the Ca(2+) currents. However, only in the case of verapamil was there a decrease of in vitro maturation efficiency. Our results suggest that, in addition to the cumulus-oocyte junctions, the plasma membrane channels provide another mode of Ca(2+) entry into bovine oocytes during meiosis.

Animals↗

PubMed1999

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.

1-Octanol↗

PubMed1999

Intercellular communication in in vivo- and in vitro-produced bovine embryos.

In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.

Animals↗

PubMed1998

Non-specific currents at fertilisation in sea urchin oocytes.

Using the whole-cell voltage-clamp technique to clamp sea urchin oocytes we show that the fertilising spermatozoon triggers an inward current of -521 +/- 56.7 pA (n = 8) at activation. Simultaneously, the plasma membrane depolarises and the conductance increases from 23.4 +/- 1.4 to 40.6 +/- 1.2 nS (n = 8). The I/V curve for the peak activation current is linear and the current reverses between 0 and +20 mV, suggesting a non-specific ion current. Since injection of inositol triphosphate induced an inward current of -1062 +/- 314 pA (n = 4), and the current was inhibited by preloading oocytes with the calcium chelator BAPTA, the non-specific activation current in sea urchin appears to be calcium dependent.

Animals↗

PubMed1997

Nitric oxide gates fertilization channels in ascidian oocytes through nicotinamide nucleotide metabolism.

In this paper we use the nitric oxide (NO) donor sodium nitroprusside to examine the response of the unfertilised oocyte of the ascidian Ciona intestinalis to nitric oxide. We show that the release of NO triggers an inward current that displays similar properties to the ascidian fertilisation current. Furthermore, the production of NO causes the release of intracellular calcium through a ruthenium-red sensitive mechanism. Our data suggest that these effects are due to the stimulation of nicotinamide nucleotide metabolism, but the active second messenger is not cyclic adenosine diphosphate ribose (cADPr). Finally, we show that NO production increases at fertilisation. The results suggest that ascidian sperm trigger the release of NO and this second messenger causes the breakdown of nicotinamide nucleotides leading to the production of a second messenger which induces the fertilisation current and may assist in the production of the increase in calcium.

Adenosine Diphosphate Ribose↗

PubMed1997

A soluble extract from human spermatozoa activates ascidian oocytes.

A soluble extract from human spermatozoa induced calcium oscillations and extrusion of the first polar body when injected into oocytes of the ascidian Ciona intestinalis. The properties of calcium oscillations and time of polar body extrusion precisely mimic oocyte activation induced by C. intestinalis sperm or sperm extracts. The data suggest that human sperm extracts can activate oocytes of different phyla by the same mechanism as homologous spermatozoa. Injection of inositol 1,4,5-trisphosphate (IP3) into C. intestinalis oocytes mimicked to some extent the initial stages of oocyte activation, but the results demonstrate that ascidian oocyte activation by human sperm extract cannot be explained solely in terms of IP3-induced calcium release. Injection of other calcium releasing second messengers, cyclic adenosine diphosphate ribose, or calcium ions, does not lead to oocyte activation or release intracellular calcium in ascidian oocyte. It was concluded that human spermatozoa contain one or more molecules than can trigger intracellular calcium release in oocytes from different phyla.

Adenosine Diphosphate Ribose↗

PubMed1997

Gap junctional units are functionally expressed before first cleavage in the early ascidian embryo.

Manually apposed ascidian zygotes established electrical communication within 50 min of fertilization and before cytokinesis. Junctional conductance between zygotes was 14.5 +/- 2.9 nS (n = 7), similar to that previously reported for ascidian two-cell-stage blastomeres, suggesting that zygotes and blastomeres express an equivalent number of gap junctional half-channels. Because puromycin at 400 microM does not inhibit the functional expression of these half-channels, they appear to be of maternal origin and their activation does not require protein synthesis. Loading zygotes with 500 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or exposing zygotes to 10 microM of the calcium ionophore A-23187 shows that these half-channels are regulated by intracellular calcium, consistent with the behavior of these channels in adult tissues. The results show that gap junctional units are expressed in the ascidian at the zygote stage.

