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Analogs of 4-androstene-3,17-dione and testosterone were tested as substrates for the aromatizing enzyme complex of human placenta. Compounds modified in rings B, C and D were found to be aromatized via a pathway similar to that postulated for 4-androstene-3,17-dione and testosterone, in which oxidation to the 19-hydroxy and 19-oxo (or corresponding gem-diol) intermediates occurs. No evidence of additional intermediates was obtained.

Activity of the steroid 7±-hydroxylase which converts androstenedione into 7±-hydroxyandrostenedione was detected in testicular microsomal fraction of adult rats, but no significant activity of the enzyme was observed in those of newborn and immature rats. Administration of gonadotropin or testosterone to adult rats reduced the 7±-hydroxylase activity as well as the 17²-hydroxysteroid dehydrogenase in their testis. By experimental bilateral cryptorchidism, the above two enzyme activities were also reduced on the basis of gland, but their specific activities in the microsomal fraction were increased. 7±-Hydroxyandrostenedione and 7±-hydroxytestosterone showed neither androgenic nor anabolic activity in castrated rats. The level of luteinizing hormone in serum elevated by castration was reduced by administration of 7±-hydroxylated C19-steroids. Activities of the adrenal 11²- and 18-hydroxylases, Δ4-5±-hydrogenase and 3±-hydroxysteroid dehydrogenase, that were significantly influenced by castration, recovere...

Testicular glands of 12-month-old male chicken were homogenized and fractionated by differential centrifugation. After subcellular fractions were incubated with several radioactive steroid precursors, the metabolic products were identified on the basis of the mobilities on the thin-layer chromatogram, chemical reactions, and recrystal-lization with authentic preparations to constant specific activities. Enzyme activities related to androgen formation from pregnenolone were found to exist mainly in the microsomal fraction (10,000–105,000g precipitate). Activities of 20α- and 20β-hydroxysteroid dehydrogenases were exclusively in supernatant fluid at 105,000g. Pregnenolone was transformed to testosterone in the microsome fraction via the following two pathways: through progesterone, 17α-hydroxyprogesterone, and androstenedione; and also through 17α-hydroxypregnenolone, dehydroepiandrosterone, and androstenedione, depending on the cofactors. The biosynthetic pathways to testosterone in chicken testes were discussed in comparison with those of other vertebrates.

The location and some characteristics of rat adrenal C(19)-steroid 5alpha-reductase were investigated by using [7alpha-(3)H]androst-4-ene-3,17-dione and [7alpha-(3)H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3alpha- and 3beta-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17beta-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C(19)-steroid 5alpha-reductase, which showed similar values for K(m) and V(max.) when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5alpha-androstane-3,17-dione and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5alpha-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5alpha-reduction of C(19)- and C(21)-steroids in the rat adrenal.

An exploration of the feasibility of using substrate disappearance as a method of enzyme assay in steroid metabolic rate studies was carried out. In addition the enzymatic capabilities of rat testis microsomes after chronic treatment with large amounts of pituitary hormones or 17 beta-estradiol were investigated. The method of measuring substrate after a reaction proved to be a method of general utility and special applicability when limited to a substrate metabolized via a single pathway. FSH (follicle-stimulating hormone) treatment did not greatly change the specific activity of any of the 3 enzymes investigated. LH (lueinizing hormone) treatment had an inhibitory effect on all 3 enzymes investigated; this may reflect inhibition of endogeneous production of all gonadotropins or formation of antibodies to injected foreign protein. Large doses of estradiol decreased the rates of progesterone disappearance and androstenedione reduction. The desmolase step in the 17 alpha-hydroxy-20-carbonyl cleavage is a rate-limiting step in the latter part of the androgen biosynthetic pathway under a variety of experimental conditions as well as in the normal animal.

