Effect of 5-thio-D-glucose on testosterone biosynthesis in vivo in mice testes

Testicular synthesis of (14C)cholesterol and (14C)testosterone from (14C)acetate were investigated in mice treated with 5-thio-D-glucose at a dose of 33 mg/kg body weight/day for 21 days. The testicular synthesis of free cholesterol as well as steroids were significantly decreased. The steroid synthesizing enzymes, cholesterol esterase, cholesterol side-chain cleaving enzyme, total alpha-hydroxysteroid dehydrogenase and total beta-hydroxysteroid dehydrogenase, were also analysed. Cholesterol esterase and total beta-hydroxysteroid dehydrogenase were significantly reduced whereas total alpha-hydroxysteroid dehydrogenase was unaffected. Hence, a decrease in free cholesterol for steroid synthesis and a decreased activity of the steroidogenic enzyme, beta-hydroxysteroid dehydrogenase, were responsible for the diminished synthesis of testosterone.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.     

Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-delta7-reductase deficiency (Smith-Lemli-Opitz syndrome)

Patients with Smith-Lemli-Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-delta(7)-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of delta(7) and delta(8) unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites. Gas chromatography/mass spectrometry (GC/MS) was used in the identification of two major metabolites as 7- and 8-dehydroversions of the well-known steroid pregnanetriol. Other steroids, such as 8-dehydro dehydroepiandrosterone (8-dehydro DHEA) and 7- or 8-dehydroandrostenediol were also identified, and several more steroids are present in urine but remain uncharacterized. As yet, the study provides no evidence for the production of ring-B unsaturated metabolites of complex steroids, such as cortisol. We believe that the following transformations can utilize ring-B dehydroprecursors: StAR transport of cholesterol, p450 side chain cleavage, 17-hydroxylase/17,20-lyase, 3beta-hydroxysteroid dehydrogenase, 3alpha-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 5beta-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11beta-hydroxylase or 5alpha-reductase towards 7- or 8-dehydroprecursors.     

Control of ovarian cholesterol ester biosynthesis

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.     

Activities of enzymes responsible for steroid biosynthesis and cholesterol ester metabolism in rabbit ovarian interstitial tissue and corpora lutea. A comparison of enzyme activities with flow rates

A method involving the use of isolated cholesterol ester-storage granules as substrate is described for the assay of cholesterol esterase in rabbit ovarian tissues. Activities of cholesterol esterase 100-200-fold higher than those previously reported in ovarian tissues were measured by using this method. In addition to that of cholesterol esterase, activities of cholesterol ester synthetase, cholesterol side-chain cleavage enzyme and 3beta-hydroxy steroid dehydrogenase were determined in rabbit ovarian interstitial tissue and corpora lutea. Activities of these enzymes are in general compatible with the flows through them measured under a variety of conditions both in vivo and in vitro. It is concluded that, in the rabbit ovarian tissues investigated, these enzymes are capable of catalysing the conversions usually attributed to them.     

The 20 alpha-hydroxysteroid dehydrogenase of Streptomyces hydrogenans

In addition to the well-known 3 alpha,20 beta-hydroxysteroid dehydrogenase ('cortisone reductase'), Streptomyces hydrogenans produces a relatively stable, NAD-dependent 20 alpha-hydroxysteroid dehydrogenase of molecular mass approximately 48 kDa. This enzyme catalyzes the transfer of hydrogen from the 4-pro-S position of NADH.     

Effect of allylisopropylacetamide and Sedormid on enzymes of steroid metabolism in rat liver

Female rats, treated with allylisopropylacetamide (AIA) showed a marked decrease of hepatic NADH-5 alpha-reductase, NADPH-5 alpha-reductase, NAD+- and NADP+-3 alpha-hydroxysteroid dehydrogenase activities and an increase of the activity of NADH- and NADPH-5 beta-reductase and NAD+ and NADP+-3 beta-hydroxysteroid dehydrogenase. Administration of Sedormid decreased the activities of 5 alpha-reductases and 3 alpha-hydroxysteroid dehydrogenases (substrate, 5 alpha-dihydrotestosterone) and increased the activity of NADH-5 beta-reductase, whereas no effect was seen on NADPH-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase.     

