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PubMed1992

Regulation of 3-ketosteroid reductase messenger ribonucleic acid levels and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase activity in rat liver by sex steroids and pituitary hormones.

We have recently characterized three types of complementary DNA clones encoding predicted isoenzymes of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family. Transient expression in nonsteroidogenic cells reveals that the type III isoenzyme specific for male liver does not display oxidative activity for classical substrates of 3 beta-HSD, in contrast to the two other 3 beta-HSD isoenzymes, thus showing exclusively 3-ketosteroid reductase (3-KSR) activity. In order to better understand the sex-specific control of 3 beta-HSD activity and type III 3-KSR gene expression in rat liver, we have studied in adult animals of both sexes the effect of sex steroids and hypophysectomy, pituitary implants, PRL, and GH on type III 3-KSR messenger RNA (mRNA) levels and 3 beta-HSD/delta 5-delta 4 isomerase activity as measured by the conversion of [14C]dehydroepiandrosterone into [14C] delta 4-androstenedione. Ribonuclease protection assay using types I-, II-, and III-specific complementary RNA probes reveals that type III transcripts are the only species detectable in liver RNA extracted from intact males, whereas no hybridization signal was detectable with any of the three probes in intact female liver RNA. In males, 15 days after castration, liver type III 3-KSR mRNA levels decreased by 80% compared to intact controls, whereas 3 beta-HSD activity was reduced by 48%. Administration of dihydrotestosterone (DHT) increased by 8.25-fold type III 3-KSR mRNA concentration and completely reversed the inhibitory effect of orchiectomy on 3 beta-HSD activity. In ovariectomized animals, treatment with DHT markedly increased type III 3-KSR mRNA accumulation and 3 beta-HSD activity, thus leading to values similar to those measured in intact males. Simultaneous treatment with 17 beta-estradiol almost completely abolished the stimulatory effect of DHT in female rats, whereas no significant effect was seen in males. Twenty-four days after hypophysectomy, type III 3-KSR mRNA levels were decreased by 50-55% in males, whereas in females these transcripts markedly increased from undetectable to 28-36% of the value measured in intact male rats. Treatment with DHT or 17 beta-estradiol for a period of 9 days starting 15 days after hypophysectomy had no effect in male and female rats. On the other hand, treatment with ovine PRL (1 mg, twice daily) had no effect in males but completely blocked the elevation of type III 3-KSR mRNA levels and 3 beta-HSD activity observed after hypophysectomy in females.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1999

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low-energy collision-induced dissociation to produce informative spectra.

The use of collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) has been shown to produce fragmentation that is useful for the structural analysis of bile acids and their conjugates. Low-energy CID using a triple quadrupole has been used to help characterise bile acid identity but the majority of work has been conducted using high-energy CID on specialised instrumentation. This paper describes the use of low-energy CID as a rapid method for identification of urinary bile acids and presents some examples of its use in the diagnosis of liver disease in infants. These include the differential diagnosis of peroxisomal disorders, identification of compounds (e.g. 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulphate) indicative of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase deficiency and the confirmation of the identity of an unusual bile acid series consisting of different conjugates of lithocholic acid. The use of lithium cationisation and derivatisation with aminosulfonic acids for the analysis of unconjugated and glycine-conjugated bile acids has also been evaluated.

Bile Acids and Salts↗
PubMed2000

Generation of stable cell lines by spontaneous immortalization of primary cultures of porcine granulosa cells.

