The mechanism of the synthesis of 16-androstenes in human testicular homogenates.

The biochemical pathway leading to the 16-unsaturated C19 steroids--known as sex pheromone (precursors) in pig and man--is still a matter of dispute. In the 16-ene-synthetase process, via which 5,16-androstadien-3 beta-ol (ADL) or 4,16-androstadien-3-one (ADN) are biosynthesized from pregnenolone (P5) or progesterone (P4), a number of 2 or even 3 step conversions have been suggested in porcine tests, including 20 beta-reduction, 21-hydroxylation and 16,17-dehydrogenation. Studying the 16-ene-synthetase reaction in human testicular homogenates, we adduced evidence for the hypothesis that ADL is synthesized from P5 in a single step, not requiring separate intermediates. Our proposal for the 16-ene-synthetase mechanism also explains why, at least in our hands, synthesis of ADL is always accompanied by co-synthesis of its satellite 5-androstene-3 beta,17 alpha-diol (epiA5): both steroids are synthesized as a mere consequence of the fact that the proposed elimination and substitution reactions for the synthesis of ADL and epiA5, respectively, are competitive processes.

Formation of 5,16-androstadien-3 beta-ol from pregnenolone in human testicular microsomes.

A microsomal fraction of testicular tissue from a patient with prostatic carcinoma was incubated with [4-14C]pregnenolone in the presence of an NADPH-generating system for different periods of time. The metabolites were separated by Sephadex LH-20 column chromatography and then identified by thin-layer chromatography, radio-gas chromatography, and crystallization studies. Pregnenolone was converted to a major metabolite, 5-androstene-3 beta,17 beta-diol via 17-hydroxypregnenolone and then dehydroepiandrosterone. Another major metabolite was 5,16-androstadien-3 beta-ol, which increased with the time of incubation and accumulated in the incubation medium. After 120 min of incubation, 34.6% of the precursor was converted to 5-androstene-3 beta,17 beta-diol and 15.1% to 5,16-androstadien-3 beta-ol. In addition to the above-mentioned steroids, 16 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, and 5-androstene-3 beta,17 alpha-diol were identified as minor metabolites of pregnenolone. From these results it was concluded that human testicular microsomes possess enzymic activities for the synthesis of 5,16-androstadien-3 beta-ol, as well as androgens from pregnenolone.

Stereospecific removal of the 16 alpha-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol from pregnenolone.

[16 alpha-2H]Pregnenolone was synthesized by catalytic deuteriation of 3 beta-hydroxy-5,16-pregnadien-20-one followed by base-catalyzed back exchange of the 17 alpha-2H atom, and [16 beta-2H]pregnenolone by catalytic hydrogenation of 3 beta-hydroxy-5,16-[16-2H]pregnadien-20-one, which had been synthesized from [16,16-2H]dehydroepiandrosterone. The labelled pregnenolones were incubated separately with the microsomal fraction of boar testis. The metabolites were analyzed by gas chromatography-mass spectrometry, and the isotope compositions of the following six metabolites were determined: 17-hydroxypregnenolone, dehydroepiandrosterone, 5-androstene-3 beta,17 alpha-diol, 5-androstene-3 beta,17 beta-diol,16 alpha-hydroxypregnenolone and 5,16-androstadien-3 beta-ol. The first four metabolites derived either from [16 alpha-2H]- or from [16 beta-2H]pregnenolone showed essentially the same isotope compositions as those of their respective precursors. The 16 alpha-hydroxypregnenolone and the 5,16-androstadien-3 beta-ol biosynthesized from [16 alpha-2H]pregnenolone lost the 2H label, while the same metabolites biosynthesized from [16 beta-2H]pregnenolone retained the albel. The result shows that the 16 alpha-hydrogen is stereospecifically removed with the retention of the 16 beta-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol.

Cytochrome b5 promotes the synthesis of delta 16-C19 steroids by homogeneous cytochrome P-450 C21 side-chain cleavage from pig testis.

