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PubMed1991

Regulation of adrenal 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase expression and activity by adrenocorticotropin and corticosterone in the rat.

This study describes the regulation of adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) expression and activity by ACTH and corticosterone, alone or in combination, in intact male and female rats as well as the effect of ACTH on 3 beta HSD expression and activity in the adrenals of hypophysectomized female animals. The effect of treatment on total 3 beta HSD mRNA levels was measured by dot blot hybridization using rat 3 beta HSD cDNA, while the specific regulation of type I and type II 3 beta HSD mRNAs was analyzed by ribonuclease protection assay. The concentration of 3 beta HSD protein was measured by Western blot, using cross-reacting antibodies raised against purified human placental 3 beta HSD, while 3 beta HSD enzymatic activity was measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione. The present data show that the trophic effect of ACTH on male and female rat adrenals is accompanied by increases in total 3 beta HSD mRNA, enzymatic activity, and protein content. Hypophysectomy, on the other hand, causes a marked decrease in 3 beta HSD mRNA levels and enzymatic activity, which is completely reversed by administration of ACTH. On the other hand, corticosterone treatment results in a marked inhibition of 3 beta HSD mRNA levels, enzymatic activity, and protein content in intact animals; this effect is probably mediated by a decrease in ACTH secretion. The present data show that ACTH and corticosterone, via its inhibitory action on ACTH secretion, have potent and opposite effects on the expression of two 3 beta HSD genes in the rat adrenal; a parallel effect is observed on both type I and II 3 beta HSD. Such data suggest that 3 beta HSD could well play a major role in the regulation of steroid formation in the adrenal cortex.

Adrenal Glands↗
PubMed1992

Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.

Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into the corresponding delta 4-3-ketosteroids, interconvert 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). When homogenate from cells transfected with a plasmid vector containing type I 3 beta-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5 alpha-androstanedione (A-dione), thus indicating an intrinsic androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity of this 3 beta-HSD isoform. Although the relative Vmax of 17 beta-HSD activity is 14.9-fold lower than that of 3 beta-HSD activity, the Km value for the 17 beta-HSD activity of type I 3 beta-HSD is 7.97 microM, a value which is in the same range as the conversion of DHT into 3 beta-diol which shows a Km value of 4.02 microM. Interestingly, this 17 beta-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this "secondary" activity. Such 17 beta-HSD activity is inhibited by the classical substrates of 3 beta-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), delta 5-androstene-3 beta,17 beta-diol (delta 5-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 microM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3 beta-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Expression of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase: regulation by luteinizing hormone and forskolin in Leydig cells of adult rats.

LH is required to maintain the activity of 3 beta-hydroxysteriod dehydrogenase/delta 5----4-isomerase (3 beta HSD) in testicular Leydig cells. The objective of the present study was to determine whether LH and effectors such as forskolin, which act via the intracellular cAMP signal transduction pathway, can regulate the expression of 3 beta HSD in rat Leydig cells in vitro. Primary cultures of Leydig cells were prepared from testes of adult rats and treated with oLH, forskolin, (Bu)2cAMP, or cholera toxin. The effects of treatment on 3 beta HSD activity were measured using [3 alpha-3H]dehydroepiandrosterone as substrate. Immunoreactive 3 beta HSD was quantified by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a polyclonal antiserum against 3 beta HSD. The synthesis of 3 beta HSD was quantified after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated cellular lysates of Leydig cells radiolabeled with L-[35S]methionine. The levels of 3 beta HSD mRNA were quantified by Northern analysis and hybridization with a cDNA encoding testicular 3 beta HSD (rat type I). A cell-free protein-synthesizing system was used to test the ability of 3 beta HSD mRNA to be translated into immunoreactive 3 beta HSD. 3 beta HSD activity increased 3.5- and 5.0-fold in Leydig cell cultures treated with forskolin (1 microM) and (Bu)2cAMP (1 mM), respectively, compared with control cultures. Maximal activity was attained after 48-72 h and maintained through 120 h of treatment. The increase in 3 beta HSD activity could be accounted for quantitatively by increases in the steady state levels and the rates of synthesis of 3 beta HSD. The cellular levels of immunoreactive 3 beta HSD increased 4.0- and 7.6-fold in Leydig cells treated with forskolin and (Bu)2cAMP, respectively. Moreover, both of these effectors increased by 6- to 8-fold the levels of newly synthesized 3 beta HSD after 24-72 h of treatment. Ovine LH, forskolin, cholera toxin, and (Bu)2cAMP increased the cellular levels of 3 beta HSD mRNA in a dose-dependent manner. The magnitude of the increases ranged from 2- to 42-fold, compared with that in control cultures, after 12 h of treatment. Maximal responses were effected by 1 ng/ml ovine LH, 1 microM forskolin, 1 ng/ml cholera toxin, and 1 mM (Bu)2cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.

