Steroid biosynthesis by the ovary of the European eel, Anguilla anguilla L., at the silver stage.

The ovary of the European eel, Anguilla anguilla, at the silver stage, was incubated either as an intact tissue preparation or as a homogenate with and without cofactors in the presence of [4-¹⁴C] pregnenolone and [4-¹⁴C]progesterone. Intact tissue incubates displayed a more complex metabolite profile than reinforced homogenates, and deprivation of exogenous cofactors reduced the profile even further. Among the metabolites derived from pregnenolone, the following steroids were identified by their isopolarity and isomorphicity with standard compounds: 17α-hydroxypregnenolone; dehydroepiandrosterone; progesterone; 17α-hydroxyprogesterone; and androstenedione. The last three steroids plus testosterone, 17β-hydroxyandrostenedione, and adrenosterone were identified using progesterone as a precursor. Metopirone inhibited the formation of 11-oxygenated androgens. 11-Deoxycorticosteroids were not found, indicating the absence of steroid 21-hydroxylase activity in the eel ovary. Integration of the product yield-time curves demonstrates that in vitro the activities of the enzymes 3β-, 17β-, and 11β-hydroxysteroid dehydrogenases were less apparent than those of steroid 17α,20-C₂₁-desmolase, and 17α-, and to a lesser extent 11β-hydroxylase. Irrespective of the incubation conditions, pregnenolone produced more Δ⁵-3β-hydroxy-thanΔ⁴-3-ketosteroids, suggesting a predominance of the former biosynthetic pathway. Among the unidentified metabolites, water-soluble compounds were formed from both precursors in intact tissue incubates.     

Androgen biosynthesis by marmoset testes in vitro.

These studies were undertaken to determine the major steroid metabolites formed from selected androgen precursors by the testis of the marmoset, Saguinus oedipus, a New World primate of the family Callitricadae. Testicular fragments (50 mg) were incubated for 3.0 hr with pregnenolone-7-³H or with progesterone-7-³H. The major metabolites formed from pregnenolone were 17α-hydroxyprogesterone (42.7%), testosterone (20.5%), androstenedione (11.4%) and progesterone (9.2%). Nonmetabolized substrate was 6.8% of radioactivity. For porgesterone incubations, 17α-hydroxyprogesterone was the major matabolite (49.0%), with testosterone (21.2%) and androstenedione (10.7%) as lesser metabolites. Unreacted progesterone accounted for 14.9% of all radioactivity. The unusually high levels of 17α-hydroxyprogesterone in marmosets is in contrast to that observed in other mammalian species.     

Aromatization and 5α-reduction in brain and nonneural tissues of a cyclostome,Petromyzon marinus

Aromatization and 5α-reduction appear to be essential for the expression of certain androgen actions in the central nervous system. Although 5α-reductase activity occurs in neural tissues of invertebrates and all major vertebrate groups, brain aromatase has not been traced below elasmobranch. Using the lamprey (Petromyzon marinus), androgen metabolism was investigated in brain, gonad, and other tissues by incubating homogenates with [³H]androstenedione and cofactors. After extraction, chromatography, and phenolic partition, products were identified by dilution with authentic unlabeled steroid and recrystallization to constant specific activity. Estrone was formed in ovary, testis, and kidney but not in liver, muscle, or tissue-free control incubates. Testicular samples also synthesized estradiol-17β. Identification of estrogen in brain incubates was not definitive. All tissues except muscle had 5α-reductase activity as shown by accumulation of 5α-androstandione. No 5α-dihydrotestosterone or estradiol-17α were identified in any samples. The presence of 5α-reductase in cyclostome brain is consistent with the widespread phylogenetic distribution of this enzyme although the occurrence of central aromatase activity in Agnatha has yet to be demonstrated.     

The influence of mammalian and avian gonadotropins on in vitro ovarian steroid synthesis in the turtle (Chrysemys picta).

