Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.     

Minor triterpenoids of Fomes officinalis

The degraded triterpenoid, 3β,6β-dihydroxy-4,4,14α-trimethyl-Δ⁸-5α-pregnene-20-one (II) and 3-keto-dehydrosulfurenic acid (III), have been isolated from the extracts of Fomes officinalis and their structures determined.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

High performance liquid chromatography of steroid metabolites in the pregnenolone and progesterone pathways

Rapid separation of gonadal steroids in the progesterone(Δ⁴) and pregnenolone pathway (Δ⁵) has been accomplished by the use of high performance liquid chromatography (HPLC). Two HPLC systems are utilized. The first requires the use of two separate radial compression columns (C-18 and C-8), with steroids being eluted with a methanol-water gradient. The second employs a stainless steel C-18 (reversed phase) column with a 12% octadecylsilane coating. The latter system separates seven of the eight steroids in the Δ⁴ and Δ⁵ pathways in thirty-five minutes. For the quantitation of steroids directly, integration of the peak areas, using 254 nm absorption for the Δ⁴ pathway steroids (5 ng minimum limit), and 210 nm absorption for the Δ ⁵ pathway steroids (25 ng minimum limit) is used. For the quantitation of radiolabeled metabolites resulting from incubation of gonadal tissue with radiolabeled steroid precursors, either one of two methods is used: (1) the eluent can be recovered from the HPLC using a fraction collector, and counted in liquid scintillation counter or (2) the entire eluent (or a portion of it) can be counted immediately by directing the flow through a radioactivity detector.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

A radioimmunoassay for the measurement of 5-androstene-3β,17β-diol in plasma

A reliable radioimmunoassay for the determination of 5-androstene-3β, 17β-diol in plasma is described. Antisera were obtained by immunization of rabbits with 3β,17β-dihydroxy-5-androsten-16-one coupled to bovine serum albumin in position 16. The antiserum was characterized by titer, affinity, and specificity. Only dehydroepi-androsterone (24.3 %) and pregnenolone (2.7 %) showed a small crossreactivity. The assay method consisted of extraction with ether, thin-layer chromatography and endpoint determination.The reliability of the method was studied. The interassay variability was 7.5 % at a concentration of 1.22 μg/l. The limit of detection was 0.068 μg/l. Specificity was achieved by Chromatographic separation of the crossreacting steroids. Mass recovery experiments with 250 and 500 pg were performed, in which 99.0 ± 4.6 % of the smaller and 97.6 ± 11.3 % of the greater mass were recovered. In 45 healthy adult males plasma concentrations between 0.44 and 1.80 μg/l were found. The median was 1.06 μg/l. Stimulation of the Leydig cells with human chorionic gonadotropin (HCG) increased plasma concentrations by 93 % (average in 12 males). Therapeutic castration in 8 men caused an average decrease of 55.4 % in plasma values.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Stereospecific reduction of progesterone by Pisum sativum

[4 -¹⁴C]-Progesterone was applied to the leaves of growing pea plants, Pisum sativum. After 3 weeks, about 50% of the administered steroid was reduced, about 20% being reduced to 5α-pregnane-3α,20β-diol as the major metabolite. The radioactivities of 5α-pregnane-3α,20α-diol and 5α-pregnane-3α,20β-diol after 3 weeks were more than twice those after one week. The following radioactive metabolises were also isolated: 5α-pregnane-3,20-dione; 20α-hydroxy-4- pregnen-3-one; 20β-hydroxy-4-pregnen-3-one; 3α-hydroxy-5α-pregnan-20-one; 3α-hydroxy-5β-pregnan-20-one; 3β-hydroxy- 5α-pregnan-20-one; 20β-hydroxy-5α-pregnan-3-one; 5α-pregnane-3β,20β-diol; and 5β-pregnane-3α,20β-diol. The radioactivities of the 5α-pregnane derivatives were considerably higher than those of the corresponding 5β-pregnane derivatives.     

Thein vitro effect of theophylline on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown in the catfish,Clarias batrachus

The effects of theophylline (a phosphodiesterase inhibitor) and cAMP on 17α, 20ß-dihydroxy-4-pregnen-3-one-induced germinal vesicle breakdown was investigatedin vitro in catfish (Clarias batrachus) oocytes. Folliculated oocytes incubated with 17α, 20ß-dihydroxy-4-pregnen-3-one at the concentration of 1 μg/ml induced 93.2 ± 2.23% germinal vesicle breakdown. When the oocytes were prestimulated with 17α,20ß-dihydroxy-4-pregnen-3-one for 6 h and then treated with different concentrations of theophylline, there was a significant drop in the frequency of germinal vesicle breakdown at the concentrations 2.0, 1.5 and 1.0 mM. However, theophylline was found to be incapable of inhibiting germinal vesicle breakdown at its lowest concentration (0.5 inM). In the time course study, significant inhibition of germinal vesicle breakdown was recorded when 1 mM theophylline was added up to 30 h of 17α,20ß-dihydroxy-4-pregnen-3-one Stimulation but the inhibitory effect of theophylline gradually (time dependent manner) declined if the stimulatory time of 17α,20ß-dihydroxy-4-pregnen-3-one was increased. A similar inhibition of germinal vesicle breakdown was also recorded with various concentrations of cAMP. Except 0.5 mM, all the higher concentrations of cAMP significantly inhibited 17α,20ß-dihydroxy-4-pregnen-3-one induced germinal vesicle breakdown.     

Dynamics of excretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate, and of the glucuronides of testosterone and 17β-oestradiol, by urine of reproductively mature male and female rainbow trout (Oncorhynchus myklss)

Levels of sulphated 17α20β-dihydroxy-4-pregnen-3-one (17,20 β -P; the oocyte maturation inducing steroid) in blood plasmas of sexually mature male and female rainbow trout Oncorhynchus mykiss , were very low in comparison to those of the free steroid. However, relatively large amounts were found in urine of both sexes.
Catheters were inserted into the urinary bladders of unovulated and ovulated females and of ripe-running males, and the fish then placed in spawning channels. Three-hourly urine samples were collected between 09.00 and 18.00 hours and then a 15-h sample between 18.00 and 09.00 hours the next morning. Measurements were made of 17,20 β -P-sulphale, testosterone glucuronide (T-G) and 17 β -oestradiol glucuronide (E2-G). In females, the highest rates of excretion of E2-G, T-G and 17,20 β -P-sulphate were found in unovulated, ovulating and ovulated females, respectively. The rates of excretion of 17,20 β -P-sulphate, T-G and E2-G in ovulated females were unaffected by the presence of a male. id males, however, there was a sharp increase in the rate of excretion of 17,20 β -P-suiphate and T-G in fish which were paired with an ovutated (nesting) female. A similar increase was found in males injected with male trout pituitary extract.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Studies on steroid conjugates: IX. Urinary excretion of sulfate conjugated metabolites of cortisol in man.

Following i.v. administration of [4-14C]cortisol, various sulfate conjugated metabolites of cortisol in urine were identified and their respective excretion rates measured. The results obtained demonstrated the following: 1) sulfate conjugates as a group are excreted considerably slower than glucuronide conjugates; 2) sulfate conjugates of steroids with non-reduced ring-A (C-21 sulfates) are excreted (and presumably formed) much faster than steroid-3-sulfates, which require reduction of the ring-A prior to the conjugation; 3) the excretion of C-3 sulfates of ring-A reduced steroids with glycerol side-chain (cortols and cortolones) is significantly faster than those of the corresponding steroids with dihydroxyacetone side-chain (THF, THE and their 5alpha-isomers); 4) the relative concentrations of C-21 sulfates of steroids with ring-A intact (FK, EK, ER, epiER and 6beta-hydroxycortisol) are much higher than the concentrations of C-21 glucuronides of these steroids.     

Solubilization and partial purification of 4,16-androstadien-3-one synthesizing enzyme from boar testis microsomes and requirements for the enzymatic activity

The enzyme related to the synthesis of 4,16- androstadien-3-one from progesterone was investigated using boar testis. To elucidate the activity, in the soluble form, the lyophilized powder of 12,000 g supernatant from homogenate was first treated with n-butanol after which the enzyme could be extracted with 1 mM ethylenediaminete-traacetic acid(EDTA), 1 mM dithiothreitol(DTT) and 20 % glycerol. The enzyme was stable in this medium.The enzyme filtered through a column of Sephacryl S-200 super fine gel exhibited requirements of NADPH-cytochrome C reductase and phosphatidylcholine for maximum enzymatic activity. The requirements of reductase and phosphatidylcholine were not observed in the crude extract fraction. The enzyme separated by column chromatography with DEAE-cellulose required phosphatidylcholine for the synthesis but the reductase had no effect. These lines of evidence suggest that the activity of the enzyme, as related to synthesis of 4,16-androstadien-3-one from progesterone might be regulated by phosphatidylcholine and reductase, $\text{in situ}$.     

Failure to detect delta 5-3 beta-hydroxysteroid oxidoreductase activity in the preimplantation rabbit embryo

Preimplantation rabbit embryos were incubated with pregnenolone and dehydroisoandrosterone under conditions which gave formazan precipitation by the histochemical technique. The metabolic fate of the labeled steroids was assessed simultaneously. There was no concomitant transformation of pregnenolone to progesterone and dehydroisoandrosterone was not transformed to androstenedione. It is concluded that the formazan precipitation is coupled with an "activity" other than delta 5-3 beta-hydroxysteroid oxidoreductase.     

Intermediates in 16-androstene biosynthesis in neonatal porcine testis

The metabolism of pregnenolone has been studied in microsomal preparations of testes from 3-week old piglets. Metabolites identified after derivatization by capillary gas-chromatography and further by combined gas chromatography-mass spectrometry were: 17-hydroxy- and 16 alpha-hydroxypregnenolone, dehydroepiandrosterone, 5-androstenediols, 5-pregnenediol and andien-beta. Pregnenolone and 16-dehydro-pregnenolone, previously not separable by other methods, were successfully resolved as methyl oximetrimethylsilyl ethers using capillary column gas-chromatography.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.