A simple procedure for solubilizing 3beta-hydroxysteroid dehydrogenase from microsomes of rat adrenals and testis interstitial cells

A simple procedure is described for solubilizing microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Microsomes from rat adrenals or from testicular interstitial cells were incubated for 1 or 2 h at 0 C in a buffer containing NaCl followed by overnight storage at -20 C. Maximum solubilization of 3beta-hydroxy-5beta-androstan-17-one-HSD (androstane-3beta-HSD) was obtained by incubating adrenal microsomes with 1 M NaCl and interstitial cell microsomes with 2 M NaCl. Incubation with NaCl for 1 or 2 h resulted in maximum solubilization; incubation with NaCl for 4, 8 or 24 h did not change the amount of enzyme solubilized. From adrenal microsomes incubated with 1 M NaCl, up to 80% (105.7 millimicron/mg microsomes) of the total androstane-3beta-HSD activity was recovered in the supernatant following centrifugation at 130,000 x g for 1 h. The maximum amount of androstane-3beta-HSD solubilized from interstitial cell microsomes was 56% (29.5 millimicron/mg microsomes) at 2 M NaCl. The "solubilized" androstane-3beta-HSD was retarded when chromatographed on a Sephadex G-200 column and it did not pellet out when centrifuged at 130,000 x g for 15 h. KCL appeared to be equally effective in solubilizing androstane-3beta-HSD from microsomes. Other steroid dehydrogenase activities such as pregnanolone-HSD and 3beta-hydroxy-5alpha-androstan-17-one-HSD were also found in the 130,000 x g supernatant.     

Structure and steroidogenic activity of the granulosa layer of F1 preovulatory ovarian follicles of the hen (Gallus domesticus)

The study was performed to determine the structure and steroidogenic activity of granulosa cells derived from the germinal disc region, proximal region and distal region of the largest preovulatory ovarian follicle (F1) of the hen. The study was carried out on 34 Hy-Line Brown egg-laying hens aged 40 weeks. Morphology of the granulosa cells was studied by histological assessment and scanning electron microscopy. Moreover, the level of P4, histochemical activity of 3beta-HSD and expression of 3beta-HSD gene mRNA in granulosa cells of F1 follicle were determined. The findings indicate that the morphology and steroidogenic activity of the granulosa layer in F1 preovulatory ovarian follicle are associated with the region of the follicle. This is consistent with earlier studies. In the germinal disc region the granulosa cells form a multilayer while in the proximal and distal regions granulosa cells form a single layer. Analysis of P4 concentration revealed that its level in granulosa cells was markedly reduced closer to the germinal disc. Moreover, our study demonstrates for the first time the lower histochemical activity of 3beta-HSD and expression of 3beta-HSD mRNA in granulosa cells from the germinal disc region compared with the proximal and distal region.     

Response to gonadotropic stimulation of cultured rat luteal cells isolated from corpora lutea of early pregnancy. Cytochemical and biochemical studies

The hormonal activity of corpora lutea isolated from pregnant rat was examined on 1, 2, 3, 4, 5, 6, 15, and 20th day of pregnancy. The cells were grown as a monolayers up to 6 days at 37 degrees C in Medium 199 supplemented with 10% calf serum. The concentrations of progesterone and estrogens were measured using appropriate radioimmunoassays [1, 7] respectively. Luteal cells were cultured with the addition of the following amounts of hormones: 100 ng LH, 10 i.u. HCG, 100 ng PRL and 150 ng estradiol 17 beta. Cytochemical and histochemical observation of the activity of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD) were also carried out. The addition of LH and HCG to culture medium of cells collected on day 1 and 2 of pregnancy caused increased histochemical reaction for delta 5, 3 beta-HSD and progesterone secretion. It was only on day 3 of pregnancy that the influence of PRL was observed. On day 4 corpus luteum cells began to respond to exogenous estradiol. On day 5 the sensitivity of corpus luteum to exogenous hormones disappeared but the intensive hormonal activity of the corpus luteum marked by the high level of progesterone, was maintained.     

Distribution of 3 beta-hydroxysteroid dehydrogenase during development of the rat adrenal cortex and capsule

