Biomedical subjects

J I Mason

Publications and source records attributed to J I Mason.

At least 73 records · Page 4Linked to original sources

PubMed1995

Absence of molecular defect in the type II 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) gene in premature pubarche children and hirsute female patients with moderately decreased adrenal 3 beta-HSD activity.

To date the molecular basis and hormonal criteria for inherited mild late-onset 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency congenital adrenal hyperplasia (CAH) have not been defined. We have thus investigated the presence or absence of mutation in the type II 3 beta-HSD gene encoding adrenal/gonadal 3 beta-HSD in each of five premature pubarche children and hirsute female patients manifesting moderately decreased adrenal 3 beta-HSD activity. ACTH-stimulated hormonal levels in all patients compared with mean levels in pubertal stage-matched normal subjects were between 2.5 and 6.5 SD for 17-hydroxypregnenolone levels, and between 2.5 and 7 SD for dehydroepiandrosterone levels in all except one patient. 17-Hydroxypregnenolone to cortisol ratios were between 2.5 and 4.3 SD, and dehydroepiandrosterone to androstenedione ratios were between 3 and 8.6 SD. The type II 3 beta-HSD gene regions of a putative promoter, exons I, II, III, and IV, and exon-intron boundaries in all subjects were amplified by polymerase chain reaction and then sequenced. All patients had normal sequences of the type II 3 beta-HSD gene in both alleles. Three female patients heterozygotic for severe 3 beta-HSD deficiency CAH with one allele mutation of the gene demonstrated normal ACTH-stimulated hormone profiles. These data indicate that moderately decreased adrenal 3 beta-HSD activity resulting in modestly increased delta 5 precursor steroid levels and delta 5 to delta 4 steroid ratios in premature pubarche and hirsute patients is not caused by a mutation in the type II 3 beta-HSD gene.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells.

Although changes in the expression of key steroidogenic enzymes such as cytochrome P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase (P450c17), aldosterone synthase, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the human adrenal cortex are known to be controlled by factors activating the protein kinase A or protein kinase C signaling pathways, little is known concerning the effects of increased intracellular Ca2+. In this study we describe the effects of K+, an agent known to increase intracellular Ca2+ through the opening of voltage-sensitive Ca2+ channels, on steroidogenesis in H295R human adrenocortical cells and corresponding changes in expression of these vital steroidogenic enzymes. Treatment of cells for 48 h with K+ (14 mM) resulted in an increase in aldosterone (3.5-fold) as well as the 17 alpha-hydroxylated steroids cortisol (2.9-fold) and dehydroepiandrosterone (DHEA; 3.7-fold). This action of K+ was accompanied by a dose-dependent (P < 0.05 at 6 mM K+ or above) and time-dependent (P < 0.05 at 24 h and beyond) increase in expression of P450c17 and, to a lesser extent, cytochrome P450 cholesterol side-chain cleavage messenger RNA (mRNA). Treatment with K+ also caused a time-dependent increase in aldosterone synthase mRNA levels, which were detectable by 12 h. Treatment with K+, however, was without effect on 3 beta HSD expression. These effects contrast with those of (Bu)2cAMP, which stimulated a greater increase in cortisol and DHEA secretion as well as P450c17 expression. The effects of K+ treatment also differ from those of AII, which promoted a greater aldosterone secretory response (5.7-fold), but a lesser effect on DHEA secretion (2.2-fold) and P450c17 expression. Although AII and TPA (known activators of protein kinase C) as well as forskolin and (Bu)2cAMP (known activators of protein kinase A) increased the expression of 3 beta HSD mRNA, K+ treatment was without effect, suggesting that elevation of [Ca2+]i in response to K+ did not activate the protein kinase C or protein kinase A signaling pathways. Furthermore, the effects of K+ on steroid secretion and 17 alpha-hydroxylase activity were reproduced by the voltage-sensitive Ca2+ channel activator BAYK 8644, and increases in P450c17 mRNA in response to K+ were reversed by the Ca2+ channel antagonist, nifedipine. We conclude that K+ can modulate the expression of key steroidogenic enzymes in H295R cells through the Ca2+ signaling pathway without involvement of the protein kinase A or protein kinase C pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Type 2 11 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid and activity in human placenta and fetal membranes: its relationship to birth weight and putative role in fetal adrenal steroidogenesis.

Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) have been described which catalyze the interconversion of cortisol (F) to cortisone (E). 11 beta HSD activity has previously been reported in placenta and fetal membranes, where its role may be to protect the developing fetus from glucocorticoid excess. Furthermore, in the rat, an association between placental 11 beta HSD activity and the subsequent development of hypertension in the offspring has been reported. We have characterized the isoforms of 11 beta HSD in human fetal membranes and dissected placental tissue at term and investigated the relationship between placental 11 beta HSD activity and fetal and placental weights. 11 beta HSD activity studies in the presence of 0.1 mumol/L F and NAD (indicative of type 2 isoform activity) revealed high levels of activity in trophoblast dissected free of vessels (561 +/- 87 pmol E/h.mg protein; n = 4) > undissected placenta > cotyledenous vessels dissected away from trophoblast > placental and reflected amnion. In contrast, in the presence of 2.5 mumol/L F and NADP (indicative of type 1 isoform activity), only decidua and chorion demonstrated significant levels of 11 beta HSD activity. Type 1 11 beta HSD activity in chorion was probably due to decidual contamination, in that it was absent in decidua-free fused chorion obtained from a twin pregnancy. In keeping with these data, type 1 11 beta HSD messenger ribonucleic acid (1.5 kilobases) was detected in decidua, but in no other tissue, and high levels of type 2 11 beta HSD messenger ribonucleic acid (1.9 kilobases) were found in undissected placenta and trophoblast. In 27 term placentas, 11 beta HSD activity varied from 194-448 pmol E/h.mg protein. There was a weak, but significant, positive correlation between term placental 11 beta HSD activity and fetal weight (r = 0.408; P = 0.034), but no correlation with placental weight. Thus, in man, the reported association of a small fetus and a large placenta predisposing to adult hypertension cannot be explained on the basis of defective 11 beta HSD activity. However, the placenta offers an immense reservoir for F clearance (1.73-7.95 mumol/min.placenta) and may be a principal factor driving fetal ACTH secretion and, hence, fetal adrenal steroidogenesis.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

Ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase of rainbow trout: its cDNA cloning and properties of the enzyme expressed in a mammalian cell.

A cDNA clone encoding 3 beta-hydroxysteroid dehydrogenase delta 5-4-isomerase (3 beta-HSD) was isolated from a cDNA library of rainbow trout ovarian thecal cells. Southern hybridization analysis of trout genomic DNA with the cDNA suggested the presence of a single gene encoding 3 beta-HSD in the rainbow trout showing a total genomic size of less than 4 kilobases (kb). The cDNA hybridized to a species of mRNA isolated from rainbow trout ovaries; the 1.4 kb transcripts were most abundant in trout ovaries during the later stages of oogenesis. The trout 3 beta-HSD expressed in non-steroidogenic monkey kidney tumor (COS-1) cells showed a unique enzymatic 3 beta-HSD activity. Dehydroepiandrosterone was a more favoral substrate of the trout 3 beta-HSD than 1- alpha-hydroxypregnenolone. Interestingly, the trout 3 beta-HSD expressed in COS-1 cells exhibited minimal ability to convert pregnenolone to progesterone.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

The NCI-H295 cell line: a pluripotent model for human adrenocortical studies.

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones. Indeed, the lack of a human adrenocortical cell line retaining the ability to produce any of the major adrenal steroid products has slowed studies on normal and abnormal adrenal function. This obstacle has now been largely overcome with the availability of H295 cells, which represents the first adrenocortical cell line to maintain the ability, under specified conditions, to produce all the adrenocortical steroids (i.e., mineralocorticoids, glucocorticoids, and adrenal androgens). Thus, H295 cells appear to act as pluripotent adrenocortical cells capable of being directed to produce each of the zone-specific steroids. The H295 cell line should prove to be of value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

The presence of two cytochrome P450 aldosterone synthase mRNAs in the hamster adrenal.