Androstane-3,17-diol↗
PubMed1993

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse tissues: male-specific isoforms are expressed in the gonads and liver.

Multiple isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD) are expressed in various mouse tissues in a tissue-specific, sex-specific, and developmental manner. Three distinct immunoreactive species [molecular masses, 47, 44, and 42 kilodaltons (kDa)] are detectable by Western immunoblot analysis using a 3 beta HSD antiserum. Different immunoreactive isoforms are expressed in steroidogenic (44 and 47 kDa in gonads) and nonsteroidogenic (42 and 47 kDa in liver and kidney) tissues. Two of these isoforms are sex-specific in the gonads (47 kDa) and liver (42 kDa), because they are detectable only in male mice. Sex-specific expression in the liver is developmentally regulated. Low levels of this male-specific hepatic isoform are first detectable at 23-25 days of age, but its level of expression increases progressively during sexual maturation to adult levels. NAD(+)-dependent 3 beta HSD activity is detectable in homogenates of all tissues examined, but the kinetic characteristics of this activity differ among tissues and are sexually dimorphic in the liver. Apparent Michaelis constants for dehydroepiandrosterone are much lower in steroidogenic (0.24 +/- 0.07 microM for testis) than in nonsteroidogenic (range, 10-100 microM for liver and kidney) tissues and are lower in male mouse liver (16 +/- 1 microM) than in female mouse liver (82 +/- 20 microM). Oligonucleotides with unique sequences but encoding homologous regions of the mouse type I, II, and III 3 beta HSD cDNAs were used for Northern blot analyses. A type I oligomer hybridizes with RNA from steroidogenic (adrenal, ovary, and testis) tissues, and a type III oligomer hybridizes with RNA from nonsteroidogenic (liver and kidney) tissues. A type II oligomer, however, hybridizes specifically with RNA from testis and liver of male mice, tissues that express a male-specific 3 beta HSD. These results suggest that type II-like transcripts may encode a 47-kDa sex-specific 3 beta HSD in testis and a 42-kDa sex-specific 3 beta HSD in liver of male mice. It is unclear how many members of subfamilies of the 3 beta HSD gene family will be discovered. The mouse may prove to be a valuable experimental model, as this is the first species in which multiple immunoreactive isoforms can be identified in a single tissue. This multiplicity makes it difficult to correlate the size and number of immunoreactive isoforms with the diverse kinetic characteristics of NAD(+)-dependent 3 beta HSD activities in tissue homogenates and to develop isoform-specific probes.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1993

Growth hormone transgenes regulate the expression of sex-specific isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in mouse liver and gonads.

The sexually dimorphic pattern of GH secretion regulates the expression of several steroidogenic enzymes in rat liver, including a male-specific 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD). Recently, we identified male-specific isoforms of immunoreactive 3 beta HSD in mouse liver [42 kilodaltons (kDa)] and gonads (47 kDa). To test whether GH can regulate the expression of these murine 3 beta HSDs, endogenous forms of 3 beta HSD were studied in transgenic mice expressing heterologous GH transgene products. Mice from five transgenic lines were used; two expressed GH transgenes encoding the phosphoenolpyruvate carboxykinase (PEPCK) promoter fused to either the human (h) GH (somatogenic and lactogenic) or bovine (b) GH (somatogenic) structural genes, and three expressed GH transgenes encoding the mouse metallothionein-1 (MT1) promoter fused to the hGH, hGH variant (hGHv), or bGH structural genes. Control mice were normal nontransgenic littermates. Expression of a male-specific (42 kDa) isoform of hepatic 3 beta HSD is dramatically suppressed in all transgenic mouse lines, as detected on Western immunoblots, without affecting a 47-kDa isoform expressed in livers of both male and female mice. This negative regulation was not observed in mouse kidney, which normally expresses two 3 beta HSD isoforms (in both sexes) with molecular masses similar to those in liver. Considering that PEPCK and MT1 promoters direct expression of GH fusion genes in both tissues, the inhibition of hepatic, but not renal, 3 beta HSD immunoreactivity suggests that GH affects sex-specific, rather than tissue-specific, expression of 3 beta HSD. As in the liver, sex-specific expression of 3 beta HSD in the testis is also suppressed by heterologous GH, but with one notable difference. Only human-derived GH (MT1-hGH and MT1-hGHv) effectively inhibits expression of the 47-kDa sex-specific isoform of testicular 3 beta HSD, without affecting the 44-kDa isoform expressed in gonads of both male and female mice. These results suggest that the negative effects of heterologous GH on sex-specific 3 beta HSDs may be mediated by PRL receptors in the testis and GH receptors in the liver. PEPCK-GH transgenes had little effect on testicular 3 beta HSD, possibly because this promoter (unlike MT1) is relatively inactive in this tissue. In the liver of male transgenics (PEPCK-hGH), loss of the sex-specific (42-kDa) 3 beta HSD has little effect on the Km for dehydroepiandrosterone (DHEA; 11 microM) compared with that in normal controls (16 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in adrenal cortex mitochondria and microsomes.