The synthesis of steroids from 7α[³H]cholesterol and 7α[³H]pregnenolone by turtle ovarian tissues in vitro was studied. Pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, estrone, estradiol 17β, estriol, and 16-epiestriol were identified as products. All estrogens were detectable in incubates of preovulatory follicular tissue, but only small quantities of estrone were found in incubates of follicular tissue from postovulatory animals and luteal tissue. The effects of mammalian and avian gonadotropins on the metabolism of tritiated precursors were studied. Both mammalian and avian LH were stimulatory when conversion of cholesterol or pregnenolone to major steroid products was examined. In particular, enhancement of estrogen biosynthesis predominated in preovulatory follicular tissue, whereas increased progestin yield was the major effect in follicular and luteal tissue from postovulatory animals. The effects of FSH were minimal compared to the same dose of LH. Thus, a slight increase in estrogen yield was only noted when preovulatory follicular tissue was incubated with cholesterol and mammalian FSH, and neither mammalian nor avian FSH had an effect on pregnenolone conversion by follicles from postovulatory animals. Prolactin had no effect on luteal progesterone synthesis when used alone, but reduced the stimulatory effect of mammalian LH on progesterone synthesis. 11-Desoxycorticosterone was not found to be a product of the turtle ovary under normal conditions or after in vitro ACTH stimulation.     

The in vitro biosynthesis of steroids by the gonad of the cuttlefish (Sepia officinalis L.)

In vitro steroid biosynthesis in the male and female gonads of Sepia officinalis was investigated. Using tritium- and ¹⁴C-labeled precursors, seven enzyme systems were demonstrated: a C_20,22-lyase (cholesterol→pregnenolone), a 3β-hydroxysteroid dehydrogenase-Δ_5?4-isomerase complex (pregnenolone → progesterone; 17α-hydroxypregnenolone → 17α-hydroxyprogesterone; dehydroepiandrosterone → androstenedione), a 17α-hydroxylase (pregnenolone → 17α-hydroxypregnenolone; progesterone → 17α-hydroxyprogesterone), a C_17,20-lyase (17α-hydroxypregnenolone → dehydroepiandrosterone), a 17β-hydroxysteroid dehydrogenase (dehydroepiandrosterone → androstenediol; androstenedione → testosterone; estrone→estradiol-17β), a 20α-ol dehydrogenase (progesterone→20α-dihydroprogesterone), and a 20β-ol dehydrogenase (progesterone→20β-dihydroprogesterone). The yields were rather low (0.01–3%), except when pregnenolone was used (8%). Radioimmunoassay techniques indicate the presence of testosterone in the plasma of the cuttlefish but the absence of estrogens.     

In vitro biosynthesis of steroids from progesterone by the ovaries and pyloric ceca of the starfish Asterias rubens

In vitro biosynthesis of steroids from progesterone in ovaries and pyloric ceca of Asterias rubens has been investigated. The biosynthesis of 17α-hydroxyprogesterone, androstenedione, testosterone, 20α-dihydroprogesterone, 11-desoxycorticosterone, and 5α-pregnane-3,20-dione could be demonstrated to take place in the tissues of both organs by using [1,2-³H]progesterone as a precursor. The yields of intermediates of the Δ⁴-pathway and of 11-desoxycorticosterone are small, being higher in the ovaries than in the pyloric ceca. The yields of 20α-dihydroprogesterone are low, those of 5α-pregnane-3,20-dione are high. In both cases the yields in the pyloric ceca exceed those in the ovaries. The results indicate the presence of the following biosynthetic enzyme systems in ovaries and pyloric ceca of Asterias rubens: 17α-hydroxylase, C₁₇C₂₀-lyase, 17β-HSD, 20β-HSD, 21-hydroxylase, and 5α-reductase. The importance of these enzymes for the metabolism of progesterone, i.e., the biosynthesis of C₁₉-steroids, 20α-dihydroprogesterone, and corticosteroids, will be discussed.     

Studies on the reproductive cycle on the female cobra, Naja naja. II. In vitro ovarian steroid synthesis.