Fibroblasts of the adult adrenal cortex are considered to be nonsteroidogenic connective-tissue cells. However, it has been reported that in response to regenerative stimuli, adrenocorticotropic hormone (ACTH), and transformation to malignancy, these cells acquire characteristics of parenchymal cells, which includes delta 5, 3 beta-hydroxysteroid-dehydrogenase (delta 5, 3 beta-HSD) activity. To determine whether such delta 5, 3 beta-HSD activity in adult adrenocortical fibroblasts was due to the activation or augmentation of gene expression normally occurring during embryogenesis, a histochemical study of adrenocortical development, with particular attention to the connective-tissue capsule, was undertaken. Cryostat sections of rat embryos, from 14-days postconception (PC) to birth, and of adrenal glands 1-8, 44 and 90 days after birth were tested histochemically for delta 5, 3 beta-HSD. The same or adjacent sections were stained for PAS-positive material and reticulin, and with hematoxylin and eosin. delta 5, 3 beta-HSD activity overlapped with fibroblast-like cells and with extracellular connective-tissue components in the periphery of the glands from day-17 PC onward. delta 5, 3 beta-HSD activity over the capsule diminished shortly after birth and was absent in the adult. Appropriate controls showed that the staining within the capsule was specific and not an artifact. 3 beta-HSD activity in the capsule was more intensive when dehydroepiandrosterone (DHEA) was replaced by etiocholan-3 beta-ol-17-one (ETIO) as the steroid substrate. Furthermore, the spatial distribution of 3 beta-HSD activity in the cortex differed depending on the substrate used, and the distribution patterns changed with developmental age. (ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of testosterone secretion from individual rat Leydig cells.

The purpose of the present study was to analyze testosterone secretion from individual purified Leydig cells, using a reverse hemolytic plaque assay (RHPA) as an approach for identifying and characterizing subtypes of Leydig cells. Leydig cells from adult rats and protein A-coated ovine erythrocytes were mixed and incubated for appropriate lengths of time in the presence or absence of antitestosterone antibody, hormones or an analog of cyclic AMP. The slides from RHPA were histochemically stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Results show that testosterone secreting cells can be clearly identified by the formation of hemolytic plaques. The proportion of plaque-forming cells increases with incubation time, reaching a plateau at 60 min in the presence of gonadotropin. It was observed that not all 3 beta-HSD positive cells form plaques. It is concluded that the purified Leydig cell population has cells with differential steroidogenic and androgen-secretory activities.     

Glycyrrhetinic acid bound to 11 beta-hydroxysteroid dehydrogenase in rat liver microsomes.

A binding protein which exhibits high affinity to [3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11 beta-HSD activity. These results show that the purified [3H]glycyrrhetinic-acid binding protein is 11 beta-HSD. From the molecular mass of 11 beta-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11 beta-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [3H]glycyrrhetinic acid binding and 11 beta-HSD activity indicate that the C30-carboxyl and C11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11 beta-HSD inhibition.     

Histochemistry of 3beta-hydroxysteroid dehydrogenase in rat ovary. I. Amethodological study

By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3beta-HSD was investigated: 1. Prefixation of small tissue blocks with 1% W/V methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced. 2. Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% W/V for 5 min) thereby enhancing the Nothing dehydrogenase reaction. 3. Preextraction of lipids with acetone for 20 min at -30 degree C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells). 4. No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20 degree C. 5. By using the double-section incubation method no diffusion of 3beta-HSD or rediffusion of NADH or PMSH could be noticed withn 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0mg/ml). 6. The utlity of different inhibitors of alkaline phosphomonoesterase was tested and discussed. 7. By inhibiting alkaline phosphomonoesterase with 0.1 mM of L-p-bromotetramisole or 16 mM of beta-glycerophosphate, 3beta-HSD was shown to be exclusively NAD-linked. 8. Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3 beta-HSD, but not of NADPH-tetrazolium reductase. 9. 3beta-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited by N-ethyl maleimide. 10. Whether alcohol dehydrogenases may use steroids as substrate is discussed; It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed. 11. Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase. 12. Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF). 13. The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone. 14. If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.     

Histochemical changes in the Leydig cells of rats drinking the aqueous hollyhock extract

The effect of aqueous hollyhock flower (Althaea rosea Cav. var. nigra) extract on the rat Leydig cell metabolism and morphology was studied using histochemical, morphometric and radioimmunological methods. The rats were drinking the extract for 30 days (group A1) and for 180 days (group A2). Leydig cells of group A1 manifested marked increase in the 3beta-HSD, G6PD and NADPD activities and in the Khanolkar reaction intensity. These findings were accompanied by the increase in the volume of Leydig cells and their nuclei. In group A2 Leydig cells, statistically insignificant changes in the G6PD and NADPD activities were observed, however, the significant increase in the 3beta-HSD activity and the Khanolkar reaction intensity indicated compensatory changes. The statistically significant elevation of the androgen level accompanied by a decrease in estrogen content in homogenates of group A2 testes pointed to weak antiestrogenic effect of the extract. The obtained results indicate an influence of the hollyhock extract on steroid metabolism.     

Membrane-bound human 3beta-hydroxysteroid dehydrogenase: overexpression with His-tag using a baculovirus system and single-step purification

The membrane-bound human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni(2+)-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C(12)E(8). High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K(m) of 1.7 microM and a V(max) of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme.     

Regulation by dexamethasone of the 3 beta-hydroxysteroid dehydrogenase activity in adult rat Leydig cells.

