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Biomedical subjects

F F Rommerts

Publications and source records attributed to F F Rommerts.

At least 73 records · Page 4Linked to original sources
PubMed1985

Rapid and sensitive detection of oestrogen receptors in cells and tissue sections by autoradiography with 125I-oestradiol.

The presence of receptors for steroid hormones in individual cells and tissue sections was assessed within 4-24 h using dry mount autoradiography with radio-iodinated oestradiol. Low affinity and nonspecific binding of steroids were significantly reduced by washing the cells or sections with diluted antiserum to oestradiol. For cells of the MCF-7 cell line variations in grain density were observed, indicating that cells of the MCF-7 cell line are heterogeneous with respect to their cellular receptor concentrations of oestrogen receptors. Receptor-negative cells, such as peritoneal macrophages, did not retain oestradiol label. In tissue sections of rat and calf uterus, predominant labelling was observed on the endometrial gland cells and stroma. Oestradiol receptor binding in the uterus cytosol for both radio-iodinated and tritiated oestradiol showed the same qualitative characteristics as determined by sucrose gradient sedimentation profiles and a comparable amount of binding sites was found for both labels. The relative binding affinity of 125I-oestradiol compared to [3H]oestradiol is about 70-80%. The dry mount autoradiographic technique as presented can be used for rapid screening of heterogeneity in oestrogen receptor distribution in cells and tissue sections, since this technique reveals differences in receptor concentrations on the single cell level.

Animals↗
PubMed1985

Are polyphosphorylated phospholipids involved in the hormonal control of cholesterol side-chain cleavage activity in tumour Leydig cells?

The possible role of LH or dcAMP induced changes in polyphosphorylated phospholipid metabolism in the regulation of cholesterol side-chain cleavage activity has been studied in tumour Leydig cells. Mitochondria isolated from LH-stimulated Leydig cells were 400% more active in pregnenolone production than mitochondria from control cells. Steroid production in isolated mitochondria from control cells could be stimulated only 25% by cytosol fractions from stimulated cells and 100 microM phosphatidyl inositol-4'-phosphate (PtdIns4P). Other polyphosphorylated phospholipids were either inactive or showed aspecific effects. During a preincubation period tumour cells were labelled with [32P]phosphate and steady-state labelling was obtained for the pholyphosphorylated phospholipids after 40-60 min. [32P]Phosphate incorporation in Ptd Ins4P, phosphatidyl inositol (PtdIns), phosphatidyl choline (PtChl), phosphatidyl ethanolamine (PtdEtn) and cardiolipin (CL) was not affected by treatment of the Leydig cells with LH which stimulated (6-fold), or with cycloheximide which suppressed (4-fold) steroid production. A 25% increase of phosphate incorporation by LH was observed only in phosphatidyl inositol-4',5'-biphosphate (PtdIns4,5P2). 32P Incorporation in PtdIns4,5P2, PtdIns,PtdEtn and CL was stimulated by quinacrine 50 microM. Under these conditions the LH-stimulated pregnenolone production but not the 25-hydroxycholesterol dependent pregnenolone production, was completely inhibited. The results obtained with isolated mitochondria and intact cells indicate that increased levels of polyphosphorylated phospholipids are not consistently correlated with increased mitochondrial pregnenolone production. This argues against an important role of polyphosphorylated phospholipids in the hormonal regulation of cholesterol side-chain cleavage activity in tumour Leydig cells.

Animals↗
PubMed1985

Changes of oestrogen receptor levels in Leydig cells from mice and rats during culture.

Two functional properties of Leydig cells in culture, i.e. LH-stimulated steroidogenesis and nuclear oestrogen receptor levels have been investigated. Leydig cells isolated from testes of immature rats and mature mice maintained their responsiveness to LH during 48-72 h of cell culture, although the mouse Leydig cells appeared to be less responsive to LH after 72 h of culture. In contrast, nuclear oestrogen receptor levels in both types of Leydig cells declined to 10-20% of the initial value after 24 h in culture. In the 48-72 h culture period nuclear oestrogen receptor levels recovered to 75% of the initial value only in Leydig cells from immature rats, whereas the nuclear oestrogen receptor levels in Leydig cells from mature mice remained low. These data demonstrate that during in vitro culture of Leydig cells, preservation of LH responsiveness does not necessarily warrant that other Leydig cell parameters e.g. nuclear oestrogen receptors also remain unaltered.

Animals↗
PubMed1985

Lutropin stimulates de novo synthesis of short-lived proteins required for lutropin-dependent steroid production in tumour Leydig cells.