We report the generation of stable cell lines obtained by spontaneous immortalization of primary cultures of porcine granulosa cells. Three hundred stable cell lines were obtained from three independent immortalization trials. Two of these cell lines retained the steroidogenic capabilities characteristic of granulosa cells, such as de novo synthesis of progesterone and conversion of androstenedione into estradiol-17beta. All the stable cell lines expressed the P450arom and 3betaHSD genes, confirming their granulosa origin. Moreover, the steroidogenic stable granulosa cells also expressed StAR and P450scc genes. Stable cells were developed in cultures using Medium 199 supplemented with 5% newborn calf serum (NBCS). The surviving cells overcame the senescent phase and entered a stage of continuous growth for over one hundred generations. No stable colonies were obtained from cultures grown in MEM or DMEM or media supplemented with 10% NBCS or 5 and 10% fetal calf serum (FCS). Medium 199 is a formulation richer in nutrients compared to MEM or DMEM and the cell growth capability of NBCS is lower than that of FCS, probably due to deficiency of growth factors. We speculate that spontaneous immortalization of granulosa cells may be facilitated by using a rich culture formulation supplemented with low concentrations of serum deficient in growth factors. We have validated the stable cell lines for studying the effect of hormonal steroids on granulosa cell steroidogenesis and the expression of the steroidogenic genes. Therefore, we believe that they are useful models to study the molecular mechanism involved in granulosa cell differentiation and steroidogenesis.

Animals↗
PubMed2001

Expression of connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development.

Connexin 43 (Cx43) is the most abundant and ubiquitously distributed gap junction protein in testicular cells. Lack of Cx43 expression results in male infertility. We investigated whether Cx43 is expressed and regulated in Leydig, Sertoli and germinal cells at different stages of postnatal development. Cx43 was detected using three different antibodies shown by immunoblotting to be highly specific. At different postnatal ages Cx43 localization was compared in serial or double labeled testicular cryosections with immunocytochemical distribution of steroidogenic enzyme, 3 betahydroxysteroid-dehydrogenase (3betaHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GNCA1), which are specific markers of interstitial Leydig, Sertoli and germinal cells, respectively. In the interstitium, round cell clumps (RCC) with lipid droplets positive for 3betaHSD and Cx43 were frequently found at intertubular areas at birth and Cx43 was mainly localized at cell membrane appositions. From day 3, the number and size of 3betaHSD-positive RCC started to decrease, and reached a minimum at 7-14 dpp; Cx43 expressed by them is progressively downregulated. From day 21 an increase in the size and number of RCC positive for Cx43 and 3betaHSD was found that continued at 24, 26 and 28 days and reached a maximum at 35 and 60 dpp. Biphasic expression of interstitial Cx43 and 3betaHSD was also found to be positively and temporally correlated with fluctuations in intratesticular testosterone content at all ages studied. In the seminiferous cord (SC), Cx43 was expressed at birth between adjacent Sertoli cells (MIH positive) localized at the periphery, as well as in their cytoplasm projections that surround centrally localized gonocytes. From days 3 to 7, Cx43 labeling increased in Sertoli cells mainly at their apical border. At day 14, Cx43 distribution in Sertoli cells changed from apical to basal in parallel to migration of germinal (GNCA1-positive) cells from the periphery to the center of the SC. At all these ages, Cx43 was also localized at cell borders between Sertoli and germinal cells. In conclusion, this study demonstrates that Cx43 in Leydig cells is regulated during postnatal development in an age and functional dependent manner. In the tubule, it is demonstrated that Cx43 is modulated in Sertoli cells during the neonatal and prepubertal period. We also provide evidence for the first time that Cx43-gap junctions communicate between Sertoli and germinal cells before and during the first wave of spermatogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

A colorimetric assay method for 3beta-hydroxy-delta5-steroid dehydrogenase.

3beta-Hydroxy-Delta5-steroid dehydrogenase is an important enzyme of steroid hormone biosynthesis present in steroidogenic tissues like adrenal, testis, and ovary of vertebrates. The enzyme is assayed mainly by radiochemical substrates. Spectrophotometric assay is not adequately sensitive to detect the enzyme activity since it is often present in low levels. We have developed a simple colorimetric assay based on formazan formation due to the reduction of the tetrazolium salt. The reaction mixture containing the substrate, pregnenolone or dehydroepiandrosterone, NAD and iodonitrotetrazolium in 0.1 M Tris-HCl buffer (pH 7.8), and the enzyme extract is incubated for 1 h at 37 degrees C. Absorbance at 490 nm is read in a spectrophotometer. The enzyme activity was linear with time and protein concentration. The assay works well with adrenal tissue extract, whereas in the case of testis, Leydig cell preparation may be required. We have assayed the enzyme activity in the adrenal of rat, mouse, and gerbil. The method is two- to threefold more sensitive than the spectrophotometric assay.