When progesterone labeled with one isotope and 17-hydroxyprogesterone labeled with another were incubated together in the presence of an 800 ×g × 10 min supernatant fraction of mouse testis homogenate, the ratio of progesterone label to 17-hydroxyprogesterone label in androstenedione and testosterone was greater than in the original mixture. No evidence of a pathway from progesterone to androstenedione not involving 17-hydroxy progesterone could be found. When the same substrate mixture was incubated with a similar homogenate fraction in the absence of both oxygen and cofactors, very little metabolism occurred. The mitochondrial and microsomal fractions were separated from the incubated mixture and the proportion of the 2 substrates in each was determined. The ratios of the 2 in the particulate fractions resembled those in androstenedione and testosterone under normal incubation conditions rather than the ratio added. Thus, it seems that there is a differential distribution of the 2 steroids into the micr...

The appearance of intermediates and products in the transformation of 4-androstene-3,17-dione and testosterone to estrone and 17β-estradiol by human placental microsomes was studied. The variables investigated were time, temperature and the concentrations of reduced pyridine nucleotides and of testosterone. The relative amounts of metabolites were markedly influenced by all of the factors studied except temperature, although the reaction velocity at 37 °C was about twice that at 27 °C. Oxidoreduction at C-17 in starting material, intermediates and products was examined using a mixture of [4-14C]testosterone and [7β, 17α-2H2]testosterone as substrate. Oxidation and reduction at C-17 was most prominent at the beginning and the end of the reaction sequence. 17α-Hydroxy-5α-androst-1-en-3-one was found to be an inhibitor of the C19 17β-hydroxysteroid dehydrogenase activity of human placental microsomes.

Steroid Δ4-5α-hydrogenase was intracellularly localized in the nuclear and microsomal fractions of the rat ventral prostate (Nozu Sc Tamaoki 1973). The nuclear Δ4-5α-hydrogenase was found to have the following enzymatic properties essentially similar to the microsomal Δ4-5α-hydrogenase, with regard to the metabolism of [4-14C] testosterone in the presence of NADPH: The optimum pH of the enzymes in the two fractions was around 7.0 and the maximal conversion rates were obtained at a temperature of 35 to 40°C. The apparent Km values of the nuclear and microsomal Δ4-5α-hydrogenases were estimated respectively as 1.05 and 0.90 μmol/l, while the Km value of the hepatic microsomal Δ4-5α-hydrogenase was simultaneously estimated as 154 μmol/l. Among steroids, the most potent inhibitor group on the enzymatic 5α-hydrogenation of testosterone was Δ 4-3-oxo-C-21 steroids such as progesterone and 17α-hydroxyprogesterone, which were competitively converted into their 5α-hydrogenated metabolites in the highest rates. Among some anti-androgens, cyproterone and its acetate hardly inhibited the activities of the nuclear and microsomal enzymes, whereas etienic acid (4-androsten-3-one-17β-carboxylic acid), oestradiol-17β and diethylstilboestrol markedly inhibited both prostatic enzymes in a competitive manner. The Ki values of etienic acid, oestradiol-17β and diethylstilboestrol for the nuclear Δ4-5α-hydrogenase were respectively 1.50, 0.49 and 1.02 μmol/l, indicating similar affinities of these for the nuclear enzyme to that of testosterone.

In the cell-free homogenates (supernatant fluid at 800× g) of canine testes, the following Δ5-pathway was suggested as the major biosynthetic route of testosterone in vitro: Pregnenolone → 17α-hydroxypregnenolone → dehydroepiandrosterone → 5-androstenediol → testosterone. This pathway was found to be partly dependent on a predominant cofactor present in the medium. In addition to the androgen formation, 17α,20α-dihydroxy-4-pregnen-3-one and 11 -deoxycortisol were produced in significant yields from 17α-hydroxyprogesterone by the cell-free homogenates. The activities of 17α-hydroxylase, 21-hydroxylase, C17—C20 lyase and 17β-hydroxysteroid dehydrogenase were concentrated in the microsomal fraction (10 000-105 000× g precipitate) of the testicular homogenates. By a time-course study on the metabolism of 17α-hydroxypregnenolone and dehydroepiandrosterone with the testicular microsomal fraction in the presence of NAD and NADPH, testosterone was formed from 17α-hydroxypregnenolone via dehydroepiandrosterone and subsequently 5-androstenediol or androstenedione. The biosynthetic pathways of androgens among vertebrates are discussed from the comparative endocrinological point of view.

