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M Ascoli

Publications and source records attributed to M Ascoli.

At least 91 records · Page 5Linked to original sources
PubMed1983

An improved method for the solubilization of stable gonadotropin receptors.

The studies presented herein describe the usefulness of glycerol in enhancing the binding activity and stability of detergent-solubilized gonadotropin (LH/hCG) receptors. The data presented show that when Leydig tumor cells (designated MA-10) are extracted with Triton X-100 in the presence of glycerol, one can quantitatively solubilize the cellular LH/hCG receptors without losing hormone-binding activity. It is also shown that in the presence of glycerol, the free soluble receptors can be maintained for at least 3 days with little or no reduction in their hormone-binding activity. These findings should prove useful for future studies on the structure and properties of gonadotropin receptors.

Animals↗
PubMed1982

Studies on the source of cholesterol used for steroid biosynthesis in cultured Leydig tumor cells.

The studies presented herein were designed to determine the source(s) of cholesterol used for steroid biosynthesis in a clonal strain of cultured Leydig tumor cells (designated MA-10). Our results show that, when the cells are placed in medium devoid of extracellular cholesterol and acutely stimulated with human choriogonadotropin, most of the cholesterol used for steroid biosynthesis is derived from intracellular stores. Upon prolonged stimulation, when the intracellular stores are depleted, the cells meet the enhanced demand for cholesterol by increasing de novo synthesis. The presence of low density lipoprotein has little or no effect on the amount of steroid synthesized during acute stimulation, but it enhances the amount of steroid produced during prolonged stimulation by directly providing cholesterol substrate to the cells.

Acetates↗
PubMed1982

Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits.

The studies presented herein were aimed at characterizing the pathway involved in the internalization and degradation of human choriogonadotropin by cultured Leydig tumor cells. A quick biochemical method that differentiates between the surface-bound and internalized hormone was developed. Using this method and two hormone derivatives labeled exclusively (with 125I) in the alpha or beta subunits, it was possible to follow the fate of each hormone subunit during hormone binding, internalization, and degradation. The results show that the hormone is internalized in the intact form and that it reaches its place of degradation (presumably the lysosomes) in the intact form. The pathway for degradation of the internalized hormone is complex, and it appears to involve processing of one or both subunits of the intact hormone, followed by subunit dissociation and further degradation of the individual subunits. The alpha subunit is quickly degraded by the cells. The only detectable degradation products are extracellular amino acids. The beta subunit is degraded slower, and several intracellular degradation products are detectable before amino acids appear in the medium.

Animals↗
PubMed1982

Desensitization of steroidogenesis in cultured Leydig tumor cells: role of cholesterol.

We have reported previously that the human choriogonadotropin (hCG) receptors of cultured Leydig tumor cells can be down-regulated with hCG or mouse epidermal growth factor (mEGF) and that such down-regulation results in a loss of the steroidogenic response of the cells to hCG. Exposure of the cells to hCG, but not to mEGF, also resulted in a decrease in steroidogenic responses to cholera toxin and cAMP. The results presented herein show that the hCG-induced loss of steroidogenic response to cAMP is due to the depletion of intracellular cholesterol and that this depletion can be prevented by the addition of low density lipoprotein. Our results also show that after exposure of the cells to mEGF or hCG their steroidogenic response to hCG is limited by the number of hCG receptors, regardless of the presence of low density lipoprotein.

Animals↗
PubMed1981

Removal of the surface-bound human choriogonadotropin results in the cessation of hormonal responses in cultured Leydig tumor cells.

The relationship between the binding and internalization of human choriogonadotropin and the stimulation of cAMP and steroid production was studied in cultured Leydig tumor cells. It was found that removal of the surface-bound hormone results in a rapid cessation of both cAMP and steroid production. We propose that the surface-bound hormone is responsible for the activation of steroidogenesis and that hormone internalization is involved in the deactivation of this process.

Cell Line↗
PubMed1981

Characterization of the desensitized state of Leydig tumor cells.

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusions with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor.

