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W Staib

Publications and source records attributed to W Staib.

At least 37 records · Page 2Linked to original sources
PubMed1984

Comparison of androgen biosynthesis in isolated leydig cells from rat and mouse testis: incubation and superfusion studies.

Production of testosterone by highly purified Leydig cells prepared from rat and mouse testes is compared. Testosterone formation is improved to a higher degree in rat (2.7-fold) than in mouse (1.7-fold) cells by collagenase treatment of the testis compared with mechanical isolation. Mouse Leydig cells respond to exogenous stimuli (choriogonadotropin, dibutyryl cyclic AMP) with 2.4-fold higher testosterone secretion than rat cells. A 1.7-fold increased conversion of androgen precursors to testosterone by mouse compared with rat Leydig cells is demonstrated in static incubations as well as in steady-state superfusion experiments and can be derived from enhanced androstenedione reduction and a less inhibitory effect of progesterone on this process in mouse Leydig cells.

Androgens↗
PubMed1984

Effect of streptozotocin-induced hyperglycaemia on androgen-binding protein in rat testis and epididymis.

In adult male rats treated with streptozotocin 6 weeks before the experiments, androgen-binding protein concentration was increased in testicular tissue by 33% (p less than 0.01) and reduced in epididymal tissue by 25% (p less than 0.005) in animals exhibiting severe hyperglycaemia as compared with animals remaining in normoglycaemia or moderate hyperglycaemia. Androgen-binding protein content was diminished in epididymal tissue by 40% (p less than 0.0005) but not changed in testicular tissue. If related to constant body weight, the sum of testicular and epididymal androgen-binding protein was identical in both normo- and hyperglycaemic animals. This disturbance in androgen-binding protein distribution may be the consequence of altered testicular secretion or impaired transport of androgen-binding protein from testes to epididymides.

Androgen-Binding Protein↗
PubMed1984

Estimation of kinetic parameters of androgen-synthesizing enzyme activities in superfused Leydig cells from rat testes: difference between endogenous and exogenous substrates.

Kinetic parameters of 3 beta-hydroxysteroid dehydrogenase/isomerase, steroid-17 alpha-monooxygenase, and steroid-17,20-lyase activities were estimated under steady-state conditions. Purified Leydig cells from rat testes were superfused with pregnenolone, progesterone, or 17 alpha-hydroxyprogesterone. The Km values for both the monooxygenase- and the lyase-catalyzed reactions were by factors of five to ten higher if analyzed with the exogenously added substrate (0.98 and 0.65 microM, respectively) than if calculated from endogenous substrate derived from a precursor (0.10 and 0.13 microM, respectively). This discrepancy may be explained by different substrate partition between the intra- and extracellular spaces and by different substrate concentration at the active site of the respective enzyme, depending on whether the actual substrate is of exogenous or endogenous source.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

A carrier-free cell superfusion system for the study of androgen formation from precursor steroids by isolated rat testis Leydig cells under steady-state conditions.

A cell superfusion system is presented in which cells can be kept in suspension if there is an equilibrium between cell sedimentation velocity and superfusion medium flow velocity. Since the cross-section area of the central core increases gradually, theoretical considerations and experimental results demonstrate that single isolated cells as well as small cell aggregates are not able to leave the superfusion chamber. The whole apparatus is constructed from glass to avoid adsorption of steroids. As an application of the system, androgen formation from progestin precursors by isolated rat testis Leydig cells is shown. Under steady-state conditions, constant concentrations of substrates, intermediates, and products are measured in the collected effluent. In contrast to conventional cell incubation, constant testosterone formation rates and linearly increasing cumulative testosterone production are achieved.

Animals↗
PubMed1984

Oxidative determination of 14C-labeled 2-oxo acids.

A simple and rapid assay for the determination of 1-14C- or U-14C-labeled 2-oxo acids is described. It is based on the selective and complete oxidation of the carboxyl group to 14CO2. Preceding purification procedures are not necessary. In rat hindlimb perfusion studies, the procedure was used to develop an indirect method for the estimation of the intracellular dilution of [1-14C]pyruvate and to determine the relationship between the transamination and decarboxylation rates of leucine in the perfused tissue by the use of tracer doses of L-[1-14C]leucine.

