Biosynthesis and metabolism of testosterone by sertoli cell-enriched seminiferous tubules

Sertoli cell-enriched tubules isolated from rats which had been treated with 1,4-dimethyl sulfonyloxybutane were incubated with either [¹⁴C] progesterone or [¹⁴C] testosterone for 2 hours. Tubules of normal rats and fragments of Sertoli cell-enriched testes were incubated under the same conditions. Sertoli cell-enriched tubules converted progesterone to 20α-dihydroprogesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. The major metabolite was 20α-dihydroprogesterone. The percentage conversion of progesterone into testosterone corresponded to a production of 10–20 ng testosterone. Sertoli cell-enriched tubules converted testosterone to dihydrotestosterone, androstenedione, 3α-androstanediol and 3β-androstanediol. Under our experimental conditions, dihydrotestosterone was the major 5α-reduced metabolite. Normal tubules converted progesterone and testosterone to the same metabolites as Sertoli cell-enriched tubules. Fragments of Sertoli cell-enriched testes were much more active than isolated tubules in metabolizing progesterone. They produced the same amounts of 5α-reduced metabolites of testosterone.     

Bioconversion of the androgen precursors by pre- and post-pubertal rat testes with special reference to local irradiation and gonadotropin stimulation

Pubertal changes in the testicular steroid enzyme activities, responsible for the androgen production, were studied in rats in relation to the effects of testicular irradiation, followed by gonadotropin stimulation and cyproterone suppression. Five groups of pro-pubertal and adult rats were used in this study. The $\text{in vitro}$ bioconversion from progesterone-4-14C and 17-hydroxyprogesterone-44C to testosterone, androstenedione, androstanediol, dihydrotestosterone and androsterone, demonstrated the effect of age in all cases of drug response investigations. The sexually immature animals in the control group had higher levels of androstenedione than testosterone, in contrast to the findings in the adults. With irradiation, androgen biosynthesis was suppressed in both age groups, which did not recover, under gonadotropin stimulation, in spite of the generation of new cells caused by the treatment. The irradiated adult testes demonstrated ‘pre-pubertal’ type bioconversion by catabolizing the substrates more towards 5α-reduced androgens, like androstanediol (5α-androstane-3α 17β-diol) and androsterone. With cyproterone the 17α-hydroxylase activities were found to be diminished.     

The effects of hypophysectomy and human chorionic gonadotropin administration on the in vitro metabolism of progesterone by the rat testis

Changes in the $\text{in}$$\text{vitro}$ capacity to convert progesterone to its metabolites were studied in testes of adult rats hypophysectomized for varying lengths of time. After 30 days of hypophysectomy rats were injected for periods of 10 and 20 days with 100 i.u. of HCG daily to observe what changes could be induced in the testicular conversion of progesterone. Hypophysectomy increased the formation of 20α-hydroxy-4-pregnen-3-one and decreased the formation of testosterone. In hypophysectomized animals injected with HCG there was an immediate decrease in the 20α-hydroxy-4-pregnen-3-one formation, but no appreciable accumulation of testosterone, as the animals demonstrated an immature pattern of testicular function. The results indicate that 20α-hydroxy-4-pregnen-3-one may act as a positive feedback agent to prolong and heighten gonadotropin discharge, and confirm the importance of metabolites of testosterone prior to adulthood.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Hydroxysteroid dehydrogenase activity in tissues of two insect species

• 1.1. The metabolism of two tritium labelled vertebrate-type steroids was studied in two insect species, i.e. the fleshfly, Sarcophaga bullata, and the Colorado potato beetle, Leptinotarsa decemlineata.
• 2.2. After injection of [³H]androstenedione into Sarcophaga bullata pharate adults, testosterone (both as free steroid and as conjugate) could be identified as a metabolic product. This indicates the presence of the 17β-hydroxysteroid dehydrogenase (HSD) enzyme in the fleshfly.
• 3.3. Injection of 17α-hydroxy[³H]progesterone into Leptinotarsa decemlineata last instar larvae resulted in the formation of 17α-hydroxy-20α-dihydroprogesterone, 17α-hydroxy-20β-dihydroprogesterone and their conjugates. This indicates the presence of both the 20α-HSD and the 20β-HSD enzyme in Leptinotarsa.
• 4.4. Important conversions in the biosynthetic pathway of steroids in vertebrates, such as the conversion of 17α-hydroxyprogesterone to androgens (Leptinotarsa) and the aromatization of androgens to estrogens (Sarcophaga), were not demonstrated in the metabolic studies.

