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Growth of Pseudomonas testosteroni on testosterone was optimal at 30°C. Induction of steroid-binding activity occurred optimally at 25°C to 30°C. Binding activity first appeared after 9 h of growth on testosterone and reached its maximum at 30 h. Dehydrogenation activities, on the other hand, reached a maximum at 12 h. Steroid-binding activity was induced by growth on several C19 and C21 steroids. At incubation temperatures of 37°C or 45°C there was minimal induction of binding activity. Binding activity induced at 30°C was irreversibly destroyed at 45°C and reversibly inhibited at 37°C.

Recent studies concerning the biosynthesis, metabolism, and possible functions of steroids other than sterols in plants are reviewed and discussed. These studies embrace the saponins, glycoalkaloids, pregnane derivatives, cardiac glycosides, as well as their aglycones, and the sex hormones.

The fully developed follicular wall consisting of theca interna and granulosa cells after antrim formation, granulosa cells alone, corpus hemorrhagicum, corpus luteum, corpus albicans and stromal tissue were collected from ovaries of different mares. All cell preparations were incubated with progesterone-4- 14 C and some were also incubated with 17-hydroxyprogesterone-4- 14 C androstenedione-4- 14 C or testosterone-4- 14 C to study the relative activities of the different enzymes involved in the biosynthesis of estrogens from progesterone. Because of the differences in amount of extracellular fluid and connective tissue the yields of the different metabolites were expressed per mg DNA as well as per unit weight of tissue. The follicular walls showed active 17α-hydroxylase, 17,20-lyase, 17β-dehydrogenase and aromatizing enzyme systems. Granulosa cells alone showed a similar pattern except that the aromatizing system did not seem to be proportionately as active. The recently ovulated corpus hemorrhagicum showed high 17α-hydroxylase, 17,20-lyase and 17β-dehydrogenase activities but little aromatizing activity, while the corpora lutea showed 17α-hydroxylase and (17-hydroxyprogesterone)-20-oxido-reductase activities but low 17,20-lyase and no measurable formation of estrogens. When androstenedione or testosterone were substrates, both follicular walls and corpora lutea produced estrogens in similar amounts; the decreased lyase activity in the luteal cells, therefore, appears to account for the lack of estrogen production from C 21 precursors.

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.

Randomly labeled cholesterol-C 14 was oxidized with chromic acid and from the oxidation products t -dehydroandrosterone-C 14 , Δ 5 -pregnen-3-ol-20-one-C 14 , and 3-hydroxy-Δ 5 -etiocholenic-C 14 acid were isolated. Conversion of t -dehydroandrosterone-C 14 into Δ 4 -androstene-3,17-dione-C 14 and testosterone-C 14 , of Δ 5 -pregnen-3-ol-20-one-C 14 into progesterone-C 14 , and of 3-hydroxy-Δ 5 -etiocholenic-C 14 acid into desoxycorticosterone-C 14 is described.

The metabolism of testosterone, androstenedione and dehydroepiandrosterone in the rat preputial gland has been studied. A high activity of 5α-reductase is present as shown by the formation of 17β hydroxy-5α-androstan-3-one and 5α-androstan-3, 17-dione as the major products from testosterone and androstenedione respectively. Other enzyme activities are present including 17β-hydroxy steroid dehydrogenase, but the amounts of testosterone and 17β-hydroxy-5α-androstan-3-one formed from androstenedione and dehydroepiandrosterone are low. The main product of dehydroepiandrosterone metabolism was androstenedione indicating a high level of 3β-hydroxy steroid dehydrogenase 4-5 isomerase activity. The metabolism was compared with that in rat skin where it was found that the extent of metabolism was much less. The possible significance of the various products formed and of differences between skin and preputial gland metabolism is discussed. Some differences were noted between the metabolism of androgens by rat skin and preputial gland and the metabolism of androgens by human skin.

