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PubMed1993

Changes in the abundance of mRNA for type-I 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4 isomerase in the human placenta and fetal membranes during pregnancy and labor.

A local decrease in progesterone synthesis in the placenta and fetal membranes has long been proposed as a possible mechanism in the control of human labor. We have examined whether changes occur in the abundance of mRNA for 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4 isomerase (3 beta-HSD), the enzyme which catalyzes the conversion of pregnenolone to progesterone in human placenta and fetal membranes, by Northern blot analysis using a cDNA probe to human placental type-I 3 beta-HSD, the predominant isoenzyme in the placenta. The abundance of 3 beta-HSD mRNA (1.7-kb transcript) was about 10-fold greater in term placenta than in chorio-decidua, but undetectable in total RNA from amnion. There was no change in the abundance of 3 beta-HSD mRNA in either placenta or chorio-decidua obtained after elective cesarean section at term, after preterm labor, or after term or postterm vaginal delivery. We conclude that the abundance of 3 beta-HSD mRNA does not change in the placenta or fetal membranes with labor, consistent with the view that changes in 3 beta-HSD gene expression and decreased progesterone production are unlikely to effect intrauterine paracrine/autocrine regulatory mechanisms leading to term or preterm labor in women.

Blotting, Northern↗
PubMed2005

Endogenous ouabain: upregulation of steroidogenic genes in hypertensive hypothalamus but not adrenal.

BACKGROUND: Mammalian tissues contain a presumed endogenous Na+, K(+)-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides. METHODS AND RESULTS: Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and Delta5-3beta-hydroxysteroid dehydrogenase/Delta5-Delta4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue. CONCLUSIONS: Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.

Adrenal Glands↗
PubMedDate not supplied

Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[14C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkylated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. RESULTS: According to the sequence analyses, NADH shifted radioalkylation by 2 alpha-[14C]BAP away from the Arg-250 peptide (251GQFYYISDDTPHQSYDNLNYTLSK274) and toward the Lys-135 tryptic peptide (136EIIQNGHEEEPLENTWPAPYPHSK159). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His262 in the Arg-250 peptide by 8.2-fold. His142 in the Lys-135 peptide was radiolabeled by 2 alpha-[14C]BAP only in the presence of NADH. CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[14C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.

Amino Acid Sequence↗
PubMed2003

Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

Adrenal Cortex Neoplasms↗
PubMed2003

Developmental expression of genes involved in neurosteroidogenesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in the rat brain.

In the central nervous system, neurosteroids, in particular progesterone, have neurotrophic and neuroprotective effects. We thus decided to study the developmental expression of 3beta-hydroxysteroid-dehydrogenase/Delta(5)-Delta(4) isomerase (3betaHSD), an enzyme that converts pregnenolone to progesterone, in the male rat brain at 0, 7, 14, and 70 d after birth. 3betaHSD mRNA was widely distributed throughout the brain, as shown by in situ hybridization. At all ages, the same cerebral structures were labeled, but the intensity of the hybridization signal constantly decreased during postnatal development. As the hippocampus is of particular interest because of its neuronal plasticity, we chose to quantify the changes in 3betaHSD mRNA levels as well as progesterone and pregnenolone concentrations in this structure. Quantitative in situ hybridization confirmed a decrease in the expression of 3betaHSD mRNA with progressing age, as revealed by a significant reduction in the density of silver grains per cell in the CA1 layer. This decrease was confirmed by semiquantitative RT-PCR on hippocampal samples. Concentrations of hippocampal pregnenolone and progesterone measured by gas chromatography/mass spectrometry were highest on the day of birth and lower at the other ages. Plasma concentrations of these steroids were lower than those in the hippocampus, suggesting that they may have been mostly synthesized in situ since the day of birth. These results demonstrate variations in the expression of a gene coding for an enzyme critically involved in progesterone synthesis in the hippocampus throughout postnatal development.

