Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.

Prevalence and Prognostic Value of the Polymorphic Variant 1245A>C of HSD3B1 in Castration-resistant Prostate Cancer.

INTRODUCTION: The purpose of this study was to investigate the prevalence and prognostic value of the polymorphic variant (1245A>C), a single nucleotide polymorphism (SNP) of the HSD3B1 gene, in the tumors of patients with castration-resistant prostate cancer (CRPC). MATERIALS AND METHODS: We retrospectively evaluated 44 patients with CRPC who underwent palliative transurethral resection of the prostate. Genomic DNA was extracted from formalin-fixed and paraffin-embedded material, and 1245A>C SNP of the HSD3B1 gene was analyzed via Sanger sequencing. Cox regression analysis was used to assess the prognostic value of the respective SNP with time to progression as well as cancer-specific and overall survival in the subgroup of patients receiving second systemic treatment. RESULTS: The SNP was present in 20 patients (51.2%) who received second line systemic treatment additionally to androgen deprivation, of which 16 (80%) patients were heterozygous and 4 (20%) were homozygous. Correlation analysis revealed no association of the SNP with any clinical characteristics at initiation of second-line systemic treatment. Moreover, the presence of the variant (1245A>C) of HSD3B1 was not associated with any survival endpoint. CONCLUSIONS: The variant allele 1245C of the HSD3B1 gene is present in approximately one-half of patients with CRPC; however, it is not associated with oncologic outcomes. These findings, however, need to be interpreted with caution as the sample size is small. Further research on biomarkers is needed to help tailor clinical decision making in prostate cancer, especially in the increasingly complex therapeutic landscape of CRPC.

HSD3B1 Genotypes Conferring Adrenal-Restrictive and Adrenal-Permissive Phenotypes in Prostate Cancer and Beyond.

Castration-resistant prostate cancer (PCa) almost invariably occurs after androgen deprivation therapy for metastatic disease and is driven in part by androgen synthesis within the tumor. 3ß-hydroxysteroid dehydrogenase isoenzyme-1 catalyzes the conversion of adrenal precursor steroids into potent androgens essential for PCa progression. A common 1245 A→C missense-encoding single nucleotide polymorphism in HSD3B1 (rs1047303), the gene that encodes this enzyme, leads to a more stable protein that is resistant to degradation and thus increased production of potent androgens from adrenal precursors, facilitating castration-resistant PCa development. Consistent with this mechanism, this adrenal-permissive HSD3B1(1245C) genotype is associated with inferior outcomes after androgen deprivation therapy for advanced PCa, and increased sensitivity to pharmacologic blockade of adrenal precursors in metastatic disease. Herein, we review current knowledge of the mechanisms conferred by HSD3B1 genotype to alter androgen physiology and accelerate development of castration-resistant disease and its associations with clinical PCa outcomes. In light of its effect on steroid physiology, we also discuss its potential associations with non-PCa phenotypes.

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

Gossypol inhibits LH-induced steroidogenesis in bovine theca cells.

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0-25 µg/mL) for 24 h. Gossypol inhibited LH-stimulated theca cell A4 and P4 production in a dose-dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down-regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down-regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.

Steroidogenic differential effects in neonatal porcine Leydig cells exposed to persistent organic pollutants derived from cod liver oil.

Seafood products, including fish and fish oils, are major sources of persistent organic pollutants (POPs) which may cause endocrine disruption related to reproductive dysfunction in males. Primary porcine neonatal Leydig cells were exposed to three extracts of POPs obtained from different stages in production of cod liver oil dietary supplement, in the absence and presence of luteinizing hormone (LH). No reduced viability was observed and all POP extracts showed increased testosterone and estradiol levels in unstimulated cells and decreased testosterone and estradiol secretion in LH-stimulated cells. A decrease in central steriodogenic genes including STAR, CYP11A1, HSD3B and CYP17A1 was obtained in both culture conditions with all POP extracts. We implicate both small differences in composition and concentration of compounds as well as "old" POPs to be important for the observed steroidogenic effects.

Characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase expressed in the adrenal gland and gonads.

The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C(19) steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3beta-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3beta-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17beta-HSD type II-like activity was also observed. A phylogenetical analysis of the 3beta-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.

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.

Neurosteroids and female reproduction: estrogen increases 3beta-HSD mRNA and activity in rat hypothalamus.

