Kinetic mechanism of reduction of testosterone by hepatic 5 beta-reductase of chicken and inhibition of the reductase activity by a secosteroid, an azasteroid and glycyrrhetinic acid.

The present studies on initial velocity of testosterone reduction by hepatic 5 beta-reductase (4-en-3-oxosteroid 5 beta-reductase) of chicken and mode of inhibition of the 5 beta-reduction by 5 beta-dihydrotestosterone and NADP+ indicated that the reduction of testosterone occurred after the 5 beta-reductase bound firstly to NADPH and then to testosterone, forming a ternary complex. After 5 beta-reduction, 5 beta-dihydrotestosterone and then NADP+ were liberated from the complex, following a mechanism of "ordered Bi-Bi". Effect of (4R)-5,10-seco-19-norpregna-4,5-diene-3,10,20-trione (a steroidal 5 alpha-reductase-inhibitor or Secosteroid), diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (the other steroidal 5 alpha-reductase-inhibitor or 4-MA), and glycyrrhetinic acid (3 beta-hydroxy-11-oxoolean-12-en-30-oic acid, a 5 beta-reductase-inhibitor) was examined upon the 5 beta-reductase activity by double reciprocal plots. The mode of inhibition against testosterone by 4-MA and glycyrrhetinic acid was found to be competitive, while that by Secosteroid was non-competitive.

Purification of 5 beta-reductase from hepatic cytosol fraction of chicken.

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5 beta-reduced metabolites derived from [4-14C]testosterone. During the purification procedures, 17 beta-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5 beta-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were able to be removed from 5 beta-reductase fraction. The final enzyme preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5 beta-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. Km of 5 beta-reductase for NADPH was estimated as 4.22 x 10(-6) M and for testosterone, 4.60 x 10(-6) M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5 beta-reductase were examined.

In vitro metabolism of testosterone on hepatic tissue of chicken (Gallus domesticus).

Among the subcellular fractions of chicken liver homogenates, the microsomal and cytosol fractions were most active in metabolism of testosterone with mutually different enzymological features. On the other hand, the nuclear and mitochondrial fractions had far lower activity of metabolizing the steroid. Metabolism by the cytosol fraction: the following steroids were identified as the metabolites of testosterone. 5 beta-Dihydrotestosterone (17 beta-hydroxy-5 beta-androstan-3-one), 5 beta-androstane-3 alpha,17 beta-diol and its 3 beta-epimer, 3 alpha-hydroxy-5 beta-androstan-17-one and its 3 beta-epimer and 5 beta-androstanedione. Metabolism by the microsomal fraction: from testosterone under aerobic condition, androstenedione was obtained as the major metabolite, besides the minor polar metabolites, production of which diminished when incubated in the atmosphere of carbon monoxide. From the results, testosterone was accepted to be firstly converted by the cytosol fraction into 5 beta-dihydrotestosterone which was then reduced to 5 beta-androstane-3 alpha,17 beta-diol and its 3 beta-epimer. These diols were further converted partially to 3 alpha -and 3 beta-hydroxy-5 beta-androstan-17-ones. These pathways were supported by the results of our incubation study with 5 beta-dihydrotestosterone and 5 beta-androstanedione as substrates. By the microsomes, testosterone was aerobically and anaerobically transformed to androstenedione as the major metabolite. Throughout our incubation experiments, no 5 alpha-reduction of a delta 4-3-oxo-steroid was detected in the chicken liver.

Change of steroidogenic pathways in the ovary of a tropical catfish, Clarias macrocephalus, Gunther, after hCG treatment.

