Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.     

Steroid metabolism in human teratocarcinoma cell line PA 1

Human ovarian teratocarcinoma cells of line PA 1, (Zeuthen et al., 1979[1]) used as model for early embryonic cells, were analyzed for their in vitro capacity to convert steroids. The cells were incubated for 20 h with radioactive pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone or estradiol-17 beta, or with non-radioactive progesterone, 6 alpha- or 6 beta-hydroxyprogesterone, 3 beta-hydroxy-5 alpha-pregnan-20-one, dehydroepiandrosterone or estradiol-17 beta. The metabolites were analyzed by thin layer chromatography or studied by gas chromatography-mass spectrometry. The results indicate that PA 1 cells are able to metabolize, although to a restricted amount, a variety of steroids, most markedly progesterone. The metabolites were almost exclusively found in the medium. The main metabolite of progesterone was 3 beta, 6 alpha-dihydroxy-5 alpha-pregnan-20-one. Minor formation of progesterone from pregnenolone could be detected. Human chorionic gonadotropin did not have any effect on pregnenolone metabolism. No formation of estradiol-17 beta or estrone from dehydroepiandrosterone, androstenedione or testosterone could be detected. However, estradiol-17 beta was shown to be converted mainly to estrone. These findings indicate that undifferentiated PA 1 teratocarcinoma cells like certain mouse teratocarcinoma cells, seem not to be steroidogenic but are capable of metabolizing naturally occurring steroid hormones and their precursors.     

Catabolism of cholesterol by bovine adrenal-cortex enzymes: in vitro formation of oxygenated sterols and side-chain cleavage products.

The identification of sterols and steroids formed by incubation of [7alph-3H, 26-14C]cholesterol with mitochondrial enzymes from bovine adrenal cortex is reported. Only 17% of the radioactivity associated with cholesterol metabolites was found in pregnenolone, whereas 15% was reliable to oxygenated sterols and 6% to steroid compounds. The significance of the formation of these compounds is discussed particularly as regards oxygenated cholesterol derivatives.     

Concerted metabolism of steroid hormones produced by cocultured ovarian cell types

Ovaries of immature, intact rats were dispersed by collagenase-DNase treatment and cultured in serum-free medium (ovarian cell culture). The hormonal responsiveness of the ovarian cell was compared to that exhibited by pure granulosa cells isolated via needle puncturing. Surprisingly, despite the fact that the majority of the cultured cells should have been comprised of granulosa type, no follicle-stimulating hormone-inducible progesterone or 20 alpha-OH-progesterone (20 alpha-OH-P) could be detected by radioimmunoassay, as typically occurs in cultures of pure granulosa cells. Therefore, in order to unravel the cause for the different responsiveness between the granulosa and the ovarian cell, we applied thin-layer chromatography analysis to follow the metabolic fate of added radioactive pregnenolone to intact monolayers in culture. Such TLC analysis revealed that, after priming with follicle-stimulating hormone, added [3H]pregnenolone was converted to progesterone which was rapidly reduced and finally accumulated as 5 alpha-pregnane-3 alpha,20 alpha-diol(pregnanediol). In addition to pregnanediol, a second class of steroid hormones accumulated in the coculture medium, namely androsterone and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol (pregnanetriol). These latter two were specific products of the ovarian coculture, indicating the presence of theca-interstitial cells bearing 17 alpha-hydroxylase and 17,20-lyase activities. Pregnanediol, rather than progesterone, was found to be the progestin precursor for androgen formation. We thus conclude that due to exchange of steroid metabolites between the cocultured cell types, the final steroid products are different by far from the expected contributions of each individually cultured cell type. Moreover, these findings reveal an additional aspect of the "two-cell theory," suggesting a granulosa-thecal concerted metabolism of progestin steroids, rather than exchange of aromatizable androgens.     

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.     

Inhibition of rat testicular microsomal steroidogenesis by oxytocin and metyrapone

Capillary gas chromatographic 'steroid profiling' has been utilised to separate and quantify the metabolites (derivatized as methyloximes and/or trimethylsilyl ethers) formed from pregnenolone after incubation with rat testicular microsomes. A wide range of steroid metabolites was found, indicating that both the 5-ene and 4-ene pathways of testosterone biosynthesis were operating, as well as 16 alpha-hydroxylation, 20 beta-reduction and the formation of several C19 steroids (the 16-androstenes). At the concentration used, Metyrapone markedly inhibited 16 alpha- and 17-hydroxylation and side-chain cleavage of 17-hydroxylated C21 steroids. 16-Androstene production was also markedly inhibited and the formation of other metabolites was affected to lesser extents. Oxytocin abolished the formation of all C21 and C19 metabolites of pregnenolone.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Evidence for the biosynthesis of DHEA from cholesterol by first-trimester human placental tissue: source of androgens.

