Biomedical subjects

R Talevi

Publications and source records attributed to R Talevi.

At least 19 recordsLinked to original sources

PubMed2003

Selection of highly fertilization-competent bovine spermatozoa through adhesion to the Fallopian tube epithelium in vitro.

Mammalian spermatozoa undergo a marked reduction in number during their journey through the female reproductive tract. One of the checkpoints in the selection of fertilizing spermatozoa may be the transient adhesion to the Fallopian tube epithelium, an event previously shown to play a key role in sperm storage. Bovine spermatozoa adhering to the Fallopian tube epithelium in vitro may be synchronously released by sulphated glycoconjugates. In the present study, experiments were designed to quantify the number of spermatozoa selected through adhesion, and to compare the zona pellucida (ZP) binding and fertilization competence of the initial sperm suspension versus the bound and unbound sperm subpopulations. Results showed that: (1) a fraction accounting for about 30% of the initial sperm suspension was selected by in vitro adhesion to oviductal epithelial cell monolayers; (2) selected spermatozoa, collected after heparin-induced release, had a significantly superior ZP binding and fertilization competence (mean +/- SD: 110 +/- 28 bound spermatozoa per oocyte; % cleavage, mean +/- SEM: 89 +/- 4) compared with both the initial sperm suspension (45 +/- 10 bound spermatozoa per oocyte, P < 0.001; % cleavage: 69 +/- 3, P < 0.05) and the unselected subpopulation (30 +/- 4 bound spermatozoa per oocyte, P < 0.001; % cleavage: 58 +/- 3, P < 0.01). These findings support the hypothesis that binding to oviductal cells is not only beneficial for sperm survival but also represents a crucial step for the selection of spermatozoa endowed with superior fertilization competence.

Animals↗

PubMed2001

Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer.

This paper describes the morphological and biochemical changes in Discoglossus pictus coelomic oocyte envelope (CE) following passage through the oviduct. As in other anurans, in this species, the transformation of the envelope into vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein. In addition, several features, typical of Discoglossus pictus, were observed. A new layer, VE-D, forms underneath the VE region facing the site of sperm entrance, the dimple. In the VE, arrowhead-like bundles of fibrils are perpendicularly oriented toward the dimple. Ultrastructural observations and staining with UEA-I suggested that VE-D might have a role in supporting sperm penetration into the dimple by orienting VE bundles and exposing sugar residues such as fucose. In 'in vitro' tests, VE binding of sperm occurs only if sperm are exposed to A23187, in agreement with previous data (Campanella et al., 1997: Mol Reprod Dev 47:323-333). Sperm binding occurs all over the VE. Accordingly, extracts of the VE covering the animal or the vegetal hemisphere have the same affinity to lectins (DBA, DSA, GNA, MAA, SBA, SNA, UEA-I, WGA). The CE contains six main glycoproteins. Peptide mapping indicated that during CE transformation into VE, gp 42 shifts to an apparent M(r) of 40 and gp 61 is converted to an apparent M(r) of 63 kDa. Lectin blot analyses showed extensive changes in cross-reactivity of most glycoproteins during the CE-->VE transition. The fact that DBA and UEA-I stain gp 63 rather than gp 61 and that this change is related only to gp 63, suggested that O-glycosylation and terminal fucose might be acquired by gp 63 in preparation of fertilization. Gp 63 has recently been cloned (Vaccaro et al., submitted) and shown to exhibit high homology to Xenopus gp 69/64, a VE sperm ligand (Tian et al., 1997a: J. Cell Biol. 136: 1099-1108; Tian et al., 1997b: Dev Biol 187:143-153), and to ZP2 of mammals.

Animals↗

PubMed2001

Presence of a 31-kD protein band in human cumulus--corona radiata--conditioned media and pregnancy outcome.

