Search

Multiple sclerosis (MS) is an autoimmune disease that usually occurs during the reproductive years in both sexes. Many male patients with MS show lower blood testosterone levels, which was also observed in male rats during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. To better understand the causes of decreased testosterone production during EAE, we investigated the expression status of genes and proteins associated with steroidogenesis in the testes. No changes in the number of interstitial cells were observed in EAE animals, but the expression of the insulin-like 3 gene was reduced at the peak of the disease, implying that the Leydig cell functional capacity was affected. Consistent with this finding, the expression of most steroidogenic enzyme genes and proteins was reduced during EAE, including StAR, CYP11A1, CYP17A1 and HSD3B. No signs of testicular inflammation were observed. Recovery of steroidogenesis was observed after injection of hCG, the placental gonadotropin, or buserelin acetate, a gonadotropin-releasing hormone analogue, at the peak of EAE. Together, our results are consistent with the hypothesis that impaired testicular steroidogenesis originates upstream of the testes and that low serum LH is the main cause of decreased testosterone levels during EAE.

Placenta produces progesterone and estradiol for maintaining pregnancy. Two critical enzymes are responsible for their production: 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) that catalyzes the formation of progesterone from pregnenolone and aromatase that catalyzes the production of estradiol from testosterone. Fungicide ziram may disrupt the placental steroid production. In the present study, we investigated the effects of ziram on steroid formation in human placental cell line JEG-3 cells and on HSD3B1 and aromatase in the human placenta. Ziram did not inhibit progesterone production in JEG-3 cells and HSD3B1 activity at 100μM. Ziram was a potent aromatase inhibitor with the half maximal inhibitory concentration (IC 50 ) value of 333.8nM. When testosterone was used to determine the mode of action, ziram was found to be a competitive inhibitor. Docking study showed that ziram binds to the testosterone binding pocket of the aromatase. In conclusion, ziram is a potent inhibitor of human aromatase., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We examined whether variants in genes related to sex hormone biosynthesis and metabolism were associated with hypospadias in humans. We examined 332 relatively common tag single-nucleotide polymorphisms (tagSNPs) in 20 genes. Analyses included 633 cases (84 mild, 322 moderate, 212 severe and 15 undetermined severity) and 855 population-based non-malformed male controls born in California from 1990 to 2003. We used logistic regression models to estimate odds ratios (OR) and 95% confidence intervals (CI) for each SNP. Several of the 332 studied SNPs had p < 0.01: one in CYP3A4, four in HSD17B3, one in HSD3B1, two in STARD3, 10 in SRD5A2 and seven in STS. In addition, haplotype analyses gave several associations with p < 0.01. For HSD17B3, 14-SNP and 5-SNP blocks had ORs of 1.5 (95% CI 1.1, 2.0, p < 0.001) and 2.8 (95% CI 1.6, 4.8, p < 0.001) respectively. For SRD5A2, 9-SNP, 3-SNP and 8-SNP blocks had ORs of 1.7 (95% CI 1.3, 2.2, p < 0.001), 1.4 (95% CI 1.1, 1.8, p = 0.008) and 1.5 (95% CI 1.2, 1.9, p = 0.002) respectively. Our study indicates that several genes that contribute to sex hormone biosynthesis and metabolism are associated with hypospadias risk., (© 2013 American Society of Andrology and European Academy of Andrology.)

There are three enzymes involved in the biosynthesis of the adrenal androgen dehydroepiandrosterone (DHEA) sulfate. Cholesterol side-chain cleavage (CYP11A1) and 17alpha-hydroxylase/17,20-lyase (CYP17) metabolize cholesterol into DHEA, whereas steroid sulfotransferase family 2A1 (SULT2A1) is responsible for conversion of DHEA to DHEA sulfate. We previously examined the mechanisms regulating CYP11A1, CYP17, and SULT2A1 transcription and found that each is regulated, in part, by the transcription factor GATA-6. Previous studies suggested that mediator complex subunit 1 (MED1, also called PPARBP or TRAP220) is a cofactor involved in not only the regulation of nuclear receptors but also the activation of GATA-6 transcription. Herein we demonstrated a role for MED1 in the regulation of CYP11A1, CYP17, and SULT2A1 transcription. Transient transfection assays with SULT2A1 deletion and mutation promoter constructs allowed the determination of specific the GATA-6 binding cis-regulatory elements necessary for transactivation of SULT2A1 transcription. Binding of MED1 and GATA-6 was confirmed by coimmunoprecipitation/Western analysis and chromatin immunoprecipitation assay. We demonstrated expression of MED1 mRNA and protein in the human adrenal and determined that knockdown of MED1 expression via specific small interfering RNA attenuated CYP11A1, CYP17, and SULT2A1 expression levels in H295R cells. In addition, we demonstrated that MED1 enhanced GATA-6 stimulated transcription of promoter constructs for each of these genes. Moreover, the activity of MED1 for SULT2A1 promoter was mediated by GATA-6 via the -190 GATA-binding site. These data support the hypothesis that MED1 and GATA-6 are key regulators of SULT2A1 expression, and they play important roles in adrenal androgen production.