Adrenal Glands↗
PubMed1996

Mitochondrial localization of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase in interrenals of the toad Bufo arenarum H.

The enzymatic activity of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones including progesterone, mineralocorticoids, glucocorticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal. The present study demonstrates that in the interrenal of Bufo aernarum H., 3 Beta HSD/I activity localizes in mitochondria and micromes. It also shows that the two distinct pathways to aldosterone previously demonstrated for interrenals of B. arenarum H. exhibited differential subcellular localizations, microsomal for the 4-ene route and mitochondrial for the 5-ene route. Kinetic constants of 3 Beta HSD/I were determined for the oxidation of pregnenolone and the recently described 3 Beta-hydroxy analogue of aldosterone (3 Beta AA). The preferred substrate of the mitochondrial 3 Beta HSD/I enzyme was 3 Beta AA (Km = 0.7 microM and 14.0 microM for 3 Beta AA and pregnenolone, respectively). However, the microsomal enzyme has a greater affinity for pregnenolone (Km = 0.8 microM) than for 3 Beta AA (Km = 17.0). Enzymes from both localizations have similar nucleotide (NAD+) requirements, activities being higher in summer. This dual localization opens novel possibilities for the regulation of interrenal functions.

Aldosterone↗
PubMed1997

Subcellular localization of 3 beta hydroxysteroid dehydrogenase isomerase in testis of Bufo arenarum H.

3 Beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones and is usually considered to be mainly microsomal, although there is a dual distribution of the enzyme in toad interrenals. The present study demonstrates that in the testicular tissue, as in interrenals of Bufo arenarum H., 3 beta HSD/I is both mitochondrial and microsomal. The conversion of dehydroepiandrosterone to androstenedione takes place only in microsomes while pregnenolone is converted to progesterone in both microsomes and mitochondria. Kinetic constants of 3 beta HSD/I were determined by the oxidation of pregnenolone and dehydroepiandrosterone. The preferred substrate of the microsomal 3 beta HSD/I enzyme was dehydroepiandrosterone (K(m) = 0.17 microM and 0.53 microM for dehydroepiandrosterone and pregnenolone, respectively) not only during the breeding season but also in the non-breeding period (K(m) = 0.49 microM and 2.9 microM for dehydroepiandrosterone and pregnenolone, respectively).

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1999

Thyroid hormone regulation of perch ovarian 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase activity: involvement of a 52-kDa protein.

Ovarian follicles were collected from perch belonging to the prespawning (vitellogenic) stage and incubated in vitro for 5 h in the absence (control) and presence of 3, 5, 3'-triiodothyronine (T3). Addition of increasing concentrations of T3 from 12.5 to 100 ng/ml caused a linear increase of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3 beta-HSD) activity to 50 ng and then it leveled off indicating a saturation of enzyme activity with 50 ng T3. T3 stimulation of 3beta-HSD activity could be blocked by cycloheximide indicating the involvement of T3-induced protein (TIP) isolated and purified earlier from this laboratory. Addition of fish TIP purified from perch ovarian follicle (fTIP) or rat granulosa cell TIP to ovarian follicular incubation at a dose of 5 microg/ml significantly increased (P < 0.01) 3beta-HSD activity. To observe whether TIP acts directly on the enzyme or not, 3beta-HSD from perch ovarian follicle was purified to homogeneity by the following steps: (i) Sephadex G 75 gel filtration, (ii) DEAE-Sephacel chromatography, and (iii) NAD-affinity column chromatography. Purified 3beta-HSD gave a clear single band on an SDS gel and its molecular weight is 45 kDa. Addition of fTIP to an assay mixture containing purified 3beta-HSD resulted in a fourfold increase of the enzyme activity. fTIP alone did not show enzyme activity when incubated with the radiolabeled substrate. Addition of T3 (50 ng) to the 3beta-HSD assay mixture had no effect on the enzyme activity. Determination of Vmax and Km of the purified enzyme in the absence (control) and presence of fTIP demonstrated a considerable increase of 3beta-HSD affinity and rate of enzyme reaction.