The MA-10 line is a clonal strain of Leydig tumor cells that has receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). These cells respond to hCG, cholera toxin, and 8-Br-adenosine 3':5'-monophosphate with increased steroid production. It is reported herein that exposure of the MA-10 cells to mEGF results in a substantial (80 to 90%) reduction in the number of hCG receptors per cell. The loss of hCG receptors is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. The steroidogenic responses to cholera toxin and 8-Br-adenosine 3':5'-monophosphate, however, are not affected. Other results presented show that mEGF is not a mitogen for these cells.

20-alpha-Dihydroprogesterone↗
PubMed1981

Desensitization to gonadotropins in cultured Leydig tumor cells involves loss of gonadotropin receptors and decreased capacity for steroidogenesis.

The ability of human choriogonadotropin (hCG) to regulate its receptors and target cell responses has been studied in a clonal strain of cultured Leydig tumor cells (MA-10). Exposure of the MA-10 cells to hCG results in decrease in hCG binding activity which is dependent on time and the concentration of hCG. This decrease is due to a change in the number of receptors rather than in the affinity of the receptors, and it is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. Exposure of the MA-10 cells to hCG also resulted in a reduction of the steroidogenic responses to cholera toxin and 8-Br-adenosine cyclic 3',5'-monophosphate. The hCG-induced loss of steroidogenic responses to these stimuli seems to be due to the stimulation of steroidogenesis rather than to the decrease in hCG receptors because it also can be induced when steroidogenesis is stimulated with cholera toxin or 8-Br-adenosine 3',5'-monophosphate under conditions such that the number of hCG receptors is not reduced.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses.

Several clonal lines of cultured Leydig tumor cells have been established and characterized in terms of gonadotropin receptors and steroid production. Although freshly isolated cells derived from the M5480P tumor have functional hCG receptors, only two of the five clonal lines established were shown to bind significant quantities of hCG. In these clones, steroid production can be stimulated to the same extent by hCG, cholera toxin, and 8-Br-cAMP. The other three clones bind a small amount of hCG and respond to the hormone with a marginal increase in steroidogenesis. Steroid production, however, is significantly stimulated by cholera toxin or 8-Br-cAMP. A comparison of the steroids produced by freshly isolated cells and two of the clones revealed some changes in the steroidogenic pathway. The most obvious change is an increase in the ability of the cultured cells to synthesize 20 alpha-dihydroprogesterone (20 alpha-hydroxypregn-4-en-3-one). These clonal lines may provide a suitable model system for the study of gonadotropin actions and regulation of the expression of differentiated functions of Leydig cells.

20-alpha-Dihydroprogesterone↗
PubMed1981

The dynamics of the steroidogenic response of perifused Leydig tumor cells to human chorionic gonadotropin, ovine luteinizing hormone, cholera toxin, and adenosine 3',5'-cyclic monophosphate.

A perifusion system has been developed in which a dose-dependent response of isolated Leydig tumor cells to steroidogenic stimuli, as assessed by the rates of steroid production, can be measured as a function of time. In response to a continuous perifusion for 340 min with a saturating concentration of hCG, ovine LH (oLH), cholera toxin, or 8-Br-cAMP, there is a rapid increase in the rate of progesterone production, which reaches a maximum about 100 min after the onset of stimulus in the medium and then declines to a rate somewhat higher than basal. Thus cholera toxin and 8-Br-cAMP as well as hCG and oLH are able to desensitize the Leydig tumor cells to further stimulation by the same agent. Although a 10-min pulse of a saturating concentration of hCG yields the same steroidogenic response as that elicited by continuous perifusion with saturating hCG, a pulse of a saturating concentration of oLH yields a steroidogenic response only when oLH is maintained in the perifusate. These data establish a substantial difference in the actions of hCG and oLH. The results could be explained by the higher apparent affinity of hCG for the gonadotropin receptor, such that, upon removal of hormone from the perifusate, oLH would be more readily eluted from the receptor. These findings support the hypothesis that oLH and hCG exert their stimulatory effects only while bound to the cell surface. (Endocrinology 108: 632, 1981)

Animals↗
PubMed1981

Characterization of steroid production in cultured human choriocarcinoma cells.