3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is a NAD(+)-dependent membrane-bound enzyme that catalyzes the oxidation of delta 5-3 beta-hydroxysteroids to delta 4-3-keto structures during adrenal, gonadal, and placental steroidogenesis. Enzyme activity is located in both microsomes and mitochondria. In these experiments we examined the membrane topologies of 3 beta HSD in rat and calf adrenal microsomes and mitochondria by comparing access to the active sites of coenzyme and the inhibitor mersalyl, a nonpenetrant organic mercurial anion. Microsomal activity required exogenous NAD+ and was inhibited by mersalyl, indicating that the active site faced the medium in vitro and the cytoplasm in vivo. In contrast, mitochondrial 3 beta HSD used matrix space NAD+, was inhibited by reduction of intramitochondrial NAD(P)+, and was insensitive to mersalyl. Mitochondrial activity was decreased by exogenous NADH (apparent Ki, 2.8 microM) and increased by added NAD+ (apparent Ka, 2.4 microM). However, mersalyl blocked the effects of exogenous NADH and NAD+ and returned the activity to that observed before coenzyme addition. The membrane-sidedness of the NAD+ activation was examined further in submitochondrial particles prepared by sonication of pyridine nucleotide-depleted calf adrenal cortex mitochondria. Particles were prepared in the absence or presence of 10 mM NAD+ and contained none or 2.9-7.3 nmol NAD+/mg protein, respectively. Both groups of submitochondrial particles required exogenous NAD+ for 3 beta HSD activity, indicating that the active site faced the medium (the particles were everted), and the contained NAD+ was inside the particles. However, 3 beta HSD activity was increased 12-140% in particles that contained NAD+. The results suggest that mitochondrial 3 beta HSD is an integral inner membrane protein, that the active site faces the matrix space and is influenced by coenzyme availability, and that a regulatory site(s) faces the intermembrane space. Binding of NAD+ or NADH to this external site increases or decreases, respectively, the rate of catalysis at the active site. Mitochondrial 3 beta HSD activity may be enhanced by oxidation of intermembrane space NADH via an active rotenone- and antimycin-a-insensitive NADH oxidase.

Adrenal Cortex↗
PubMed1995

Regulation of 3 beta-hydroxysteroid dehydrogenase delta 5/delta 4-isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells.

Activin is a dimeric protein implicated in the local control of follicular steroidogenesis. Using cultured rat granulosa cells, we previously showed that the effect of activin on FSH-induced progesterone synthesis changes with preovulatory follicular development, from positive regulation in nondifferentiated (immature) granulosa cells to negative regulation in preovulatory (mature) granulosa cells. The aim of the present study was to assess development-related effects of activin on the expression of enzymes crucial to progesterone synthesis: cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase (3 beta HSD). Nondifferentiated granulosa cells were isolated from the ovaries of estrogen-pretreated immature female rats that received no other treatment; differentiated granulosa cells were obtained from similar animals treated for 48 h with human FSH to induce preovulatory follicular development. Cells were cultured for 48 h in serum-free medium with and without human FSH and/or recombinant activin-A, and medium was collected for measurement of progestagens (progesterone, pregnenolone, and 20 alpha-dihydroprogesterone). In cultures of nondifferentiated granulosa cells, activin augmented the FSH-induced production of all three steroids. In differentiated granulosa cells, activin suppressed the FSH-stimulated production of progesterone and 20 alpha-dihydroprogesterone, but had no effect on pregnenolone. The presence of exogenous pregnenolone increased the overall production of progesterone, but did not alter qualitative steroidogenic responses to activin. To assess the interaction between FSH and activin on 3 beta HSD and P450scc messenger RNA (mRNA) expression, Northern blot analyses were performed on total RNA isolated from cultured granulosa cells. Treatment in vitro with FSH alone markedly enhanced the abundance of both the 3 beta HSD and P450scc mRNA transcripts in nondifferentiated and differentiated granulosa cells. FSH-stimulated expression of P450scc mRNA was further enhanced by cotreatment of nondifferentiated granulosa cells with activin. However, activin had no consistent effect on FSH-stimulated expression of 3 beta HSD mRNA in nondifferentiated cells. In differentiated granulosa cells, both mRNAs were suppressed more than 50% by the presence of activin. We conclude that the in vitro effects of activin on FSH-induced expression of 3 beta HSD and P450scc mRNAs in rat GC are similar: initially stimulatory (P450scc) or without effect (3 beta HSD), then becoming completely inhibitory. The mechanism of this development-dependent change in the granulosa cell response to activin remains to be elucidated.