Animals↗
PubMed1993

The genes encoding gonadal and nongonadal forms of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase are closely linked on mouse chromosome 3.

The biosynthesis of steroid hormones in the gonads and adrenal glands requires the activities of the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) which catalyzes the NAD(+)-dependent dehydrogenation and subsequent delta 5-->delta 4 isomerization of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The mouse expresses four isoforms of 3 beta HSD. 3 beta HSD I is expressed in gonads and adrenal glands and appears to be the major steroidogenic form, 3 beta HSDs II and III are expressed in both liver and kidneys, and 3 beta HSD IV has been detected only in kidneys. To determine the genetic relationship between the 3 beta HSD isoforms, we have mapped the chromosomal locations of the four genes by linkage analysis using gene-specific probes derived from the 3' untranslated regions of 3 beta HSD cDNA clones. The four 3 beta HSD structural genes (Hsd3b) are closely linked within a segment of chromosome 3 that is conserved on human chromosome 1. The order of markers on Chr 3 surrounding Hsd3b is: centromere-Gba-(4.4 +/- 2.2)-Hsd3b-(3.3 +/- 1.9)-Tshb-(6.7 +/- 2.7)-Amy-1.

Animals↗
PubMed1998

Activities of aromatase and 3beta-hydroxysteroid dehydrogenase/delta4-delta5 isomerase in whole organ cultures of tissues from developing zebra finches.

The hormonal basis for masculine song development in the zebra finch remains unidentified. To understand how steroids are differentially supplied to the brains of males and females to cause sexually dimorphic development of this behavior, we have studied the steroidogenic capability of zebra finch tissues during early development (1 to 8 days posthatching). Here, we report on the use of cultures of whole gonads, adrenals, and telencephalons to measure the activities of two steroidogenic enzymes: aromatase, the enzyme that catalyzes the conversion of androgen to estrogen, and 3beta-hydroxysteroid dehydrogenase/delta4-delta5 isomerase (3beta-HSD), the enzyme that converts pregnenolone into progesterone. We also examined the effect of cAMP on aromatase activity in these tissues as this intracellular second messenger has been shown previously to regulate aromatase in both central and peripheral tissues of other species. In untreated cultures, aromatase was detected at the highest levels in male and female telencephalon and in ovary. Dibutyryl (dB)-cAMP had no significant effect on aromatase activity in any tissue. However, after dB-cAMP treatment, estrogens were regularly detected in cultures of whole testes. Although this activity was relatively low when compared to total activity found in other tissues, due to the small size of the testes at this age of development, the specific activity (per milligram of protein) might be high enough to produce some estrogen. Adrenal aromatase was unconfirmed in the presence or absence of cAMP. 3Beta-HSD activity was undetected in brain but was detected in gonads and adrenals from all birds. There were no significant differences in gonadal or adrenal 3beta-HSD activity between males and females. Although these data present the first evidence for testicular aromatase in the zebra finch, they provide no evidence to support a mechanism to generate a greater estrogenic signal in male zebra finches after hatching.

Adrenal Glands↗
PubMed2001

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.

Androstadienes↗
PubMed1991

Detection of frequent BglII polymorphism by polymerase chain reaction and TaqI restriction fragment length polymorphism for 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase at the human HSD beta 3 locus (1p11-p13).