Animals↗

PubMed1997

Polarized distribution of L-type calcium channels in early sea urchin embryos.

Using the whole cell clamp technique, we have measured calcium-dependent currents and steady-state conductance in early sea urchin blastomeres. The calcium currents in M phase decreased from 8.5 microA/cm2 at the four-cell stage to 5.4 microA/cm2 at the eight-cell stage. In 16-cell stage embryos, calcium currents were 7.4 microA/cm2 in the mesomeres, 2.3 microA/cm2 in the macromeres, and were not detected in the micromeres. In contrast, the micromeres had a two- to threefold higher steady-state conductance than the mesomeres or macromeres, which may be due to potassium ion conductivity. Nifedipine, an L-type channel antagonist, delays cleavage division at a concentration of 0.05-0.1 mM and causes developmental defects, such as poor skeletal differentiation in later sea urchin embryos.

Animals↗

PubMed1996

A soluble sperm factor gates Ca(2+)-activated K+ channels in human oocytes.

PURPOSE: Our goal was to study the activation current in physiologically competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa or aged in vitro, and, in particular, to determine whether a soluble sperm factor triggers a fertilization current comparable to that observed with intact spermatozoa and to characterize the current involved. METHODS: The whole-cell voltage-clamp technique was used on spare metaphase II oocytes, obtained with patient consent from IVF programs. In this configuration a soluble fraction from human spermatozoa was microinjected, and the current recorded. RESULTS: Metaphase II human oocytes generate bell-shaped outward currents of 400-1000 pA (X = 706 +/- 322; n = 10), following injection of a cytosolic extract from human spermatozoa. The amount of sperm extract injected was less than 10% of the total oocyte volume and was equivalent to 1-10 spermatozoa. A similar current was generated following exposure to 20 microM of the calcium ionophore A23187 (n = 10). The steady-state conductance of the oocyte increased from 10 to 19.8 nS (n = 10) following injection of the sperm factor and from 5.3 to 27.7 nS following ionophore exposure. Both sperm factor- and ionophore-induced currents were reduced in amplitude when the unfertilized oocyte was preexposed to 25-75 microM iberiotoxin (n = 8) and eliminated at a concentration of 100 microM iberiotoxin. CONCLUSIONS: The data support the hypothesis of a soluble sperm factor involved in the activation of human oocytes and shows that the initial activation response in the human oocyte is the gating of Ca(2+)-activated K+ channels.

Calcimycin↗

PubMed1996

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate.

Human oocyte development was evaluated after a reduced time exposure to spermatozoa in vitro. A total of 119 patients were assigned to two study groups in a randomized prospective study in which each patient's oocytes were exposed to spermatozoa for either 1 h (group 1 - 58 patients) or the standard 16 h incubation period (group 2 - 61 patients). The fertilization rate obtained in group 1 was higher than in group 2 (285/393, 73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyte interaction occurs within 1 h. This was confirmed in a study in vitro using fluorescently labelled spermatozoa and normal oocyte-cumulus complexes. Spermatozoa enter the cumulus complex within 15 min, traverse the cumulus layer within 3 h, and first appear in the oocyte cortex at 4 h post-insemination. The incidence of polyspermy was higher in oocytes exposed to spermatozoa for 16 h (3%) than for 1 h (1%). There was no difference in the cleavage rate or morphological characteristics of embryos from both study groups. However, when evaluating the timing of embryo development, group 1 generated a significantly higher percentage of four to five cell embryos when compared to group 2 (55 versus 39%; P < 0.001), documented at 40 h post-insemination. The implantation and pregnancy rates for group 1 were 11 and 28%, while the corresponding rates for group 2 were 8 and 15%. This suggests that a reduced exposure of oocyte to spermatozoa favours embryo viability, possibly due to a decrease in potential damage from sperm metabolic waste products.

