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C J Dix

Publications and source records attributed to C J Dix.

At least 37 records · Page 2Linked to original sources
PubMed1982

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells.

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues(10-8-10-6 M) on the functional activity (testosterone and cyclic AMP production and [125I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0-90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells. The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10-7 M) and after an initial lag period (2h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10(6) cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (p less than 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased. These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.

Animals↗
PubMed1982

Catecholamine stimulation of testosterone production via cyclic AMP in mouse Leydig cells in monolayer culture.

The effects of the beta-adrenergic hormone agonist, isoproterenol, on testosterone and cyclic AMP production in mouse Leydig cells in culture have been investigated. It was found that isoproterenol increased testosterone production on days 1, 2 and 3 of culture but not in freshly cultured cells. Cyclic AMP production was however increased on all days of culture. In subsequent studies carried out on day 2 of culture the amounts of testosterone formed during incubation with isoproterenol were 20-90% of those obtained with maximum stimulating levels of luteinizing hormone. The amounts of cyclic AMP formed were extremely low compared with those obtained with luteinizing hormone (22 +/- 5.3 and 2320 +/- 100 pmoles/10(6) cells/2 h respectively). Isoproterenol (10(-8) -10(-7) M) gave a significant increase in testosterone production and reached a maximum with 10(-6) M. Similar dose-response curves for cyclic AMP production were obtained. The stimulation of cyclic AMP and testosterone by isoproterenol was highly dependent on the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. Propranolol blocked, in a dose-dependent manner, both isoproterenol-stimulated cyclic AMP and testosterone production. In the presence of excess luteinizing hormone no additional effects of isoproterenol were detected. Epinephrine also stimulated testosterone production. It is concluded that catecholamines stimulate testosterone production in mouse Leydig cells in monolayer culture and that this effect if mediated by cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1982

The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

Purified Leydig cells were obtained from adult mouse testes by mechanical dispersion followed by Percoll density-gradient centrifugation as described by Schumacher et al. (1978). The cells were then established in monolayer culture by maintaining them in medium and 10% serum at 32 degrees C in 95% O2, 5% CO2. The cells rapidly attached to the culture dishes, gradually flattened and became epitheloid in appearance. Testosterone production by the cells in response to maximum stimulating levels of LH (100 ng/ml) and dibutyryl cyclic AMP (1 mM) was maintained for at least 2 days (approximately 1 microgram/10(6) cells/2h) and then declined to lower levels by days 3-4. Cyclic AMP production in response to LH was higher on day 1 than day 0 and then declined to lower levels by days 3-4. Binding of [125I]hCG was similar on day 0 and day 1 (approximately 20 fmoles/10(6) cells) and then declined to lower levels by days 3-4. The functional activity of the cells cultured in 0, 1 and 10% foetal calf serum was also examined; no significant effect of the serum on LH-stimulated testosterone or cyclic AMP production was found; however, a decrease of up to 50% in the binding of [125I]hCG to the Leydig cells occurred in the presence of serum. These results demonstrate that the function of differentiated adult Leydig cells can be maintained for at least 2 days in culture.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour.

An LH sensitive adenylate cyclase from a tumour Leydig cell has been investigated. The plasma membranes, prepared by a 2 phase (dextran-polyethylene glycol) centrifugation method were found to have the following properties: In the presence of LH plus p(NH)ppG (guanosine 5'-beta, gamma-imido triphosphate) or fluoride ions, maximum adenylate cyclase activity was obtained in the plasma membranes with 4 to 6 mM Mg2+ plus 0.33 to 2 mM ATP. LH alone stimulated adenylate cyclase activity 2-fold when compared with basal activity and the time course of cyclic AMP production was linear up to 45 min. With GTP (10(-5)M) and GTP plus LH, adenylate cyclase activity was increased 3 and 6-fold, respectively, for up to 20 min and thereafter declined. In contrast p(NH)ppG (10(-5)M) and p(NH)ppG plus LH increased adenylate cyclase activity 7 and 14-fold which was maintained for at least 45 min. Fluoride ions increased the enzyme activity linearly over 45 min approx 18-fold. When GTP or p(NH)ppG were added alone there was a lag time of activation of approximately 10 min which was abolished by the addition of LH. GTP but not p(NH)ppG at concentrations greater than 10(-4) inhibited basal and LH stimulated adenylate cyclase when compared with 10(-5)M GTP. The tumour Leydig cell adenylate cyclase is thus essentially similar to other hormone sensitive somatic cells. The present study makes it feasible to prepare plasma membranes by a simple method from large quantities of pure Leydig cells.