The effect of long-term in vitro treatment with dexamethasone, insulin and/or LH on the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity and the testosterone level was examined in cultures of Leydig cells from adult rats. A rapid and simple method for measuring the 3 beta-HSD activity has been developed, in which the NADH, generated by 3 beta-HSD, reduced nitroblue tetrazolium to a product with absorption maximum at 560 nm. Km for the reaction was 8.1 microM and Vmax was 12.7 nmol/min x mg protein. Addition of 0.1 or 1 microM dexamethasone for 44 h decreased the 3 beta-HSD activity to 83% and the basal testosterone level to 64% of control value after 22 and 44 h of culture. Addition of 1 nM insulin inhibited the 3 beta-HSD activity to 90% after 44 h of culture, whereas the testosterone level was increased after 3 h. Addition of 0.1 ng/ml LH did not affect the 3 beta-HSD activity in Leydig cells from adult rats. Concomitant treatment of the cells with dexamethasone and insulin inhibited the 3 beta-HSD activity to 74%, indicating an additive effect, whereas no additive effect on the testosterone level was observed. The results demonstrate that the 3 beta-HSD activity can be measured in a rapid and reliable way by measuring the reduction of nitroblue tetrazolium. Furthermore, the results suggest that dexamethasone acts on 3 beta-HSD through a mechanism different from that of insulin, as an additive effect was observed.     

3 beta-Hydroxysteroid dehydrogenase in human placental microsomes and mitochondria: co-solubilization of androstene and pregnene activities

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) was solubilized from human term-placental microsomes and mitochondria using the non-ionic detergent, polyoxyethylene 20 cetyl ether (BrijR-58). Electron photomicrographs showed microsomes and mitochondria well disrupted by the detergent. The pregnene (C-21) and androstene (C-19) activities co-solubilized over a range (0.04-0.44) of BrijR-58/protein (B/P) concentration ratios (w/w). Optimal solubilization of the C-19 and C-21 activities were 63.3 +/- 2.6% (mean +/- SEM) from mitochondria (B/P ratio 0.37) and 71.8 +/- 2.1% from microsomes (B/P ratio 0.34). Detergent treatment of microsomes and mitochondria--varying time (5-90 min, pH 7.4) or varying pH (6.0-7.8, 90 min)--yielded C-19 activities identical with C-21 activities. The C-21/C-19 specific activity ratios of 3 beta-HSD in particulate, solubilized and chromatographed preparations were 2.28 +/- 0.16 (mean +/- SEM) for mitochondria and 1.97 +/- 0.07 for microsomes. 3 beta-HSD molecular weight estimates were 208,000 (microsomes) and 220,000 (mitochondria). These studies argue that a single protein is responsible for both the C-19 and C-21 activities of 3 beta-HSD and that this protein is the same in microsomes and mitochondria.     

Evidence that Leydig precursors localize in immature band two cells isolated on Percoll gradients

The present studies examined responses to hCG and/or insulin of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase activity (3 beta-HSD) in cultured Band 2 and Band 3 cells from 25- to 40-day-old rats isolated on Percoll gradients. In Band 2 cells, from 25-day-old rats enzyme activity increased about 3- and 2.5-fold, after 6 days of exposure to hCG or insulin, respectively. However, hCG did not stimulate enzyme activity in Band 2 cells from 30-, 35- and 40-day-old animals, and responses to insulin alone or insulin plus hCG declined with age. In Band 3 cells only insulin increased enzyme activity at each age. Neither hCG or insulin altered DNA levels in Band 2 or Band 3 cells, suggesting that increased activity in Band 2 cells from 25-day-old rats was not due to cellular replication. However, hCG increased the number of cells staining positive for 3 beta-HSD about 4-fold in Band 2 cells from 25-day-old rats. Insulin did not increase the number of positive staining cells in Band 2 and Band 3 cells from 25-day-old rats, suggesting that its major effect was to increase enzyme activity in existing cells. These results suggest that during a limited period of maturation precursor cells in Band 2, which are undetected by histochemical staining for 3 beta-HSD, can be converted to Leydig cells in culture by hCG.     

A simple assay of solubilized adenosine receptors with nitrocellulose membrane filters

A rapid and simple assay of solubilized adenosine receptors with nitrocellulose membrane filters is described. This assay was sensitive and reproducible when applied to adenosine receptors solubilized from rat brainstem membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Appropriate values of dissociation constants for the solubilized adenosine receptors to their tritiated agonists were obtained by the membrane filter technique. This method should be applicable for the assay of a variety of solubilized receptors.     

The corpus luteum periodicum in the bitch: histochemical and biochemical investigations during different phases of the cycle.