     

The effect of progesterone analogues,naturally occurring steroids,and contraceptive progestins on hypothalamic and anterior pituitary Δ4-steroid (progesterone) 5α-reductase

The effects of a number of steroids on the conversion of progesterone to 5α-dihydroprogesterone by hypothalamic and pituitary progesterone 5α-reductase have been investigated. Using enzyme preparations from female rats and ³H-progesterone as substrate, 5α-reduced products (5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one) were analyzed by reverse isotopic dilution analysis. The amount of total 5α-reduced products formed was compared in the presence and absence of the test steroid. Derivatives lacking the Δ⁴ and/or the 3-keto moiety were without effect. Corticosterone had no effect. 16β-Methylprogesterone inhibited progesterone 5α-reduction in both tissues by at least 65%, while the 2α-, 6α-, and 7α-methylated derivatives had lesser effects. 3-Oxo-4-pregnene-20β-carboxaldehyde and 21-fluoroprogesterone were potent inhibitors. 17-Hydroxyprogesterone was a competitive inhibitor (substrate) with Ki's of 0.27 μM (pituitary) and 0.29 μM (hypothalamus). Medroxyprogesterone exerted little inhibitory effect. Of the 19-norsteroids examined, only norethindrone appreciably inhibited the 5α-reduction. These results suggest that some natural Δ⁴-3-ketosteroids can modify enzymatic activity. Also, inhibitory analogues may be useful for studies on the role of this 5α-reduction of progesterone.     

Identification by capillary gas chromatography-mass spectrometry of eleven non-ecdysteroid steroids in the haemolymph of larvae of Sarcophaga bullata

$\text{O-}\text{Pentafluorobenzyloxime}$ (OPFB)-heptafluorobutyrylester (HFB) derivatives and $\text{OPFB}\text{-O-}\text{methyloxime}$ (MO)-trimethylsilylether (TMS) derivatives of non-ecdysteroid steroids were prepared from haemolymph extracts of last instar larvae of the fleshfly Sarcophaga bullata. Using a negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS) technique the following steroids could be identified: progesterone, testosterone, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, androst-5-ene-3β,17β-diol, androstenedione, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, 17α-hydroxyprogesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxyprogesterone. Although the technique is very sensitive, estrogens could not be detected. These results suggest an active metabolism of progesterone and testosterone.     

The metabolism of androgens in rat preputial glands

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.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

5α-reduction of an anti-androgen TSAA-291, 16β-ethyl-17β-hydroxy-4-estren-3-one,by nuclear 5α-reductase in rat prostates

Inhibition of 5α-reduction of testosterone by an anti-androgen TSAA-291 (16β-ethyl-17β-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of ³H-TSAA-291 was examined both $\text{in vitro}$ and $\text{in vivo}$. In the $\text{in vitro}$ experiment using nuclear 5α-reductase of the prostate, 5α-dihydrotestosterone formation from ³H-testosterone was inhibited in a competitive manner by the anti-androgen. In the $\text{in vitro}$ experiment using ³H-TSAA-291, 5α-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 μCi/rat of ³H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5α-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency on the 5α-reductase activity of several other 16β-substituted androstane and estrane analogues was also examined.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

In vitro conversion of 5alpha-reduced metabolites of testosterone by the seminal vesicles, ventral prostate glands and testes of adult rats

In order to ascertain the ability of rat seminal vesicles, testes and ventral prostate glands to interconvert 5α-reduced androgens, these three organs were incubated with either tritiated 17β-hydroxy-5αandrostan-3-one (5α-dihydrotestosterone,DHT), 5α-androstane-3α, 17βdiol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol). The incubation environment utilized (Krebs-Ringer bicarbonate glucose buffer) was selected because the histologic appearance of the tissue at the conclusion of the incubation was indistinguishable from tissue fixed immediately after sacrifice of the animal, thereby approximating the physiologic conditions as closely as possible. In incubations of rat seminal vesicles, ³H.-3β-diol was not metabolized while 26.7 ± 3.8% of ³H-3α-diol appeared as DHT and 17.2 ± 1.5% of ³H-DHT was metabolized to 3α-diol. A small amount (7.5 ± 0.8%) of ³H-DHT was, however, converted to 3β-diol. In incubations of rat testes, the major metabolite, regardless of substrate, was 3α-diol. The conversion of 75.7 ± 2.1% of ³H-3β-diol to 3α-diol has demonstrated, for the first time, that this steroid can be metabolized by the rat testis. Rat ventral prostate glands metabolized $\text{18.5 ± 2.5\%}\text{of}{}^3\text{H-3β-}\text{diol}$ to DHT and 61± 2.9% of ³H-3α-diol to DHT. When ³H-DHT served as the substrate, 83.2 ± 1.5% remained unmetabolized. The prostate glands are, therefore, capable of metabolizing 3β-diol to DHT.     