Progesterone is the major steroid synthesized by the JEG-3, BeWo, and JAR cell lines of choriocarcinoma. A lesser amount of pregnenolone is produced. The 17alpha-hydroxy derivatives of these steroids are only minimally present in three lines. The addition of fetal calf serum to the culture medium modestly increases the synthesis of these steroids, but increases the quantity of 17beta-estradiol produced by 30- to 90-fold. The addition of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, androstenediol, and testosterone was shown to stimulate 17beta-estradiol synthesis. There is a clear dose-response relationship between the amount of testosterone added and the quantity of 17beta-estradiol produced. These results indicate that 3beta-hydroxysteroid dehydrogenase-isomerase, 17beta-ol dehydrogenase, and aromatase are active in cultured choriocarcinoma cells, whereas 17beta-hydroxylase and 17-20 desmolase do not appear to be functional in these cells. It is concluded that the stereoidogenic capabilities of choriocarcinoma cells in culture are similar to those of the in vivo placenta and support their use as an experimental model of placental steroidogenesis.

Adrenal Hyperplasia, Congenital↗
PubMed1981

Effects of hypocholesterolemia and chronic hormonal stimulation on sterol and steroid metabolism in a Leydig cell tumor.

The studies presented herein were done to investigate the effects of drug-induced hypocholesterolemia and chronic hormonal stimulation on cholesterol metabolism and steroid biosynthesis in a functional Leydig cell tumor. It was found that 4-aminopyrazolo(3,4-d)-pyrimidine (4-APP)-induced hypocholesterolemia had no effect on a) the amount of cholesterol present in the tumor, b) cholesterol biosynthesis, and c) steroid production. Chronic stimulation with choriogonadotropin also had no effect on the amount of cholesterol present in the tumor, but it increased steroid production and cholesterol biosynthesis. These results suggest that the Leydig tumor cells primarily use intracellular cholesterol for steroid biosynthesis. Other data show that 4-APP treatment reduces gonadotropin binding in the Leydig tumor cells.

Adenine↗
PubMed1980

Degradation of the subunits of receptor-bound human choriogonadotropin by Leydig tumor cells.

Biologically active, iodine-labeled derivatives of human choriogonadotropin in which all the iodine is localized either in the alpha or beta subunits have been prepared. It is found that upon binding to Leydig tumor cells these derivatives are ultimately degraded to 3'-monoiodotyrosine. A comparison of the rates of degradation of the derivatives labeled exclusively in the alpha or beta subunits show that the alpha subunit is degraded somewhat faster than the beta subunit. It was also found that NH4Cl, chloroquine and leupeptin inhibited the degradation of both subunits to the same extent. These results show that the Leydig tumor cells degrade both subunits of the receptor-bound human choriogonadotropin, and suggest that the two subunits are degraded by the same mechanism(s).

Animals↗
PubMed1980

Lipid composition and gonadotropin-mediated lipid metabolism of the M5480 murine Leydig cell tumor.

The effects of human choriogonadotropin (HCG) stimulation on lipid composition in the murine Leydig cell tumor M5480 grown subcutaneously were determined. The main lipids of the Leydig cell tumor were found to be largely triacylglycerols and phospholipids. Daily in vivo administration of human choriogonadotropin to tumor-bearing mice for 3 days increased the phospholipid content and altered the phospholipid composition of the tumors. There was no demonstrable change in the levels of triacylglycerols, cholesterol, and cholesteryl esters. HCG had no major effect on the fatty acid patterns of the major lipid fractions with the exception of cholesteryl esters, which had a decreased amount of arachidonic acid following HCG-treatment. Results of in vitro incubations of tumor cells prelabeled with [1-14C]arachidonate showed that the label was lost more rapidly from cholesteryl esters of HCG-treated cells than from control cells during (the 12-hour) incubation. Moreover, less [1-14C]acetate was incorporated into the cholesteryl ester fraction of hormone-treated cells than in control cells. HCG stimulated the activity of cholesteryl ester hydrolase in dispersed cells within 3 hours. These results demonstrate that an acute effect of HCG on tumor Leydig cell metabolism is to increase the metabolism of cholesteryl esters, probably by stimulating cholesteryl ester hydrolase activity. The long term effect is an accumulation of phospholipids which may be utilized for membrane synthesis.

Animals↗