The human adrenal reticularis produces the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S). As opposed to the cortisol and aldosterone little is known regarding the mechanisms that regulate the production of the adrenal androgens. Several recent studies have shown that type II 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5 (CYB5), and steroid sulfotransferase (SULT2A1) play an important role in the regulation of adrenal androgen production. Specifically, adrenal production of DHEA-S is correlated with reticularis expression of SULT2A1 and CYB5. In contrast, HSD3B2 has an inverse correlation with adrenal androgen production likely due to its unique ability to remove precursors from the pathway leading to DHEA. Therefore, its expression is limited to the adrenal glomerulosa/fasciculata but not in reticularis. The differential expression of these three proteins appears to be critical for reticularis function. In this review, we focus on studies that have begun to define the mechanisms regulating the transcription of these genes. Understanding the mechanisms controlling differential expression of these proteins should provide novel information about the human adrenal reticularis and its production of DHEA and DHEA-S.

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.

The enzyme complex 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) is involved in the biosynthesis of all classes of active steroids. The expression of 3b-HSD in human uterine endometrium during the menstrual cycle and decidua was examined in an effort to understand its role during ova implantation. 3beta-HSD was weakly expressed in the glandular epithelium of the proliferative phase and moderately expressed in the glandular epithelium of secretory phase of the endometrium. In the decidua of the ectopic pregnancy, 3beta-HSD was strongly expressed. The human uterine endometrial 3beta-HSD was identified as being the same type as the placental 3beta-HSD by RT-PCR and sequence analysis. In addition to the expression of 3beta-HSD, P450scc was expressed in the decidua of the ectopic pregnancy. These results suggest that pregnenolone might be synthesized from cholesterol by P450scc de novo and then, it is converted to progesterone by 3beta-HSD in the uterine endometrium. The data implies that the endometrial 3beta-HSD can use not only the out-coming pregnenolone from the adrenal gland but also the self- made pregnenolone to produce progesterone. The de novo synthesis of progesterone in the endometrium might be a crucial factor for implantation and maintenance of pregnancy.

3beta-hydroxysteroid dehydrogenase (3beta-HSD) is an enzyme that converts pregnenolone to progesterone. It has been believed that 3beta-HSD is simply a converting enzyme of female steroid hormone. Recently, 3beta-HSD expressing cells were identified in the spinal cord. Steroid synthesis in the nervous system may indicate that steroid plays a role in the nervous system. We report here the increased expression of 3beta-HSD mRNA in the dorsal root ganglion (DRG) after peripheral nerve injury using reverse transcription-polymerase chain reaction and in situ hybridization histochemistry techniques. We detected only a few 3beta-HSD signals in the naïve DRG, and found that 3beta-HSD mRNA expression increased 3 days after injury and this increase was still observed at 14 days. Our results suggest that progesterone may have a role in the process against neuronal injury or in regeneration in the peripheral nervous system.

Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.