Conversion of progesterone to 17 alpha-hydroxyprogesterone plus androstenedione (17 alpha-hydroxylation) and to androstadienone (delta 16 synthetase activity) by microsomes from neonatal pig testis, were both inhibited by antibodies raised against homogeneous cytochrome P-450 C21 side-chain cleavage. Inhibition of the two activities showed the same relationship to the concentration of antibody added. Analogous results were obtained with pregnenolone as substrate. In a reconstituted enzyme system consisting of the homogeneous cytochrome P-450 C21 side-chain cleavage enzyme, P-450 reductase and NADPH, addition of cytochrome b5 resulted in the synthesis of the corresponding delta 16-C19-steroid from progesterone (androstadienone) and pregnenolone (androstadienol). The effect of cytochrome b5 was concentration-dependent and prevented by anti-cytochrome b5. It is concluded that the cytochrome P-450 C21 side-chain cleavage enzyme from pig testicular microsomes is also capable of synthesizing delta 16-C19-steroids and is, therefore, likely to be responsible for the large amounts of the pherormone androstadienone produced by male pigs.

Comparative rates of formation, in vivo, of 16-androstenes, testosterone and androstenedione in boar testis.

Three mature Large White boars were anaesthetized and received [7(n)-3H]pregnenolone by continuous infusion into right and left spermatic arteries for up to 180 min. Spermatic venous blood flow was measured by separate timed collections of completely diverted outflow from each testis and blood not sampled was returned to the peripheral circulation. The total radioactivity in plasma from each testis increased markedly during the first 60 min of infusion to reach a plateau from 80 to 180 min. Radiolabelling of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 beta-ol and -3 alpha-ol showed similar patterns with ratios of mean radioactivity of 5:3:1 respectively between 80 and 180 min. In comparison, the amounts of tritiated 4,16-androstadien-3-one formed were very small. The radiolabelling of testosterone and 4-androstenedione occurred more rapidly than that of the 16-androstenes and reached maxima by 30 min. However the amounts were only one-fifth (testosterone) and one-tenth (4-androstenedione) those of the combined quantities of tritiated 16-androstenes. Addition of human chorionic gonadotrophin (hCG) to the infusate to one testis in each animal (so that 5000 i.u. hCG were delivered in 15-20 min) produced no change in the outputs of radiolabelled steroids although radioimmunoassay of spermatic venous plasma in samples from the third experiment showed a transient increase in the concentration of 4-androstene-3,17-dione during the hCG infusion. It is suggested the lack of response to hCG could be produced by saturation and down regulation of binding sites by the very high local concentrations of hCG.

Formation of [17-2H]androsta-5,16-dien-3beta-ol from [17,21,21,21]pregnenolone by the microsomal fraction of boar testis.

After incubation of [17,21,21,21-2H4]pregnenolone, which had been synthesized from nonlabeled pregnenolone by base-catalyzed exchange procedure, with the microsomal fraction of boar testis, [17-2H]androsta-5,16-dien-3beta-ol was identified as a metabolite. The identification has been performed by gas chromatography-mass spectrometry of the metabolite and of its hydrogenation product. Conclusive evidence on the location of the 2H atom at C-17 has been obtained by transforming the metabolite to androsta-4,16-dien-3-one and then to androst-4-ene-3,17-dione and by proving that the former compound contained a 2H atom while the latter did not. The result indicates that, in boar testis, androsta-5,16-dien-3beta-ol is synthesized from pregnenolone by a pathway in which 17 alpha-hydrogen atom of pregnenolone is retained throughout the reaction, excluding 17 alpha-hydroxypregnenolone and 3beta-hydroxyandrost-5-en17-one as intermediates.

Metabolic fate of cholesteryl methyl ether in Mycobacterium phlei.

Mycobacterium phlei transformed cholesteryl methyl ether into three metabolites: 3beta-methoxy-dinor-5,17(20)-choladien-22-oic methyl ester (I), 3beta-methoxy-5-androsten-17-one (II), and 3beta-methoxy-dinor-5-cholen-22-ol (III). After isolation with thin-layer chromatography, their structures were elucidated by mass, IR and NMR spectroscopy. Compound II was the major product. Compounds I and III were products of various side reactions. In the presence of 8-hydroxyquinoline that inhibits degradation of the steroid nucleus, 1,4-androstadiene-3,17-dione was formed in addition to the compounds mentioned. This indicates that a moderate splitting of the ether bond takes place.