Biomedical subjects

Susanna Dolci

Publications and source records attributed to Susanna Dolci.

9 recordsLinked to original sources

PubMed2006

Subcellular localization and regulation of type-1C and type-5 phosphodiesterases.

We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for the first time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2005

Imatinib mesylate inhibits Leydig cell tumor growth: evidence for in vitro and in vivo activity.

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

Animals↗

PubMed2005

Regulation of phosphodiesterase 5 expression and activity in human pregnant and non-pregnant myometrial cells by human chorionic gonadotropin.

OBJECTIVES: This study has a twofold aim: 1) to investigate whether protein expression and enzyme activity of phosphodiesterase 5 (PDE5) can be detected in human myometrium and undergo changes in relation to the presence of pregnancy and/or labor; 2) to evaluate whether PDE5 expression and activity in myometrial cells can be influenced by human chorionic gonadotropin (HCG). METHODS: Primary cultures of myometrial cells, obtained from non-pregnant women and from pregnant women at term, either before or during labor, were carried out in the presence of HCG or dibutyryl-cyclic AMP (db-cAMP), the non-hydrolizable analogue of cAMP. PDE5 expression in cultures of myometrial cells was detected by immunocytochemistry and western blot. PDE5 activity was detected in cell extracts by enzyme assay. RESULTS: PDE5 is expressed and is functionally active in smooth muscle cells. Treatment of cell cultures with HCG and db-cAMP results in a reduction of PDE5 expression and activity. The effects of HCG and db-cAMP are exerted irrespective of the functional status of the myometrium (non-pregnant, pregnant not in labor, pregnant in labor). CONCLUSIONS: PDE5 protein is expressed in human non-pregnant and pregnant myometrium. HCG reduces PDE5 expression and enzyme activity in smooth muscle cells, possibly through a pathway involving cAMP.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2004

Analysis of the gene expression profile of mouse male meiotic germ cells.

Wide genome analysis of difference in gene expression between spermatogonial populations from 7-day-old mice and pachytene spermatocytes from 18-day-old mice was performed using Affymetrix gene chips representing approximately 12,500 mouse known genes or EST sequences, spanning approximately 1/3rd of the mouse genome. To delineate differences in the profile of gene expression between mitotic and meiotic stages of male germ cell differentiation, expressed genes were grouped in functional clusters. The analysis confirmed the previously described pre-meiotic or meiotic expression for several genes, in particular for those involved in the regulation of the mitotic and meiotic cell cycle, and for those whose transcripts are accumulated during the meiotic stages to be translated later in post-meiotic stages. Differential expression of several additional genes was discovered. In few cases (pro-apoptotic factors Bak, Bad and Bax), data were in conflict with the previously published stage-dependent expression of genes already known to be expressed in male germ cells. Northern blot analysis of selected genes confirmed the results obtained with the microarray chips. Six of these were novel genes specifically expressed in pachytene spermatocytes: a chromatin remodeling factor (chrac1/YCL1), a homeobox gene (hmx1), a novel G-coupled receptor for an unknown ligand (Gpr19), a glycoprotein of the intestinal epithelium (mucin 3), a novel RAS activator (Ranbp9), and the A630056B21Rik gene (predicted to encode a novel zinc finger protein). These studies will help to delineate the global patterns of gene expression characterizing male germ cell differentiation for a better understanding of regulation of spermatogenesis in mammals.

Animals↗

PubMed2003

Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation.

It is well established that the c-kit gene plays an essential role in the proliferation of differentiating spermatogonia in prepuberal mice. However, the mechanisms that regulate the onset of spermatogenesis, i.e. differentiation of spermatogonial stem cells and c-kit expression, are poorly understood. Here we identify a novel signal transduction system in mouse prepuberal testis regulating this developmental event, involving bone morphogenetic protein 4 (BMP4) and its transduction machinery. BMP4 is produced by Sertoli cells very early in the postnatal life and is successively down regulated in peri-puberal Sertoli cells. Its receptor Alk3 and the R-Smad Smad5 are specifically expressed both in proliferating primordial germ cells and in postnatal spermatogonia. BMP4 stimulation of cultured spermatogonia induces Smad4/5 nuclear translocation and the formation of a DNA-binding complex with the transcriptional coactivator p300/CBP. In vitro exposure of undifferentiated spermatogonia to BMP4 exerts both mitogenic and differentiative effects, inducing [3H]thymidine incorporation and Kit expression. As a result of the latter event, Kit-negative spermatogonia acquire sensitivity to Stem Cell Factor.

Acetyltransferases↗

PubMed2003

Kit regulatory elements required for expression in developing hematopoietic and germ cell lineages.

