PubMed Health⌕ Search

Biomedical subjects

R Gualtieri

Publications and source records attributed to R Gualtieri.

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

Pyriform cell differentiation in Podarcis sicula is accompanied by the appearance of surface glycoproteins bearing alpha-galNAc terminated chains.

The present histochemical and cytochemical study using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, LCA, Con-A, DBA, MPA, BPA) has demonstrated that, in Podarcis sicula, the differentiation of small follicle cells into pyriform cells by means of intermediate cells is accompanied by the appearance of glycoproteins bearing alpha-GalNAc terminated O-linked side chains on the cell surface. The distribution of DBA- and MPA-binding sites over the follicular epithelium changed during the different stages of oocyte growth. DBA- and MPA-binding sites first appeared at the beginning of folliculogenesis within the zona pellucida (ZP) and on the surface of small cells, i.e., the stem cells of pyriform cells. Afterward, labeling was evident on the cell surfaces of intermediate cells and, later on, also of pyriform cells. On the other hand, no labeling was detected on the small cells located under the basal lamina, which, reportedly, do not differentiate into pyriform cells (Filosa et al. J. Embryol. Exp. Morphol., 1979; 15:297-316). Once pyriform cells were differentiated, the distribution of DBA- and MPA-binding sites over the follicular epithelium remained unchanged until intermediate and pyriform cells underwent apoptosis (Motta et al. J. Exp. Zool., 1996; 276:233-241) and the follicular epithelium transformed into a monolayer composed of small follicle cells only (Filosa Mon. Zool. Ital., 1973; 7:151-165). During this stage of oocyte growth, DBA and MPA labeling gradually decreased to completely disappear in the follicular epithelium of vitellogenic follicles. It is noteworthy that the observed changes in the distribution of DBA- and MPA-binding sites represent the first evidence recognized by lectins of a gradual modification of surface glycoprotein distribution over the follicular epithelium in the ovarian follicles of nonmammalian vertebrates so far studied. Finally, the zona pellucida (ZP), characterized by the presence of GalNAc, GluNAc, Man, and Gal, was demonstrated to be first synthetized by the oocyte and later on by the follicle cells.

Acetylgalactosamine↗
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↗
PubMed1999

Intercellular bridges between granulosa cells and the oocyte in the elasmobranch Raya asterias.

In the present ultrastructural study intercellular bridges, connecting somatic granulosa cells to oocyte, have been detected for the first time and their modifications have been followed during Raja oogenesis. Intercellular bridges make their first appearance in small previtellogenic follicles as connecting devices between small cells and the oocyte. Later on, when the follicular epithelium becomes polymorphic and multilayered, for the presence of small, large, and pyriform-like cells, intercellular bridges link the oocyte and the different granulosa cells. Intercellular bridges contain ribosomes, whorl of membranes, mitochondria and vacuoles. Such cytoplasmic components are present also in the cell apex of large and pyriform-like cells thus suggesting, in agreement with other species (Motta et al. J. Exp. Zool., 1996;276:223-241) they may flow toward the oocyte. In this regard the presence of intercellular bridges during the oogenesis of cartilagineous fish may represent a crucial event of the active cooperation between granulosa cells and the oocyte.

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↗
PubMed1996

Localization of egg-surface carbohydrates and fertilization in Discoglossus pictus (Anura).

The distribution of carbohydrates on the unfertilized egg surface in regions receptive or refractory to sperm penetration was analysed in the anuran amphibian Discoglossus pictus by means of a panel of lectins at the level of both the light and electron microscope. Results showed that a gradient of fucosyl-containing glycoconjugates was present at the pre-determined site of sperm entrance and topographically overlapped a similar gradient in sperm entry previously reported. Of a number of simple and complex carbohydrates examined for their ability to inhibit fertilization, fucoidan, ascophyllan and fucose affected the sperm-egg interaction. These sugars, although not decreasing sperm motility or triggering the acrosome reaction, rendered the sperm unable to penetrate the egg jelly coats. The possible involvement of fucosyl-containing glycoconjugates in the sperm-egg interaction in Discoglossus pictus is discussed.