We isolated a cDNA from a hamster adrenal cDNA library which was similar in sequence to those of the mouse and rat P450c18 cDNAs. The hamster P450c18 cDNA, however, was shorter than the rat and mouse P450c18 cDNAs at its 5'-end and the peptide leader sequence was absent. From a hamster genomic library we isolated and sequenced the first seven exons and a 5'-flanking region of the first P450c18 gene exon. With this information we were able to generate a P450c18 cDNA containing the peptide leader sequence using the polymerase chain reaction. Northern analyses were performed on adrenals from hamsters maintained on a low sodium diet for 0, 4, 7 and 10 days using a 32P-labeled sequence specific to P450c18; two mRNA bands were found at 2 and 3.4 kb. The intensity of both bands was increased about 3- to 5-fold under sodium restriction compared to controls. A distinct mRNA band of 2.3 kb hybridized with an oligonucleotide specific to P450(11) beta and its intensity did not change following low sodium intake. Immunoblotting analyses were performed using an antibovine adrenal P450(11) beta antibody that does not discriminate between P450(11) beta and P450c18 proteins. Three bands were detected at 52, 48 and 45 kDa in homogenate preparations of entire glands. Furthermore, the 45 kDa protein band was present in homogenates of the zona glomerulosa and absent in homogenates of the zone fasciculata-reticularis. In conclusion, these results show that the hamster adrenals express P450c18 as do mouse, rat and human adrenal glands. Furthermore, two P450c18 mRNAs, which are inducible by a low sodium intake, are present in the hamster adrenal vs one for the rat. The physiological role of these two hamster adrenal mRNA species remains to be elucidated.

Adrenal Glands↗

PubMed1994

Over-expression of human type I (placental) 3 beta-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus.

Human type I placental 3 beta-hydroxy-5-ene-steroid dehydrogenase/steroid 5-->4-ene-isomerase (3 beta-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3 beta-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3 beta-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3 beta-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3 beta-HSD substrate, 5 alpha-androstan-3 beta- ol-17-one, in the Sf-9 cell homogenate (Km = 17.9 microM) and placental microsomes (Km = 16.7 microM). The 3 beta-HSD activity (Vmax = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (Vmax = 9.1 nmol/min/mg). The Km values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (Km = 14.7 microM) and placental microsomes (Km = 14.4 microM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (Vmax = 25.7 nmol/min/mg) relative to placenta (Vmax = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3 beta-HSD/isomerase in human placenta.

Animals↗

PubMed1994

Evidence of a steroidogenic enzyme gene dose effect on adrenal gene expression in hereditary rabbit congenital adrenal hyperplasia.

We previously reported the gene deletion encoding cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450SCC, resulting in complete elimination of the adrenal gene expression and causing congenital adrenal hyperplasia in the rabbit. Using the rabbit congenital adrenal hyperplasia model, we investigated the wild type (wt) P-450SCC gene dose effect on gene expression in three P-450SCC genotype animals [wt/wt, wt/mutant (mt), mt/mt] identified by Southern blot analysis. Northern blots using a rabbit P-450SCC cDNA probe revealed no detectable P-450SCC mRNA in individual adrenals of animals with congenital adrenal hyperplasia (mt/mt) and approximately half or slightly less than half the levels of the mRNA in the pooled adrenals of five heterozygous (wt/mt) newborn animals compared with the mRNA levels in the pooled adrenals of five homozygous normal (wt/wt) newborn animals. Identical P-450SCC mRNA levels were found individual adrenals of adult animals with regard to the P-450SCC genotype, although at a higher expression level than in the newborn animals of the same genotype. Control Northern blots using human CPY21-B cDNA and cytoplasmic actin cDNA probes confirmed the accuracy and integrity of RNA. Western immunoblotting using anti-ovine P-450SCC antibody revealed decreased P-450SCC protein in the adrenals of wt/mt animals at approximately half the level of the P-450SCC protein in the adrenals of the wt/wt animals. Baseline and ACTH-stimulated serum corticosterone (B) levels in vivo were similar between the age-matched wt/mt and wt/wt animals, whereas ACTH-stimulated B levels in adult animals were higher than those in the newborn animals irrespective of P-450SCC genotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1994

Regulation of type 1 angiotensin II receptor messenger ribonucleic acid expression in human adrenocortical carcinoma H295 cells.

We have studied the hormonal regulation of type 1 angiotensin-II receptor (AT1-R) mRNA expression and [125I]angiotensin-II ([125I]AII) binding in human adrenocortical carcinoma H295 cells, which exhibit predominantly AT1-subtype receptors. Activation of the cAMP signaling pathway with forskolin or (Bu)2cAMP caused a rapid decrease in AT1-R mRNA levels (decreased 65% within 3 h). This preceded a time-dependent (maximal, 70% within 12 h) and dose-dependent (IC50, 2 microM forskolin) loss of [125I]AII binding together with decreased phosphoinositidase-C activation (72% decrease) on subsequent AII challenge. Thus, the decreases in AT1-R mRNA levels and functional receptor expression parallel each other in response to activation of protein kinase-A. AII treatment also caused a rapid loss in AT1-R mRNA (maximal, 80% decrease within 3 h), but 48-h treatment caused both [125I]AII binding and the subsequent phosphoinositidase-C response to decrease by only 6% (P < 0.05) and 22% (P < 0.05), respectively. The effect of AII on AT1-R mRNA levels was fully reproduced by the combination of calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol 13-acetate), suggesting that AII action was through protein kinase-C and possibly other Ca(2+)-sensitive protein kinases. The effect of AII, but not forskolin, was reversed by treatment in the presence of cycloheximide. In conclusion, control of AT1-R expression is differentially regulated by adenylate cyclase and phosphoinositidase-C signaling pathways, which act at multiple levels in human adrenocortical cells.

Adrenal Cortex Neoplasms↗

PubMed1994

Type 2 11 beta-hydroxysteroid dehydrogenase in human fetal tissues.

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of active cortisol to inactive cortisone, and regulates the access of cortisol to both the mineralocorticoid and glucocorticoid receptors. Two isoforms of 11 beta-HSD have been described, the cloned "type 1" NADP(H)-dependent dehydrogenase/oxo-reductase and a high affinity NAD-dependent dehydrogenase (type 2). In the fetus, 11 beta-HSD activity may serve to protect developing tissues from cortisol excess or may modulate the permissive actions of glucocorticoids. We have studied 11 beta-HSD activity and mRNA levels in human mid-gestational fetal tissues. Tissue homogenates were incubated with either 0.1 mumol/L cortisol and 400 mumol/L NAD, 2.5 mumol/L cortisol and 400 mumol/L NADP, or 0.1 mumol/L cortisone wither either 400 mumol/L NADPH or NADH. No activity (< 2.5% conversion) was observed in fetal tissues using either cortisone or 2.5 mumol/L cortisol as a substrate. 11-oxo-reductase activity was observed in maternally-derived decidua. In keeping with these activity studies, northern blot analysis of fetal tissue RNA and PCR-reverse transcriptase of type 1 11 beta-HSD mRNA indicated 11 beta-HSD mRNA in decidua, but failed to detect any type 1 11 beta-HSD mRNA transcripts in fetal tissues. In contrast when 0.1 mumol/L cortisol was used as a substrate in the presence of NAD, 11 beta-HSD activity was ubiquitous with highest levels seen in the kidney (131 +/- 16 (SE) pmoles cortisone formed/h/mg.protein) > lung > gonad > liver > colon. 11 beta-HSD activity in fetal tissues is mediated by the type 2, high affinity, isoform. The widespread distribution of this novel isoform suggests that it may play an important role in fetal development. Type 1 11 beta-HSD mRNA and activity are absent in mid-gestational fetal tissues, but present in maternally-derived decidua, suggesting that its ontogeny is a late-gestational of post-natal event.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

Human kidney 11 beta-hydroxysteroid dehydrogenase is a high affinity nicotinamide adenine dinucleotide-dependent enzyme and differs from the cloned type I isoform.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the conversion of cortisol to cortisone and plays an important role in the mammalian kidney in regulating cortisol access to the mineralocorticoid receptor. 11 beta HSD-deficient states, such as the syndrome of apparent mineralocorticoid excess (AME), and licorice ingestion result in hypertension in which cortisol acts as a mineralocorticoid. A gene and complementary DNA sequence encoding type I human 11 beta HSD have been described, but this gene is normal in patients with AME. Separate 11 beta HSD isoforms have been described in rat and rabbit kidney, but 11 beta HSD has not been characterized in human kidney. Kinetic analysis of 11 beta HSD activity in human fetal kidney microsomes revealed only a high affinity isoform (apparent Km, 60 nmol/L for cortisol, 13 nmol/L for corticosterone), the activity of which was exclusively nicotinamide adenine dinucleotide (NAD) dependent. No 11-oxo-reductase activity was seen in either renal homogenates or microsomes. 11 beta-Dehydrogenase activity was inhibited by glycyrrhetinic acid (the active ingredient in licorice) in a competitive fashion, with a Ki of 8.7 nmol/L. This 11 beta HSD isoform was clearly distinct from the type I h11 beta HSD enzyme, in that COS-1 cells transfected with type I h11 beta HSD complementary DNA expressed a low affinity (apparent Km, 2.13 mumol/L) isoform, the activity of which was NAD phosphate dependent. 11-Oxo-reductase activity was present in intact transfected cells (apparent Km for cortisone, 0.36 mumol/L), but not in cell lysates. In contrast to the cloned, low affinity, type I h11 beta HSD enzyme, human kidney contains a high affinity NAD-dependent 11 beta HSD isoform. It seems probable that this isoform is responsible for protecting the renal mineralocorticoid receptor from glucocorticoid excess, and a defect in its activity may explain AME.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

Immunolocalization of steroidogenic enzymes P450scc, 3 beta HSD, P450c17 and P450arom in the corpus luteum of the Hokkaido brown bear (Ursus arctos yesoensis) in relation to delayed implantation.