The Kit (White) gene encodes the transmembrane receptor of stem cell factor/Kit ligand (KL) and is essential for the normal development/maintenance of pluripotent primordial germ cells (PGCs), hematopoietic stem cells (HSCs), melanoblasts, and some of their descendants. The molecular basis for the transcriptional regulation of Kit during development of these important cell types is unknown. We investigated Kit regulation in hematopoietic cells and PGCs. We identified 6 DNase I hypersensitive sites (HS1-HS6) within the promoter and first intron of the mouse Kit gene and developed mouse lines expressing transgenic green fluorescent protein (GFP) under the control of these regulatory elements. A construct driven by the Kit promoter and including all 6 HS sites is highly expressed during mouse development in Kit+ cells including PGCs and hematopoietic progenitors (erythroid blast-forming units and mixed colony-forming units). In contrast, the Kit promoter alone (comprising HS1) is sufficient to drive low-level GFP expression in PGCs, but unable to function in hematopoietic cells. Hematopoietic expression further requires the addition of the intronproximal HS2 fragment; HS2 also greatly potentiates the activity in PGCs. Thus, HS2 acts as an enhancer integrating transcriptional signals common to 2 developmentally unrelated stem cell/progenitor lineages. Optimal hematopoietic expression further requires HS3-HS6.

Animals↗

PubMed2002

Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells.

The discovery of sterility in the descendants of telomerasenull mutant mice, owing to the lack of spermatogonia proliferation, has drawn attention to the role of telomerase activity in mouse spermatogenesis. Since spermatogonia proliferation is under Kitl control, we explored its possible role in the regulation of telomerase activity. We show that Kitl induces telomerase activity in mitotic spermatogonia and increases the mRNA levels of both the catalytic subunit form and the telomerase RNA template. The increase of telomerase activity by Kitl is blocked by the presence of the PI3K inhibitor LY294002. Kit-positive proliferating male primordial germ cells (PGCs) show low levels of telomerase activity, but they increase telomerase activity upon Kitl stimulation. Diplotene-arrested growing oocytes that reexpress Kit do not increase telomerase activity upon Kitl stimulation. Our data suggest that the induction of telomerase by Kitl may contribute to the self-renewing potential of male germ cells and of PGCs.

Animals↗

PubMed2002

Type 5 phosphodiesterase expression in the human vagina.

OBJECTIVES: It has been demonstrated that clitoral and vaginal tissues express nitric oxide synthase isoforms in a way that parallels that of the penile corpus cavernosum. Considering the role of the vagina in the female sexual response and the anatomic connection between the clitoris and the anterosuperior vaginal wall, our aim was to study the distribution of type 5 phosphodiesterase (PDE5) in the anterosuperior wall of the human vagina. METHODS: Immunohistochemistry was performed on the vaginal tissue of 14 women obtained at autopsy and on exfoliated cells of the vaginal epithelium obtained from 5 healthy female donors. Specific antibodies against PDE5 were tested on both paraffin sections and cytologic smears. Immunoblotting experiments were performed in parallel with the same antibodies. RESULTS: The histologic analysis of human cadaveric vaginal tissue revealed that PDE5 immunoreactivity was mostly localized in the smooth muscle of vessels, forming a pseudocavernous tissue in the vaginal wall and endothelium. The Skene periurethral glands and vaginal epithelium were also positive for the antibody. The latter finding was confirmed using exfoliated cells of the vaginal epithelium harvested in vivo. CONCLUSIONS: The presence and tissue distribution of PDE5 in the human vagina suggest that the integrated system of nitric oxide synthase-PDE5 may play a physiologic role not only in the male sexual response but also in female sexual arousal.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

Molecular genetics of male infertility: stem cell factor/c-kit system.

PROBLEM: Infertility, affects about 5% of human males and genetic factors are recognized in approximately 30% of them. The mouse represents a good model to study autosomal genes that might play a role in spermatogenesis. In mice, mutations in the c-kit gene and in the gene encoding stem cell factor (SCF) cause pleiotropic defects among which sterility. A possible involvement of the SCF/c-kit system in human spermatogenesis was investigated. METHODS OF STUDY: A group of 65 idiopathic azoospermic patients was screened for the presence of mutations in the human c-kit gene codon encoding tyrosine 721 (Y721), analogous to Y719 in the murine c-kit gene (a residue known to be essential for a normal spermatogonial proliferation). Furthermore we have used a mouse model for studying the molecular mechanisms that regulate the transcription of the endogenous SCF gene. RESULTS: No mutations have been detected on codon encoding Y721 of the human c-kit gene, in our group of infertile patients. CONCLUSIONS: A larger group of azoospermic patients, including preferentially patients affected by Sertoli-cell-only syndrome, should be screened in order to exclude a role of c-kit mutations in Y721 in spermatogenesis defects. In this study we also show that the murine SCF promoter is transcriptionally active and stimulated by follicle stimulating hormone (FSH), 3'-5' cyclic adenosine monophosphate (cAMP) analogs, and IBMX in primary mouse Sertoli cells, and that the cAMP effect is cell-specific, as the SCF promoter is not stimulated in other SCF-expressing cell types tested.

Animals↗