Biomedical subjects

S Dolci

Publications and source records attributed to S Dolci.

At least 37 records · Page 2Linked to original sources

PubMed1994

Expression of the Xist gene in urogenital ridges of midgestation male embryos.

We show by RT-PCR analysis that transcripts from the Xist gene, which is normally expressed from the inactive X chromosome of female somatic cells and postnatal male germ cells, are transiently expressed also in male embryos around the time of testis differentiation, mainly in somatic cells of urogenital ridges. In the postnatal testis, we find that Xist transcripts are mainly localized within the nucleus of haploid spermatids. These findings suggest that inactivation of the X-chromosome might occur not only in adult male germ cells, but also, transiently, in somatic cells of the male urogenital ridge. Both in the embryonal gonad and in differentiating germ cells Xist expression in males overlaps the pattern of expression of the testis determining gene Sry.

Animals↗

PubMed1994

Testosterone levels as a marker of prognosis to goserelin treatment in metastatic breast cancer.

Testosterone levels were measured in blood and urine of 35 premenopausal metastatic breast cancer patients before starting therapy with the gonadotrophin-releasing hormone (GnRH) analogue, goserelin. The aim of the study was to verify the reliability of testosterone measurement as a marker of prognosis. The time interval between starting therapy and progressive disease (time to progression) was chosen to assess prognosis. Univariate and multivariate analysis showed that only urinary testosterone levels were significantly associated with time to progression (Wald test 6.66, P = 0.01 for univariate and Wald test 7.93, P = 0.0049 for multivariate analysis), whereas no association was found for testosterone in blood. A statistical model is proposed to evaluate probability of progressive disease in relation to testosterone values in urine at different times. According to the model, the probability of progression decreases with increasing urinary testosterone values.

Adult↗

PubMed1994

Developmental regulation of the thyroid hormone receptor alpha 1 mRNA expression in the rat testis.

The multiplicity of thyroid hormone (TH) effects appears to be mediated by two TH receptors (THRs) encoded by two genes, alpha and beta, and, perhaps, by their various isoforms. The expression of THR beta is correlated with the presence of high affinity binding sites for TH, and all the mutations which cause the syndrome of generalized thyroid hormone resistance occur in THR beta. The function of THR alpha has not been clearly defined as yet. Another enigma in TH action is the effect on the testis. It has been shown that the testis of the adult rat does not respond to TH as measured by an increase in oxygen consumption. Furthermore, it has not been possible to demonstrate the presence of a nuclear high affinity binding site for TH in adult testis. To resolve these problems were measured the levels of THR alpha, its nonhormone binding variant, and THR beta mRNA in the testis at various stages of development. We discovered that the beta-message is absent at all times, whereas the alpha-message is expressed only from fetal through prepubertal stages and is absent in adult testis. THR alpha, but not the beta-mRNA, was detected in immature Sertoli cells in culture, and neither was found in adult Sertoli cell-enriched cultures. Furthermore, THR alpha and its variant mRNA was found, using in situ hybridization, in the seminiferous cords and seminiferous tubules of fetal and prepubertal testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Direct evidence that the mouse sex-determining gene Sry is expressed in the somatic cells of male fetal gonads and in the germ cell line in the adult testis.

Reverse transcriptase polymerase chain reaction (RT-PCR) analysis shows that Sry mRNA is expressed in male fetal urogenital ridges from 12.5 day p.c. embryos, but not in enriched populations of primordial germ cells from the same embryos, indicating that Sry is expressed in the somatic cells of the embryonal gonad at the time of testis determination. We also show that, in the adult male mouse testis, Sry mRNA is expressed at high levels in meiotic and postmeiotic germ cells and, at much lower levels, also in Sertoli cells. Treatment with cyclic adenosine monophosphate (cAMP) analogs of cultured Sertoli cells from postnatal testis completely abolishes Sry mRNA expression.

Animals↗

PubMed1993

Combined action of stem cell factor, leukemia inhibitory factor, and cAMP on in vitro proliferation of mouse primordial germ cells.

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre-formed cell feeder layers. The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 microM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5-bromo-2'-deoxyuridin (BrdU) incorporation. These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro.

Animals↗

PubMed1993

Follicle-stimulating hormone induction of steel factor (SLF) mRNA in mouse Sertoli cells and stimulation of DNA synthesis in spermatogonia by soluble SLF.