Animals↗
PubMed1984

Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis.

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.

17-alpha-Hydroxyprogesterone↗
PubMed1984

Effect of human chorionic gonadotropin and estradiol in vivo on estradiol binder and cytochrome P-450 concentrations in rat testis.

The effects of single administration to adult male rats in vivo of various amounts of human chorionic gonadotropin (HCG) and of single or repeated injections of estradiol on testicular cytoplasmic estradiol binder concentrations and on microsomal progesterone-binding cytochrome P-450 were compared. Half-life periods of HCG-induced loss of estradiol binder and cytochrome P-450 concentrations are identical (6 h) whereas a strong dissociation of these half-life periods are evident after chronic estradiol treatment (less than 2 h for the estradiol binder, about 35 h for cytochrome P-450). Depletion of cytoplasmic estradiol binder is not a sufficient condition for mediation of effects on cytochrome P-450 content. Rate of replenishment of microsomal cytochrome P-450 is similar after HCG or estradiol treatment. Both HCG- and estradiol-induced loss of cytochrome P-450 occur not only in Leydig cells but also in microsomes prepared from seminiferous tubules. Additional information is presented contradicting the hypothesis that loss of cytochrome P-450-dependent steroidogenic enzymes caused by HCG could be mediated by estrogens.

Animals↗
PubMed1984

Further characterization of estrogen binding to rat testis cytosol.

Binding of estradiol (E2), estriol (E3), RU16117, and moxestrol to testis cytosol from adult male rats was investigated. High-affinity binding sites were identified in the 8-9S region of sucrose density gradients; a second, high-capacity binding component in the 4S region was probably due to contamination with serum. Thermodynamic properties of the testicular estrogen binding site were quite similar to those of the uterine receptor. E2 had the highest affinity for testicular cytosol binding sites (Ka: E2 much greater than moxestrol greater than E3 greater than RU16117). Comparison of association rate (E2 greater than E3 greater than moxestrol = RU16117) and dissociation rate constants (E3 = RU16117 greater than E2 much greater than moxestrol) as well as studies in vivo revealed moxestrol as a long-acting and RU16117 as a short-acting compound. This difference may be useful for evaluation of the mediation of estrogen effects in the rat testis.

Animals↗
PubMed1984

In vitro and in situ skeletal muscle pyruvate dehydrogenase activity in adrenalectomized and glucocorticoid treated rats.

Skeletal muscle glucose oxidation is significantly reduced and alanine release enhanced in adrenalectomized rats after short-term glucocorticoid treatment. A possible site of regulation is the pyruvate dehydrogenase complex. Pyruvate dehydrogenase activity was measured in vitro in extracts of the gastrocnemius muscle of normal, adrenalectomized and short-term glucocorticoid treated rats by the p-nitroaniline-arylamine-acetyltransferase method and in situ in non-recirculating perfusions of isolated hindlimbs with physiological pyruvate levels and tracer doses of [1-14C] pyruvate by measuring the off-kinetic of 14CO2 wash out in the effluent. Neither method showed a direct influence of glucocorticoids on skeletal muscle pyruvate dehydrogenase activity.

Adrenal Glands↗
PubMed1984

Direct effect of androgens on progesterone binding and metabolism in rat testis microsomes.

A possible site of action at which androgens may control their own biosynthesis in rat testicular tissue in terms of an intratesticular feedback mechanism is investigated. It is shown that both progesterone (Ks = 0.45 microM) and testosterone (Ks = 14.7 microM) induce spectral changes at microsomal cytochrome P-450; these spectral effects are not additive and therefore both steroids may act on the same species of cytochrome P-450. This hypothesis is supported by the observation of competitive inhibition by testosterone of progesterone binding to solubilized microsomal proteins (Ki = 10.0 microM) and of progesterone conversion to androgens (Ki = 14.3 microM). It is concluded that rat testicular androgen biosynthesis is subject to feedback regulation not only via the pituitary-testicular axis but also by direct action of androgens on microsomal reactions dependent on progesterone-binding cytochrome P-450.