OBJECTIVE: To investigate relationships between cumulus-oocyte complex (COC) morphology, protein patterns of cumulus-corona (CC) cell-conditioned media, and pregnancy outcome in IVF-ET cycles. DESIGN: Retrospective study. SETTING: Private university IVF center. PATIENT(S): One hundred twenty infertile women who underwent IVF-ET procedures. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): COC grading, analysis of CC cell morphology and conditioned media, and pregnancy rate (PR). RESULT(S): After IVF and embryo transfer, cultured CC cells were classified as high (HA) or low (LA) on the basis of their adhesive properties. Neither adhesion activity nor fertilization rates and embryo quality were correlated with COC grading. PR in cycles with HA cells was 38%, but 14% of cycles showing LA activity also had positive outcome. To find more meaningful parameters of CC cells useful to predict fertilization and pregnancy, the electrophoretic protein patterns of media conditioned by HA or LA cells were studied. Retrospective analysis showed that all cycles in which replaced embryos were associated with the presence of a 31-kD band in conditioned media failed implantation, whereas 83% of cycles lacking this band resulted in positive, ongoing pregnancy. CONCLUSION(S): Pregnancy prediction cannot rely simply on CC cell morphological analysis. Screening of conditioned media may provide more reliable parameters.

Adult↗

PubMed2001

Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro.

The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.

Animals↗

PubMed2000

In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release.

The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm-oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.

Acrosome↗

PubMed1998

Antral follicle development influences plasma membrane organization but not cortical granule distribution in mouse oocytes.

In the present study, we evaluated the contributions of antral follicle development and antral granulosa cell-released factor(s) to the acquisition of a mature mouse oocyte plasma membrane organization and cortical granule distribution. This has been performed by comparing in-vitro matured oocytes derived from early antral follicles (here referred to as denuded oocytes) or from pre-ovulatory follicles, and cultured either as cumulus-intact or cumulus-free oocytes, with in-vivo ovulated eggs. By using scanning and transmission electron microscopy, the denuded oocyte surface appears to be characterized by the presence of long microvilli, while that of pre-ovulatory oocytes and of ovulated eggs by shorter microvilli. However, denuded oocytes can acquire a pre-ovulatory-like plasma membrane configuration when matured in vitro in the presence of early antral granulosa or cumulus cells, but not of NIH-3T3 fibroblasts. On the contrary, fluorescence and confocal microscopy analyses after labelling with fluorescent Lens culinaris agglutinin show that all the oocyte classes analysed are characterized by similar cortical granule distribution and density. Thus, complete antral follicle development plays an important role in the process of oocyte surface differentiation, probably through the action of antral granulosa cell-released factor(s), but it does not affect oocyte capacity to normally distribute cortical granules.

Animals↗

PubMed1997

Heterogeneity of the zona pellucida carbohydrate distribution in human oocytes failing to fertilize in vitro.

The mammalian zona pellucida contains several glycoproteins whose oligosaccharide moieties are known to play a key role in the interaction with spermatozoa. Since zona pellucida defects may represent one of the most likely causes of failed fertilization in human in-vitro reproduction, we have studied the carbohydrate composition and distribution over the human zona pellucida by means of lectins. Donated, not inseminated cumulus-oocyte complexes, from cohorts with high fertilization rates, and fertilization-failed oocytes from cohorts inseminated with proven fertile donor semen, were analysed using 11 fluorescein-labelled lectins, on deplasticized semi-thin epoxy sections. Results showed that wheat germ agglutinin (WGA), Maclura pomifera (MPA) and Pisum sativum (PSA) bound to the extracellular matrix bordering the zona pellucida-corona radiata interface of cumulus-oocytes complexes, while the zona pellucida was labelled by WGA, Concanavalin A (ConA) and PSA. WGA labelling and correlative electron microscopy on the cumulus-oocyte complexes demonstrated that this lectin is a useful tool to trace the cortical granule distribution in the human oocyte. Surprisingly, in the failed-fertilized oocytes the zona pellucida was also labelled by MPA and showed three different patterns: (i) labelling of the zona pellucida outer surface; (ii) uniform labelling; (iii) labelling of an outer zona pellucida layer with variable thickness. Comparative analysis of WGA and MPA labelling on single failed-fertilized oocytes demonstrated that MPA zona pellucida patterns are not related to the cortical reaction. The nature and meaning of the MPA pattern of failed-fertilized oocytes were discussed in the light of zona pellucida defects impairing sperm receptivity.

Adult↗

PubMed1994

Localisation and capacitation-dependent loss of buffalo sperm-coating antigens shared with rat sperm.

