Beta-hydroxysteroid Dehydrogenase-isomerase Activity Research Articles

Evidence that testosterone regulates Leydig cell 3 beta-hydroxysteroid dehydrogenase-isomerase activity by a trans-acting factor distal to the androgen receptor.

Leydig cells are a target for their own steroid product, testosterone, and thus could be subject to short-loop feedback regulation by androgens. The authors previously reported that 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity was higher in freshly isolated Leydig cells from C57BL/6J than those from C3H/HeJ inbred mice. To determine whether this strain-related difference in 3 beta HSD activity could be mediated by differential sensitivity to feedback effects of testosterone, Leydig cells from the two strains were cultured in the presence or absence of testosterone, the synthetic androgen receptor agonist, mibolerone, or the nonaromatizable androgen, dihydrotestosterone. After 7 days of treatment, all three androgens significantly decreased 3 beta HSD activity in Leydig cells from C57BL/6J, but not from C3H/HeJ mice. When Leydig cells were cultured with hydroxyflutamide, an androgen receptor antagonist, the effect of testosterone was negated. To determine whether the strain-related difference in sensitivity to testosterone was mediated by a difference in the androgen receptor protein, Leydig cells from reciprocal F1 hybrid lines of mice were cultured in the presence or absence of testosterone. Testosterone treatment inhibited 3 beta HSD activity in both F1 lines to the same extent as observed for Leydig cells from C57BL/6J mice. Thus, there is a strain-related difference in the response to testosterone, but it cannot account for the strain-related difference in Leydig cell 3 beta HSD activity because the high 3 beta HSD strain (C57BL/6J) is the sensitive strain. Although the effect on C57BL/6J Leydig cells is androgen receptor-mediated, the dominant effect of testosterone on both F1 lines rules out a difference in the androgen receptor protein per se. However, the data are consistent with the difference being in a trans-acting factor distal to the androgen receptor.

Evidence that the same structural gene encodes testicular and adrenal 3 beta-hydroxysteroid dehydrogenase-isomerase.

Thermostability of 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity was examined in testes and adrenal glands from several inbred lines and feral mice. A thermolabile variant of 3 beta HSD was detected in the feral Brno mice. The thermostability (t1/2) of 3 beta HSD was approximately 7 min for both testes and adrenal glands from C57BL/6J mice, compared with 4 min for both tissues from Brno mice. Comparison of testicular and adrenal 3 beta HSD thermostability in six kinds of mice indicated that the t1/2 of 3 beta HSD was correlated in the two tissues and could be classified into two distinct types, thermolabile and thermostable. In contrast, quantitative variants in 3 beta HSD activity were not correlated in the two tissues. These data are consistent with the hypothesis that testicular and adrenal 3 beta HSD is encoded by the same structural gene but that expression of 3 beta HSD activity is independently controlled in testes and adrenal glands.

Partial characterization of alcohol dehydrogenase activity in purified rat Leydig cells.

Ethanol metabolism to acetaldehyde by NAD+-dependent alcohol dehydrogenase (ADH) activity reduces, in part, androgen secretion by rat Leydig cells. ADH in Leydig cells is proposed to decrease the NAD+/NADH ratio and thereby inhibit NAD+-dependent delta 5-3 beta hydroxysteroid dehydrogenase-isomerase activity and increase NADH-dependent 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase activity. Although the reciprocal changes in these steroidogenic enzyme activities by ethanol are attributed to ADH activity, there is very little information about this enzyme in purified Leydig cells. The present studies examined specific characteristics of this enzyme in metrizamide-gradient purified Leydig cells. ADH activity was linear with respect to protein concentration and incubation time. The activity was concentrated in the soluble fraction, and the most effective cofactor was NAD+. The apparent Km for ethanol was 0.50 mM, and the Vmax was 53 nmol NADH/10 min/mg protein. When Leydig cell cytosol was incubated with a fixed ethanol concentration (50 mM) and increasing NAD+ and the data were plotted according to Lineweaver-Burk, a biphasic curve was observed with apparent Km's of 0.032 and 0.17 mM. The optimum pH for the enzyme was 8.2, and the enzyme was inhibited in a dose-dependent manner by 4-methylpyrazole. These studies further characterize ADH activity in purified Leydig cells and demonstrate that this enzyme exhibits many characteristics similar to the more widely studied liver enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Maximal testosterone production in Leydig cells from inbred mice relates to the activity of 3 beta-hydroxysteroid dehydrogenase-isomerase.