Distribution of progesterone-4-14C and 17-hydroxyprogesterone-7-3H between microsomes and sucrose-buffer when equilibrium was achieved without metabolism, and as precursors when metabolism to androstenedione occurred, indicated that the ratio of the two compounds in the rnicrosomes rather than in the medium determined the proportion of the two precursors in the C19 steroids. The isotope ratios in microsomal and supernatant 17-hydroxyprogesterone indicated that the active sites of 17α-hydroxylase and 17,20- lyase were so closely associated that 17-hydroxyprogesterone or a 17-oxy free radical intermediate formed from progesterone did not equilibrate with added 17-hydroxyprogesterone in the microsomes before being converted to androstenedione. No such relationship existed between 17,20-lyase and 17β(testosterone)-dehydrogenase. Relative concentrations of the different compounds, as distinguished from precursor ratios, indicated that 17-hydroxyprogesterone, androstenedione and testosterone distributed like 3H...

17β-Hydroxysteroid dehydrogenase, which catalyzes oxidoreduction between androstenedione and testosterone, has been solubilized by sonication from porcine testicular microsomal membrane for the first time. From the solubilized enzyme preparation, the dehydrogenase activity was precipitated by between 40 and 75% saturation of ammonium sulfate. After the precipitate was dissolved, the enzyme solution was applied to a Sephadex G-100 column and the dehydrogenase activity emerged at 1.80 void volumes. For further purification, it was subjected to DEAE-cellulose column chromatography; by eluting with a linear gradient of increasing phosphate concentration, the enzyme activity between 15 and 20 mM phosphate. Finally, when the partially purified enzyme preparation was applied to a Bio-Gel P-100 column, the dehydrogenase activity emerged at 1.46 void volumes. The final enzyme preparation was purified 1880-fold in specific activity compared to the microsomal fraction. The purified enzyme preparation approached apparent homogeneity, checked by discontinuous polyacrylamide gel electrophoresis at pH 8.3 and by thin-layer gel chromatography. The final preparation was able to produce 67.7 nmol testosterone from androstenedione per min per mg protein at 37°C in the presence of NADPH, and could be stored at–20°C for 6 months with no significant loss of the enzyme activity. The molecular weight and molecular radius of the 17β-hydroxysteroid dehydrogenase was estimated to be about 35500 and 2.37 nm, respectively, from the results of the gel filtration chromatography. In the phosphate buffer pH 7.4 maximal rate of reduction of androstenedione by the enzyme was observed at 50°C, and Q10 for the enzyme was 3.7, while the activation energy was 19500 cal. The rate of reduction of androstenedione at 37.5°C was about a half of the maximal one at 50°C in the phosphate buffer, whereas the reduction rate at 37°C was higher than the one at 50°C in the citric acid-Na2HPO4 buffer pH 7.3. The enzyme for androstenedione showed a pH optimum between 6.5 and 7.5 in the presence of NADPH at 37°C but the optimal pH of the enzyme was shifted to 8.5 by raising the incubation temperature to 50°C. Estrone was the acceptor of the hydrogen released from NADPH by the 17β-hydroxysteroid dehydrogenase and dehydroepiandrosterone served a similar purpose for androstenedione. The oxidation of testosterone by the enzyme in the presence of NADP+ proceeded at the same rate as the reduction of androstenedione. The purified enzyme preparation was devoid of any alcohol or 3-hydroxyhexobarbital dehydrogenase activities or other enzymes related to testicular steroidogenesis.

