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Biomedical subjects

G Siracusa

Publications and source records attributed to G Siracusa.

At least 19 recordsLinked to original sources
PubMed1997

Hyaluronan synthesis by mouse cumulus cells is regulated by interactions between follicle-stimulating hormone (or epidermal growth factor) and a soluble oocyte factor (or transforming growth factor beta1).

Expansion of the cumulus cell-oocyte complex (COC) in the preovulatory mammalian follicle requires a transient induction of hyaluronan (HA) synthesis by the cumulus cells. We studied the interactions of known factors that regulate this process by isolating compact COCs from mice and inducing their expansion in vitro. Maximum HA synthesis requires either follicle-stimulating hormone (FSH) or epidermal growth factor (EGF) in combination with either a soluble factor(s) produced by the oocyte or transforming growth factor beta1. FSH (or EGF) exerts its effects during the first 2 h of incubation, before HA synthesis actually begins. The oocyte factor(s) (or transforming growth factor beta1) exerts its effects from 2 h onwards and must be continuously present throughout the subsequent approximately 10 h to achieve a maximum level of HA synthesis. FSH stimulates intracellular cAMP synthesis, which correlates with net HA production up to approximately 14 fmol/COC at 5 ng/ml FSH; however, higher concentrations of FSH increase cAMP levels approximately 10-fold higher with no additional effect on HA synthesis. EGF at saturating concentrations for HA synthesis does not stimulate cAMP above basal levels. Tyrosine kinase inhibitors genistein and tyrphostin AG18 nearly abolish the HA synthesis response to EGF and inhibit the response to FSH by approximately 60%, suggesting that a tyrosine kinase activity is involved for both factors, whereas FSH also operates partially through another signaling pathway. Actinomycin D abolishes HA synthesis if added at the beginning of culture and reduces HA synthesis by approximately 50% if added between 6-12 h when HA synthesis is normally maximal. The results suggest that regulation of HA synthesis is primarily controlled at the transcriptional level.

Animals↗
PubMed1996

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylate cyclase and promotes proliferation of mouse primordial germ cells.

During migration and for about 2 days after their arrival in the gonadal ridges, primordial germ cells (the embryonic precursors of gametes of the adult animal) proliferate actively. Certain growth factors, such as stem cell factor and leukemia inhibitory factor, seem to be essential for survival, proliferation and possibly differentiation of mouse primordial germ cell in vivo and/or in vitro. Similarly, increase in intracellular cAMP is followed by a marked enhancement of primordial germ cell proliferation, at least in culture. In the present study, we show that pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38), two neuropeptides of the secretin-glucagon-vasoactive intestinal polypeptide-GH-releasing hormone family, stimulate in vitro proliferation of mouse primordial germ cells, bind to primordial germ cells and gonadal somatic cells (possibly to type I PACAP receptor) and activate adenylate cyclase in the same cells. Moreover, PACAP-like immunoreactivity was found in gonadal ridges, mostly on germ cell surface. In conclusion, evidence is provided that PGC proliferation can be stimulated by certain bioactive polypeptides, thus suggesting a novel regulatory role for such compounds in early gonad development.

Adenylyl Cyclases↗
PubMed1995

Cognitive impairment in early-onset multiple sclerosis. Pattern, predictors, and impact on everyday life in a 4-year follow-up.

OBJECTIVES: To assess the evolution of cognitive dysfunction in early-onset multiple sclerosis, to identify clinical predictors of mental decline, and to determine its impact on a patient's everyday life. DESIGN: The cognitive performance of 50 patients with multiple sclerosis on a neuropsychological battery was compared with that of 70 control subjects initially and again after a 4-year interval. Clinical predictors of cognitive impairment and its effect on daily life were analyzed by stepwise linear regression. SETTING: The research clinic of a university department of neurology. PARTICIPANTS: A consecutive sample of 50 inpatients and outpatients with multiple sclerosis (mean disease duration, 1.58 years) and 70 demographically matched healthy control subjects selected from the patients' relatives and friends. MAIN OUTCOME MEASURES: Mean psychometric test scores of both groups at the initial and follow-up testing. Regression coefficients measuring the relationship between clinical parameters and cognitive capacity and between mental decline and performance of common tasks measured by the Environmental and the Incapacity Status scales. RESULTS: Multiple sclerosis-related deficits in verbal memory and abstract reasoning on initial testing remained more or less stable on the retest, at which time linguistic disturbances on the Set and Token tests also emerged. A patient's initial disability level predicted decreased performance on only four of 13 cognitive variables, and disease duration did so on only two. Extent of intellectual decline on initial testing, initial disability level, and progressive course were independent determinants of handicap in a patient's work and social activities. CONCLUSIONS: Cognitive and neurological deficits appear not to develop in parallel. Yet cognitive dysfunction proves to be a predictor of handicap in everyday life, even in patients in the incipient phase of multiple sclerosis.

Adolescent↗
PubMed1995

Mouse oocytes inhibit plasminogen activator production by ovarian cumulus and granulosa cells.

Following the preovulatory surge of gonadotropins, the compact layer of cumulus cells in the antral follicle secretes a hyaluronic acid-enriched extracellular matrix and undergoes a morphological change referred to as cumulus expansion. It has been previously shown that a soluble factor(s) produced by the oocyte is required, in combination with FSH, to promote this process. Since such matrix is sensitive to proteases we have now studied the effect of the oocyte on another gonadotropin-controlled follicle cell function, i.e., the synthesis of plasminogen activator (PA). Our data indicate that isolated cumulus cells secrete uPA in the medium and that FSH or dbcAMP increases this production. The presence of the oocyte or the oocyte-conditioned medium greatly reduces uPA synthesis induced by FSH and dbcAMP in cumulus cells by modulating the abundance of its mRNA. The ability of the mouse oocyte to produce such a factor(s) is dependent upon its stage of development, with fully grown oocytes but not growing oocytes or two-cell embryos being able to inhibit uPA synthesis. A preliminary characterization of this factor suggests that it is a heat-unstable protein with an apparent molecular weight above 100 kDa. Thus, the mouse oocytes appear to promote preovulatory matrix accumulation that occurs just prior ovulation by modulating the gonadotropin action on both the synthesis and the degradation of specific matrix component.

Animals↗
PubMed1994

Low-affinity nerve growth factor receptor is expressed during testicular morphogenesis and in germ cells at specific stages of spermatogenesis.

Nerve growth factor (NGF) is essential for neuronal development and differentiation. Recent reports have shown that its low-affinity receptor (LNGFR) is expressed and developmentally regulated in a broad range of embryonic and adult tissues outside the nervous system, although the functions of the receptor in such tissues remain unknown. Recently, NGF and LNGFR have been detected in adult mouse, rat, and human testis. The results of the present work demonstrate that LNGFR is expressed much before the onset of spermatogenesis in both mouse and rat testis. In situ hybridization shows that the mRNA for LNGFR is expressed in the peritubular cells of the embryonic mouse testis. Immunohistochemical analysis of the rat testis shows LNGFR-expressing cells to be scattered in the intertubular compartment in the embryonic testis, and to become organized in a cellular layer that surrounds myoid cells of the seminiferous tubules during postnatal development. Furthermore, in peripuberal and adult mouse and rat testis we have identified the expression of an abundant and shorter mRNA of 3.2 kb that cross hybridizes to the low-affinity NGF receptor transcript (3.7 kb). This shorter mRNA species, which appears at the beginning of spermatogenesis in the adult, has been identified by in situ hybridization and by Northern blot with RNA isolated from homogeneous populations of meiotic germ cells to be expressed by pachytene spermatocytes and round spermatids. Our results suggest a complex developmental role for LNGFR during testicular morphogenesis and identify the expression, at specific stages of spermatogenesis, of a new germ cell-specific transcript homologous to the receptor RNA.

Animals↗
PubMed1994

Histotypic in vitro reorganization of dissociated cells from mouse fetal gonads.