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3 alpha/beta-, 20 alpha- and 20 beta-): evidence for two forms of 3 alpha/beta-hydroxysteroid dehydrogenase.

Three enzymatic activities (3 alpha/beta-hydroxysteroid dehydrogenase, 20 beta- and 20 alpha-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20 beta-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3 alpha/beta-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787-794]. We report here that neonatal pigs testis is rich in 3 alpha/beta- and 20 beta-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of 3 alpha/beta-reduction rises to high levels whereas 20 beta-reduction remains low. Activity of 20 alpha-reduction is of intermediate level in the neonate, falls to a nadir at 60 days and rises to high levels in the mature animal. Western blots of cytosolic proteins show that the bifunctional enzyme (3 alpha/beta-plus 20 beta-hydroxysteroid dehydrogenase) is high in neonatal testes and falls to low levels at maturity. It is proposed that the neonatal testis possesses the bifunctional enzyme which is replaced by a second enzyme at maturity, that is a 3 alpha/beta-hydroxysteroid dehydrogenase without 20 beta-reductase activity. The possible functional significance of these changes is considered.     

Inhibition of pyridine-nucleotide-dependent enzymes by pyrazoles. Synthesis and enzymology of a novel A-ring pyrazole steroid

A novel A-ring pyrazole steroid, 2,3-bisaza-A-nor-1,5(10)-estradien-17 beta-ol (3), was synthesized as a potential inhibitor of steroidal NAD(P)H-dependent oxidoreductases. Compound 3 proved to be a potent inhibitor of 3(17)beta-hydroxysteroid dehydrogenase (from P. testosteroni) exhibiting a Ki of 90 +/- 20 nM. The activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase (from S. hydrogenans), steroid-5 alpha-reductase (from rat prostate), and 3 alpha-hydroxysteroid dehydrogenase (from rat liver) were unaffected by pyrazole 3. Dead end inhibition studies indicate an ordered binding of cofactor prior to substrate or pyrazole inhibitor.     

Molecular cloning of a novel type of rat cytoplasmic 17beta-hydroxysteroid dehydrogenase distinct from the type 5 isozyme

Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD+ as the preferred coenzyme at pH 7.4, and showed low activity towards 20alpha- and 3alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3alpha-hydroxysteroid dehydrogenase.     

The role of the gonads and the hypophysis in the regulation of hydroxysteroid dehydrogenase activities in rat kidney.

With the exception of 3beta-hydroxy-steroid dehydrogenase all the hydroxysteroid dehydrogenases of adult male and female rat kidney show significant sex differences in their activities. Interference with the organisms endocrine balance (gonadectomy on day 25 of life, hypophysectomy on day 50, a combination of both these operations, administration of testosterone or oestradiol) demonstrates that the sexually differentiated enzyme activities may be classified as androgen or oestrogen dependent, the respective sex hormone acting either in an inductive or repressive manner. The criteria for androgen dependency (microsomal 3alpha- and 20beta-, cytoplasmic 17beta- and 20alpha- hydroxysteroid dehydrogenase) are the feminization of the enzyme activity in male animals after castration and the masculinization of the activity in male and female castrates as well as in normal female animals after administration of testosterone. This latter effect on normal females cannot be a testosterone mediated inhibition of ovarian function since ovariectomy has no effect. For 3alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase the effects of hypophysectomy parallel those of gonadectomy. However, after hypophysectomy the activity of 17beta-hydroxysteroid dehydrogenase falls significantly below the gonadectomized level. The androgen effect on 3alpha and 20beta-hydroxysteroid dehydrogenase is independent of the hypophysis, whereas that of 17beta- and 20alpha-hydroxysteroid dehydrogenase is mediated by the hypophysis.     

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.     

Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7 beta-hydroxysteroid dehydrogenase

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7 beta-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7 beta-hydroxyl groups were quantified using 7 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxyl groups (total bile acids) were quantified using 3 alpha-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7 beta-hydroxyl groups]/[3 alpha-hydroxyl groups] X 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7 beta-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8-24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.     