The in vitro and in vivo effects of some new inhibitors of 3β-hydroxy-Δ5-steroid oxidoreductase have been compared to the effects of the previously known inhibitors, 2α-cyano-4,4,17α trimethyl–5-androsten-17β-ol-3-one (I) and 17β-hydroxy- 4,4,17α-trimethyl–5-androsten-(2,3d)-isoxazole (II). All of the new inhibitors are as potent as I and II in inhibiting the conversion of dehydroepiandrosterone to androstenedione and pregnenolone to progesterone by testicular microsomes. All of the inhibitors, including I and II, also inhibit in vitro C17–20 lyase as manifested by diminished formation of androstenedione and testosterone from 17α-hydroxyprogesterone. in vivo, I, II, III (α, 16α-dicyano-4,4′-dimethyl–5-pregnene-3,20-dione) and VII (2α-cyano-4,4′-dimethyl-2′,3′-tetrahydrofuran- 2′-spiro-17–5-androsten-3-one) alters adrenal and ovarian steroidogenesis with a concomitant excretion of 3β-hydroxy-Δ5-steroidal precursors for at least as long as 7 days after a single dose. The remaining three steroids produce sim...

Earlier studies [8] demonstrated the morphological differences between chorionic villi of central and peripheral regions in placentones. Histochemical and biochemical investigations [9] revealed results corresponding to the morphological findings, that is regionally difFerentiated activity of alkaline phosphatase. They showed that centrally situated villi, which demonstrate a lower degree of maturity than peripherally situated ones, also reacted like less mature villi in respect to activity of alkaline phosphatase. The purpose of this paper is to examine whether enzymes of steroid-biosynthesis also demonstrate regional differences concerning their own specific activities. This concerns, above all, aromatising enzyme Systems and 17s-hydroxysteroid-oxidoreductase. For this reason, we measured the in vitro transformation of 2d-4-androstendione to testosterone and to oestrone/oestradiol-17j8 by microsomes fromtissue of central and peripheral regions of the placentone.

The C19-steroid 5α-reductase activity in the microsomal fraction of rat adrenal tissue under various hormonal treatments was examined. In intact control rats the activity is similar in both males and females, and after gonadectomy it is markedly increased. Treatment with oestradiol (150μg/day per animal for 7 days) or testosterone propionate (2mg/day per animal for 7 days) lowered the activity of 5α-reductase in castrated animals to approximately the values for intact animals in both sexes, and in intact animals the activity was also decreased by these treatments. The enzyme activity was also decreased by adrenocorticotrophin treatment but to a lesser extent than by the steroid hormones. The activity of the 5α-reductase enzyme in the Snell adrenocortical tumour 494 is very low when incubated as a whole homogenate, but the activity in microsomal material of the tumour was measured and unexpectedly found to be similar to that in intact controls.

From the incubation study of androstenedione-4-14C with subcellular fractions of immature rat testes in the presence of NADPH, it was concluded that Δ4-5α-hydrogenase which converted the substrate to 5α-androstanedione was localized in the microsomal fraction (10,000-105,000 ×g precipitate), while 3α-hydroxysteroid dehydrogenase was localized in the supernatant fluid at 105,000 ×g. The Km value of the microsomal Δ4-5α-hydrogenase for androstenedione was estimated as 8.7 × 10−5m. Human chorionic gonadotrophin (30 IU) and/or testosterone propionate (100 μg) were administered daily for 1 and 2 weeks to the immature rats at the 28th day after birth. When the enzymic activities related to testicular transformation of steroids were examined in the cell-free homogenates and in the microsomal fractions, Δ4-5α-hydrogenation of androstenedione and 17α-hydroxyprogesterone and also the side-chain cleavage of 17α-hydroxyprogesterone were severely inhibited by the treatment with testosterone propionate alone. After the...