A central event in mammalian reproduction is the LH surge that induces ovulation and corpus luteum formation. Typically, the LH surge is initiated in ovariectomized rats by sequential treatment with estrogen and progesterone (PROG). The traditional explanation for this paradigm is that estrogen induces PROG receptors (PR) that are activated by exogenous PROG. Recent evidence suggests that whereas exogenous estrogen is necessary, exogenous PROG is not. In ovariectomized-adrenalectomized rats, estrogen treatment increases hypothalamic PROG levels before an LH surge. This estrogen-induced LH surge was blocked by an inhibitor of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD), the proximal enzyme for PROG synthesis. These data indicate that estrogen induces de novo synthesis of PROG from cholesterol in the hypothalamus, which initiates the LH surge. The mechanism(s) by which estrogen up-regulates neuro-PROG is unknown. We investigated whether estrogen increases 1) mRNA levels for several proteins involved in PROG synthesis and/or 2) activity of 3beta-HSD in the hypothalamus. In ovariectomized-adrenalectomized rats, estrogen treatment increased 3beta-HSD mRNA in the hypothalamus, as measured by relative quantitative RT-PCR. The mRNAs for other proteins involved in steroid synthesis (sterol carrier protein 2, steroidogenic acute regulatory protein, and P450 side chain cleavage) were detectable in hypothalamus but not affected by estrogen. In a biochemical assay, estrogen treatment also increased 3beta-HSD activity. These data support the hypothesis that PROG is a neurosteroid, produced locally in the hypothalamus from cholesterol, which functions in the estrogen positive-feedback mechanism driving the LH surge.

Opposite effects of a single IIIS5 mutation on phenylalkylamine and dihydropyridine interaction with L-type Ca2+ channels.

Replacement of L-type Ca(2+) channel alpha(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca(2+) channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Ca(v)1.2DHP(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)-[(3)H]devapamil occurred to Ca(v)1.2DHP(-/-) mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (-)-verapamil but not for (-)-gallopamil. The mutation caused a pronounced slowing of (-)-[(3)H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 mum (-)-verapamil inhibited a significantly larger fraction of Ba(2+) inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca(2+) channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca(2+) channels.

[Effects of salidroside on carbohydrate metabolism and differentiation of 3T3-L1 adipocytes].

OBJECTIVE: To observe the effects of salidroside on carbohydrate metabolism and differentiation of 3T3-L1 adipocytes. METHODS: 3H-glucose uptaking rate in adipocytes treated by salidroside was detected. Adipocytes treated by salidroside in differentiation were stained by oil red O and analyzed by spectrophotography quantitatively. The expression of PPAR-gamma and C/EBP-alpha mRNA relating to differentiation of adipocytes was detected by reverse transcription PCR. RESULTS: 3H-glucose uptaking rate in the salidroside group was 110.4%, higher than that in the blank control group (P < 0.01). Salidroside suppressed the differentiation of 3T3-L1 pre-adipocytes and down-regulated the expression of PPAR-gamma and C/EBP-alpha mRNA (P < 0.01, vs control group). CONCLUSION: Salidroside promotes the 3H-glucose uptaking, suppresses the differentiation and down-regulates the expression of PPAR-gamma and C/EBP-alpha mRNA in 3T3-L1 adipocytes.

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis.

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.

Rapid modulation of ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase gene expression by prolactin and human chorionic gonadotropin in the hypophysectomized rat.

In order to better understand the role of prolactin (PRL) and luteinizing hormone (LH) on progesterone biosynthesis in the ovary, we have investigated the time course (1-9 days) of the effect of PRL and human chorionic gonadotropin (hCG) on ovarian 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) expression in the hypophysectomized rat. As evaluated by quantitative in situ hybridization using a 35S labelled type I 3 beta-HSD cDNA probe, the administration of hCG for 2, 3 and 9 days induced increases of 63%, 145% and 146% above control, respectively, in 3 beta-HSD mRNA levels in ovarian interstitial cells. The absence of apparent effect of the gonadotropin in other ovarian cell types could explain the small modulation of ovarian 3 beta-HSD protein content and enzymatic activity observed in total ovarian tissue. On the other hand, treatment with PRL caused a rapid decrease in 3 beta-HSD mRNA levels in corpus luteum by 23%, 63%, 76% and 78% (P < 0.01) following 1, 2, 5 and 9 days of treatment, respectively. The short-term inhibitory effect of PRL was also observed on ovarian immunoreactive 3 beta-HSD protein, as measured by Western blot analysis, and on 3 beta-HSD activity measured by the conversion of [14C]dehydroepiandrosterone into [14C]androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression in rat and characterization of the testis isoform.

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. 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 3 beta-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.

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.