Follicle-stimulating hormone (FSH) and its intracellular mediator, cAMP, increase the mRNA levels for the Steel factor (SLF, the c-kit ligand) in cultured primary mouse Sertoli cells. The inductive effect of cAMP is more evident in cultures from 13-day-old animals than in cultures from 18-day-old animals. Analysis through the polymerase chain reaction (PCR) indicates that (Bu)2cAMP or FSH treatment increases the levels of the mRNAs for both the potentially soluble form and the transmembrane form of SLF in cultured Sertoli cells. The ratio between mRNAs encoding the potentially soluble form and the transmembrane form of SLF increases during postnatal testis development, and it is higher in cultured Sertoli cells with respect to total testis, suggesting that, under the in vitro conditions, SLF could be produced by Sertoli cells mainly as a soluble factor. Soluble recombinant SLF stimulates, in a dose-dependent fashion, thymidine incorporation in cultures of isolated germ cell populations enriched in the mitotic stages (spermatogonia), independently of the presence of serum, whereas cAMP analogs have no effect. Autoradiographic analysis shows that SLF selectively stimulates DNA synthesis in type A spermatogonia.

Alternative Splicing↗

PubMed1993

Proliferation of mouse primordial germ cells in vitro: a key role for cAMP.

Two agents known to enhance the level of intracellular cAMP (dibutyryl cAMP and forskolin) markedly increase the number of 8.5, 10.5, and 11.5 days postcoitum (dpc) mouse primordial germ cells (PGCs) cultured on TM4 cell feeder layers. Forskolin (FRSK) caused a significant increase of PGC number also in monodispersed cell suspensions obtained from PGC-containing tissues of the three embryonic ages studied and in purified 11.5 dpc PGCs cultured without feeder layers. The addition to the culture medium of adenosine-3',5'-cyclic monophosphorothioate RP isomer (Rp-cAMPS, a competitive antagonist for cAMP-dependent protein kinases), significantly reduced the effects of FRSK. Last, FRSK stimulated PGC proliferation, as assessed by 5-bromo-2'-deoxyuridine incorporation. We conclude that cAMP-dependent mechanisms play a crucial role in the control of mitotic proliferation of mouse PGCs in culture.

Adrenocorticotropic Hormone↗

PubMed1993

Urinary testosterone as a marker of risk of recurrence in operable breast cancer.

We investigated the role of urinary testosterone levels as a marker of risk of recurrent disease in 113 operable breast cancer patients (70 premenopausal, 43 postmenopausal). Twenty-four-hour urine collections for testosterone measurement were obtained before surgical treatment, between 20-40 days thereafter, and then every 6 months for 5 years. The cutoff values to separate 'high testosterone (A+)' from 'normal testosterone (A-)' were 8.0 micrograms/24 h in premenopause and 4.9 micrograms/24 h in postmenopause. Urinary testosterone levels were considered high when they exceeded the cutoff value in at least 2 of the first 3 measurements (pretreatment, post-treatment, 6 months) of each patient. According to the aforementioned criterion, 33 patients (29.2%) had high testosterone levels, which were associated to axillary node involvement in 16 patients. Thirteen of the latter relapsed during the 5-year follow-up period (5/7 in premenopause, 8/9 in postmenopause). Relapse-free survival (RFS) curves were drawn only for node-positive patients owning to the small number of recurrences observed in the node-negative group. In premenopausal node-positive patients, RFS was significantly different for patients presenting high and normal urinary testosterone levels (77% vs 28%, respectively; logrank test, p < 0.006). In postmenopausal node-positive patients, RFS was also different between the two groups (54% vs 11% in 'high' and 'normal' excretors, respectively) but the difference was not statistically significant. The present findings suggest that urinary testosterone is a prognostic indicator of early breast cancer recurrence in node-positive patients.

Adult↗

PubMed1993

Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis).

Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30 degrees C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417-427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530-536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831-838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281-284). In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4-5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Cellular and molecular aspects of mouse primordial germ cell migration and proliferation in culture.

The development of mouse primordial germ cells is followed from their first appearance in the extraembryonic mesoderm of the posterior amniotic fold (7 dpc embryo) to their settlement in the genital ridges (12.5 dpc embryo). The role of fibronectin as adhesive substrate and/or in stimulating cell motility during PGC migration is discussed. Recent papers showing how PGCs migrate when cultured in vitro on cellular monolayers are reviewed. The process of PGC homing is proposed to be controlled by chemotaxis as well by developmentally regulated cell-to-cell interactions. Finally, evidence that survival and proliferation of PGCs is strictly dependent on growth factors such as LIF and MGF, and possibly on a cAMP-dependent mechanism is reported.

Animals↗

PubMed1991

Requirement for mast cell growth factor for primordial germ cell survival in culture.