1. 1. The steroid content of the ovaries and fat bodies of female Triturus cristatus carnifex have been analysed during the breeding season. 2. 2. Progesterone and androstenedione have been identified in the ovarian extract; the following steroids have been identified in the fat bodies: pregnenolone, progesterone, dehydroepiandrosterone, testosterone and estrone. 3. 3. Incubations of acetone powder carried out separately with ovarian and fat body tissue using cholesterol-4-C14 as the substrate yielded the following metabolites in the case of the ovary: progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone; and in the case of the fat bodies: pregnenolone, 17α-hydroxypregnenolone, 17α-hydroxprogesterone, and testosterone. 4. 4. Incubations with either tissue using testosterone-4-C14 as the substrate yielded 11-ketotestosterone, 11β-hydroxyandrostenedione, androstenedione and esterone. 5. 5. These findings support the view that steroid hormones identified in the fat bodies could be produced in situ from cholesterol.

The in vitro metabolism of steroid hormones in a masculinovoblastoma has been investigated. Studies with testosterone and androstenedione revealed a metabolic block at the aromatization step. The tumor exhibited the capacity to form small quantities of 21- but not 11-hydroxylated compounds from 4-C14-progesterone and 17α-hydroxyprogesterone. With the exception of the aromatization process, the tumor appeared to metabolize progesterone and 17α-hydroxyprogesterone in essentially the same manner as normal ovary. The similarity of the steroid metabolic pattern to that of normal ovarian tissue indicates that it was not derived from ectopic adrenal tissue. Rather, it appears to have originated from ovarian cells that have lost their capacity to aromatize ring A. A block at the aromatizing step would be expected to result in an accumulation of androstenedione and related compounds that would be conducive to the masculinizing effects observed in this disease.

Progesterone at relatively high fermentation beer concentrations was converted efficiently by three fungi tested to androstenedione or androstadienedione; the usual end products of these conversions are the corresponding D-ring lactones. With Septomyxa affinis progesterone was converted to androstadienedione in 75% yield (theoretical). Δ1-Testosterone acetate is an intermediate in this steroid side chain cleavage sequence. Furthermore, it was suggested that in the bioconversion of progesterone by S. affinis the same enzyme functions twice in the reaction sequence. First it converts Δ1-progesterone to Δ1-testosterone acetate and subsequently it converts androstadienedione to Δ1-testololactone. The lactonization reaction may be inhibited by relatively high concentrations of progesterone, the substrate for the first reaction. Progesterone was a better inducer of this enzyme than was androstadienedione, and it also appeared to be the preferred substrate for the enzyme.

Free and sulfate-conjugated neutral steroids were isolated from the pooled testes of 33 human fetuses of 8.5 – 20.0 cm crown-rump length and the compounds were identified by gas-liquid chromatography and gas chromatography — mass spectrometry. Unconjugated pregnenolone and testosterone were the main compounds present. Thus it has been established that under in , vivo conditions the fetal testis synthesizes androgens and that apparently the pathway pregnenolone → 17a-hydroxypregnenolone → dehydroepi-androsterone → androstenedione/5-androstene-3β, 17β-diol → testosterone is preferred.

Reconstituted cells were formed by haemolysis of human erythrocytes in hypotonic media followed by resealing of the membranes in isotonic solution. These so-called “resealed erythrocyte ghosts” could be filled with different protein solutions present in the haemolysing medium. Reconstituted cells containing bovine serum albumin or diluted plasma with a large amount of corticosteroid binding globulin (CBG) were prepared by this method. Uptake of steroids by reconstituted cells was measured using equilibrium dialysis and incubation studies and was compared with the uptake by cells not containing proteins. After washing of the cells containing CBG, the steroid specifically bound by this protein inside the cell could be determined. Free steroids (testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone, oestradiol, progesterone, cortisol) penetrated quickly into the reconstituted cells, compared with the rather slow penetration of conjugated steroids (dehydroepiandrosterone sulphate and oestrone 3-sulphate). A difference between the amount of cortisol and progesterone bound to CBG in resealed ghosts was only observed if plasma proteins were also present outside the reconstituted cells. Under these conditions the rate of uptake by binding sites inside the ghost was much slower for cortisol than for progesterone. This was not due to differences in membrane permeability but mainly to different distribution coefficients of the steroids between medium and ghosts.