Characterization of NADP+: 3 beta-hydroxysteroid dehydrogenase from microsomes of rat liver

A NADP(+)-dependent 3 beta-hydroxysteroid dehydrogenase activity was localized in the microsomal fraction of rat liver. This enzyme was solubilized and separated completely from 3 alpha-hydroxysteroid dehydrogenase by Matrex red A column chromatography. Partially purified 3 beta-hydroxysteroid dehydrogenase catalyzed the oxidation and reduction between the 3 beta-hydroxyl and 3-ketonic group of steroids or bile acids having no double bond in the A/B ring, but was inactive toward 3 alpha-hydroxyl group. The enzyme required NADP+ for oxidation and NADPH for reduction. The activity was inhibited by p-chloromercuribenzoic acid or p-chloromercuribenzenesulfonic acid at the concentration of 10(-4) M. The molecular weight of the enzyme was estimated to be about 43,000 by Sephadex G-200 column chromatography. From these results, it is concluded that the enzyme is a new type of microsomal NADP+:3 beta-hydroxysteroid dehydrogenase.     

Enzyme histochemistry of cultured ovarian cells. II. Histochemistry of porcine theca interna cells in tissue culture.

By means of histochemical technique the activities of delta53beta-, 17beta-, 20alpha-hydroxysteroid dehydrogenases and glucose-6-phosphate dehydrogenase, as well as alkaline and acid phosphatase were investigated in monolayer cultures of theca interna cells, isolated from preovulatory porcine ovarian follicles. It was found that theca interna cells exhibited high and constant activity of delta53beta-hydroxysteroid dehydrogenase and G6P-DH, whereas activities of both 17beta- and 20alpha-hydroxysteroid dehydrogenase were lower and showed some fluctuations during in vitro culture. Addition of LH to the medium brought about the increase of all studied dehydrogenases. FSH was less effective. Estradiol showed quite and inhibiting effect. All the hormones mentioned above caused the increase of alkaline and acid phosphatase activity in cultured porcine theca interna cells.     

Molecular characterization of the cynomolgus monkey Macaca fascicularis steroidogenic enzymes belonging to the aldo-keto reductase family

Steroidogenic enzymes belonging to the aldo-keto reductase family (AKR) possess highly homologous sequences while having different activities. To gain further knowledge about the function as well as the regulation of these enzymes in the monkey, we have isolated cDNA sequences encoding monkey type 5 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 3alpha-hydroxysteroid dehydrogenase, and characterized their enzymatic activity and mRNA tissue distribution. Sequence analysis indicates that these enzymes share approximately 94 and 76% amino acid identity with human and mouse homologs, respectively. Monkey type 5 17beta-HSD possesses 95.9% amino acid sequence identity with human type 5 17beta-HSD. It catalyzes the transformation of 4-androstenedione into testosterone, but it lacks 20alpha-hydroxysteroid dehydrogenase activity that is present in the human enzyme. This activity seems to be specific to human, since mouse type 5 17beta-HSD does not show significant 20alpha-HSD activity. In addition, monkey and mouse 20alpha-HSD possess relatively high 20alpha-, 3alpha-, and 17beta-HSD activities, while their human counterpart is confined to 20alpha-HSD activity. The monkey 3alpha-HSD possesses relatively high 3alpha-, 17beta-, and 20alpha-HSD activities; human type 1 3alpha-HSD exerts 3alpha- and 20alpha-HSD activities; the mouse 3alpha-HSD displays a unique 3alpha-HSD activity. Quantification of mRNA expression shows that the monkey 3alpha-HSD is exclusively expressed in the liver, while the type 5 17beta-HSD is predominately found in the kidney, with lower levels observed in the stomach, liver, and colon. Monkey 20alpha-HSD mRNA is highly expressed in the kidney, stomach, and liver. Our study provides the basis for future investigations on the regulation and function of these enzymes in the monkey.     

Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase.     