Activins↗
PubMed1996

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Adrenal Cortex Hormones↗
PubMed1997

Inhibition of fetal adrenal adrenocorticotropin receptor messenger ribonucleic acid expression by betamethasone administration to the baboon fetus in late gestation.

Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzyme delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3beta-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 microl; n = 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 +/- 0.07 arbitrary densitometric units) than in untreated controls (0.63 +/- 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of beta-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 +/- 0.02) than in untreated controls (0.84 +/- 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (microm) of the definitive zone (183 +/- 14 vs. 128 +/- 7; determined by immunohistochemical expression of 3beta-HSD), as well as the levels of the mRNA and protein for 3beta-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17alpha-hydroxylase-C(17,20) lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3beta-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.

Adrenal Glands↗
PubMed1999

Expression of steroidogenic genes in maternal and extraembryonic cells during early pregnancy in mice.

The ontogeny and functional role of steroidogenesis during early gestation in rodents is poorly understood. In previous studies, we have shown that expression of messenger RNAs (mRNAs) encoding two key enzymes indispensable for de novo synthesis of steroid hormones, i.e. cholesterol side chain cleavage cytochrome P450 (P450scc) and a newly identified isoform of murine 3beta-hydroxysteroid dehydrogenase/isomerase type VI (3betaHSD VI), is initiated upon decidualization of the uterine wall induced by implantation. In situ hybridization and immunohistochemical visualization of 3betaHSD VI mRNA and protein shows high expression of this enzyme in the antimesometrial cells of the decidua of days 6.5-7.5 post coitum (p.c.). Thereafter, expression of 3betaHSD VI in the decidual zones disappears and is replaced by a high expression of mRNA and protein in the embryonal giant trophoblast cells. At the peak of their development on day 9.5 p.c., the mouse giant trophoblast cells also express Steroidogenic Acute Regulatory (StAR) protein, which is required for steroidogenesis in the gonads and adrenal cortex. Our findings also suggest that the declining levels of P450scc, 3betaHSD VI, and StAR proteins between days 10.5-14.5 p.c. in the developing placenta is consistent with previous reports that the mouse placenta is not involved in de novo synthesis of steroids during the second half of pregnancy. Collectively, the results of the present study suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy. We propose that the local production of progesterone acts as an immunosuppressant at the fetal maternal interface preventing the rejection of the fetal allograft.

Amino Acid Sequence↗
PubMed2005

Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.

The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.

Adrenal Glands↗
PubMed2003

The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway.

Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.

17-alpha-Hydroxyprogesterone↗
PubMed1976

Pregnenolone, 17-OH-pregnenolone, and testosterone in plasma of patients with congenital adrenal hyperplasia.

Both pregnenolone and 17-OH-pregnenolone were found to be higher in the plasma of patients with poorly controlled congential adrenal hyperplasia than in normal subjects. The plasma levels of these precursor steroids were significantly correlated with urinary 17-ketosteroid and pregnanetriol excretion and with plasma testosterone. The mechanism where by plasma pregnenolone and 17-OH-pregnenolone levels are elevated in patients with 21-hydroxylase deficiency is unknown, but the phenomenon of product inhibition is suggested as a possible explanation. As 17-OH-pregnenolone in plasma is almost entirely of adrenal origin, its measurement promises to be useful in the management of patients with congenital adrenal hyperplasia. Acute stimulation with ACTH caused negligible changes in the plasma levels of pregnenolone and 17-OH-pregnenolone and failed to distinguish between overly, appropriately, and under-treated patients. However, following repeated stimulation with repository ACTH, the steroid levels rose. These findings indicate limited adrenal responsiveness to ACTH following chronic glucocorticoid treatment of congenital adrenal hyperplasia, even in under-treated patients, and suggest that normal precursor steroid levels in plasma and normal 17-ketosteroid and pregnanetriol excretion can only be achieved by the suppression of total steroidogenesis to less than that occurring in normal subjects.