The production of androgens and estradiol from 17α-hydroxy[4-14C]progesterone and [17α-3H]dehydroepiandrosterone by the dog ovary in vitro has been investigated using homogenates and slices of ovaries from dogs pretreated with gonadotrophins. Androstenedione, testosterone and estradiol were isolated and quantitated, and the ratio 3H/14C and specific radioactivity of these steroids were determined. 1. 1. The dog ovary is able to convert 17α-hydroxyprogesterone and dehydroepiandrosterone to androstenedione, testosterone and estradiol in vitro. 2. 2. Dehydroepiandrosterone is the preferred substrate for the formation of the isolated steroids. 3. 3. Reduced TPN stimulates ovarian 17-desmolase and 17β-reductase and promotes the production of androstenedione and testosterone. 4. 4. The results obtained with tissue slices in vitro in the presence of TPN and a TPN-reducing system are similar to those obtained with ovarian perfusions in vivo. 5. 5. The dog ovary can form 19-hydroxyandrostenedione in vitro. Conversion in vivo to this steroid by the trophic stimulated dog ovary has not been observed.

The steroid composition of and steroid conjugation in human cadaver and boar testes were investigated by analyzing the endogenous steroids. Gas-liquid chromatography and gas chromatography-mass spectrometry were used to identify the steroids and to determine the position of the sulfate group in sulfate conjugates. For the latter purpose, the steroids were first acetylated and subsequently solvolyzed and converted to trimethylsilyl ethers. In addition to the compounds previously identified as endogenous components, human testis was also found to contain progesterone, 17α-hydroxyprogesterone and 20α-hydroxy-4-pregnen-3-one in the free steroid fraction and 3β-hydroxy-17α-5-pregnen-20-one in the monosulfate fraction.

1. 1. The testis and vas deferens contain a 17β-hydroxysteroid oxido-reductase which actively reduces radioactive C 19 and C 18 17-ketosteroids to 17β-hydroxysteroids. 2. 2. Androstenedione, dehydroepiandrosterone and estrone were reduced respectively to testosterone, androst-5-ene-3β,17β-diol and estradiol-17β. 3. 3. Testosterone and estradiol-17β were not oxidized in our experimental conditions to androstenedione and estrone. 4. 4. A minor 17α-hydroxysteroid oxidoreductase reduced androstenedione to epitestosterone (2 per cent) and estrone to estradiol-17α (8 per cent) in organ cultures of vas deferens. 5. 5. Androgenic gland was less active than testis or vas deferens to synthesize testosterone. 6. 6. A 21-hydroxylase catalysed the conversion of progesterone to 11-deoxycorticosterone, and 17-hydroxyprogesterone to 11-deoxycortisol. 7. 7. Steroid metabolism is mainly in the testis and vas deferens which are the main target tissues of the androgenic hormone.

The methods in use in the authors laboratory for the determination of plasma testosterone, androstenedione and dihydrotestosterone are described and possible pitfalls are discussed. The gas-chromatographic methods are compared to methods based on competitive protein binding and the pros and cons of each are discussed. It appears that in expert hands GLC methods for plasma androgens are not inferior to methods based on CPB, the only advantage of the latter being their greater practicability, which is however largely neutralized by the lack of specificity of CPB methods, requiring extensive preparatory purification of material used.