The synthesis of steroids in vitro by minced ovarian tissue from the cobra, Naja naja, using [³H]pregnenolone and [³H]dehydroepiandrosterone([³H]DHA) as precursors was studied. From [³H]pregnenolone the major products were progesterone, pregnanolone (3α-hydroxy-5β-pregnan-20-one), 17α-hydroxyprogesterone, androstenedione, and testosterone. DHA and 17α-hydroxypregnenolone were tentatively identified, but insufficient material was available for positive characterization. From incubations using [³H]DHA as precursor, the only products identified were testosterone, androstenedione, and estradiol-17β. Significant amounts of radioactivity were associated with an estriol fraction from both the pregnenolone and the DHA incubations but were not further characterized. Time-lapse studies revealed an extremely rapid conversion of [³H] pregnenolone to progesterone, with a maximum occurring after 15 min in tissue taken from a cobra in April at the height of the reproductive period. Addition of cofactors to the medium markedly stimulated the synthesis of progesterone and pregnanolone from [³H]pregnenolone, but appeared to inhibit the production of other ovarian steroids. Mammalian LH, when added to the incubation medium, was found to stimulate the biosynthesis of 17α-hydroxyprogesterone, androstenedione, and testosterone from [³H]pregnenolone. Addition of fresh, homogenized snake pituitary or mammalian FSH appeared to increase the yield of testosterone but none of the precursors in the pathway, and there was a suggestion that FSH alone increased the rate of aromatization.     

The in vitro formation of 5 alpha-dihydrotestosterone by the testis of Discoglossus pictus

Testes from sexually mature amtran amphibian Discoglossus pictus were incubated in vitro with testosterone-4-¹⁴C (5 × 10^?6M) at 30°C for 2 hr, without cofactor. The following metabolites were identified: 5α-dihydrotestosterone, 5α-androstane-3β, 17β-diol, androstenedione, 5α-androstanedione. The 5α-DHT is the main metabolite formed. These results indicated the presence of a Δ4-3-ketosteroid-5α-oxydoreductase in the testes of Discoglossus. One hundred and fifty nanograms of DHT were isolated after incubation of 100 mg of tissue with testosterone-4-¹⁴C for 2 hr.A significant amount of testosterone (30 40%) was metabolized in conjugated and hydrosoluble compounds. Steroid glucuronides and sulfates were identified by enzymatic hydrolysis. Less than 4% of incubated testosterone-4-¹⁴C is transformed to testosterone glucuronide and 5% to testosterone sulfate.Testosterone metabolism, studied in vitro, varied according to different physiological states: hydrosoluble steroids formation is about 30% during the period of reproduction: hypophysectomy decreased the conjugation rate, whereas estrogen-treatment increased it. The formation of free 5α-DHT from testosterone-4-¹⁴C by testes of Discoglossus can be experimentally modified; 1060 ± 335 pmoles of 5α-reduced steroids were produced in the normal testes from a sexually mature animal: HCG-injection increased the activity of the steroid-Δ4–5α-reductase, the amount of 5α-reduced steroids formed was 1692 ± 759 pmoles per testis in 2 hr: testes from a hypophysectomized animal and from an animal during the sexual inactivity period produced smaller amounts, 752 ± 82 pmoles and 734 ± 241 pmoles per testis in 2 hr: estrogen treatment introduced no change in the 5α-reductase activity, 1011 ± 377 pmoles per testis in 2 hr.Experiments with cellular fractions demonstrated the presence of 5α-reductase activity in the nuclear and microsomal fractions. Most of the activity uper milligramm of protein: was recovered in the microsomal fraction. The 5α-reductase is NADPH dependent.     