Continuous protein synthesis is required for the hormonal regulation of cholesterol side chain cleavage activity. A protein with a short half-life (t1/2 = 2-13 min) is believed to play an important role, but the regulation of the synthesis of this putative rapidly-turning-over protein is largely unknown. The steroid production rate in tumour Leydig cells can be increased more than 4-fold after addition of lutropin. However, steroid production by cells preincubated for 60 min with medium containing cycloheximide (89 microM) could not be stimulated when lutropin was added to the medium. Thus, the putative protein with the short half-life is apparently not derived from a stable precursor protein. Moreover, in tumour Leydig cells incubated with low concentrations of cycloheximide (0.2-0.8 microM), inhibition of steroid production was significantly greater in lutropin-stimulated cells than in control cells. These results support the hypothesis that lutropin regulates the de novo synthesis of rapidly-turning-over proteins by increasing the rate of initiation of the translation step of protein synthesis.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Ethane dimethane sulphonate (EDS) specifically inhibits LH stimulated steroidogenesis in Leydig cells isolated from mature rats but not in cells from immature rats.

Effects of ethane dimethane sulphonate (EDS) on the pattern of protein synthesis, steroid production and ATP levels in isolated Leydig cells have been investigated. After incubation of Leydig cells isolated from mature rats with EDS (75 micrograms/ml) for 3-5 h, the synthesis of a 33 kDA and 50 dKa protein and LH stimulated steroid production was inhibited, but the LH stimulated cAMP production and conversion of 22R-hydroxycholesterol to testosterone were not affected. Busulphan or ethyl methyl sulphonate (EMS) at similar molar concentrations had no effect on steroid production. After 24 h incubation with EDS Leydig cells were detached from the plastic surface and had rounded up, but the cellular ATP levels were the same as in control cells. Leydig cells were destroyed after incubation with EMS 2000 micrograms/ml for 24 h. EDS had no detectable effects on steroid production by isolated Leydig cells from mice, from Leydig cell tumour tissue or from immature rats, nor on rat adrenal cells or on LH and FSH secreting pituitary cells. The data indicate that EDS specifically inhibits LH regulated functional properties of mature Leydig cells possibly via alkylation of proteins. EDS could be a valuable tool to study possible regulator proteins for control of steroidogenesis in Leydig cells from adult rats.

Alkylating Agents↗
PubMed1985

Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate.

Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.

3-Hydroxysteroid Dehydrogenases↗
PubMed1985

The importance of the Leydig cells in the vascular response to hCG in the rat testis.

The increase in permeability of the testicular blood vessels following an injection of hCG into rats is abolished completely if the animals are treated 3 days earlier with ethane dimethane sulphonate (EDS), a compound that effectively eliminates Leydig cells from the testes. As there is other evidence that androgens or prostaglandins are not involved in this vascular response, further studies will be necessary to determine whether these data mean that another vasoactive substance is secreted by the Leydig cells or whether the EDS also eliminates other cells besides the Leydig cells, for example the mast cells found in the vicinity of the testicular artery.

Animals↗
PubMed1984

Fluorescent androgen derivatives do not discriminate between androgen receptor-positive and -negative human tumor cell lines.

For the evaluation of histochemical procedures for detection of androgen receptors, three human tumor cell lines have been used: PC-93 and NHIK-3025, both biochemically characterized as androgen receptor-positive, and EB-33, biochemically characterized as androgen receptor-negative. The binding of three fluorescent ligands, testosterone-17 beta-hemisuccinate-bovine serum albumin-fluorescein isothiocyanate, testosterone-17 beta-hemisuccinate-fluoresceinamine, and 5 alpha-dihydrotestosterone-17 beta-hemisuccinate-fluoresceinamine, to the cells was evaluated. The relative binding affinities of the ligands for the androgen receptors were low (less than 5% when compared to methyltrienolone). Treatment of the cells with the androgen-fluoresceinamine derivatives resulted in a fluorescent labeling of the cytoplasm in both intact and "freeze-damaged" cells of the three cell lines. This staining was independent of the presence of receptors. Nuclei were not stained. Incubation of intact cels with the protein-linked conjugate did not result in significant cellular fluorescence. Only cells with damaged membranes showed a positive histochemical reaction, both in nucleus and cytoplasm, irrespective of the receptor content of the cells. The fluorescence intensity was not suppressed with excess 5 alpha-dihydrotestosterone or methyltrienolone, which are known to prevent binding of low affinity ligands to androgen receptors. From these results it is concluded that androgen receptors cannot be detected by these fluorescent ligands with low affinity for the receptor. The observed fluorescence of the cells is therefore due to binding of the ligands to other binding sites. The visualization/histochemical demonstration of these binding sites does not appear to be related to the presence of androgen receptors.

Adenocarcinoma↗
PubMed1984

Fluorescent ligands, used in histocytochemistry, do not discriminate between estrogen receptor-positive and receptor-negative human tumor cell lines.

A cell line containing estrogen receptors (MCF-7) and a cell line lacking estrogen receptors (PC-93) were used for a comparison of biochemical and histochemical procedures to detect estrogen receptors. We evaluated three different fluorescent estrogen derivatives: 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, 17 beta-estradiol-17-hemisuccinate-fluoresceinamine, and coumestrol. The main results were: The relative binding affinities of these ligands for the estrogen receptor were between 0.1 and 2% of the affinity of estradiol. Fluorescent staining of the cells showed no relation to the presence of estrogen receptors. Staining was not suppressed with excess estradiol-17 beta, which is known to prevent binding of low affinity ligands to estrogen receptors. Cells with intact membranes were not stained after treatment with the albumin-linked estrogen derivative; only cells with damaged cell membranes were stained. Treatment of cells with 17 beta-estradiol-17-hemisuccinate-fluoresceinamine resulted in a fluorescent labeling of the cytoplasm in intact and artificially damaged cells. Coumestrol caused only fluorescence of the cytoplasm in intact cells. It is concluded that estrogen receptors cannot be detected with these low affinity ligands. Fluorescence of these cells is probably due to binding of the ligands to low affinity binding sites. The presence of these low affinity binding sites appears not to be related to the presence or absence of estrogen receptors and can therefore not be used to discriminate between estrogen receptor-positive and receptor-negative tumor cells.

Breast Neoplasms↗
PubMed1984

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats.

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.

Aging↗
PubMed1984

Development of adenosine responsiveness after isolation of Leydig cells.

Effects of adenosine and related compounds on the regulation of steroid production by isolated Leydig cells have been investigated. Steroid production by freshly isolated Leydig cells from testes of immature or mature rats and mice, or from Leydig tumor tissue could not be stimulated with adenosine, nicotinamide-adenine dinucleotide (phosphate) [NADPH, NAD(P)] or N6-(1-2-phenylisopropyl)-adenosine (PIA) (50 microM), whereas luteinizing hormone (LH) stimulated steroid production more than 10-fold. After 24 h incubation all adenosine-related compounds, but not inosine, stimulated steroid production to 20-100% of the maximal LH-stimulated activity. LH- or 22R -hydroxycholesterol-stimulated steroidogenesis in Leydig cells from immature rats did not decrease during the 24-h culture period, whereas ATP levels increased. The first significant effect of adenosine on steroid production in these cells was found after an incubation period of 3 h. In cells incubated for 1 h and 24 h, LH stimulated cyclic adenosine 3':5'-monophosphoric acid (cAMP) production 10-fold. Significant effects of adenosine and PIA on cAMP production or protein phosphorylation could only be shown in cells incubated for 24 h. Effects of adenosine on Leydig cells in intact testis tissue of immature rats could not be determined. The results suggest that after isolation of Leydig cells, specific alterations in the cell membrane occur, causing increased sensitivity to adenosine and related compounds. Adenosine apparently does not play a role in the role of steroid production in Leydig cells in vivo.

Adenosine↗
PubMed1983

Subcellular localization of LH-dependent phosphoproteins and their possible role in regulation of steroidogenesis in rat tumour Leydig cells.

Stimulation of rat tumour Leydig cells with LH resulted in phosphorylation of 7 proteins of 17, 22, 24, 33, 43, 57 and 76 kDa, and in dephosphorylation of a single protein of 20 kDa. The subcellular localization of these LH-dependent phosphoproteins in combination with effects of inhibitors of microfilament formation and protein synthesis, suggest that phosphoproteins of 20, 43 and 76 kDa present in the cytosol may be involved in the action of microfilaments, whilst phosphoproteins of 24 and 33 kDa present in microsomes may be involved in specific protein synthesis.

Animals↗
PubMed1983

LH-dependent steroid production and protein phosphorylation in culture of rat tumour Leydig cells.

The possible cause of the decreased responsiveness of cultured Leydig cells to stimulation with LH was investigated with rat tumour Leydig cells cultured at 32 degrees C for 2 days. Pregnenolone production and phosphorylation of proteins were studied in combination with the activity of cyclic AMP-dependent protein kinase. Pregnenolone production in cultured tumour Leydig cells decreased from 45 +/- 16 on day 0 to 14 +/- 7 on day 2 (ng/60 min/mg protein) under basal conditions and from 226 +/- 108 on day 0 to 30 +/- 24 on day 2 (ng/60 min/ng protein) under LH-stimulated conditions (means +/- SD, n = 4). This decrease may be accounted for by decreased capacity of cholesterol side-chain cleavage which decreased (in the presence of 25-hydroxycholesterol) from 1.68 +/- 0.26 on day 0 to 0.74 +/- 0.48 on day 2 (microgram/60 min/mg protein; mean +/- SD, n = 4) and decreased activity of cyclic AMP-dependent protein kinase (as apparent from a direct assay of protein kinase activity and the extent of phosphorylation of LH-dependent phosphoproteins).

1-Methyl-3-isobutylxanthine↗