The corpus luteum periodicum (c.l.p.) of the bitch was studied using enzyme histochemistry, clinical and biochemical methods. Assessment of variations of the serum progesterone level during different stages of the ovarian cycle and the study of enzymatic activity of the luteal tissue furnished informations on vitality and biosynthetic capacity of luteinizing cells: The period of functional activity of the c.l.p. nearly ceased about day 60 of the cycle (proestrus included) in the animals examined. In the light microscope the neo-synthesis of progesterone in the c.l.-tissue can be made visible by histochemical demonstration of the activity of Delta(5)-3 beta-hydroxysteroiddehydrogenase (3 beta-HSD) only up to a progesterone level exceeding 6.7 ng/ml plasma. The data obtained contribute to our baseline knowledge of the mechanism involved in the etiology of pseudopregnancylike conditions in the bitch.     

Hormonal secretion and enzyme activity of cultured roe-deer (Capreolus capreolus L.) Leydig cells, as measured by radio-immunological and histochemical assays

Leydig cells from roe-deer collected according to Steinberger's (1975) technique were cultured as monolayers in Leighton tubes for 10 days. Cultures were grown in medium 199 supplemented with 10% calf serum. Androgen and oestrogen secretion by Leydig cells into the culture medium was measured using appropriate radio-immunoassays. Using histochemical tests the activity of the following oxydoreductive enzymes in cultured Leydig cells was shown: delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), succinate and lactate dehydrogenases (SDH and LDH). Strong activity of the enzymes investigated during the first 4 days of culture was observed. The androgen level was high throughout the second and fourth day of culture. A decrease in hormone secretion after day 4 occurred, and this was closely correlated with enzyme activity. The oestrogen level was very low during culture. The direct effect of the luteinizing hormone (LH) added into the culture medium caused an increase in not only enzyme activity but also androgen and oestrogen levels.     

Development of a High-Throughput Cell-Based Assay for 11β-Hydroxysteroid Dehydrogenase Type 1 Using BacMam Technology

Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11beta-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11beta-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11beta-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11beta-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.     

Cloning and characterization of human form 2 type 7 17beta-hydroxysteroid dehydrogenase, a primarily 3beta-keto reductase and estrogen activating and androgen inactivating enzyme

Type 7 17beta-HSD catalyzes the transformation of estrone (E1) into estradiol (E2) and dihydrotestosterone (DHT) into 5alpha -androstane-3beta,17beta-diol (3beta-diol) as well as zymosterone into zymosterol. This suggests that in addition to cholesterol metabolism, the enzyme could play a critical role in estrogen-sensitive cells, since it inactivates DHT that generally shows antagonistic effect in the cells, while producing active E2 for cell proliferation. In this report, we describe the cloning and characterization of a second form of type 7 17beta-HSD (17beta-HSD7_2) that shares 95.6% identity with 17beta-HSD7_1. Using a 7.5kb genomic DNA fragment of 17beta-HSD7_1 as probe, we have obtained 7 BAC clones: three clones containing the 17beta-HSD7_1 gene and four containing the 17beta-HSD7_2 gene. The corresponding 17beta-HSD7_2 cDNA fragments of the coding region were obtained by amplification using RT-PCR and subcloned into pCMV expression vector and stably transfected into human embryonic kidney (HEK-293) cells. The overexpressed 17beta-HSD7_2 catalyzes efficiently the transformation of E1 into E2 and of DHT into 3beta-diol. Ribonuclease protection assays (RPA) indicate that 17beta-HSD7_2 is expressed in the liver, prostate, uterus and placenta. FISH mapping using the 7.5kb genomic DNA fragment as well as 2 BAC clones of each form allowed us to map the 17beta-HSD7_1 gene on chromosome band 1q23, and 17beta-HSD7_2 on band 10p11.2. These results contrast with a previous report that the 17beta-HSD7_1 gene was mapped to chromosomal band 10p11.2. This newly identified form of 17beta-HSD7 could have a significant role by modulating active hormone levels in estrogen-sensitive cells or tissues.     

A histochemical study on the adrenal components of the teleost Cirrhinus mrigala (Ham.)

The adrenal components of C. mrigala are embedded in the pronephric cephalic kidney around the post cardinal vein. The cortical cells responded positively to the lipids, ascorbic acid, delta 5-3 beta-HSD, G-6-PD, MAO, acid and alkaline phosphatase tests. The presence of intense MAO activity may suggest the possible involvement of monoamines in the adrenocortical function. Localization of lipids and delta 5-3 beta-HSD show the sites of corticosteroid synthesis. In the chromaffin cells, MAO, acid and alkaline phosphatase activity was moderate whereas they gave a strong reaction to ascorbic acid test in comparison to the cortical cells. Noradrenaline (NA) and adrenaline (A) storing cells were differentiated adopting glutaraldehyde silver, dichromate and iodate techniques. NA and A storing cells are almost totally depleted of their contents after reserpine treatment. The histochemical response of the adrenal gland of this species is largely comparable to that of higher vertebrates.