Steroid biosynthesis by the testes of the dogfish Scyliorhinus caniculus

Dogfish testes were incubated with radioactive progesterone, pregnenolone, and testosterone, and both free and conjugated metabolites were examined. In the free fraction, which contained 42–70% of the incubated radioactivity, progesterone, androstenedione, and testosterone were identified as incubation products of both progesterone and pregnenolone. In addition, a small amount of 17α-hydroxyprogesterone was identified as a metabolite of progesterone in one fish. Testosterone and androstenedione were the only free steroids isolated from incubations of testosterone. Although steroid glucuronide formation was insignificant, very large amounts of solvolysable steroids were isolated from all incubations. With pregnenolone and progesterone, 10–30% of the incubated radioactivity was recovered in this solvolysable fraction, in which the major products were identified as testosterone and 17α,20β-dihydroxy-4-pregnen-3-one. With two fish incubated with [¹⁴C]testosterone, 5α-androstane-3β, 17β-diol was isolated in low yield from the solvolysable fraction in addition to testosterone, but in one incubation with [³H]testosterone, the sole component of this fraction was testosterone which accounted for 21% of the initial radioactivity.     

The in vitro biosynthesis of steroids by the gonad of the mussel (Mytilus edulis)

In vitro steroid biosynthesis in the male and female gonad of Mytilus edulis was investigated. Using tritium and ¹⁴C-labeled precursors, four enzyme systems were demonstrated: a 3β-hydroxysteroid dehydrogenase-Δ_5,4-isomerase comples (pregnenolone→progesterone; 17α-hydroxypregnenolone→17α-hydroxyprogesterone; dehydroepiandrosterone→androstenedione), a C_17,20-lyase (17α-hydroxypregnenolone→dehydroepiandrosterone; 17α-hydroxyprogesterone→androstenedione), a 17β-hydroxysteroid dehydrogenase (dehydroepiandrosterone→androstenediol; androstenedioneαtestosterone; estradiol-17βαestrone) and a 5α-reductase (testosterone→5α-dihydrotestosterone). The yields were very low (0.2–1%) except when estrogens were used (10–50%). Radioimmunoassay techniques indicate the presence of testosterone and possibly estrogens in the gonad of the mussel at two different stages in its gametogenic cycle.     

Suggestion of abnormal testicular steroidogenesis in some oligospermic men

Androgen biosynthesis in the testis may be analyzed in some detail by means of techniques of in vitro incubation of small testicular biopsy specimens with suitable radiolabelled precursors. Sixty-six tissue specimens from 33 patients who underwent bilateral testicular biopsies because of infertility were incubated in vitro with [3H]pregnenolone in order to investigate the possibility of abnormalities in their steroid biosynthetic activity. As a normal control, testicular tissue obtained by testicular biopsy from a young normal volunteer was used. The distribution of metabolites in the incubates of testes from 8 infertile men differed greatly from the remaining 25 patients and the normal control. The major steroids formed from pregnenolone by the testes of those 8 men were 17-hydroxypregnenolone, dehydroepiandrosterone, 20alpha-dihydropregnenolone and and 20alpha-dihydro-17-hydroxypregnenolone. Very small amounts of delta4-3 oxo products (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) were formed suggesting a deficiency of 3beta-hydroxy-steroid-dehydrogenase activity in the testes of these 8 men, possibly related to the derangement of their spermatogenic function.     

Testicular steroid biosynthesis by the green lizard Lacerta viridis

Testes from the green lizard Lacerta viridis were incubated with [³H]pregnenolone or [³H]testosterone and the products were identified by chromatography, microchemical reaction, and crystallisation to constant specific activity or isotope ratio. The major metabolites of pregnenolone were testosterone (40.8%), androstenedione (5.5%), 5α-androstane-3β,17β-diol (4.4%), and 5α-pregnane-3β,17α,20ξ-triol (15.2%). Androstenedione was the only identifiable metabolite (4.8%) of testosterone.     

In vitro corticosteroidogenesis by the adrenal gland of the chicken (Gallus domesticus)

[4-¹⁴C]Pregnenolone, [4-¹⁴C]progesterone, and [4-¹⁴C]11-deoxycorticosterone were indubated with chicken adrenal tissue slices, whole homogenates, and subcellular fractions, with and without the addition of ACTH to the incubation medium. