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Biomedical subjects

L Monaco

Publications and source records attributed to L Monaco.

At least 73 records · Page 4Linked to original sources
PubMed1986

Sertoli cell membrane polypeptide composition is modulated by germ cells.

The polypeptide composition of Sertoli cell enriched cultures (SCEC) and Sertoli cell only cultures (SCOC) obtained after germ cell removal was investigated. Cells were labelled with [35S]methionine and analyzed by one and/or two-dimensional gel electrophoresis. The one-dimensional electrophoretic analysis of SCEC and SCOC particulate fraction did not show any appreciable difference between the two profiles. The more detailed two-dimensional electrophoretic analysis showed the appearance in SCOC of three polypeptides undetectable in SCEC. The molecular weight of these molecules ranged between 44 and 48 kDa. The effect of FSH on the Sertoli cell membrane composition was also investigated. No qualitative differences were detected, although the hormone increased many molecular species including the polypeptides appearing in SCOC. On the basis of these results, the hypothesis that germ cells influence Sertoli cell metabolic parameters is discussed.

Animals↗
PubMed1986

Putative second messengers affect cell coupling in the seminiferous tubules.

Junctional transfer of ions in monolayer primary cultures of Sertoli cells and in intact seminiferous tubules from 20 day-old rats has been investigated by using electrophysiological techniques. The electrotonic coupling of both intact tubule and monolayer culture was inhibited by the presence of dibutyryl cyclic AMP (10(-4) M) in the medium. In contrast, 1-oleoyl-2-acetylglycerol (10(-5) M) and 12-O-tetradecanoyl-phorbol-13-acetate (10(-7) M) decreased the junctional resistivity and increased the extent of the coupling in immature tubules. The significance of this modulation of the cell coupling in the seminiferous epithelium is discussed.

Animals↗
PubMed1986

Localization of adenosine receptors in rat testicular cells.

The localization of adenosine receptors in rat testicular cells was investigated by evaluating the binding of cyclohexyladenosine (CHA) to somatic and germ cells and by assessing the effect of purine nucleosides on the function of testicular cells. Most of the specific binding of CHA in the testicular homogenate was found in the seminiferous tubule fraction, with little specific binding present in the interstitial compartment. Specific binding was also detected in a total-cell suspension prepared by dispersion of the seminiferous tubules. However, the specific activity of the binding did not increase when such a suspension was fractionated to produce germ cells at different stages of spermatogenesis. Adenosine receptors were present in the crude particulate fraction prepared from testes of rats from 10 to 90 days of age. While the affinity of these receptors did not vary as the testis matured, the number of binding sites per testis increased markedly up to 60 days after birth. Conversely, when binding activity was expressed per mg of testicular protein, no major changes in the specific activity of the binding were detected in testes up to 90 days of age. Sertoli cell-enriched cultures possessed a large number of binding sites, with a receptor density of 722 +/- 147 fmoles bound/mg protein (mean +/- SE; n = 5) and an affinity constant (Kd) of 1-2 nM. Among the characteristics of this binding site were kinetics constants and specificity similar to those measured in total particulate fractions of the testis and in the brain. Specific binding of CHA was not detected in cultures of testicular peritubular cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1986

Effect of phosphodiesterase inhibitors on Sertoli cell refractoriness: reversal of the impaired androgen aromatization.

Responsiveness of the Sertoli cell after FSH pretreatment was evaluated in terms of androgen aromatization. Sertoli cell cultures were preincubated with FSH for 24 h, then cells were washed free of hormone and reincubated with FSH in the presence of androstendione. The estrogen accumulated in the medium was measured by RIA. Gonadotropin pretreatment produced a marked refractory state, and a second challenge with FSH did not produce an increase in androgen aromatization. A dose-response study showed that FSH pretreatment produced three separate effects on Sertoli cell steroidogenesis: an increased basal production of estrogen; a decreased maximal response when doses of 10 ng/ml FSH or higher were employed in the preincubation; and a decreased sensitivity of the Sertoli cell to FSH. In the last case, the ED50 was reduced approximately 3- to 5-fold. Such an impaired stimulation of androgen aromatization was no longer present when cells were incubated with the phosphodiesterase inhibitors methyl-isobutyl-xanthine (MIX). In the presence of this inhibitor, refractory cells responded to FSH better than the control cells. The possibility that MIX stimulated cAMP accumulation by acting as antagonist of purine receptor was ruled out by the finding that the nonxanthine phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone (Ro 20-1724) also reverted the refractory state. Pretreatment of the Sertoli cells with FSH produced an impaired response in the second incubation also to isoproterenol, cholera toxin, and forskolin. The response to these compounds was apparently normal when cells were incubated in the presence of MIX or Ro 20-1724. Conversely, refractory cells responded to (Bu)2cAMP in a manner indistinguishable from the fully responsive control cells. These data demonstrate that FSH induces homologous and heterologous refractory states of the Sertoli cell reflected by an impaired estrogen production. The finding that phosphodiesterase inhibitors fully restore the FSH response suggests an important role of phosphodiesterase in the induction and/or maintenance of such refractoriness.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Plasmid pFCE4: a new system of Escherichia coli expression-modification vectors.

Two versatile expression-modification vectors were obtained by inserting the origin of replication (ori) of phage f1 into the expression vector pOTS. The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4+ and pFCE4-) and excrete it into the medium as virus-like particles following infection with phage f1. These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations. The human IFN alpha-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Base Sequence↗
PubMed1984

Morphological and biochemical effects of lonidamine on cultured Sertoli cells.

Lonidamine, an antispermatogenic compound derivative of indazole-3-carboxylic acid, administered to rat Sertoli cells induced morphological changes of the cells which rapidly shrinked forming cytoplasmic projections. Some biochemical parameters of Sertoli cells measured after drug administration were their aromatizing ability, protein synthesis, and ribonucleic acid synthesis. The amount of estradiol produced by treated cells was significantly lower than the amount produced by control cells, whereas protein and ribonucleic acid synthesis were not affected by the drug. Our results demonstrate the change of a specific metabolic parameter of Sertoli cells induced by this antispermatogenic compound.

Animals↗
PubMed1984

Study of the interaction between germ cells and Sertoli cells in vitro.

The nature of membrane components involved in the binding between Sertoli cells and pachytene spermatocytes in culture and the metabolic requirements for the binding to occur have been studied. Mild proteolytic digestion of germ cells by trypsin completely inhibited adhesion of germ cells to somatic monolayer. Protein synthesis and glycosylation were required to restore the adhesive properties of trypsin-treated germ cells, showing that surface molecules involved in the binding are glycoproteins. Trypsinization of germ cells after labelling causes a great reduction of several bands which become detectable again after 12 h of recovery from trypsin digestion. Among these, two bands with apparent molecular weight (MW) of 78 000 and 51 000 could be candidate components in cell adhesion.

Animals↗
PubMed1984

Purine modulation of the hormonal response of the rat Sertoli cell in culture.

To investigate the possibility that purines modulate the response of testicular cells to gonadotropin, binding of adenosine analogs and biological responses to adenosine were evaluated in Sertoli cell-enriched cultures. The adenosine analog cyclohexyladenosine bound specifically to a crude particulate fraction prepared from such cultures. Binding was saturable, and steady state studies showed the presence of a high affinity binding site (Kd = 2.1 +/- 0.3 nM; n = 4) and a receptor density of 200-300 fmol/mg protein. The bound radioactive ligand was displaced by N6-phenylisopropyladenosine (PIA), adenosine, and methylisobutylxanthine. In addition to the presence of a specific binding site, purines modulated the biological function of the Sertoli cell. The adenosine analog PIA inhibited both the FSH-dependent cAMP response and the FSH-stimulated androgen aromatization. Under all experimental conditions, the IC50 of PIA was 1-3 nM, and maximal effects were observed at 10-100 nM PIA. Adenosine itself inhibited the FSH-dependent response of the Sertoli cell, but was less potent than PIA. In addition, purine inhibition of the FSH response was antagonized by methylisobutylxanthine, while the nonxanthine phosphodiesterase inhibitor Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone] was without effect. Purine modulation was evident not only when cells were stimulated with FSH, but also when the androgen aromatization was augmented by the beta-adrenergic agonist isoproterenol, cholera toxin, and forskolin. On the contrary, the purines had no effect when cells were stimulated with (Bu)2cAMP. The data reported are consistent with the presence of purine inhibitory receptors in Sertoli cell-enriched cultures and show that purines can regulate the response of the immature Sertoli cell in vitro.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Fibroblast interferon from a human amniotic cell line: a strategy for rapid molecular cloning.

A strategy for rapid cloning and identification of cDNA clones was employed to obtain fragments of the gene for human fibroblast (beta) interferon. A human amniotic cell line (UAC), widely used for interferon assay, yielded high titers of beta-interferon and was used as a source for cDNA synthesis. Recombinant clones were screened by a combination of hybridization conditions: (a) comparing clones that hybridized with radioactive cDNA from induced UAC cells vs cDNA from cells not induced for beta-IFN; (b) using a synthetic oligonucleotide proble complementary to a region of the beta-IFN gene. Among clones selected on the basis of their positive response to both screening procedure, one (4F7) was sequenced and shown to be highly homologous to the beta-IFN gene. This clone was used as a probe to screen a human chromosomal DNA library, yielding the entire chromosomal beta-IFN gene. A "Northern" hybridization analysis of UAC mRNA from uninduced and induced cells revealed the existence of a single molecular species of mRNA for beta-IFN with the expected molecular weight.

Amnion↗
PubMed1983

Changes of Sertoli cell glycoproteins induced by removal of the associated germ cells.

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.

Animals↗
PubMed1983

Calmodulin in mouse male germ cells: a qualitative and quantitative study.

Isolated male germ cells of the mouse possess a heat-stable stimulatory activity of Ca2+-dependent, calmodulin-free phosphodiesterase. Ionic exchange chromatography allowed partial purification of the activator and the isolation of multiple forms of phosphodiesterase stimulation inhibitor. The activator has been identified as calmodulin on the basis of chromatographic behaviour and electrophoretic mobility. Quantitative analysis showed variations of calmodulin levels at different stages of spermatogenesis. Quantitative analysis of cyclic nucleotide hydrolysis in germ cell cytosol showed that the activity of Ca2+-dependent phosphodiesterase is different in meiotic and post-meiotic mouse male germ cells. These data suggest that calcium-dependent pathway and a Ca2+-dependent regulation of cyclic nucleotides are present in developing germ cells.

Animals↗
PubMed1983

Evidence of protein secretion by cultured pachytene spermatocytes.

Pachytene spermatocytes isolated from immature rat testis were cultured for 6 h in the presence of [35S]methionine and the macromolecules present in the culture medium were analyzed by one-dimensional and two-dimensional gel electrophoresis. The electrophoretic profiles obtained showed a limited number of polypeptides, some of them consisting of families of spots with the same molecular weight but a different isoelectric point. The reproducibility of the results and the unaltered metabolic activity of the cells during culture time, indicate that the macromolecules present in the medium do not represent degradative products of the cells. Part of the detected macromolecules are considered secreted proteins since the addition of monensin to the cells induces their disappearance from the culture medium.

Animals↗
PubMed1983

Retinol increases synthesis and secretion of Sertoli cell mannose-containing glycoproteins.

Sertoli cell isolated from prepuberal rats were cultured in a chemically defined medium in the presence or in the absence or retinol. In the two experimental conditions the incorporation of 3H-mannose into cellular glycoproteins was measured. The results obtained demonstrated a 3 fold increase of mannose incorporation into cellular glycoproteins after retinol addition without any qualitative variation of their electrophoretic profile. Sertoli cell secretory activity increased of approximately 50% after retinol administration; the electrophoretic analysis showed that two of the secretory products were selectively enhanced by the vitamin addition.

Animals↗
PubMed1981

Alterations in lipid incorporation in Duchenne muscular dystrophy. Studies of fresh and cultured muscle.

The incorporation of [3H] glycerol into lipids of fresh and cultured skeletal muscle obtained from patients with Duchenne muscular dystrophy (DMD), patients with myotonic dystrophy (My Dyst), controls, and aborted fetuses (10-12 weeks old) was studied. A significant increase of specific incorporation of [3H] glycerol into phosphatidylcholine (PCh), phosphatidylserine (PS), phosphatidylinositol (PI), and triglycerides (TRI) was found in DMD and fetal muscle in both fresh and cultured muscle. No significant differences, however, were noted between the values of glycerol incorporation in DMD and fetal muscle. The ratio between phospholipids and TRI changed significantly for fresh muscle in DMD (3.5) and fetal muscle (4.9) versus controls (24). The incorporation of glycerol into these lipids in My Dyst fresh and cultured muscle showed the same value as controls when expressed both as incorporation/mg protein and ratio between phospholipids and TRI.

Adolescent↗