Adenosine Triphosphate↗
PubMed1982

Guanine nucleotide mediated desensitization of adenylate cyclase in cell free preparations from a Leydig cell tumour.

Cell free desensitization of a tumour Leydig cell plasma membrane adenylate cyclase has been demonstrated in the presence of guanine nucleotides. In experiments in which the membranes were pre-incubated with various nucleotides and LH, it was shown that this decreased adenylate cyclase activity was dependent on the presence of GTP and occurred both in the presence and absence of ATP. While pre-treatment with LH alone appeared to enhance subsequent adenylate cyclase activity, this hormone was able to potentiate the desensitizing effect of GTP. The desensitizing effect of GTP was not inhibited by sodium fluoride. In contrast, the GTP analogue p(NH)ppG (guanosine 5'beta, gamma-imido triphosphate) caused a persistent activation of the adenylate cyclase. GMP and guanosine also initially inhibited the adenylate cyclase activity, but this was entirely reversed by p(NH)ppG plus LH. GDP in addition to GTP caused desensitization but this was only partially reversed by p(NH)ppG plus LH. It is proposed that in similarity with the ovary (Bockaert et al. 1976; Ezra & Salomon 1982a) desensitization of Leydig tumour cell plasma membrane adenylate cyclase may involve a GTP-mediated phosphorylation step.

Adenylyl Cyclases↗
PubMed1981

Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.

Animals↗
PubMed1981

The heterogeneity of Leydig cells from mouse and rat testes--evidence for a Leydig cell cycle?

A method for purifying Leydig cells by centrifugation of testes cells on continuous density gradients of Percoll has been investigated. The distribution of Leydig cells in the separated bands of cells obtained and their receptor content and testosterone production after addition of lutropin (LH) has been measured. In agreement with previous work (Schumacher, Schäfer, Holstein & Hilz 1978) it was found that highly pure mouse Leydig cells (average density 1.070 g/ml) could be prepared by this method. These cells responded to LH and produced high amounts of testosterone (1 - 4 microgram/10(6) cells/2 h), and bound [125]hCG specifically (25 - 64 fmols hCG bound/10(6)). Similarly from rat testes, Leydig cells (average density 1.072 g/ml) were purified. These cells also responded to LH and produced 5 - 25 ng testosterone/10(6) cells/2 h and bound [125]hCG specifically (3 - 18 fmols hCG bound/10(6)cells). Two other bands of nucleated cells of lower density (approximately 1.045 and 1.052 g/ml) were formed on the gradients from both mouse and rat testes. Both these bands of cells were found to contain Leydig cells which bound [125]hCG specifically but little or not stimulation of testosterone production could be demonstrated. Fractionation of the gradients after separation of the cells into small aliquots demonstrated that fractions containing up to 100% Leydig cells could be isolated which were not stimulated to produce testosterone after addition of LH. It is concluded that in both the adult rat and mouse testes, Leydig cells of different densities and steroidogenic responsiveness to LH exist. The data obtained in this and other studies suggest that Leydig cells in the rat and mouse testes are not a homogeneous population and that they may be undergoing a continuous cycle of activity which involves changes in density and steroidogenic capacity.

Animals↗
PubMed1980

Modulation of rat uterine steroid hormone receptors by estrogen and antiestrogen.

The effect of sc injections (25 microgram) of estradiol benzoate, monohydroxytamoxifen [1-(4 beta-dimethylaminoethoxyphenyl)1-(4-hydroxyphenyl)-2-phenylbut-1-ene] and tamoxifen [trans-1-(4 beta-dimethylaminoethoxyphenyl)1,2-diphenylbut-1-ene] every 12 h on uterine wet weight, DNA, and cytoplasmic estrogen and progesterone receptor levels has been studied in the immature rat for up to 90 h. Estradiol benzoate produced a 4-fold rise in uterine wet weight, with a doubling of uterine DNA within 48 h. Tamoxifen and monohydroxytamoxifen doubled uterine weight, with a small rise in uterine DNA. Cytoplasmic estrogen receptor levels were reduced within 24 h by all treatments. Estradiol benzoate and monohydroxytamoxifen produced a maximal rise in nuclear estrogen receptor levels within 8 h, followed by a rapid decline to control levels within 80 h. Tamoxifen produced a slower rise in nuclear estrogen receptor levels and never reached the levels achieved by estradiol or monohydroxytamoxifen. The level of nuclear tamoxifen-estrogen receptor complexes slowly decreased with time. In each case, the cytoplasmic progesterone receptor levels increased as nuclear estrogen receptor levels decreased. Cycloheximide (5 microgram/2 h for 8 h before and 20 h after the first estrogen or antiestrogen injection) was used to determine the effect of protein synthesis inhibition on the hormone receptor profiles. Progesterone receptor synthesis was inhibited by cycloheximide. Cycloheximide did not affect translocation, but produced a rapid decrease in nuclear estrogen receptor levels. The results suggest that without the continual translocation of estrogen receptors from the cytoplasm, the antiestrogen-estrogen receptor levels in the nuclear compartment decrease because of destruction or processing. The nuclear antiestrogen-estrogen receptor pool is therefore not static but dynamic. High affinity ligand-estrogen receptor complexes are readily processed in the nucleus to effect progesterone receptor synthesis in the cytoplasm; however, this series of biochemical reactions is only secondary to the fundamental events essential for cell division. After an initial increase in cytoplasmic steroid receptor synthesis by estrogen or antiestrogen, there is a gradual reduction in total cytoplasmic and nuclear estrogen receptor complexes in response to continual nuclear stimulation. This reduction, in turn, reduces progesterone receptor synthesis.

Animals↗
PubMed1980

Subcellular effects of monohydroxytamoxifen in the rat uterus: steroid receptors and mitosis.

The administration of either monohydroxytamoxifen (25 micrograms) or oestradiol benzoate (25 micrograms) to immature rats resulted in similar depletion of the cytoplasmic oestrogen-receptor pool, with a transient (approx. 48 h) increase in nuclear oestrogen-receptor levels. Oestradiol benzoate increased uterine wet weight with a corresponding increase in uterine cytoplasmic progesterone-receptor levels, DNA content and cell division at 48 h. In contrast, monohydroxytamoxifen produced only a partial increase in uterine wet weight. Although the increase in concentration of uterine progesterone receptors (per mg DNA) by monohydroxytamoxifen was comparable to that produced by oestradiol benzoate, the nuclear antioestrogen-oestrogen-receptor complexes were unable to stimulate a large rise in whole uterine DNA content and cell division. Examination of stromal and epithelial mitotic activity 48 h after administration of 0.25, 2.5 and 25 micrograms oestradiol benzoate, monohydroxytamoxifen or tamoxifen showed that the inability of antioestrogens to stimulate oestrogen-like mitotic activity was not related to the dose of antioestrogen that was administered. It is suggested that the inability of the antioestrogen-oestrogen-receptor complexes to initiate the nuclear events which lead to cell division should be exploited in the investigation of the nuclear mechanism of oestrogen action. The high potency of monohydroxytamoxifen and its inherent biological activity, in that it is not metabolically activated before exerting its effects, provide clear advantages for its future use as a pharmacological probe.

Animals↗
PubMed1979

Inhibition of cell division and stimulation of progesterone receptor synthesis in rat oestrogen target tissues by non-steroidal antioestrogens.

Recent studies from this laboratory have demonstrated oestradiol-oestrogen receptors associated with the nuclear compartment of the rat uterine cell will initiate protein synthesis, as evidenced by a rise in progesterone receptor concentrations, and cell division whereas the anti-oestrogen - oestrogen receptor complex causes protein synthesis and cellular hypertrophy rather than hyperplasia. It is probable that this separation of biological activities resides in the intrinsic activity of the respective receptor complexes. We have demonstrated that caution should be exercised in the interpretation of low affinity ligand-hormone receptor interactions undertaken in vitro. Simple tests for ligand specificity for a binding protein are clearly insufficient evidence to characterise a hormone receptor complex using a conventional 15 hr technique of sucrose density gradient analysis. Oestrogens and anti-oestrogens do not seem to disrupt the subunit integrity of the cytoplasmic oestrogen receptor and it appears likely that the ligand plays a fundamental role in confering the correct biological properties to the hormone receptor complex.

Animals↗