The heterodimeric sperm-coating protein CFS was previously localised on the middle-piece region of rat spermatozoa by anti-CFS rabbit antibodies. CFS-immunorelated antigens were detected in the secretion of the water buffalo seminal vesicle by protein electrophoresis and Western blotting. Spermatozoa from buffalo epididymal cauda were incubated with the rat antigen and, upon immunostaining with anti-CFS antibodies and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgGs, CFS was found attached on both the post-acrosomal region and the tail. Indirect immunofluorescence analysis permitted the localisation of CFS-related antigens on the same domains of buffalo ejaculated spermatozoa. These results suggest that the buffalo antigens not only share some epitopes with the homologous rat antigen but may also have some of its functional properties. Ejaculated spermatozoa were capacitated in vitro and then assayed for their content of CFS-like antigens. An inverse relationship was found between the levels of capacitation and the amounts of antigens detected, thus suggesting that the in vitro treatment was effective at removing CFS-related proteins from the cell surface. Titration of these proteins to monitor plasma membrane changes during sperm manipulation or to evaluate sperm quality is proposed.

Animals↗

PubMed1994

Association of cumulus-oocyte complexes with the intrafollicular levels of a blood protein in Bubalus bubalis.

The protein pattern of the follicular fluid (FF) and the ultrastructure of the inner cumulus-oocyte complex (COC) has been analysed in single antral follicles (n = 146) of buffalo B. bubalis ovaries. The protein population of FF was fractionated by SDS-PAGE; the resulting pattern was Coomassie stained and processed for densitometry. Comparative analysis of sera and autologous FFs showed a marked difference in the level (measured as the percentage of total proteins) of one 21 kDa polypeptide band, called 'L'. Concentration of L, which was mainly higher in the serum (2.05 +/- 1.5%) than in the surrounding FF (0.98 +/- 0.94%), fluctuated widely in fluids from the same ovary. On gel filtration of FF and SDS-PAGE of the fractions collected, the L polypeptide was found and eluted together with a 36 kDa polypeptide, called 'H', with an exclusion volume lower than that of albumin. The levels of both polypeptides in the eluted fractions were measured by gel densitometry, and the same ratio H/L was found (2:1). These data suggest that H and L are subunits of a complex high-molecular-weight protein. The presence of L levels in male sera comparable to those detected in females indicates that this putative protein does not originate in the ovary but is transported from the blood. Moreover, a correlation between the increase in the percentage of Lf (calculated as %L in FF/%L in serum) and atresia was observed. COCs (n = 86) obtained during the collection of the single FF samples were processed for transmission electron microscopy. The ultrastructure of each COC was compared with the SDS-PAGE data of the associated FF. Healthy COCs were found to be related to very low levels of Lf (between 0 and 14% of those measured in serum). COCs with an early atretic ultrastructure undetectable at the dissection microscope, were associated with FFs having Lf levels between 24% and 60%; advanced atresia was associated with Lf values up to 70%. Finally, the acrosome reaction of buffalo precipicitated spermatozoa in vitro was monitored by adding one volume of FF with high (FF+; Lf = 80%) or undetectable (FF-) values of Lf to the sperm suspension.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosome↗

PubMed1991

Intercellular communication in the early human embryo.

A preliminary study on intercellular communicative devices in the early human embryo has been made using dye-coupling techniques and electron microscopy (EM). Lucifer yellow injected into single blastomeres of embryos at the 4-cell stage up to the late morula stage did not spread to neighbouring cells, indicating that gap junctions and cytoplasmic bridges are not significant pathways for information transfer. Dye spread was first observed in the blastocyst stage, where trophectoderm cells and inner mass cells were shown to be in communication through gap junctions. Studies at the EM level confirmed this finding. Tight junctions and desmosome-like structures, apparent from the 6-cell stage onward, were located both peripherally and centrally and were initially nonzonular. The role of intercellular devices in the primary differentiation of the human embryo is discussed.

Blastocyst↗

PubMed1991

L-type Ca2+ currents in ascidian eggs.

We have studied Ca2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at -30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn2+ and Nifedipine, while the T-type current is inhibited in Ni2+. When Ba2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca2+ during early developmental processes.

Animals↗

PubMed1989

Distribution of ion channels in ascidian eggs and zygotes.

Ascidian eggs and zygotes were whole-cell voltage-clamped and inward membrane currents, generated by stepping the membrane potential, studied from fertilization up to cytokinesis. Currents, induced by changing the voltage in steps from -80 to -30 mV, or to 0 mV, had maximum amplitudes which ranged from 400 to 1200 pA in the unfertilized egg and 100 to 1300 pA in the zygote. At 5 to 10 min after fertilization it was not possible to generate inward currents owing to the activity of nonspecific fertilization channels. Preceding cytokinesis, we observed a reduction in amplitude of the inward currents. By cutting eggs and zygotes into fragments, we have shown that the ion channels generating these inward currents are symmetrically distributed over the egg plasma membrane, but regionalized in the zygote with a maximum density at the animal pole.

Animals↗

PubMed1988

Fertilization in Discoglossus pictus (Anura). I. Sperm-egg interactions in distinct regions of the dimple and occurrence of a late stage of sperm penetration.

The heterogeneity of the egg surface with respect to receptivity to sperm was investigated in Discoglossus pictus; in this species fertilization occurs only in an indentation called the dimple, at the center of the animal hemisphere. Following insemination sperm are seen in the outermost jelly layers and in the lens-shaped jelly plug, converging to the dimple center, D1. A fertilization potential (FP) is recorded 30 sec following insemination. About 30 min after fertilization, when fertilization cones can be detected easily, immotile sperm are found at the center of the cone, where 10 min later they accomplish penetration. After 15 min the cone regresses and the second polar body is extruded. In eggs where the plug was experimentally displaced with respect to the dimple, spermatozoa contacted the sides of the dimple and simple protrusions formed but not cones. Spermatozoa do not elicit a normal FP in these regions but small step depolarizations which may be followed by a gradual rise to a positive plateau potential. Such eggs do not develop. In the protrusions, sperm may be only partially incorporated and the unpenetrated portion appears to degenerate. We conclude that at least two regions exist in the dimple: D1, where the FP is triggered, cones are formed, sperm penetration is fully accomplished and development is initiated; and D2 + D3 where the electrical response is not a normal FP, cones do not form, total sperm penetration does not occur, and development is not initiated.

Animals↗

PubMed1988

The cortical reaction in the egg of Discoglossus pictus: a study of the changes in the endoplasmic reticulum at activation.

In Discoglossus pictus previous ultrastructural observations have shown that at the animal dimple, where sperm fuse with the egg, cortical granules (CG), vacuoles, and tightly packed clusters of small cisternae are present. At fertilization the clusters open (i.e., become loose) and give rise to longer cisternae arranged in whorls and chains which migrate toward the plasma membrane. The vacuoles fuse to form cisternae and exocytose along with the CG. In the rest of the egg periphery, while exocytosis occurs, the clusters do not open as a result of activation (C. Campanella, R. Talevi, U. Atripaldi, and L. Quaglia (1986). In "Molecular and Cellular Biology of Fertilization" (J.L. Hedrick, Ed.). Plenum, New York). We have recently conducted electrophysiological studies which have detected inward currents at the dimple center, outward current at the rest of the egg surface, and an eightfold increase in [Ca2+]i which propagates from the site of activation throughout the egg (R. Nuccitelli, D. Kline, W. Busa, R. Talevi, and C. Campanella (1988). Dev. Biol. 130, 120-132). In this paper we have asked whether the anionic current and the Ca2+ increase could be causally related to the changes of the smooth endoplasmic reticulum (SER) at activation. The results obtained by activating the eggs in ion-substituted Ringers indicate that (1) the migration of cisternae is not dependent on the polarity of the activation current crossing the dimple, but is strongly impaired, together with CG exocytosis, by 5 x Cl- Ringer; (2) TMB-8, a drug which partially blocks calcium release (C. Y. Choiu and M. J. Malagodi (1975). Brit. J. Pharmacol. 53, 279-288), partially inhibits opening of cisternae clusters and the formation of an SER network in the dimple. This suggests a causal relationship between the Ca2+ rise and the cluster transformation at activation.

Animals↗

PubMed1988

A highly localized activation current yet widespread intracellular calcium increase in the egg of the frog, Discoglossus pictus.