Age Factors↗
PubMed2004

The human 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase type 2 promoter is a novel target for the immediate early orphan nuclear receptor Nur77 in steroidogenic cells.

The human (h) 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase type 2 (3beta-HSD2) enzyme, encoded by the hHSD3B2 gene, is mainly found in gonads and adrenals. This enzyme catalyzes an essential early step in the biosynthesis of all classes of steroid hormones. The critical nature of the enzyme is supported by the occurrence of human syndromes that are associated with insufficient 3beta-HSD2 expression and/or activity. Although the need for a functional 3beta-HSD2 enzyme is indisputable, the molecular mechanisms that regulate HSD3B2 expression (both basal and hormone-induced) in steroidogenic cells remain poorly understood. A role for the Nur77 family of immediate-early orphan nuclear receptors in steroidogenesis has received recent interest. For example, Nur77 is present in gonads and adrenals, where its expression is robustly and rapidly induced by hormones that stimulate steroidogenic gene expression. Moreover, the expression patterns of Nur77 and at least one key steroidogenic gene (hHSD3B2) closely parallel one another. We now report that the hHSD3B2 promoter is indeed a novel target for Nur77 in both testicular Leydig cells and adrenal cells. We have mapped a novel response element located at -130 bp specific for Nur77 and not other orphan nuclear receptors (steroidogenic factor-1 and liver receptor homolog-1) previously shown to regulate hHSD3B2 promoter activity. This Nur77 element is essential and sufficient to confer Nur77 responsiveness to the hHSD3B2 promoter, and its mutation blunts basal and hormone-induced hHSD3B2 promoter activity in steroidogenic cells. We also show that Nur77 synergizes with all members of the steroid receptor coactivator family of coactivators on the hHSD3B2 promoter. Taken together, our identification of Nur77 as an important regulator of HSD3B2 promoter activity helps us to better define the tissue-specific and hormonal regulation of the HSD3B2 gene in steroidogenic cells.

Animals↗
PubMed1978

Progesterone metabolism by cultured Sertoli cells.

The ability of Sertoli cells to metabolize progesterone to testosterone (T) and 5alpha-dihydrotestosterone (DHT) was investigated in vitro. Cultures of Sertoli cells isolated from testes of 80-day-old rats were incubated with [7(n)-3H]progesterone (10 muCi/3.0 nmol) for 0.5, 1, 2, and 3 h. The amount of progesterone converted to T, androstenedione (A), DHT, 17alpha-hydroxyprogesterone, and 20alpha-dihydroprogesterone was calculated on the basis of crystallization data. The amount of substrate converted to the various steroids increased between 0.5-3 h of incubation. During this period, T increased 6-fold; A, 2-fold; DHT, 4-fold; 17alpha-hydroxyprogesterone, 3-fold; and 20alpha-dihydroprogesterone, 12-fold. The amount of substrate converted to C-19 steroids (T, A, and DHT) increased linearly with time; 19.4 ng androgen/1 x 10(6) cells were formed within 3 h, the largest amount (11.83 ng) being DHT. Although the amount of 3H-labeled C-19 steroids formed from [3H]-progesterone by Sertoli cells is relatively small (1.8% conversion) compared to T formation by whole testicular tissue or by isolated interstitial cells, the ability of Sertoli cells to form T and DHT from progesterone may be physiologically important in the local regulation of Sertoli cell function and spermatogenesis.

Animals↗
PubMed1980

Effect of chronic alcohol ingestion on the biosynthesis of steroids in rat testicular homogenate in vitro.

The activity and kinetics of delta 5-3 beta-dehydrogenase and 17 alpha-hydroxylase, using labeled pregnenolone as substrate, were measured in gonadal homogenates from rats fed alcohol or isocalorically substituted carbohydrate for 40 days. There was no difference in the rate or reaction kinetics for either enzyme noted between control and alcohol-treated animals when the assays were carried out in the presence of saturating amounts of exogenous pyridine nucleotide cofactors. However, when exogenous cofactors were omitted from the reaction mixture, there was decreased activity of the delta 5-3 beta-dehydrogenase system and increased activity of the 17 alpha-hydroxylase reaction. Furthermore, a cofactor-specific inhibiting effect on delta 5-3 beta-dehydrogenase activity by NADH and NADPH was found. Incubation of gonadal homogenates from the alcohol-treated animals with pyruvate on lactate (in the absence of exogenous cofactors) resulted in an increase and a decrease, respectively, in enzyme activity. These studies indicate that chronic alcohol use decreases gonadal delta 5-3 beta-dehydrogenase activity and that this is most likely due to an effect of the agent on the concentration and/or availability of pyridine nucleotide cofactors rather than to a direct effect on the enzyme. This phenomenon may account for the mechanism by which alcohol decreases testosterone secretion in these animals.

Animals↗
PubMed1982

Mechanism of induction of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in rat adrenocortical cells by corticotropin.

The regulation of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) was studied in primary cultures of rat adrenocortical cells. In the absence of ACTH, this enzymic activity was found to decay with a half-life of 3.1 days, which was similar to the half-life of the enzyme activity induced by ACTH in vitro (3.5 days). The increase in 3 beta-HSD activity was highly specific for ACTH and dibutyryl cAMP; the activity was not increased by other hormones known to affect adrenocortical growth or function. The induction of 3 beta-HSD activity by ACTH or dibutyryl cAMP required a lag period of approximately 4 h and was dependent on RNA and protein syntheses. The increase in 3 beta-HSD activity observed after ACTH treatment was not a result of ACTH-induced inhibition of degradation of the enzyme, nor was it due to the synthesis of a soluble intermediate which could directly activate the enzyme. ACTH stimulated the incorporation of [35S]methionine into a protein associated with 3 beta-HSD activity detected on polyacrylamide gels after electrophoresis of Triton X-100 extracts of adrenocortical cells. The induction of this protein by ACTH was inhibited by actinomycin D. A protein band of a partially purified preparation of rat adrenal 3 beta-HSD was found to comigrate with the ACTH-induced protein on sodium dodecyl sulfate-polyacrylamide gel. These results suggest that ACTH caused the de novo synthesis of 3 beta-HSD by a mechanism dependent on RNA synthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland.

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids.

The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

17-alpha-Hydroxypregnenolone↗
PubMed1990

Light microscopic immunocytochemical localization of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in the gonads and adrenal glands of the guinea pig.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex hormone steroids. To obtain precise information about the cellular and subcellular localization of 3 beta HSD in the gonads and adrenals of the guinea pig, we have proceeded to immunocytochemical localization of the enzyme using antibodies developed against purified human placental 3 beta HSD. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells. In the ovary, on the other hand, immunostaining was found in the cytoplasm of the cells of the corpus luteum and theca interna. The granulosa cells of the follicles were not immunolabeled, with the exception of one layer of cells in contact with the zona pellucida. This restricted labeling was observed in growing and mature follicles, but not in primordial and small primary follicles. In the adrenals, the three zones of the cortex were labeled, whereas no staining could be detected in the medulla. Contrary to findings in the testis and ovary, staining was found in both the cytoplasm and nuclei of adrenocortical cells. The present data suggest a functional interaction between the ovocyte and the specialized layer of 3 beta HSD-containing granulosa cells which might play a role in ovocyte maturation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in rat gonads and adrenal glands by immunocytochemistry and in situ hybridization.

The enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) is involved in the biosynthesis of all classes of steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain information on the precise localization of 3 beta HSD in rat gonads and adrenal glands, two complementary cytochemical techniques were used; immunocytochemical localization was achieved with antibodies developed against purified human placental 3 beta HSD, while 3 beta HSD mRNA localization was achieved by in situ hybridization performed with a recently cloned rat 3 beta HSD cDNA. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells, while by in situ hybridization, specific silver grains were also seen over the interstitial cells. In the ovary, immunostaining was found in the cytoplasm of cells of the corpus luteum and theca interna, while the granulosa cells of the follicles showed no positive reaction. By in situ hybridization, a specific hybridization signal was observed over granulosa cells of the corpus luteum, which are mainly responsible for progesterone secretion, and to a lesser extent over theca interna cells, known for their role in secreting C19 androgens. In the adrenals, the three zones of the cortex were equally immunolabeled, whereas no staining could be detected in the medulla. Similarly, by in situ hybridization, silver grains were located over the zona glomerulosa, fasciculata, and reticularis, while no specific autoradiographic reaction could be observed on the chromaffin cells of the medulla. The present study provides new information about the precise cellular localization of 3 beta HSD in the adrenal glands and gonads in the rat, thus providing useful information about the site of action of 3 beta HSD, especially in the gonads. Moreover, the approaches used for localization studies, especially quantitative in situ hybridization, should provide a useful tool for assessing the role of hormones on 3 beta HSD expression in the different compartments of the gonads and adrenal glands.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Changes in 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase messenger ribonucleic acid, activity and protein levels during the estrous cycle in the bovine ovary.

A 1169 base pair fragment of bovine 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) cDNA was used to quantitate 3 beta-HSD messenger RNA (mRNA) levels in the bovine ovary during the estrous cycle. The content of 3 beta-HSD protein was measured by immunoblot analysis using an antiserum developed in rabbits against human 3 beta-HSD, whereas 3 beta-HSD activity was measured using [3H]pregnenolone, [3H] dehydroepiandrosterone, and [3H]androst-5-ene-3 beta,17 beta-diol as substrates. There was a parallel increase in 3 beta-HSD mRNA, protein content, and enzymatic activity levels from days 1-3 after estrus to maximal values at 50-100% above control on days 8-11 after estrus. Thereafter, all values decreased progressively until days 16-17 before a dramatic fall to 5% or less than maximal values on days 18-20 after estrus. Almost superimposable results of enzymatic activity were obtained with the three substrates, thus suggesting a unique 3 beta-HSD or parallel changes in the activity of multiple 3 beta-HSDs. The above-described changes observed during the luteal phase are almost exclusively due to variations in corpora lutea. In fact, 3 beta-HSD activity in ovarian follicles was approximately 10,000 lower than that measured in corpora lutea. The close correlation observed over a wide range of 3 beta-HSD mRNA, protein content, and activity levels suggests that changes of ovarian 3 beta-HSD activity are controlled at the level of 3 beta-HSD gene expression and/or 3 beta-HSD mRNA stability.