Adult female catfish received an im injection of 454 IU hCG in 0.2 ml saline. Sixteen hours later, the ovarian tissue from the hCG-treated or control fish was aerobically incubated in vitro with 4-[14C]progesterone or 17 alpha-hydroxyprogesterone at 30 degrees for 60 min. When progesterone was employed as the substrate, significant production of androstenedione and testosterone was observed in the control group. However, after the hCG injection, a markedly higher amount of 20 beta-hydroxy-4-pregnen-3-one was produced. Furthermore, the androgen production was diminished, and the production of 5 beta-reduced C21 metabolites such as 5 beta-pregnane-3,20-dione and 3 alpha-hydroxy-5 beta-pregnan-20-one was also reduced in the hCG-treated group. From 17 alpha-hydroxyprogesterone as a substrate, considerable amounts of androstenedione and testosterone were obtained as the metabolites in the control group. However, after the hCG treatment, production of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOHprog) and its 5 beta-reduced metabolite was markedly stimulated, while the androgen production was reduced drastically. By evaluating the yield of each product, it was suggested that the tentatively calculated activity of 17 alpha-hydroxylase and C-17-C-20 lyase was diminished by the hCG treatment and that 20 beta-hydroxysteroid dehydrogenase was activated. It indicates that hCG changed the ovarian steroidogenic pathway from androgen production to formation of 17 alpha, 20 beta-diOHprog, an inducer of germinal vesicle breakdown.

Structure and steroidogenic enzymes of the seminal vesicles of the urohaze-goby (Glossogobius olivaceus).

The in vitro steroid metabolism in the seminal vesicles of the brackish water goby (urohaze-goby, Glossogobius olivaceus) was studied using males in the breeding season. The moderate activity of delta 5-3 beta-hydroxysteroid dehydrogenase was histochemically detected only in the epithelial cells of the organ, though these cells have the characteristics of secretory cells ultrastructurally. Cell-free homogenates (800 g supernatant fluid) of the whole tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, or testosterone in the presence of NAD+ or NADPH. Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product) and 17 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was metabolized into androstenedione (main product) and 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione, and androstenedione. These results indicate that the steroid metabolic patterns in the seminal vesicles of G. olivaceus are closely resembled to those in the testes.

Biosynthetic pathways of testosterone and estradiol-17 beta in slices of the embryonic ovary and testis of the chicken (Gallus domesticus).

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.

Developmental changes of steroidogenic enzyme activities in the embryonic gonads of the chicken: the sexual difference.

Steroidogenic enzyme activities in the left ovary and the testes of 9- to 15-day-old chicken embryos were measured, and development of the activities was compared between sexes. Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary and in the testis was comparable, and did not change throughout the period examined. Activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the ovary was similar to or higher than that in the testis, depending on substrates employed. In both gonads, 17 beta-HSD activity did not change or tended to decrease from 9 to 15 days of development. On the other hand, activities of 17 alpha-hydroxylase, C-17--C-20 lyase in the ovary were three to eight times those in the testis, and aromatase activity in the ovary was definitely higher than that in the testis at all stages examined. The activities of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase significantly increased from 9 to 11 days only in the ovary. From 13 to 15 days, the activities of 17 alpha-hydroxylase and C-17--C-20 lyase markedly increased only in the testis. These results suggest that, in the gonads of developing chicken embryos, there are sexual differences in the regulation of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase activities.

Inactivation of rat testicular NADPH-cytochrome P-450 reductase by 2,4,6-trinitrobenzenesulfonate.

Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus).

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.

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.

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and its relation to the ultrastructure of steroidogenic cells in immature and mature rat ovaries.

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and its relation to the ultrastructure of steroidogenic cells were examined in mature and immature rat ovaries. In mature (8-10 weeks old) rat ovaries, the theca interna cells of secondary as well as Graafian follicles, and the interstitial gland cells were all strongly stained with anti-17 beta-HSD antibody. However, granulosa cells, corpus luteum cells, oocytes and peritoneal epithelial cells were negative against this staining. In the ovaries of 1-week-old rats, all these cells were negative to immunostaining for 17 beta-HSD. In the ovaries of 2-week-old rats, the theca interna cells of secondary follicles and the interstitial gland cells showed a positive reaction for the 17 beta-HSD activity. Electron microscopic examination demonstrated the presence of characteristic structures for steroid secretory cells such as many lipid droplets, well developed smooth endoplasmic reticulum, and oval mitochondria with tubular cristae in the theca interna cell of secondary as well as Graafian follicles and in the interstitial gland cell of mature rat ovaries. In the ovaries of 1-week-old rats, all the theca cells of the primary and secondary follicles were fibroblast-like in their shape and fine structure, and typical interstitial cells were not recognized. In the 2-week-old rats, some of the theca interna cells and interstitial cells were well differentiated in ultrastructure, showing characteristic features for steroid secretory cells. These findings indicate that by 2 weeks after birth, theca interna cells and interstitial gland cells acquire the ability for testosterone production as seen in mature rat ovaries.