Pathway of testosterone biosynthesis in the testis of the marmoset Saguinus oedipus

These studies were undertaken to determine the principal pathway of androgen biosynthesis by the testis of the marmoset $\text{Saguinus oedipus}$. Testicular fragments (25 mg) were incubated at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing pregnenolone-7-³H (3β-hydroxy-5-pregnen-20-one) or progesterone-7-³H. Duplicate fragments were incubated with each substrate for 30 min, one hr, three hr, or five hr, for a total of 16 separate incubations. Metabolites were separated by paper and thin-layer chromatography, with identity established by recrystallization to constant specific activities and ³H/¹⁴C ratios. Pregnenolone was readily metabolized to progesterone, 17α-hydroxyprogesterone, androstenedione (4-androstene-3, 17-dione) and testosterone. Progesterone was converted to 17α-hydroxyprogesterone, androstenedione and testosterone. 17α-hydroxyprogesterone was the predominant metabolite obtained from both substrates at one, three and five hrs of incubation. Neither 17α-hydroxypregnenolone (3β-17-dihydroxy-5-pregnen-20-one) nor dehydroepiandrosterone (3β-hydroxy-5-androsten17-one) was detected in the incubates. These data suggest a predominant Δ⁴ pathway with accumulation of 17α-hydroxyprogesterone in the testis of this primate specie.     

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Steroid biosynthesis by reptilian adrenals

Incubations of whole homogenates of. the tiju lizard ($\text{Tupinambis sp}$.) adrenals tissue were carried out using ¹⁴C-labelled progesterone^1*, pregnenolone and cholesterol. ¹⁴C-progesterone was metabolized to labelled 18-hydroxycorticosterone, aldosterone, corticosterone and 11-deoxycorticosterone. Identical metabolites plus ¹⁴C-progesterone were obtained from pregnenolone. Cholesterol-4-¹⁴C was transformed into products similar to those obtained from progesterone. In all these studies the elaboration of cortisol or any other 17-hydroxylated steroids could not be demonstrated. In another set of experiments, whole homogenate preparations from adrenals of the green lizard ($\text{lacerta viridis}$) were incubated with ¹⁴C-labelled androstenedione and testosterone. Ahdrostenedione was converted to testosterone and 11β-hydroxyandrostenedione. Testosterone was metabolized to 11β-hydroxyandrostenedione and androstenedione. The results indicate that the $\text{in vitro}$ transformation of C-27 or C-21 radioactive substrate by lizard adrenals is similar to the other reptiles studied. However, it appears to possess 17β-hydroxysteroid oxido-reductase, though the adrenal tissue itself lacks 17α-hydroxylase activity.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

In vitro on an HCG responsive, testosterone secreting adrenal cortical adenoma

Clinical and biochemical investigation of a virilized woman has shown an adrenal cortical adenoma to be the source of elevated plasma testosterone levels and to be responsive to gonadotropin administration $\text{in vivo}$ (Givens et al.) (2). In the present study, the gonadotropin responsiveness and biosynthetic potential of the adenoma were evaluated $\text{in vitro}$. Incubation of minced adrenal tumor with hCG resulted in increased ¹⁴C-acetate incorporation into pregnenolone, progesterone, dehydroepiandrosterone (DHA), androstenedione, and testosterone. Androstenedione and testosterone were the major products, accounting for 27% and 20% respectively of the total radio-activity added, when ³H-pregnenolone was incubated with homogenized tissue. Estrogen synthesis was not observed in the tumor. The adenoma contained 9.0 μg/g testosterone and 1.9 μg/g androstenedione as determined by radio immunoassay. 17β-hydroxysteroid oxido-reductase was active in the adenoma. Androstenedione was reduced to testosterone at a rate of 0.6 μg/100mg/hr. Under the same conditions, reduction of estrone to estradiol was undetectable. The reductase activity was present in both the mitochondrial and microsomal fractions. NADPH was the required cofactor. When NADH was substituted, the rate was less than 10% of that with NADPH in both particulate fractions.The experimental results indicate the presence of steroid path-way(s) necessary to synthesize testosterone, and represent the first $\text{in vitro}$ demonstration of gonadotropin sensitive steroidogenesis in an adrenal cortical adenoma.     