17-alpha-Hydroxyprogesterone↗
PubMed1984

Inhibition of 17 beta-hydroxysteroid-oxidoreductase activity in purified rat Leydig cells by single injection of human choriogonadotrophin.

A marked decrease of progesterone conversion to androgens in vitro by purified rat testis Leydig cells is observed from 1/2 to 3 days after treatment of the rats with 100 IU human choriogonadotrophin (hCG) in vivo. The maximal inhibition results 2 days after hCG injection and is denoted by a 72% reduction of androstenedione formation and a 78% reduction of testosterone formation from 2.5 mumol progesterone X 1(-1). The testosterone/androstenedione ratio (about 0.4), however, is not changed after hCG treatment, indicating that the 17-ketosteroid-reductase activity is not rate-limiting under these conditions. Nevertheless, testosterone formation from 2.5 mumol androstenedione X 1(-1) is reduced by 67% and androstenedione formation from 2.5 mumol testosterone X 1(-1) is reduced by 79% 1 and 2 days after hCG treatment. The time-course of this hCG-induced decrease in both reductase and oxidase activities of the Leydig cell 17 beta-hydroxysteroid-oxidoreductase parallels the decrease of androgen formation from progesterone Kinetic analyses reveal that the Vmax of the oxidase is reduced to a significantly (P less than 0.05) greater extent than the Vmax of the reductase (79 and 62%, respectively), whereas the respective Km values remain unchanged. From these results it may be concluded that either a loss, or an inactivation of the enzyme protein, or the formation of non-competitive inhibitor occur during hCG action and that hCG may affect different enzyme activities involved in testicular androgen biosynthesis in a similar way.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Evidence for the compartmentation of pyruvate metabolism in perfused rat skeletal muscle.

In rat hindlimbs perfused with [1-14C]pyruvate and 5 mM-dichloroacetate, the calculated apparent rate of pyruvate decarboxylation was decreased with increasing perfusate pyruvate concentrations. However, in the absence of dichloroacetate the apparent rate of decarboxylation increased under these conditions. Dichloroacetate enhanced [1-14C]pyruvate uptake, but decreased the specific radioactivity of effluent lactate. Glycogen metabolism remained unaffected. The results were not consistent with a common pyruvate pool, but provide evidence for the compartmentation of pyruvate metabolism.

Animals↗
PubMed1983

Estimation of pyruvate decarboxylation in perfused rat skeletal muscle.

By the determination of pyruvate dehydrogenase activity in tissue homogenates only limited information is gained on the actual metabolic flux. We therefore determined pyruvate decarboxylation in isolated rat hindlimbs non recirculating perfused with physiological (1-14C)pyruvate levels. On the basis of perfusate pyruvate specific activity a 14CO2 production of 15.8 +/- 0.5 nmol/min per g muscle was measured. However, by this method the actual pyruvate flux through the enzyme complex is underestimated by a factor of 7 due to the intracellular dilution of label.

Animals↗
PubMed1983

A device for the liberation and determination of 14CO2.

A simple closed system for the serial determination of 14CO2 in small volumes of fluid samples, is described. The device consists of commercially available scintillation vials and silicone tube seals. 14CO2 is selectively liberated by citric acid and absorbed in a scintillation vial by Hyamine. Experiments on the effect of dichloroacetate on pyruvate dehydrogenase activity in rat hindlimbs perfused with [1-14C]pyruvate demonstrate the applicability of the method.

Animals↗
PubMed1983

Distribution of progesterone-binding cytochrome P450 and steroid-17 alpha-hydroxylase/C-17,20-lyase within different compartments of the rat testis.

Progesterone-binding capacity of cytochrome P450 and rate of progesterone consumption by cytochrome P450-dependent hydroxylases are highest in the Leydig cell fraction but are also detectable in the non-Leydig cell fraction of interstitial cells as well as in seminiferous tubules from rat testis. The Leydig cell compartment, however, contributes only to a minor extent to the total progesterone binding and hydroxylation within the whole testis.

Aldehyde-Lyases↗