In vitro decrease of lyase activity in rat ovarian cells during incubation: effect of hCG.

From PMSG-pretreated immature rats, dispersed ovarian cells were prepared with collagenase and DNase and incubated at 37 degrees C in McCoy's 5a medium under 95% air-5% CO2 atmosphere for 4 h. The activities of C17-C20 lyase measured in the 10,000 x g supernatant fluid of the cell homogenates decreased spontaneously with the lapse of time of the incubation. N,N'-Diphenyl-p-phenylenediamine (DPPD, an antioxidant) and actinomycin D inhibited the decrease most effectively. Cycloheximide was also an effective protector. Accordingly, the spontaneous decrease of the lyase activity was caused partly by an oxygen radical-mediated process and partly by a mechanism involving de novo synthesis of RNA and protein. Addition of hCG to the cells further decreased the lyase activity to about half of the control group at 4 h. DPPD itself did not affect the hCG-induced decrease of the lyase activity. However, actinomycin D and cycloheximide prevented the effect of hCG. These results indicate that de novo synthesis of RNA and protein is involved in the latter mechanism, while oxygen radical is not concerned in this process. The decrease of the enzyme activity by hCG during incubation is in agreement with the in vivo effect of hCG upon the lyase activity. On the contrary, at the end of incubation the activity of delta 5-3 beta-hydroxysteroid dehydrogenase (coupled with delta 5-delta 4 isomerase) was more than 89% of that before incubation, and the change of the enzyme activity according to the various treatments was less than 16%.

Conversion of androstenedione to 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-4-androsten-17-one by the testicular microsomal fraction of Sprague-Dawley rats.

When androstenedione was incubated with testicular microsomes of Sprague-Dawley rats in the presence of reduced nicotinamide-adenine dinucleotide (NADH), unknown metabolites were produced, in addition to testosterone and 7 alpha-hydroxyandrostenedione. The metabolites were identified as 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one (3:1) by biochemical and radiochemical methods. These results confirmed the occurrence of the reverse reactions from androstenedione to 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one catalyzed by the 3 beta-hydroxysteroid dehydrogenase and 5-ene-4-ene isomerase in the microsomal fraction of Sprague-Dawley rat testes.

Testicular and adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.

The purified multifunctional enzyme, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3 beta-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.

Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase.

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.

Purification of NADPH-cytochrome P-450 reductase from microsomal fraction of rat testes, and its chemical modification by tetranitromethane.

NADPH-cytochrome P-450 reductase in rat testicular microsomal fraction was solubilized by trypsin, and purified to apparent homogeneity in polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis. Km values were estimated as 18 microM for cytochrome c, 17 microM for dichlorophenol indophenol (DCPIP), 50 microM for K3Fe (CN)6 and 1.7 microM for NADPH. The cytochrome c reducing activity of the purified preparation was decreased by tetranitromethane (TNM), a reagent for nitration of tyrosine residues in a protein. The inactivation exhibited pseudo-first-order kinetics. A plot of log kapp vs log [TNM] gave a straight line with slope = 1.05, indicating the reaction of one modifier molecule in the inactivation process. The decrease of the reducing activities for DCPIP and K3Fe(CN)6 by TNM progressed more slowly than that for cytochrome c. The inactivation of cytochrome c reduction was protected completely by 0.1 mM NADP(H) and partially by 0.1 mM DCPIP and cytochrome c. No preventive change of the inactivation by TNM was observed by addition of NAD+ or testosterone. On the other hand, the differential modification by DTNB, TNM and DTT indicated that there were amino acid residues modified by TNM, such as tyrosine residues, at or near the active-site of the NADPH-cytochrome P-450 reductase.