We previously described significant differences in maximal testosterone production by Leydig cells from the following strains of inbred mice: C57BL/10J, C57BL/6J, DBA/2J, and C3H/HeJ. To evaluate whether these differences in maximal testosterone production related to the activities of the steroidogenic enzymes of the smooth endoplasmic reticulum of Leydig cells from these strains, the activities of 3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-hydroxylase, C17-20 lyase, and 17-ketosteroid reductase were measured in homogenates of purified Leydig cells using 3H-labeled substrates and measuring the amounts of 3H products formed. Maximal and basal testosterone production were determined by incubating aliquots of Leydig cells for 2 h in the presence or absence of 30 pM hCG. Maximal testosterone production by Leydig cells from C57BL/10J and C57BL/6J mice was significantly greater than that by Leydig cells from DBA/2J and C3H/HeJ mice. No difference in basal testosterone production was observed among the strains. Among the four enzymes studied, only 3 beta-hydroxysteroid dehydrogenase-isomerase activity was significantly correlated with hCG-stimulated testosterone production by Leydig cells from the four strains of mice. In addition, when equivalent numbers of Leydig cells from each strain were incubated with an equal concentration of [3H]pregnenolone, a similar difference in [3H] testosterone production was observed among the four strains, as was seen with hCG-stimulation. Leydig cells from C57BL/10 and C57BL/6J mice left less [3H]pregnenolone unmetabolized and produced higher amounts of [3H]testosterone than Leydig cells from DBA/2J and C3H/HeJ mice. There was a significant negative correlation between the amount of pregnenolone unmetabolized and the amount of testosterone produced. These data suggest that 3 beta-hydroxysteroid dehydrogenase-isomerase may be important in determining the differences in hCG-stimulated testosterone production by Leydig cells from the four strains of mice.

Determination of delta 5 3 beta-hydroxysteroid dehydrogenase activity in intact isolated rat Leydig cells.

A method for the determination of delta 5 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) activity in intact isolated Leydig cells was established. This method utilizes the conversion of [7-3H]dehydroepiandrosterone (1.04 mumole) to androstenedione and expresses the activity of the enzyme as mumoles of androstenedione produced/microgram DNA/h. The reaction is limited to 0.5 - 4 micrograms DNA of Leydig cells/ml (equivalent to 0.1-0.8 million of Leydig cells/ml) and to 1 h of incubation at 34 degree C. The 3 beta-HSD activity of 44 suspensions of Leydig cells isolated from adult rats was found to be 1.13 +/- 0.03 (SE) mumoles/microgram DNA/h. This new method for direct measurement of 3 beta-HSD activity in intact Leydig cells was found to be rapid, easy to perform and highly reproducible.

The developmental changes in plasma adrenal androgens during infancy and adrenarche are associated with changing activities of adrenal microsomal 17-hydroxylase and 17,20-desmolase.

The plasma concentrations of dehydroepiandrosterone, androstenedione, and dehydroepiandrosterone sulfate decrease during the first year of life, remain low during childhood, and then increase during adrenarche. To determine whether alterations in adrenal enzyme activity might explain the changing secretory pattern of the adrenal androgens, we measured human adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase, 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase activities. 12 adrenals from individuals aged 3 mo to 60 yr were studied. The patients were divided into three groups based upon the age of the patient when the adrenal glands were obtained: group 1, infants aged 3--8 mo (n = 3); group 2, preadrenarchal or early adrenarchal children aged 2--9 yr (n = 4); and group 3, adults aged 20--60 yr (n = 5). The mean activity of the 17,20-desmolase, 17-hydroxylase, and 21-hydroxylase fell by 50% and that of 3 beta-hydroxysteroid dehydrogenase-isomerase activity rose 80% from group 1 to 2. A fourfold increase in 17,20-desmolase (P less than 0.002) and 17-hydroxylase (P less than 0.001) activity and a doubling in 21-hydroxylase activity (P less than 0.005) occurred between groups 2 and 3. We conclude that the decline in plasma adrenal androgens after birth appears to be associated with a rise in 3 beta-hydroxysteroid dehydrogenase-isomerase and a fall in 17,20-desmolase and 17-hydroxylase activity. The subsequent increase in plasma adrenal androgen concentration during adrenarche is coincident with a rise in 17,20-desmolase and 17-hydroxylase activity.