Unilateral orchidectomy was performed on patients with prostatic cancer without any previous hormonal treatment and then, following the administration of 30 to 100 mg of hexestrol for 21 days, their remaining testes were resected. After incubation of progesterone-4-14C with cell-free homogenates and subcellular fractions of testes resected before the hexestrol administration, 16α-hydroxyprogesterone was obtained as one of the main metabolites. It was identified by comparison with an authentic sample on thin layer chromatogram, through chemical reactions, and by the constant specific activity of crystals obtained by repeated crystallization. Besides 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, and 20α-hydroxypregn-4-en-3-one were obtained as the metabolites, and the sum of the radioactivities of the metabolites and the recovered progesterone was always over 80% of the radioactivity initially added as substrate; no significant amount of any other radioactive prod...

[4- 14 C + 7-D 0.44 ]Androstenedione and [4- 14 C + 7β-D 0.42 ]testosterone were prepared. When they were examined by mass spectrometry, the above proportion of deuterium and protium forms resulted in mass spectra in which the molecular ion (M + ) and (M + + 1) were of equal intensity. Fragment ions that contained deuterium were also twins. When doubly-labeled androstenedione and testosterone were used as substrates for the aromatizing enzymes of human placenta, the mass spectra of metabolites were characteristically labeled and thus readily distinguished from unlabeled material. Metabolites were quantitated by counting 14 C. 17β,19-Dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy-3-oxoandrost-4-en-19-al, 3,17-dioxoandrost-4-en-19-al, estradiol-17β, and estrone were isolated, identified by their mass spectra, and quantitated following incubation of doubly-labeled androstenedione and testosterone with human placental microsomes.

A double isotope derivative method is described for the simultaneous determination of non-conjugated testosterone, androstenedione, and dehydroepiandrosterone in 5–10 ml of human plasma. Essentially the method consists of chromatographic separation of the three androgens, aromatization to estradiol with a microsomal fraction from human term placenta, and halogenation of estradiol with bromine-82 to form its 2,4-dibromo derivative. The latter is acetylated and rechromatographed for additional purification. C 14 -labelled androgens are used to correct for recoveries and Br 82 to obtain the mass. Recoveries for the three androgens have averaged 11 per cent. After subtracting the non-specific water blank (equivalent to 0.1 — 0.2 mμg estradiol) the mean plasma testosterone, androstenedione, and DHEA levels were 620, 98, and 621 mμg% for normal adult males, and 68, 150, and 650 mμg% for normal adult females, respectively. The average androgen levels (mμg%) in three adrenalectomized-ovariectomized female subjects were testosterone: 8.2, androstenedione: 21.1, and dehydroepiandrosterone: 13.6.

Subcellular fractions of testes of rainbow trout (Salmo gairdneri) were prepared by a differential centrifugation, examined under an electron microscope, and incubated with 4-14C-labeled androstenedione, testosterone, 11-deoxycorticosterone, or 11-deoxycortisol at 16°C for 60 min under an aerobic condition. After identification of the metabolites obtained, activities of 11β-hydroxylase, 17α-hydroxylase, and 17β-hydroxysteroid dehydrogenase per unit weight of protein were estimated from the quantities of the metabolites. Among the subcellular fractions, the activity of 11β-hydroxylase upon testosterone, 11-deoxycorticosterone, and 11-deoxycortisol was found concentrated in the mitochondrial fraction, while the activities of 17α-hydroxylase upon 11-deoxycorticosterone and 17β-hydroxysteroid dehydrogenase upon androstenedione and testosterone were localized mostly in the microsomal fraction. Moreover, the 11β-hydroxylase in the rainbow trout testes preferred 11-deoxycorticosterone most and 11-deoxycortisol next as the substrate, whereas testosterone was hydroxylated at 11β-position in low yield and androstenedione was not. By raising incubation temperature from 16°C to 37°C, 11β-hydroxylation of testosterone was not enhanced at all, while the 17β-hydroxysteroid dehydrogenase activity was increased more than two times as much as the activity at 16°C. From the results obtained, the biosynthetic pathway of 11-oxygenated androgens in the teleostean testes were discussed.