The objective was the immunocytochemical localization of steroidogenic enzymes in the corpus luteum of Hokkaido brown bears during the period of delayed implantation. Cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) were localized as biosynthetic sites of pregnenolone, progesterone, androgens, and oestrogens, respectively. Ovaries containing corpora lutea were obtained from three mature bears during the expected delayed implantation period and ovarian sections were immunostained by the avidin-biotin-peroxidase complex method using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. P450scc and 3 beta HSD were localized in all luteal cells, whereas P450c17 (0.4-5.1% of 1000 cells) and P450arom (7.1-11.2% of 1000 cells) were localized in only a few luteal cells. These data suggest that luteal cells contain steroidogenic enzymes required for progesterone synthesis but also have a minimum capability for synthesizing androgen and oestrogen during the delayed implantation period in Hokkaido brown bears.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Ontogeny of steroidogenic enzyme expression in the porcine conceptus.

This study examined fetal steroidogenic enzyme expression and function during pregnancy in the pig. Northern and Western analyses were performed to detect the cytochrome P450 enzyme 17 alpha-hydroxylase/17-20 lyase (P450c17) and that for cholesterol side-chain cleavage (P450scc), as well as 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) expression in several porcine fetal tissues. The data demonstrate higher steroidogenic enzyme expression in the fetal adrenal glands and testes than in the placenta at all stages of development examined. Although steroidogenic enzyme expression was maintained throughout gestation in both the fetal adrenals and the testes, adrenal P450c17 expression was higher in the early and late stages when compared with the intermediate stages of fetal development. The stimulation of fetal adrenal steroidogenic enzyme expression in the later stage fetuses was accompanied by increased expression of P450c17 in both the fetal testes and placenta. The expression of 3 beta-HSD by porcine fetal testes was low compared with that of the fetal adrenal gland at all stages of development. Adrenal explants and cultured cells secreted cortisol and androstenedione but much lower amounts of corticosterone, dehydroepiandrosterone and aldosterone. Secretion of cortisol and androstenedione by adrenal explants was maintained by ACTH for 5 days of culture but declined in controls. In cultured porcine fetal adrenal cells, ACTH and angiotensin II stimulated the secretion of multiple steroids. Porcine fetal testis explants and cultured cells secreted testosterone, dehydroepiandrosterone and androstenedione, but were only moderately responsive to trophic stimulation by LH. In general, the data suggest that the fetal adrenal glands and the fetal testes have the potential to contribute significantly to the production of steroids during pregnancy in pigs.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Steroid secretory characteristics of a virilizing adrenal adenoma in a woman.

A tumour of the left adrenal gland was identified in a woman who presented with virilization and secondary amenorrhea. Preoperatively, the plasma levels of dehydroepiandrosterone sulphate, dehydroepiandrosterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and 5-androstene-3 beta,17 beta-diol were elevated two- to fourfold whereas those of urinary 17-ketosteroids were elevated more than tenfold. The production rate of dehydroepiandrosterone sulphate was more than 16 times that in normal women whereas those of dehydroepiandrosterone, testosterone and androstenedione were approximately twofold greater; plasma testosterone was derived almost entirely from the peripheral conversion of androstenedione. Blood was obtained by catheterization of the ovarian veins, left adrenal gland vein and inferior vena cava (at two different sites) and plasma steroid levels were determined: testosterone and cortisol levels were elevated in all blood samples whereas those of androstenedione, dehydroepiandrosterone sulphate and 11-desoxycortisol were approximately six- to eightfold, 1.5-fold and nine- to 22-fold higher in the effluent on the left adrenal gland/tumour compared with the levels in the other compartments. Blood was collected hourly for 24 h to determine steroid levels under basal conditions and, also, after ACTH treatment. Plasma cortisol levels increased markedly upon ACTH administration and fell to very low levels 11 h later, but those of androstenedione, testosterone, dehydroepiandrosterone, 5-androstene-3 beta,17 beta-diol and dehydroepiandrosterone sulphate were not affected by ACTH treatment. A histological diagnosis of cortical adenoma of the extirpated tumour was made. Tissue explants and adenoma cells were maintained in culture to characterize the steroid-metabolizing properties of the tumour. The secretion of dehydroepiandrosterone sulphate by tissue explants was highly initially, but declined to almost undetectable levels after 5 days in culture. In the presence of ACTH, dehydroepiandrosterone sulphate secretion remained elevated throughout the entire study up to 5 days. Basal secretion of dehydroepiandrosterone sulphate, androstenedione, 11-desoxycortisol, cortisol, testosterone and 11 beta-hydroxyandrostenedione by adenoma cells was either very low or undetectable. In the presence of ACTH, dibutyryl cyclic AMP or cholera toxin the secretion of dehydroepiandrosterone sulphate, androstenedione and 11-desoxycortisol increased markedly with time in culture up to 3 days, whereas the other steroids were undetected in the medium. A homogenate of adenoma tissue metabolized testosterone to androstenedione, but the conversion of androstenedione to testosterone was minimal. The findings of this study served to establish that virilization in this woman was due at least in part, to excess testosterone--and testosterone-derived 5 alpha-dihydrotestosterone--produced at extra-adrenal tissue sites almost exclusively through metabolism of tumour-secreted androstenedione.(ABSTRACT TRUNCATED AT 400 WORDS)