Cell suspensions obtained from the testes of 13.5-14.5 day post coitum (dpc) mouse embryos reaggregate in cord-like structures following in vitro culture for 24 h on a reconstituted basement membrane (Matrigel). Ovarian cells of the same fetal age or cell suspensions from sex indifferent gonadal ridges (11.5 dpc embryos) form an organized cell network but not cord-like structures. Antibodies directed against laminin or against the alpha 6 subunit of its integrin receptor prevent such morphogenetic processes. The addition of 5 micrograms/ml cycloheximide, cytochalasin B or tunicamycin also inhibits the phenomenon. Interestingly, compounds that increase intracellular cyclic AMP (cAMP) (dbcAMP, forskolin and isobutyl-1-methylxanthine) induce embryonic testicular cells to organize into structures similar to those assembled by ovarian or sex indifferent cell suspensions. These findings offer a simple in vitro model for studying some aspects of early gonad development and provide novel experimental evidence that cell motility and cell-cell adhesion, possibly regulated by cAMP dependent mechanisms, are likely to play an important role in gonad morphogenesis.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Oocytes preserve the ability of mouse cumulus cells in culture to synthesize hyaluronic acid and dermatan sulfate.

A soluble factor(s) produced by fully grown oocytes is essential, together with follicle stimulating hormone (FSH), to stimulate in vitro hyaluronic acid (HA) synthesis by mouse cumulus cells (CCs). The stability of the response to this stimulus by CCs in culture was investigated. The data showed that preculture for 8 hr in basal medium reduced to approximately 30% the ability of CCs to synthesize HA in response to FSH or dibutyryl cyclic AMP (Bt2cAMP) and soluble oocyte factor(s). However, if CCs were precultured for the same period of time as intact cumulus cell-oocyte complexes, or in the presence of fully grown oocytes, or in medium conditioned by fully grown oocytes, their ability to synthesize HA was 75-95% preserved. In vitro stimulation of dermatan sulfate (DS) synthesis by CCs does not require oocyte factors and is induced by FSH or Bt2cAMP treatment alone. However, the preservation of such activity, like that of HA synthesis, depended on the presence of a soluble oocyte factor(s) during preculture. The presence of isolated oocytes or of oocyte-conditioned medium also prevented the spreading of CCs in culture. However, inhibiting CC spreading by culture on agar-coated plates or in serum-free medium did not preserve their HA or DS synthetic activity, thus suggesting that the two oocyte actions on CCs are independent. Growing oocytes were unable both to induce HA synthesis in freshly isolated CCs stimulated with FSH and to preserve the ability to synthesize HA and DS in 8-hr precultured CCs. The results suggest that the stability of the differentiated state of mouse CCs in vitro depends upon continued exposure to a soluble factor(s) produced by fully grown oocytes.

Animals↗
PubMed1993

Long-term safety of azathioprine therapy in multiple sclerosis.

We compared the frequency of malignancies in 207 multiple sclerosis patients (mean age 35.75 years, SD 10.60) who took 2.0 mg/kg azathioprine daily (mean duration 4.16 years; SD 2.38) and in 247 nontreated patients (mean age 35.44 years; SD 11.94). Five malignancies were diagnosed in the azathioprine group compared with seven in the control group. The age-adjusted occurrence rate was 3.62/1,000 person-years (95% CI, 1.17 to 8.43) in the treated and 4.24/1,000 person-years (95% CI, 1.70 to 8.73) in the nontreated group; the age-adjusted relative risk of developing a tumor was 0.85.

Adenocarcinoma↗
PubMed1993

Expression of beta 1 integrin complexes on the surface of unfertilized mouse oocyte.