De novo synthesis of steroids (from acetate) by isolated rat Sertoli cells.

Sertoli cells from 17 day old rats were shown to convert [¹⁴C]acetate to [¹⁴C]-labelled cholesterol, pregnenolone and 17α-hydroxypregnenolone$\text{in}$$\text{vitro}$. Identification was by several systems of thin layer and gas chromatography of the extracted steroids and their sylil and acetyl derivatives and by recrystallizations with authentic and acetylated unlabelled steroids. Several other steroids formed from acetate were tentatively identified. No androstenedione or testosterone were formed. That the Sertoli cell cultures were free of Leydig cells was established by the absence of histochemically detectable 3β-hydroxysteroid dehydrogenase activity and the inability of the cultures to oxidize the 3β-hydroxyl group of [¹⁴C]pregnenolone. This is the first direct evidence that Sertoli cells have the capacity to synthesize steroids $\text{de}$$\text{novo}$ from acetate.     

The in vivo metabolism of androgens by muscle and adipose tissue of normal men

Androstenedione and testosterone labeled with ³H and ¹⁴C were infused simultaneously at constant rates into the brachial arm vein of 10 normal men. During the infusions blood samples were obtained from the brachial artery, a deep vein draining primarily muscle and a superficial vein draining primarily adipose tissue of the arm contralateral to the infusion. In the 10 men the mean ± SE value for the fractional metabolism of androstenedione by muscle is 0.20 ± 0.30 which is not different from the mean value for the fractional metabolism of androstenedione by adipose tissue, 0.29 ± 0.04. The mean value for the metabolism of testosterone by muscle, 0.04 ± 0.01, is significantly less than the metabolism by adipose tissue, 0.11 ± 0.01. Intercpnversion between androstenedione and testosterone occurs in both tissues. The mean value for ρ^A,T_A,M is 0.024 ± 0.005 and for ρ^A,T_A,AT is 0.024 ± 0.005. The mean value for ρ^T,A_A,M is 0.005 ± 0.003 and for ρ^T,A_A,AT is 0.008 ± 0.003. The fractional metabolism of these androgens by these tissues is similar to the fractional metabolism of estrone and estradiol by these same tissues. Muscle appears to contribute about 5–12% of the overall metabolism of androstenedione and testosterone and 10–15% to the overall conversion of androstenedione to testosterone. Adipose tissue contributes about 2–7% of the overall metabolism of these androgens and 5–10% of the overall conversion of androstenedione to testosterone, but < 2% to the overall conversion of testosterone to androstenedione. In normal men, muscle appears to be more important to the metabolism of androstenedione and testosterone than is adipose tissue.     

In vitro androgen metabolism in mouse kidney: high 3-keto-reductase (3alpha-hydroxysteroid dehydrogenase) activity relative to 5alpha-reductase

The $\text{in vitro}$ metabolism of testosterone and dihydrotestosterone was studied in slices and cell fractions of mouse kidney. When testosterone was used as substrate, very little metabolism to dihydrotestosterone occurred suggesting very low 5α-reductase activity. When dihydrotestosterone was substrate, it was rapidly converted to 5α-androstane-3α, 17β-diol by a potent 3-keto-reductase. Ninety-five percent of this latter enzyme is located in cytosol and it requires NADPH as cofactor. The 3-keto-reductase may exist in two molecular forms which can be separated by polyacrylamide gel electrophoresis. Form A and B have mean molecular radii which correspond to molecular weights of 38,700 and 28,700, respectively. Sufficient 3-keto-reductase activity is present in cytosol at 0°C to reduce physiological concentrations (2×10^?9 M) of dihydrotestosterone without the addition of cofactor. 3-Keto-reductase activity is higher in intact male than in castrate male or female mice and increases with androgen treatment.From these studies we conclude: (a) The virtual absence of 5α-reductase in mouse kidney is consistent with the thesis that testosterone rather than dihydrotestosterone may be the intracellular androgen in this organ. (b) Kinetic studies which depend upon the $\text{in vitro}$ uptake and retention of dihydrotestosterone by receptor proteins may be difficult to interpret due to rapid metabolism of ligand.