Animals↗
PubMed1995

Silver enhancement of colloidal gold particles in deplasticized semithin epoxy sections.

A simple method for light microscope screening of pre-embedded, gold-labeled samples, by silver enhancing deplasticized semithin epoxy sections is presented. This method is of great help in visualizing the topography of gold labeled sites at low magnification and for selecting labeled sample regions for subsequent electron microscope analysis on serial ultrathin sections. The method was developed during a study of lectin binding sites on the egg surface of the anuran amphibian Discoglossus pictus.

Animals↗
PubMed1992

Tight junctions and cavitation in the human pre-embryo.

In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intracellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-like bodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocytes, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed.

Blastocyst↗
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↗
PubMed1990

Different cytoskeletal organization in two maturation stages of Discoglossus pictus (Anura) oocytes: thickness and stability of actin microfilaments and tropomyosin immunolocalization.

In Discoglossus pictus oocytes, the germinative area (GA) contains long and irregular microvilli where actin microfilaments are located. In the egg, the funnel-shaped dimple that originates by invagination of the GA is present. In the dimple both microvilli and microfilament bundles have a very orderly appearance. This report extends previous observations (Campanella and Gabbiani, Gamete Res 3:99-114, 1980) and shows that GA microfilaments are thinner (36 A average) than dimple microfilaments (60 A average), as measured in ultrathin section. Moreover, the interfilament distance is smaller in GA bundles than in the dimple bundles. To get an insight into actin organization in oocytes and eggs, we used an actin-depolymerizing factor (ADF) in which cryostat sections were incubated prior to immunofluorescent staining with antiactin antibodies. The microfilaments of the GA microvilli and partially of the oocyte cortex are resistant to ADF when compared to those in the dimple and the rest of the egg cortex. We also investigated immunocytochemically the presence of tropomyosin and found that this protein is localized in the dimple and in the cortex of oocytes and eggs but is absent in the GA.

Actin Cytoskeleton↗
PubMed1990

Variable number of tandem repeats in zygosity diagnosis in twins.

The use of DNA restriction fragment length polymorphisms (RFLP) to analyze variable number of tandem repeat (VNTR) sequences dispersed in the human genome, has become a powerful tool for the study of population genetics due to the very substantial polymorphism involved. Because the markers usually employed for twin zygosity determination (such as sex combination, placentation, HLA typing, blood group antigens, etc) may not be uniformly informative, we propose the use of synthetic olygonucleotides, representing VNTR "core" sequences, for the determination of zygosity in twins.

Base Sequence↗
PubMed1987

Unusual vascular malformation of the kidney in both twins of a monozygotic pair: short case report.

A case of double bilateral renal vessels in both twins of a MZ pair is reported for the first time. Even in non-twins, the anomalies reported so far involved the inferior polar arteries in agreement with the embryological development. The two male twins, examined at the age of 14 years, had simultaneously developed a marked hypertension at the age of 7 years. Zygosity was determined by blood group and HLA analysis and various clinical tests were carried out to diagnose the condition. It is suggested that the anomaly is the result of a genetically induced early block in embryonic development.

Adolescent↗
PubMed1985

Studies on the regulation of hemopoiesis.

Normal hemopoietic cell differentiation and proliferation is critically dependent upon marrow stromal elements. Long-term liquid culture of marrow provides a model for the study of stromal function. We evaluated the effects of radiation and 5-fluorouracil (5-FU) on various aspects of long-term murine hemopoietic cell growth and stromal function. Exposure of C57BL/6J murine adherent cells from long-term marrow cultures to varying doses of irradiation (0-1000 rad) in vitro resulted in the elaboration of growth factors stimulating granulocyte, macrophage, megakaryocyte, mixed megakaryocyte-granulocyte macrophage, and blast colonies. This increased production of growth factors appears to be related to the ablation of normal granulocyte production in the culture system since addition of normal stroma to irradiated stroma blocks growth factor production. Two cell types appear to be mediating stromal factor production and support of liquid culture hemopoiesis: a macrophagelike cell and an alkaline-phosphatase-positive epithelioid cell. Exposure of these two cell types to pokeweed mitogen results in marked enhancement of growth factor production. Furthermore, a cell line isolated from normal murine stroma produced an activity capable of acting at an early hemopoietic stem cell level and of inducing secondary marrow cell lines. The establishment of cultures from 5-FU-treated animals revealed that chemotherapy-depleted marrow was capable of establishing adequate stromal function and that the residual surviving stem cells had a higher than normal proliferative rate. In addition, the function of granulocytes derived from this post-5-FU marrow was normal. Thus, it appears that both chemotherapy and radiation exposure of marrow results in an enhanced capacity of stromal elements to produce growth factors and support hemopoiesis and that post-5-FU marrow represents an enriched source of high proliferative potential bone marrow stem cells.

Animals↗
PubMed1985

Bone marrow adherent cell hemopoietic growth factor production.

These data suggest that two types of radioresistant adherent stromal cells are adequate for maintenance of long term hemopoiesis in the Dexter culture system. One cell type appears to be a typical adherent macrophage while the other is an alkaline phosphatase positive epitheloid cell possibly representative of the adventitial reticular cell of the bone marrow. These two cell types seem to be clearly defined by the in-vivo irradiation studies and less clearly defined in the in-vitro irradiation experiments. These data do not exclude other cell types as playing important roles in modulating hemopoiesis but do suggest that these major types are probably playing important roles in maintaining stem cells in long term liquid cultures. In addition, data suggests that the epitheloid cell directly nurtures hemopoietic cells on its surface while the macrophages may serve a separate function. A number of growth factors are produced by these two cell types which appear to include CSF-1, a granulocyte CSA separate from CSF-1 and a megakaryocyte CSA separate from both the GM-CSA and CSF-1 (Table 5). Thus the present data suggest that there are at least three (formula; see text) separate hemopoietic growth factors produced. The FDC-P1 activity produced by irradiated stroma would appear most likely to be GM-CSA-II. The fact that lectins enhanced production of these activities is intriguing but the cell type on which the lectins are acting has not as yet been defined. It appears that lithium also acts upon adherent marrow stromal cells to induce production of myeloid regulatory growth factors. Lithium appears to stimulate both normal and irradiated stroma to produce hemopoietic maintenance and growth factors and the present data suggests that factors active on pre IL-3 cells, HPP-CFC, CFU-D, CFU-meg, and CFU-S are all induced from these stromal cells by lithium. Whether the lithium induced factors and the factors derived from irradiated stroma represent a number of different growth factors or one or two critical regulatory molecules is at present unclear. The synergistic activity derived from the TC-1 cell line is of particular interest in this regard. This CSF-1 dependent activity is capable of acting on a very primitive stem cell to induce impressive proliferation and differentiation. In addition an activity in TC-1 conditioned media which may be synergistic activity appears capable of inducing adherent marrow cell lines which then can subsequently produce more of the same factor, a classic autocrine system.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Synthesis of oxytocin using iodine for oxidative cyclization and silica gel adsorption chromatography for purification.

Oxytocin (OT) was synthesized employing the solid phase method. Resins made of copolymers of polystyrene-1%-crosslinked with divinylbenzene gave better yields (73-95%) of Z-Cys(Bzl)-Tyr(Bzl)-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (I) than 2%-crosslinked resins (10--56%). Reduction of I with Na-liq.NH3 and oxidation with I2-MeOH at -40 degrees minimized dimer and polymer formation, and resulted in good yields (49--54%) of OT. The large volumes of MeOH required when several grams of I are reduced and then oxidized were rapidly evaporated in vacuo, and the residue was desalted by dissolving the peptide in a small volume of glacial acetic acid and filtering to remove the salt. OT was purified by adsorption chromatography on a silica gel column with combinations of MeOH-CHCl3 of graded polarity. Oxytocin elutes with 33% MeOH-CHCl3. After two purification steps by adsorption chromatography, the resulting OT was found to be homogeneous. The hormone was characterized chemically and found to be active biologically.

Adsorption↗