Animals↗
PubMed1990

Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase expression and activity in the hypophysectomized rat ovary: interactions between the stimulatory effect of human chorionic gonadotropin and the luteolytic effect of prolactin.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes an obligatory step in the conversion of pregnenolone and other 5-ene-3 beta-hydroxysteroids into progesterone as well as precursors of all androgens and estrogens in the ovary. Since 3 beta-HSD is likely to be an important target for regulation by pituitary hormones, we have studied the effect of chronic treatment with LH (hCG), FSH, and PRL on ovarian 3 beta-HSD expression and activity in hypophysectomized adult female rats. Human CG (hCG) [10 IU, twice a day (bid)], ovine FSH (0.5 microgram, bid), and ovine PRL (1 mg, bid) were administered, singly or in combination, for a period of 10 days starting 15 days after hypophysectomy. In hypophysectomized rats, PRL exerted a potent inhibitory effect on all the parameters studied. In fact, PRL caused a 81% decrease in ovarian 3 beta-HSD mRNA content accompanied by a similar decrease in 3 beta-HSD activity and protein levels. In addition, ovarian weight decreased by 40% whereas serum progesterone fell dramatically from 1.92 nmol/liter to undetectable levels after treatment with PRL. Whereas hCG alone had only slight stimulatory effects on 3 beta-HSD mRNA, protein content and activity levels, treatment with the gonadotropin partially or completely reversed the potent inhibitory effects of oPRL on all the parameters measured. FSH, on the other hand, had no significant effect on 3 beta-HSD expression and activity. In situ hybridization experiments using the 35S-labeled rat ovary 3 beta-HSD cDNA probe show that the inhibitory effect of PRL is exerted primarily on luteal cell 3 beta-HSD expression and activity. On the other hand, it can be seen that hCG stimulates 3 beta-HSD mRNA accumulation in interstitial cells. The present data show that hCG and PRL exert potent and opposite cell-specific effects on ovarian 3 beta-HSD expression, activity, and content in the rat ovary. Moreover, the present study could suggest that female infertility associated with hyperprolactinemia in women could well be related, at least in part, to the potent inhibitory effect of PRL on ovarian 3 beta-HSD expression and activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Structure and sexual dimorphic expression of a liver-specific rat 3 beta-hydroxysteroid dehydrogenase/isomerase.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes the obligatory oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-3-ketosteroids which lead to the formation of all classes of steroid hormones. We report the molecular cloning of a third type of cDNA clone encoding rat 3 beta- HSD isolated from a rat liver lambda gt11 cDNA library. The nucleotide sequence of 1955 bp determined from overlapping cDNA clones predicts a protein of 372 amino acids which displays 80% similarity with that of rat type I and type II 3 beta-HSD proteins. RNA blot analysis reveals the presence of mRNA transcripts of 2.1 kb in male liver in contrast to the 1.7 kb mRNA species detected in adrenal and gonad poly(A)+ RNA. Ribonuclease protection assays using type I, type II and type III specific cRNA probes demonstrate the liver-specific expression of type III mRNA while the two others are expressed in adrenals and gonads. The type III mRNA species was below the detection limit in intact female liver while in hypophysectomized females, its accumulation was restored to 55% of the levels measured in intact or hypophysectomized male rats. The present data describe the presence of a third type of 3 beta-HSD mRNA species and its marked sexual dimorphic gene expression in the liver which apparently results from pituitary hormone-induced gene repression in female rat liver tissue.

Amino Acid Sequence↗
PubMed1991

Regulation of 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression by adrenocorticotropin in bovine adrenocortical cells.

In the steroidogenic pathway, 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) catalyzes the formation of hormonally active delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. In the present study the regulation of 3 beta HSD by ACTH action on bovine adrenocortical (BAC) cells in primary culture was evaluated. Western blot analysis was accomplished using an antibody against human placental 3 beta HSD. The relative molecular mass of 3 beta HSD in these cells was 45K, which was similar to that in human placenta. A significant effect of ACTH was not detected until day 6 of culture due to the high basal levels of the enzyme in BAC cells. Treatment of cells with ACTH on day 8 of culture resulted in a marked increase in the amount of 3 beta HSD protein, and this effect was correlated directly with enzymatic activity. The effects of ACTH were time and dose dependent, with an increase detectable only after 48 h of treatment; the maximal response was obtained with 10(-9) M ACTH. As demonstrated by Northern analysis, ACTH action was manifested by increasing the steady state level of 3 beta HSD mRNA. A human 3 beta HSD cDNA probe, which was used in this study, hybridized to a 1.7-kilobase species of BAC RNA. The effects of ACTH on 3 beta HSD activity and increases in 3 beta HSD protein and mRNA in BAC cells were mimicked by treatment with (Bu)2cAMP. The findings of this study suggest that ACTH controls 3 beta HSD gene expression in BAC cells by a cAMP-dependent mechanism similar to that involved in the expression of steroid hydroxylase genes. However, because the different stabilities of 3 beta HSD and hydroxylase proteins and/or mRNAs may play a critical role in determining the zone-specific steroids secreted from the adrenal cortex, other cAMP-dependent or independent regulatory mechanisms may also be important in regulating the expression of adrenal 3 beta HSD.

Adrenal Cortex↗