Sperm entry in the egg of the painted frog, Discoglossus pictus, occurs only at a specialized region of the animal hemisphere called the animal dimple, a structure not found in other species of frog. An extracellular vibrating electrode was used to measure the activation current to determine if the ion channels that open to generate the fertilization potential are localized in this region. Eggs that were activated by microinjecting inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) exhibited activation potentials very similar to those of fertilized eggs. There was a delay between the time of Ins(1,4,5)P3 injection and the initiation of the activation potential that was proportional to the distance between the site of the activating stimulus and the animal dimple, similar to the delay previously observed in prick-activated eggs (R. Talevi, B. Dale, and C. Campanella (1985). Dev. Biol. 111, 316-323). The delay lasted 30 sec when the stimulus site was 20 degrees (300 micron) from the animal dimple and 14 min when it was 150 degrees C from the dimple. Once the activation potential was initiated, there was an excellent temporal correlation between the time of depolarization and the time of the first detectable current entering the dimple region. This inward current was typically 60 microA/cm2 in amplitude and was found only in the central 200 micron of the dimple region. The outward current was distributed over the remainder of the egg surface and was much smaller in amplitude. The activation current was carried by Cl- efflux in the animal dimple region, and was reduced by DIDS and reversed by high external Cl- or I-. The occurrence of inward current only at the dimple region indicates that Cl- channels which open to produce the activation potential are localized there. Using Ca2+-specific microelectrodes, we found that [Ca2+]i increased from 0.25 to 2 microM following both fertilization and activation and returned to the unactivated level after about 37 min. Immature oocytes of D. pictus were also studied with the vibrating probe and the inward current in these cells was much less localized than that in the activating egg. A steady transcellular current of up to 4 microA/cm2 entered the entire animal hemisphere of the oocyte and exited the vegetal hemisphere.

Animals↗

PubMed1986

Distribution of fertilization channels in ascidian oocyte membranes.

Fertilization currents are similar in ascidian oocytes and fragments, irrespective of the size and global origin of the fragments; this result suggests that fertilization channels are distributed uniformly over the oocyte surface. Because no correlation exists between peak current and surface area of the cell, it is probable that the fertilizing spermatozoon does not open all available precursor fertilization channels, but a fixed number limited to an area around its point of entry.

Animals↗

PubMed1986

The cortical endoplasmic reticulum and its possible role in activation of Discoglossus pictis (Anura) eggs.

The role of endoplasmic reticulum was investigated in the egg of Discoglossus pictus; recent findings suggest that this organelle is the source of Ca2+ sequestration and release at activation. In the egg of Discoglossus the dimple is the only site where sperm-egg fusion occurs. Microvilli containing microfilament bundles penetrate into the dimple cytoplasm and thus define a cortical layer containing cortical granules, tubular cisternae 35 nm thick and vacuoles. In the underlying cytoplasm are clusters of small cisternae and mitochondria. In the region of the egg cortex outside of the dimple, clusters of cisternae, some vacuoles and a heterogeneous population of small granules are found. In eggs activated by pricking contraction starts from the site of pricking and travels to the antipode. Sections of eggs, fixed 20 to 60 s following fertilization or pricking, show that the tubular cisternae have disappeared and the clusters of cisternae have opened to give rise to longer cisternae arranged in chains. These chains pile up below the vacuoles which are now flat and long because of multiple fusion. A network of cisternae is thus formed whose constituents lean against the microfilament bundles and within 5 min from activation approach the plasma membrane. The flattened vacuoles and the cortical granules are exocytozed. Some cisternae fuse with the microvillar membrane thus participating in microvilli elongation. The microfilament rootlets grandually rearrange and become shorter. In the cortex outside the dimple the cisternae clusters do not open, whereas exocytosis of granules occurs. In eggs treated with A23187, vacuoles and cortical granules undergo exocytosis; however a cisternal network is not formed. The possible stimuli that cause the cisternae network to form at activation only in the dimple and in a matter of seconds are discussed. Our observations, namely exocytosis, formation of cortical wave of contraction and changes in the cytoskeleton organization at activation are consistent with an increase of free Ca2+. Vacuoles and cisternae appear to be good candidates for the sequestration and release of Ca2+ as well an important source for the increase in total plasma membrane at activation.

Animals↗

PubMed1986

Electrical characteristics of ascidian egg fragments.

Fragments of ascidian eggs, but at random in any plane and ranging in size from 10 to 90% of the total egg volume, displayed the electrical characteristics of the intact egg, having a resting potential of -86 mV and giving rise to an action potential upon stimulation by electrical current injection. Following insemination, the fragments generated fertilization potentials, comparable to those of intact eggs, although the repolarization phase was shorter. Our data show that there are sufficient ion channels throughout the egg surface to generate action potentials and fertilization potentials in excised egg fragments, irrespective of their global origin. Furthermore, the fertilizing spermatozoon is capable of activating fertilization channels in areas of the egg plasma membrane not destined for sperm entry.

Action Potentials↗