In adult male rats, 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) expressing cells were identified in the spinal cord from the cervical to the sacral segments. An in situ hybridization study, using an oligonucleotide common to the four known isoforms of rat 3beta-HSD, revealed its mRNA in gray matter. Measurements of optical densities in autoradiograms showed the following regional distribution: dorsal horn (layers I-III) > central canal (layer X) > or = ventral horn (layers VIII-IX) > ventral funiculus = lateral funiculus. At the cellular level, the number of grains was higher on the large motoneurons than on small neurons of the dorsal horn, but the grain density per cell was similar. Further evidence for the expression of 3beta-HSD in the spinal cord was obtained by western blot analysis, which revealed an immunoreactive protein of approximately 45 kDa in the dorsal and ventral parts of the spinal cord. Castration and adrenalectomy did not influence the expression of 3beta-HSD mRNA and protein. Gas chromatography/mass spectrometry measurements showed higher levels of pregnenolone and progesterone in the spinal cord than in the plasma. After castration and adrenalectomy, their levels remained elevated in the spinal cord, suggesting that these neurosteroids may be synthesized locally. The wide distribution of 3beta-HSD, and the high levels of pregnenolone and progesterone in the spinal cord even after castration and adrenalectomy, strongly suggest a potential endogenous production of progesterone and an important signalling function of this steroid in the spinal cord., (Copyright 2002 IBRO)

The 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) isoenzymes catalyze an essential step in the formation of all classes of active steroid hormones. We have recently shown that 3beta-HSD type 1 gene expression is specifically induced by interleukin (IL)-4 and IL-13 in several human cancer cell lines and in normal human mammary and prostatic epithelial cells in primary culture. There is evidence that IL-4 stimulates bifurcating signaling pathways in which the Stat6-signal pathway is involved in differentiation and gene regulation, whereas insulin receptor substrate (IRS) proteins mediate the mitogenic action of IL-4. As a matter of fact, we have shown that IL-4-activated Stat6 in all cell lines studied, where IL-4 induced 3beta-HSD type 1 expression but not in those cell lines that failed to respond to IL-4. The mechanism of the induction of 3beta-HSD type 1 gene expression was further characterized in ZR-75-1 human breast cancer cells. We have also found that IL-4 rapidly induced IRS-1 and IRS-2 phosphorylation in these cell lines. Moreover, insulin-like growth factor (IGF)-1 and insulin, which are well known to cause IRS-1 and IRS-2 phosphorylation, increased the stimulatory effect of IL-4 on 3beta-HSD activity. IRS-1 and IRS-2 are adapter molecules that provide docking sites for different SH2 domain-containing proteins, leading to the activation of multiple pathways, such as the phosphatidylinositol (PI) 3-kinase and the mitogen-activated protein (MAP) pathways. The inhibition of IL-4-induced 3beta-HSD expression by PI 3-kinase inhibitors (wortmannin and LY294002) as well as an inhibitor of MAP kinase activation (PD98059), indicates the involvement of those pathways in this response to IL-4. Wortmannin also blocked MAP kinase activation by IL-4, insulin and IGF-1 suggesting that the MAP kinase cascade acts as a downstream effector of PI 3-kinases. Furthermore, we showed that the PKC activator phorbol-12-myristate-13-acetate (PMA) also potentiated the IL-4-induced 3beta-HSD activity, thus suggesting that one signaling molecule that is involved in the signal transduction of the IL-4 action on 3beta-HSD type 1 expression is also a substrate for PKC. Taken together, these findings suggest the existence of a novel mechanism of gene regulation by IL-4. This mechanism would involve in the phosphorylation of IRS-1 and IRS-2, which transduce the IL-4 signal through a PI 3-kinase- and MAP kinase-dependent signaling pathway. However, the inability of IGF-1, insulin and PMA to stimulate 3beta-HSD type 1 expression by themselves in the absence of IL-4 indicates that the multiple pathways downstream of IRS-1 and IRS-2 must act in cooperation with an IL-4-specific signaling molecule, such as the transcription factor Stat6. It is also of interest to note that there also appear to be differences between the regulation of the 3beta-HSD type 1 and type 2 promoters.

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.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.

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)