Integrins are a family of cell surface receptors that mediate cell-cell and cell-matrix interactions in a variety of different cellular systems. Here we show that unfertilized mouse oocytes express beta 1 class integrins both at mRNA and protein levels. Using the reverse transcription polymerase chain reaction and oligonucleotide primers based on the DNA sequence of mouse integrins, the RNA transcripts for the beta 1, alpha 5 and alpha 6 subunits were detected in unfertilized oocytes. The expression of the mRNAs is paralleled by the expression of the corresponding proteins, in fact, the alpha 5/beta 1 and the alpha 6/beta 1 complexes can be immunoprecipitated with specific antibodies form 125I-surface-labeled oocytes. Using subunit-specific antibodies we also demonstrate the presence of the alpha 3/beta 1 at the oocyte surface but alpha 1, alpha 2, alpha 4 or alpha V subunits were not detectable. Since the mouse alpha 3 DNA sequence is not available, we have not tested for the corresponding transcript. Integrin subunits alpha 6 and beta 1 were differently distributed on the oocyte surface, as visualized by immunofluorescence staining and by immunoelectron microscopy. alpha 6 antigen was mainly confined to the microvillous area of the oocyte surface, while beta 1 was more homogeneously distributed over the whole oolemma. These data demonstrate for the first time the expression of three beta 1 integrin complexes in unfertilized mouse oocytes. Such proteins may have a role in sperm-egg interaction or during very early steps of embryogenesis.

Animals↗
PubMed1992

Effects of 2,5-hexanedione on the ovary and fertility. An experimental study in mice.

Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 11.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.

Animals↗
PubMed1992

Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads.

When mouse spermatozoa were briefly exposed in culture to radioactively labelled DNA (pSV2CAT plasmid), radioactivity could be detected by high-resolution autoradiography on the surface and within the nucleus of the spermatozoa. Spermatozoa from other mammalian species (boar, bull, man) could also bind foreign DNA. With the exclusion of human spermatozoa, which in most experiments showed very low labelling values, labelling percentages (evaluated by light microscope autoradiography) ranged between 39 and 78%. In all four species the DNA-binding ability was mainly confined to a specific region of the sperm head (equatorial segment and postacrosomal region), and the sperm-DNA association kinetics were rapid (maximum values were reached within 20-40 min). The data also indicate that factor(s) in seminal plasma might protect spermatozoa from accidental transfection by foreign DNA that may be present in the genital tracts from bacterial or viral sources.

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

Intrathecal synthesis of free immunoglobulin light chains and IgM in initial multiple sclerosis.

We studied the intrathecal synthesis of free kappa, free lambda immunoglobulin light chains and of IgM in 33 consecutive patients with possible or probable MS at the time of their first diagnosis. Nineteen patients presented free kappa or lambda light chain bands in CSF after agarose isoelectric focusing, protein transfer to nitrocellulose and immunostaining with specific antisera. Nineteen patients had increased CSF levels of free kappa light chains as measured with a competitive ELISA. Fourteen had an increased IgM index, as evaluated with a sandwich ELISA. Twenty-six patients displayed CSF oligoclonal IgG bands in CSF and 25 had cerebral magnetic resonance imaging lesions suggestive of MS. The local production of free immunoglobulin light chains and IgM is often detected in the CSF of patients with early MS.

Adolescent↗
PubMed1990

Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components.

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2-7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9-15.6 sperm per bead) and with collagen type I (6.1-18.5). Human spermatozoa bound to collagen-coated beads (15.4-22.6 sperm per bead) and, to a much lower extent, to chondroitin-sulfate-coated beads (3.2-4.7); they were also able to bind heparin-coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin-coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.

Amino Acid Sequence↗
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

Fetal germ cells establish cell coupling with follicle cells in vitro.

In the present paper we investigate whether the adhesion that occurs in vitro between mouse fetal germ cells and follicle cells is accompanied by the establishment of cell coupling between the two cell types. The possible coupling between germ cells that spontaneously aggregate in culture has also been monitored. When germ cells, isolated from the gonads of 12.5-13.5-day-post coitum embryos, were loaded with 6-carboxyfluorescein diacetate and seeded onto follicle cell monolayers dye was transferred from the germ cells to the adjacent somatic cells within 2-3 h of culture. No dye transfer was observed when germ cells were seeded on fibroblast monolayers or between members of homotypic germ cell aggregates. These results indicate that, following cell to cell adhesion, fetal germ cells are able to establish heterocellular gap junctions with somatic cells of gonadal origin in culture.

Animals↗