Mast-cell growth factor (MGF) is encoded by the murine steel (Sl) locus and is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit at the murine dominant white spotting (W) locus. Mutations at both these loci affect mast cells, primordial germ cells (PGCs), haemopoietic stem cells and melanocytes. In many Sl and W mutants, the rapid proliferation of PGC that normally occurs between day 7 and 13.5 of embryonic development fails to occur. As c-kit is expressed in PGCs while MGF is expressed in the surrounding mesenchyme, MGF might promote the proliferation of PGCs. Here we report that MGF is essential for PGC survival in culture, but does not stimulate PGC proliferation. Moreover, whereas both the transmembrane and soluble proteolytic cleavage forms of MGF stimulate mast-cell proliferation, soluble MGF has a relatively limited ability to support survival of PGCs in culture, thus explaining the sterility in mice carrying the steel-dickie (Sld) mutation, which encodes only a soluble form of MGF, and providing a functional role for a transmembrane growth factor.

Animals↗

PubMed1991

An increase of intracellular free Ca2+ is essential for spontaneous meiotic resumption by mouse oocytes.

The involvement of calcium ions in the mechanism of meiotic resumption has been studied in mouse oocytes made resistant to the lethal effects of calcium-free medium (CFM) by zona pellucida removal (De Felici et al., '89). We show here that such oocytes undergo meiotic resumption in CFM (as evaluated by germinal vesicle breakdown, GVBD) at a rate comparable to that shown by oocytes cultured in medium containing 1.7 mM Ca2+. The addition to CFM of 50 u M Quin2/AM (a membrane permeable, high affinity Ca2+ chelator) totally prevents GVBD, while purported antagonists of Ca2+ release from intracellular stores, such as 150 uM 8-(N,N-diethylamino)octyl-3-4-5 trimethoxybenzoate (TMB-8) or 300 uM chlortetracycline, only cause a slight meiotic delay. On the other hand, if the oocytes are pre-incubated for 30 min in CFM supplemented with 100 uM TBM-8 plus 0.2 mM dibutyryl-cyclic AMP (dbcAMP, a reversible inhibitor of GVBD), and then cultured in the same medium, without dbcAMP, a sustained inhibition of meiotic maturation is obtained. Our observations suggest that an increase in intracellular free Ca2+ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca2+, release of the cation from internal stores is sufficient to allow meiotic resumption.

Animals↗

PubMed1991

Involvement of carbohydrates in the hardening of the zona pellucida of mouse oocytes.

The effect of lectins with different saccharide specificity (ConA, LCA, DBA, WGA and PNA) on enzymatic digestion of the zona pellucida (ZP) of mouse oocytes was studied. All lectins tested, except PNA, induced ZP hardening with different degrees of efficiency. Moreover, extensive ZP digestion with mixed exoglycosidase prevented "spontaneous" ZP hardening. These observations suggest that changes of the carbohydrate moieties can be involved in the hardening of the zona pellucida of mouse oocytes.

Animals↗

PubMed1991

Leukemia inhibitory factor sustains the survival of mouse primordial germ cells cultured on TM4 feeder layers.

Various growth factors and cytokines were tested for their effects on survival and proliferation of mouse primordial germ cells (PGCs) cultured on TM4 cell feeder layers. Leukemia inhibitory factor was able to sustain the survival of PGCs from 10.5 dpc embryos for at least 3 days and to slow down degeneration of PGCs from 11.5 dpc embryos cultured on TM4 feeder layers.

Animals↗

PubMed1990

A study of meiosis in chimeric mouse fetal gonads.

The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments. Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex. Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus. In contrast, a smaller number (about 19%) were in meiosis in chimeric testes. None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues. Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells. These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.

Animals↗

PubMed1989

Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice.

Mature mouse sperm cells incubated in an isotonic buffer with cloned DNA capture DNA molecules over a 15 min period. Spermatozoa incubated with pSV2CAT plasmid in either circular or linear form were used to fertilize mouse eggs in vitro. Sequences complementary to pSV2CAT were identified in approximately 30% of 250 progeny by Southern blotting. A genomic library was constructed from the DNA of a positive mouse. Three positive clones were identified and two adjacent HincII restriction fragments of 240 and 370 bp showed identical sequences to the corresponding fragments of the pSV2CAT plasmid. F1 progeny showed paternal and maternal transmission of the transgenes from founders. CAT gene expression was detected on tissues of adult F1 individuals, preferentially on tails and muscle. We conclude that transgenic mice can be obtained using sperm cells as foreign DNA vectors.

Animals↗

PubMed1989

Influence of cumulus cell processes on oolemma permeability and lethality of isolated mouse oocytes cultured in Ca2+-free medium.

Cumulus cell processes remaining in the zona pellucida of mouse oocytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl-phalloidin. If the isolation of the oocytes is performed in Ca2+-free medium, the presence of such processes allows the entry into the cell of low molecular weight molecules (such as 5-6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions. Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+-free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is discussed.

Actin Cytoskeleton↗

PubMed1989

In vitro adhesion of mouse fetal germ cells to extracellular matrix components.

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5-12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitin-sulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5-14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.

Animals↗