Structures of cDNA clones encoding three members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family were characterized. To search for potential new types of 3 beta-HSD, rat types I and II 3 beta-HSD cDNAs were used as probes to screen a rat genomic DNA library. Among the clones isolated, one encodes a novel predicted rat 3 beta-HSD isoenzyme, chronologically designated type IV. The corresponding full-length cDNA was thereafter isolated by selective polymerase chain reaction amplification from rat ovary and day-15 placenta cDNA libraries. The rat type IV 3 beta-HSD cDNA encodes a predicted 372-amino acid protein of 41,854 daltons, which shares 90.9, 87.9, and 78.8% sequence identity with rat types I, II, and III proteins, respectively. Ribonuclease protection assay reveals that type IV 3 beta-HSD is the sole 3 beta-HSD mRNA species detectable in the skin and represents the predominant species in the placenta while being also detectable in the ovary and, to a lower degree, in the adrenal gland. Transient expression of type IV cDNA in SW-13 cells indicates 3 beta-HSD activity similar to that of rat type I 3 beta-HSD. The presence of multiple 3 beta-HSD genes should permit differential and tissue-specific regulation of this rate-limiting enzymatic activity essential in the biosynthesis of all classes of steroid hormones in both classical steroidogenic and intracrine peripheral tissues.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5-4-ene isomerase (3 beta-HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, and sex steroids. To obtain more information about the age-specific expression and localization of 3 beta-HSD during development in the rat ovary, two complementary cytochemical techniques were used, immunocytochemical localization with antibodies against purified human placental 3 beta-HSD, and 3 beta-HSD mRNA localization achieved by in situ hybridization with a [35S]-labeled cDNA encoding 3 beta-HSD. In the fetal ovary, no significant immunolabeling or hybridization signal could be observed. The first expression of the enzyme was observed 6 days after birth, immunolabeling as well as autoradiographic reaction being found in a few interstitial gland cells. At the 10th day of postnatal life, the theca interna cells of growing follicles appeared to be labeled with both techniques. The granulosa cells of growing follicles also exhibited hybridization signal, whereas no immunostaining could be detected in these cells at any of the time intervals studied. At puberty, 3 beta-HSD was localized in theca interna and granulosa cells of the growing and mature follicles as well as in the interstitial gland and corpus luteum cells. Similar results were obtained in 40-day-old and adult animals. The present data indicate a progressive expression of 3 beta-HSD in different morphological structures during ovarian development in the rat.

Mice differ in their adult reproductive characteristics as a function of whether they developed in utero between two male fetuses (2M males), which have higher testosterone levels, or between two female fetuses (0M males), which have higher estradiol levels. The present study was designed to further characterize biochemical parameters of 2M and 0M adult male mice. Activities of testicular steroidogenic enzymes, namely delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, and C17,20-lyase (C21SCC P450), were measured by means of radiometric assays and HPLC fractionation of substrate and products. Activity of 5 alpha-reductase in both seminal vesicle and prostate was measured by similar techniques. Estrogen and androgen receptor concentrations, which indicate capacity to respond to steroid hormones, were also examined in the accessory sex organs. For both seminal vesicle and prostate, 5 alpha-reductase activities were approximately 60% greater in 2M males than in 0M males, indicating greater capacity to form dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in testicular steroidogenic enzymes between 2M and 0M animals, whereas the trend for all three activities was higher for 2M males than for 0M males. While no differences were found in estrogen receptor concentrations, 0M prostates had three times the concentration of androgen receptors (occupied receptors) compared to 2M prostates. Our findings suggest that intrauterine fetal position exerts a significant influence on subsequent adult androgen metabolism and androgen responsiveness in reproductive organs of adult male mice.

The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues. To develop a model more closely related to the human, we have isolated and characterized cDNA clones encoding macaque 3 beta-HSD by screening a rhesus monkey ovary lambda gt11 cDNA library using a human 3 beta-HSD cDNA probe. Nucleotide sequence of 1629 bp from overlapping cDNA clones predicts a protein of 372 amino acids with a calculated molecular mass of 41,874 (excluding the first Met). The deduced amino acid sequence of macaque 3 beta-HSD displays 79.4% and 93.9% similarity with that of bovine and human 3 beta-HSD, respectively. RNA blot analysis performed under high stringency conditions of macaque poly(A)+ RNA samples using full-length 32P-labeled macaque 3 beta-HSD cDNA revealed the presence of an approximately 1.7 kb mRNA species in classical steroidogenic tissues, namely the ovary, testis and adrenal glands as well as in several peripheral tissues including the liver, kidney and epididymis. Computer analysis of the deduced macaque 3 beta-HSD protein sequence predicts the presence of an NH2-terminal membrane-associated segment as well as four additional membrane-spanning segments, thus suggesting that 3 beta-HSD is an integral protein. The availability of macaque cDNA should permit detailed studies concerning the tissue-specific expression as well as the hormonal regulation of 3 beta-HSD mRNA in classical steroidogenic glands as well as in peripheral tissues which are an important site of steroidogenesis in primates.

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.

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.

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.