Analysis of amplified polymerase chain reaction products of 575 bp from the fourth exon of the human type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase gene at locus HSD beta 3 1p11-p13, reveals a frequent two-allele polymorphism at codon Leu338 due to a silent substitution of T by C, thus creating a BglII site leading to 371- and 204-bp fragments. Southern blot analysis of BglII-digested DNA from 57 individuals using a genomic probe detects two allelic fragments of 5.3 kb and 0.77 kb, respectively, while two allelic fragments of 3.7 kb and 3.4 kb are obtained in TaqI digests with multiple constant bands, as also observed with BglII digests.

Bacterial Proteins↗
PubMed1993

Chick gonadogenesis following early surgical bursectomy. I. Histological, morphometric and histochemical data on the embryonic left ovary.

Left ovaries of bursectomized chick embryos were examined on the 17th day of incubation in comparison to normal and sham-operated controls, by histological and histochemical observations. The results show that in bursectomized embryos the cortex appears irregularly developed, with a significant decrease in the mean thickness and in the percentage of the secondary sex cords in the total cortical area. Furthermore, the germinal epithelium appears thicker and the subcortical medulla and the tunica albuginea more compact. The greater activity of the enzyme delta 5-3 beta-hydroxysteroid dehydrogenase (delta (5)3 beta HSD) found in ovaries of bursectomized embryos (histochemical method) could be related to an endocrine dismetabolism affecting the cortical development. On the basis of these results and those of other authors, some hypotheses are advanced. In particular, an action of the bursal factor on GTH receptors could be the factor responsible of the enhanced steroidogenic activity altering the hormonal environment.

Animals↗
PubMed1977

Ultracytochemical demonstration and probable localization of 3beta-hydroxysteroid dehydrogenase activity with a ferricyanide technique.

In order to localize 3beta-hydroxysteriod dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3beta-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium. A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. the addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.

Adrenal Glands↗
PubMed1988

Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells.

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed1976

Studies on the testis of the camel (Camelus dromedarius). III. Histochemical observations.

The histochemical localization of carbohydrates and lipids and some oxidative, hydrolytic and steroid-linked enzymes has been studied in the testis of the camel with particular reference to the effect of the season on the distribution of these substances. PAS-positive, but diastase-resistant, material was seen mainly in the wall of blood vessels and in the boundary tissues of the seminiferous tubules, tubuli recti and rete testis. Clear cyclical changes were seen for glycogen in the lining epithelium of the seminiferous tubules. Glycogen was most abundant in early stages and very scanty or absent in the late stages of the cycle of the seminiferous epithelium. Numerous small lipid droplets were seen in the interstitial cells and towards the lumen of the seminiferous tubules that contain elongate spermatids or spermatozoa. Large lipid droplets were also demonstrable in the basal layer of the seminiferous epithelium and in the cytoplasmic debri. Alkaline phosphatase was demonstrated in the boundary tissues of the seminiferous tubules, tubuli recti and reti testis and in the cells bordering the lumen of the seminiferous tubules. Succinate and lactic dehydrogenases showed similar patterns of distribution in the interstitial elements and intratubularly. delta5-3beta hydroxysteroid dehydrogenase was exclusively demonstrated in the interstitial cells. 17beta-hydroxysteroid dehydrogenase could not be demonstrated. The season seems to have no effect on the distribution of all these substances. The possible significance of all these findings is discussed.

Alkaline Phosphatase↗
PubMed1976

Steroid dehydrogenases in the adrenal gland of four species of birds: a histochemical study.

The presence of 5-3deltabeta-hydroxysteroid dehydrogenase, IIbeta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase has been demonstrated histochemically in the adrenal gland of the rain quail Coturnix coromendalica, barn owl Tyto alba, brown crake Amaurornis akool and painted partidge Francholinus pictus. All these enzymes occurred in the inter-renal cells. No activity was observed in the chromaffin cells. It is suggested that the inter-renal cells of these four species of birds are capable of synthesizing both corticosteroids and sex steroids.

Adrenal Glands↗