17-Ketosteroids↗

PubMed1993

Immunolocalization of steroidogenic enzymes, P450scc, 3 beta-HSD, P450c17, and P450arom in the Hokkaido brown bear (Ursus arctos yesoensis) testis.

Hokkaido brown bears (Ursus arctos yesoensis) are seasonal breeders and the profile of their serum testosterone concentrations undergoes annual changes. However, precise sites of steroidogenesis in the bear testis have not been identified. Therefore, our objective was to localize steroidogenic enzymes by immunocytochemistry using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. The steroidogenic enzymes localized were cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) as biosynthetic sites of pregnenolone, progesterone or androstenedione, androgens, and estrogens, respectively. Testes were collected from three adult bears prior to the mating season (April, Hokkaido, Japan) and prepared for immunostaining with primary antibodies, followed by an avidin-biotin-peroxidase method. P450scc was localized in Leydig cells and spermatids. 3 beta-HSD was found only in Leydig cells. P450c17 was identified in Leydig cells and spermatids. Finally, P450arom was found in Leydig cells and in spermatids that stained very intensely. Therefore, Leydig cells appear to be a site of progestin (both delta 4 and delta 5 C21 steroids), androgen, and estrogen production, whereas spermatids appear to be a site of pregnenolone, androgen, and estrogen production. Immunolocalization of steroidogenic enzymes suggests that steroidogenesis may occur not only in Leydig cells, but also in spermatids of the Hokkaido brown bear testis prior to the mating season. We also suggest that Leydig cells and spermatids are the predominant sites of androgen and estrogen synthesis, respectively, in the Hokkaido brown bear testis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Temporal and spatial localization of steroidogenic enzymes in premenopausal human ovaries: in situ hybridization and immunohistochemical study.

In situ hybridization and immunohistochemical localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P450 17 alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) was performed in 50 morphologically normal human premenopausal ovaries, and correlated these findings with their endometrial phase. In general, mRNA expression of these enzymes examined by in situ hybridization were in good agreement with immunolocalization examined by immunohistochemistry. Expression of P450scc, 3 beta HSD and P450c17 was observed in large-sized preantral follicles, consisting of more than five layers of granulosa cells, preovulatory follicles, corpora lutea, and some degenerating corpora lutea and atretic follicles in all endometrial phases. Several follicles and/or corpora lutea positive for these enzymes were observed in the same ovary. Expression of P450arom was generally observed in only one follicle (antral or preovulatory follicle) or corpus luteum per case in mid proliferative to premenstrual phase, and was not observed in menstrual to early proliferative phase. These findings indicated that (1) expression of steroidogenic enzymes was associated with the continual human ovarian process including follicular development and atresia, and (2) especially, P450arom expression may occur only in a selected antral follicle and may have an important role in dominant follicular development.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Regulation of expression of the 3 beta-hydroxysteroid dehydrogenases of human placenta and fetal adrenal.

The appropriate expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3 beta-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3 beta-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3 beta-HSD expression and, moreover, that 3 beta-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3 beta-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3 beta-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3 beta-HSD expression. Moreover, 3 beta-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3 beta-HSD expressed in these cytotrophoblastic populations was the 3 beta-HSD type I gene product (M(r), 45K) and not 3 beta-HSD type II (M(r), 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3 beta-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3 beta-HSD type II activity and mRNA expression. The higher level of 3 beta-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