Characterization of a monoclonal antibody for human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase); immunohistochemical detection in breast and prostate

Human aldo-keto reductase AKR1C3 (type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase) catalyzes the reduction of Delta(4)-androstene-3,17-dione to yield testosterone, the reduction of 5alpha-dihydrotestosterone to yield 3alpha- and 3beta-androstanediol, and the reduction of estrone to yield 17beta-estradiol. Relatively, high mRNA expression of AKR1C3 was found in human prostate and mammary gland where it is implicated in regulating ligand access to the androgen and estrogen receptor, respectively. AKR1C3 shares high sequence identity >86% with related plastic human 20alpha-hydroxysteroid dehydrogenases (AKR1C1), type 3 3alpha-hydroxysteroid dehydrogenase (AKR1C2) and type 1 3alpha-hydroxysteroid dehydrogenase (AKR1C4), and reagents are urgently needed to discriminate between these enzymes at the mRNA, protein and functional level. We describe the characterization of a high-titer isoform specific monoclonal antibody (Ab) for AKR1C3. It does not cross react with human AKR1C1, AKR1C2 or AKR1C4, human aldehyde reductase AKR1A1 or rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9) on immunoblot analysis. The monoclonal Ab can be used to detect AKR1C3 expression by immunohistochemistry in sections of paraffin-embedded mammary gland and prostate. In the breast enzyme staining was detected in ductal carcinoma in situ where the cancerous cells were strongly immunoreactive. In normal prostate immunoreactivity was limited to stromal cells with only faint staining in the epithelial cells. In adenocarcinoma of the prostate elevated staining was observed in the endothelial cells and carcinoma cells. The reagent thus has utility to access the localized expression of AKR1C3 in hormonal dependent malignancies of the breast and prostate.     

The hypophysis in the regulation of androgen and oestrogen dependent enzyme activities of steroid hormone metabolism in rat liver cytosol.

In order to determine whether the gonadal and hypophyseal modes of regulation recently reported for the microsomal enzymes of hepatic steroid metabolism are also valid for cytoplasmic enzymes, three enzymes whose activities exhibit sex differences (male:female activity ratio shown in brackets), 5beta-reductase(1.7:1), 20alpha-hydroxysteroid dehydrogenase(5 : 1) and 17beta-hydroxysteroid dehydrogenase (4:1), as well as one enzyme whose activity shows no sex difference, 3beta-hydroxy-delta5-steroid dehydrogenase, were investigated after various interferences with the endocrine balance (gonadectomy, hypophysectomy, combination of both operations, administration of testosterone or oestradiol). From the results of this and a previous study the following statements can be made about the endocrine control of hepatic enzyme activities. Those enzymes whose activities show sex differences are either androgen or oestrogen dependent; the sex hormone acts in either an inductive or repressive manner. 1) Criteria for androgen dependency are the feminization of enzyme activity after testectomy or inhibition of testicular function by administration of oestradiol; masculinization of the enzyme activity after administration of testosterone to male or female castrates. Using these criteria the following enzymes investigated in this laboratory fall into this category: all microsomal enzymes which show sex differences in their activity (3alpha-, 3beta-, delta4-3beta, 20-hydroxysteroid dehydrogenase; cortisone alpha-reductase; steroid hydroxylases and 16alpha-hydroxylase) as well as the cytoplasmic 20alpha-hydroxysteroid dehydrogenase. Apart from the single exception of 20alpha-hydroxy-steroid dehydrogenase the presence of the hypophysis is obligatory for the androgen to be effective. The hypophysis does not only work in a permissive manner, but participates in establishing the sex specific activity levels in a manner which is antagonistic to the androgen action. 2) Criteria for oestrogen dependency are that the female animal reacts to gonadectomy, as well as to the inhibition of ovarian function after testosterone administration, by a masculinization of the enzyme activities. After administration of oestradiol, but not gonadectomy, the male animal exhibits typical female activity. Using these criteria the cytoplasmic 5beta-reductase and 17beta-hydroxysteroid dehydrogenase are oestrogen dependent. The repressive oestrogen effect observed on 17beta-hydroxysteroid dehydrogenase is antagonistic to hypophyseal action, whereas in the case of 5beta-reductase it is synergistic. 3) The activities of cytoplasmic 3beta-hydroxy-delta5-steroid dehydrogenase and microsomal 7alpha-hydroxylase show no sex differences and are not influenced by any interference with the endocrine balance.     

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.