17-Ketosteroids↗
PubMed1982

Steroid enzyme activities in extraadrenal pheochromocytomas.

The adrenal medulla and tumors derived from it have been shown to be capable of converting radioactive steroid intermediates into glucocorticoid end products in vitro. This capacity for partial steroid synthesis was explored in two large extraadrenal pheochromocytomas and compared to the results of parallel studies with intraadrenal pheochromocytomas and adrenal cortex; in one experiment, all three types of tissue were obtained from a single patient and studied simultaneously. Slice preparations of extraadrenal pheochromocytoma transformed 14C-labeled pregnenolone (3 beta-hydroxypregn-5-en-20-one) into corticosterone and cortisol. Incubations of subcellular fractions demonstrated that, as in the adrenal cortex and intraadrenal chromaffin tissue, delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-hydroxylase, and 21-hydroxylase activities in extraadrenal pheochromocytomas were associated with the microsomes and 11 beta-hydroxylase was associated with the mitochondria, where metyrapone was an effective inhibitor, these findings tend to dissociate steroid-metabolizing activity in the adjacent chromaffin tissue perfused by glucocorticoid intermediates in high concentrations.

Adenoma↗
PubMed1986

A new inherited variant of the 3 beta-hydroxysteroid dehydrogenase-isomerase deficiency syndrome: evidence for the existence of two isoenzymes.

The clinical and endocrine features of a unique form of adrenal insufficiency secondary to an inherited deficiency of 3 beta-hydroxysteroid dehydrogenase-isomerase (3-HSD) were studied. The propositus was a 19-yr-old man with a history of repeated episodes of acute adrenal crisis. Family study disclosed that a 6-yr-old female sibling also was affected, and a third sibling had died during the course of an adrenal crisis. The diagnosis of adrenal insufficiency was established on the basis of extremely low serum cortisol levels and urinary 17-hydroxycorticosteroid excretion with concomitantly elevated serum ACTH levels and lack of cortisol response to ACTH administration. Impairment of C-21 steroid 3-HSD activity was strongly suggested by persistency elevated serum 17-hydroxypregnenolone to 17-hydroxyprogesterone and pregnenolone to progesterone ratios, their significant increase after ACTH administration, and their return to normal during cortisol therapy in both patients. Nevertheless, the serum dehydroepiandrosterone to androstenedione ratio, both basally and after ACTH and/or hCG stimulation, was normal. These findings coupled with the normal phenotypic development and onset of puberty in the two patients indicated intact C-19 steroid 3-HSD activity. The overall results indicate an inherited impairment of 3-HSD activity confined only to C-21 steroid substrates and, thus, suggest the existence of at least two 3-HSD isoenzymes under independent genetic regulation.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Immunocytochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human ovary.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid delta 5----4-ene-isomerase (3 beta HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain more information about the age-specific expression of 3 beta HSD in the human ovary, we have localized this enzyme by immunocytochemistry at the light microscopic level during fetal and postnatal periods of development in the human. Immunocytochemical localization was achieved using specific polyclonal antibodies developed against purified human placental 3 beta HSD. In the fetal ovary of 28-34 weeks, specific immunostaining for 3 beta HSD was exclusively detected in thecal cells surrounding primary follicles and in interstitial cells. From birth until puberty, no significant immunostaining for 3 beta HSD could be observed, while from puberty to menopause, staining was detected in theca interna cells as well as granulosa cells of growing follicles. The intensity of staining in the theca interna cells was always much higher than that in granulosa cells. Immunostaining was also found in the cytoplasm of luteinized granulosa and theca interna cells of the corpus luteum. Interestingly, there was an absence of immunoreactivity for 3 beta HSD in one to several layers of theca interna cells lying just beneath the basement membrane. These negative cells may correspond to fibroblast-like cells that are devoid of 3 beta HSD activity. In postmenopausal ovaries, immunolabeling was only found in dispersed interstitial cells, thus suggesting that the ovaries of postmenopausal women might be involved in sex steroid hormone secretion.

Aged↗