With a view to establishing whether first-trimester human placentas possess the ability to synthesize DHEA from cholesterol, homogenates of this tissue obtained from two groups of women undergoing elective termination of normally progressing pregnancy between 10 - 12 weeks gestation (n = 5, age 23 - 29 years and n = 5, age 21 - 27 years) were incubated separately with [26-(14)C]cholesterol for the generation of [14C]isocaproic acid + pregnenolone and [7n-3H]pregnenolone for the biosynthesis of [3H]DHEA. Controls consisted of homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution analysis, desmolase efficiency expressed as mean specific activity of [14C]isocaproic acid varied from 282 to 725 dpm/mmol, while that of 17 alpha-hydroxylase and steroid C-17,20-lyase, catalyzed conversion of [7n-3H]pregnenolone to [3H]DHEA varied from 3498 to 26 258 dpm/mmol. The corresponding efficiencies of enzymicconversion varied between 5.8 x 10( -2) and 1.5 x 10( -1) % for [14C]isocaproic acid, but between 5.5 x 10( -2) and 4.1 x 10( -1) % for [3H]DHEA. No such metabolite was evident in the controls of heat-denatured homogenates. These are the first study results to demonstrate that early placentas are capable of converting cholesterol to pregnenolone to DHEA, contrary to the widely held concept of DHEA production by fetal and maternal adrenal glands. This finding has important physiological implications and could provide a new dimension to the concept of fetoplacental steroidogenesis.     

Inhibition of rat ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase and 17,20 lyase by progestins and danazol

The site of action of synthetic progestins or danazol in the treatment of endometriosis is considered to be mainly the hypothalamo-pituitary level, but the direct action to the uterine endometrium and the ovary is also suggested. We investigated the effect of these synthetic steroids to rat ovarian steroidogenic enzymes. The effect of norethisterone, levonorgestrel, danazol, gestrinone, desogestrel and 3-keto-desogestrel was studied in vitro. The sources of the enzymes were prepared from ovaries of immature rats treated either with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) for 3 beta-hydroxy steroid dehydrogenase (3 beta-HSD), or with PMS for 17 alpha-hydroxylase and 17,20 lyase. The substrates used were pregnenolone (P5) for 3 beta-HSD, progesterone (P4) for 17 alpha-hydroxylase, and 17 alpha-hydroxy-progesterone (17 alpha-OH-P4) for 17,20 lyase. The substrates were incubated with the enzyme sources and coenzymes, and the products formed were measured. All the steroids inhibited 3 beta-HSD, and the inhibition by gestrinone (Ki = 3.0 microM) and 3-keto-desogestrel (17.5 microM) was particularly marked. Only desogestrel (Ki = 30.3 microM) and danazol (168 microM) inhibited 17 alpha-hydroxylase. All the steroids inhibited 17,20 lyase, and the inhibition by desogestrel (Ki = 0.70 microM), danazol (0.80 microM), and gestrinone (30 microM) was particularly marked.     

Measurement of testosterone and dehydroepiandrosterone 16alpha-hydroxylase activities by a tritium exchange method.

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16alpha-hydroxylase activity. Specifically 16-tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16-3H]progesterone and [16-3H]pregnenolone. The incubation medium consists of a phosphate buffer (pH7; 150mM), NADPH (0.1 mM), nicotinamide (10mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]testosterone (50 micron) or [16-3H]dehydroepiandrosterone (100 micron). The enzymatically released tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform, very sensitive (50 pmol of 16alpha-hydroxylated metabolites) and is independent of any further metabolism of the 16alpha-hydroxylated products.     

Phospholipases modulate immature pig testicular androgen and 16-androstene biosynthetic pathways in vitro.

The role of membrane phospholipids in porcine testicular androgen and 16-androstene biosynthesis was examined by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Untreated (control) microsomes from immature pig testes converted pregnenolone to 17-hydroxypregnenolone and DHA to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadien-3-one (dienone) in the 16-androstene pathway, these metabolites accounting for most (65%) of the pregnenolone converted. The 4-ene steroids in the androgen pathway (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) totalled less than 10% of the pregnenolone metabolites. No estrogens or 5 alpha-reduced metabolites were detected. Treatment with phospholipase A2 or C, decreased the conversion of pregnenolone to 4-ene-3-oxo steroids but did not decrease the quantities of 5-ene-3 beta-hydroxysteroids. Confirmation of these findings was obtained by measuring the individual enzymatic steps. Phospholipases A2 and C significantly reduced the conversion of DHA to androstenedione and andien-beta to dienone but did not affect 17-hydroxylase or 'andien-beta-synthetase'. However, when the C-17, 20 lyase step was measured alone, phospholipase C decreased the quantity of androstenedione produced indicating that the side-chain cleavage reaction may involve a lipid component. The different effects of phospholipases on these enzymes suggests that pregnenolone metabolism may be regulated by alterations in the membrane microenvironment.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

The effects of steroid hormones and gonadotropins on in vitro placental conversion of pregnenolone to progesterone

Using human term placental mitochondrial preparations, optimal conversion of [3H]pregnenolone to [3H]progesterone was obtained at 30 min incubation and with a mitochondrial protein content of 2.5-3.5 mg/ml. Estradiol, estrone, progesterone and testosterone in a dose range of 0.03-8.66 mumol inhibited the in vitro conversion of [3H]pregnenolone to [3H]progesterone by placental homogenates. All four steroids inhibited the pregnenolone to progesterone conversion in a dose-dependent manner. The ID50 (dose required to inhibit conversion of pregnenolone to progesterone by 50%) was 0.04 mumol for estradiol, 0.13 mumol for testosterone, 0.3 mumol for progesterone and 1.0 mumol for estriol. Neither gonadotropin releasing hormone (50-1000 ng) nor human chorionic gonadotropin (5-500 IU) affected the placental basal conversion rate of pregnenolone to progesterone in vitro. Our findings indicate that steroid hormones such as estradiol, estrone, testosterone and progesterone can inhibit local placental progesterone biosynthesis through inhibition of the enzyme complex 5-ene-3 beta-hydroxysteroid dehydrogenase.     

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum

Sliced testis tissue from Bufo arenarum was incubated in the presence of [³H]pregnenolone. Testis fragments were also used for double isotope experiments using [³H]pregnenolone and [¹⁴C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C₁₉ steroids such as dehydroepiandrosterone, 5-androsten-3β,17β diol, testosterone, 5α-dihydrotestosterone and a C₂₁ steroid, 5α-pregnan-3,20 dione. Progesterone mainly converts to 5α-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3β,17β diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis. Accepted: 17 April 1998     

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.     

Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7 alpha-hydroxylase.

To study the effect of steroid hormones on bile acid synthesis by cultured rat hepatocytes, cells were incubated with various amounts of these compounds during 72 h and conversion of [4-14C]cholesterol into bile acids was measured. Bile acid synthesis was stimulated in a dose-dependent way by glucocorticoids, but not by sex steroid hormones, pregnenolone or the mineralocorticoid aldosterone in concentrations up to 10 microM. Dexamethasone proved to be the most efficacious inducer, giving 3-fold and 7-fold increases in bile acid synthesis during the second and third 24 h incubation periods respectively, at a concentration of 50 nM. Mass production of bile acids as measured by g.l.c. during the second day of culture (28-52 h) was 2.2-fold enhanced by 1 microM-dexamethasone. No change in the ratio of bile acids produced was observed during this period in the presence of dexamethasone. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of the bile acid pathway, to bile acids was not affected by dexamethasone. Measurement of cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes, incubated with 1 microM-dexamethasone, showed 10-fold and 90-fold increases after 48 and 72 h respectively, as compared with control cells. As with bile acid synthesis from [14C]cholesterol, no change in enzyme activity was found in hepatocytes cultured in the presence of 10 microM steroid hormones other than glucocorticoids. Addition of inhibitors of protein and mRNA synthesis lowered bile acid production and cholesterol 7 alpha-hydroxylase activity and prevented the rise of both parameters with dexamethasone, suggesting regulation at the mRNA level. We conclude that glucocorticoids regulate bile acid synthesis in rat hepatocytes by induction of enzyme activity of cholesterol 7 alpha-hydroxylase.     

Modulation of 17 alpha-hydroxylase/C17,20-lyase activity in porcine theca cells

The major source of ovarian androgen is the theca cells. Androgens are produced by the conversion of progestins by the 17 alpha-hydroxylase/C17,20 lyase enzymatic system (lyase). The 3 beta-hydroxysteroid dehydrogenase and aromatase enzymes in the theca cells are modulated by gonadotropins as well as by steroids produced locally. Therefore, the combined effects of hCG plus progesterone, estradiol, or dihydrotestosterone (DHT) on microsomal lyase activity in theca cells from large and medium-sized follicles were determined. Theca cells (3 x 10(6) cells/6 ml/well) were cultured in Medium 199 (M199) containing only insulin (10 micrograms/ml) and transferrin (5 micrograms/ml). At 24 h, theca cells were treated with M199, hCG (15 ng/ml), progesterone, estradiol, or DHT (100 ng/ml) or a combination of hCG + one of the three steroids. Media were removed at various times of culture (27-72 h) and levels of androgen determined by RIA. Microsomes were incubated with 1 microCi [3H]progesterone +0.5 mM NADPH and radioactive conversion products were measured after purification by thin layer chromatography. Administration of progesterone, estradiol, or DHT alone had little effect on lyase activity in theca cells from medium-sized follicles whereas the addition of hCG alone significantly increased lyase activity in these cells. However, concomitant addition of any steroid with hCG inhibited the increase in lyase activity after the addition of hCG alone. Theca cells from large porcine follicles had a higher basal level of lyase activity compared to theca cells from the smaller follicles. Lyase activity in theca cells from large follicles was enhanced by progesterone; estradiol was inhibitory. DHT initially stimulated lyase activity in theca cells from large follicles, but was inhibitory later in culture. In contrast to its marked effect on theca cells from medium follicles, hCG had only a small effect on lyase activity in theca cells from large follicles. Thus, thecal lyase activity increased as the follicle matured, providing more androgen substrate for the production of estrogen. Lyase activity in theca cells of medium follicles appears to be regulated predominantly by gonadotropin from the pituitary while intraovarian regulation of lyase activity by steroids may be more important in larger follicles.     

Steroid metabolism in the corpus luteum of the ferret

Implantation in the ferret is believed to be induced by a luteal substance which acts in concert with progesterone (P4) and which is secreted sometime between Days 6 and 8 of pregnancy. This experiment was designed to identify the steroid products synthesized by ferret corpora lutea (CL) on these 2 days of pregnancy. CL were dissected from ferrets on Day 6 or 8 of pregnancy and incubated with [3H] pregnenolone (P3), [3H] P4, or [3H] dehydroepiandrosterone (DHEA). Controls with no tissue or with 50 microliters packed blood cells were incubated at the same time. After incubation of Day 6 CL with [3H] P3 for 180 min, 39% of the added label was found incorporated into P4, 3% into 17 alpha-hydroxyprogesterone (17 alpha-OHP4) and 1% into androstenedione (A). Incubation of Day 8 CL with the same precursor resulted in 35%, 1% and 0.65% of the label being incorporated into the previously mentioned products, respectively. Incubations of Days 6 and 8 ferret CL with [3H] P4 or [3H] DHEA confirmed these results, demonstrating activity of C21-steroid, 17 alpha-hydroxylase and delta 5-isomerase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). These results suggest that ferret CL primarily accumulate steroids of the delta4 pathway on both Days 6 and 8 of pregnancy, with P4, 17 alpha-OHP4, A and testosterone (T) being the most abundant products after in vitro incubation. Thus, ferret CL appear to metabolize steroids in a manner similar to that observed in rats, sows and mares.     

Structural and functional changes in ovaries from adult mice treated with diethylstilboestrol in the neonatal period

Ovaries from 8-week-old female NMRI mice in different stages of the oestrous cycle, or from females neonatally treated with the synthetic oestrogen diethylstilboestrol (DES; 5-10(-6) micrograms daily for 5 days), were studied histologically and for the ability to synthesize steroids from [3H]pregnenolone in vitro. Daily doses of 10(-4) micrograms DES or higher resulted in absence of corpora lutea. In ovaries lacking corpora lutea, the interstitial tissue dominated and the cells in this compartment were large with a clear cytoplasm. The steroids synthesized in ovarian homogenates were separated with thin-layer chromatography. The homogeneity of the steroids was checked in recrystallization experiments. Daily doses of 5-10(-4) micrograms DES in the neonatal period resulted in pronounced deviations in the pattern of ovarian steroids synthesized as compared with control ovaries. In DES-exposed ovaries, the synthesis of androstenedione and, above all, progesterone was high while the synthesis of 17 alpha-hydroxyprogesterone and testosterone was reduced compared with controls. These results could argue for a difference in activities of 17 alpha-hydroxylase and 17 beta-ol-dehydrogenase in ovaries from DES-treated females compared with controls. After transplantation of DES-exposed ovaries to ovariectomized control females, the steroid pattern changed to that typical for control ovaries. Control ovaries transplanted to DES-treated females had a steroid pattern similar to that of DES-exposed ovaries.     

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.