Adult↗

PubMed1996

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II.

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1-2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 x 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

Amino Acid Sequence↗

PubMed1995

Is the plasma membrane of the human oocyte reorganised following fertilisation and early cleavage?

The purpose of the present study was to determine whether the plasma membrane of the human oocyte is reorganised following fertilisation and during early cleavage. In order to characterise and localise the major sugar moieties on surface glycoproteins, oocytes and embryos were labelled with a range of fluorescent lectins. Regional organisation of plasma membrane microvilli in oocytes and embryos was also studied using scanning electron microscopy (SEM). The plasma membrane of human oocytes, zygotes and early blastomeres stained strongly and homogeneously with concanavalin A and Triticum vulgaris lectin (WGA), indicating the presence of plasma membrane glycoconjugates with alpha-D-mannosyl residues, sialic acid and beta-NAc-glucosaminyl groups. We did not observe regional domains in oocytes and zygotes, suggesting that the plasma membrane is not topographically reorganised following fertilisation. SEM shows the surface of the human zygote to be organised into short microvilli 0.2-3.0 microns in length and at a density of 5-20/microns2. In early cleavage stages the microvilli are shorter and less frequent (0.2-1.0 microns; 1-5/microns2); however, there is no evidence of polarisation at this level of organisation, at either stage of development. The surface of cell fragments, common in the human embryo in vitro, differs in having few microvilli and numerous cytoplasmic blebs. In conclusion, there are no obvious morphological signs of regionalisation in the plasma membrane of the human embryo before the 8-cell stage.

Blastomeres↗

PubMed1994

Regulation of the fertilization current in ascidian oocytes by intracellular second messengers.

Neomycin, injected into ascidian oocytes to a final concentration of 10-50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm-activated fertilization currents are not inhibited in the presence of 100 micrograms/ml intracellular heparin, suggesting that these currents are not directly gated by InsP3. The sulfhydryl reagent thimerosal at 100 microM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP3 receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores.

Animals↗

PubMed1994

Fertilization current in the human oocyte.

In this report we show that the first event of activation in the human oocyte, the Fertilization current (FC), is a slow transient outward current of 300 pA, which induces a gradual hyperpolarization of the plasma membrane from -20mV to -60mV, 60-120 min after insemination, followed by a repolarization to -20mV. Activation currents (AC) of 600-2,500 pA, induced by exposure to the calcium ionophore A23187 or by microinjection of InsP3 into the cytosol, are also outward. The AC are inhibited by preloading oocytes with EGTA suggesting they are calcium dependent. Since AC are 2-10-fold the amplitude of the FC the fertilizing spermatozoon in the human only activates a portion of the primary elements stored in the oocyte for triggering metabolic depression. Oocyte activation in the human resembles that in the hamster rather than other mammals or invertebrates studied to date.

Calcimycin↗

PubMed1994

A morphological and functional study of fusibility in round-headed spermatozoa in the human.

OBJECTIVE: To study the molecular origin and functionality of the plasma membrane of round-head spermatozoa in the human. DESIGN: Clinical and laboratory study. SETTING: Patients in a clinical and academic environment. PATIENTS: Men with round-head spermatozoa. RESULTS: Pisum sativum lectin homogeneously stains the surface of round sperm; however, the staining pattern and transmission electron microscopy show that the plasma membrane does not alter after exposure to the calcium ionophore A23187. In a clinical program, round-head spermatozoa injected subzonally into metaphase II oocytes with or without pretreatment with the fusogen polyethylene glycol did not bind or fuse to the oocyte surface. CONCLUSION: The data suggests that plasma membrane fusion in human gametes is regulated by specific surface molecules and that exposure of these molecules on the sperm surface cannot be triggered by elevating intracellular calcium alone.

Adult↗