Minced tissue from a feminizing adrenal carcinoma was incubated for 1 hr with cofactors and equimolar amounts of 7α-3H-pregnenolone and 4-14C-progesterone. Seventeen compounds were isolated and recrystallized to constant specific activity. The isotopic ratios indicated that the conversion of pregnenolone to progesterone was negligible and that 3H in 17-hydroxyprogesterone came primarily by way of 17-hydroxypregnenolone. The 16-hydroxy derivatives of progesterone, testosterone and androstenedione were derived exclusively from 14C-progesterone, as were desoxycorticosterone, corticosterone, corticosterone, compound A and aldosterone. Pregnenolone gave rise to a high proportion of isotopically labeled 17-hydroxyprogesterone, testosterone, 2-hydroxytestosterone and estrone. The series 17-hydroxyprogesterone→compound S→cortisol→Reichstein's V showed a 3H/14C ratio which decreased successively: 12.2, 8.1, 6.5, 2.6. All these findings confirm the predominant role of pregnenolone as precursor for C19 and ...

The role of DPNH and TPNH in the reduction of [4- 14 C]-testosterone or [4- 14 C] and rostenedione by female rat liver homogenate and its subcellular fractions was studied. The extracted metabolites were separated by thin layer and paper chromatography. 5α-Androstane-3α, 17β-diol was the major metabolite. Androsterone and 5α-dihydrotestosterone occurred in lesser quantities and 5α-androstane-3β, 17β-diol, 5α-androstanedione and epiandrosterone in small amounts. Androstenedione (or testosterone) was produced only in the presence of DPNH. The omission of the coenzymes or TPNH-generating system resulted in only a trace amount of the metabolites.

Radioactive pregnenolone ( 14 C or 3 H labeled) was infused at a constant rate via the spermatic artery of the isolated dog testis in order to study the quantitative significance of pathways used for the biosynthesis and secretion of testosterone in this organ. The amount of steroids and their content of radioactivity were estimated in spermatic venous blood during pregnenolone infusion and in the testis tissue at the end of such infusions. During continuous pregnenolone infusion the true specific radioactivity (μC/ mmole) of several steroids in spermatic venous blood plasma was different from the spermatic radioactivity of the same steroids in the infused testicular tissue. This observation supports the view that steroids may be present in different testicular compartments which are not equally supplied with infused pregnenolone and that these compartments do not add to steroid secretion into the spermatic venous blood in a homogeneous fashion. Concomitant infusion of human chorionic gonadotrophin or cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with radioactive pregnenolone via the spermatic artery resulted in increased secretion of all known intermediates between pregnenolone and testosterone. The specific radioactivity of all these steroids decreased in such experiments. Thus the major effect of human chorionic gonadotrophin or cyclic AMP on the formation of testicular steroids occurred at step(s) prior to the formation of pregnenolone. The specific radioactivity of dehydroepiandrosterone decreased more than that of pregnenolone or 17α-hydroxypregnenolone, particularly in human chorionic gonadotrophin or cyclic AMP stimulated testes, and it is possible that dehydroepiandrosterone was also formed via metabolic pathways which did not use pregnenolone and 17α-hydroxypregnenolone as intermediates. Dehydroepiandrosterone appeared to be an important steroid for testosterone formation in vivo and metabolism of dehydroepiandrosterone to testosterone proceeded both via androstenediol and androstenedione. The present results clearly demonstrate that pregnenolone was converted to testosterone in the canine testis both via a. Δ 4 - and a Δ 5 -pathway.

Unconjugated testosterone (T) and androstenedione were isolated from cord plasma obtained from mixed arterial and venous cord blood collected after vaginal deliveries at term. The identification of both hormones is based upon double labelled derivative formation and reverse isotope dilution. Only the latter procedure gave constant 3 H 14 C ratios for the acetate of testosterone. Androstenedione was also biotransformed to 17β-estradiol. The same procedures did not provide sufficient proof for the existence of 16α-hydroxyandrostenedione and 16α-hydroxytestosterone in cord blood. Tentative evidence for their being present is given.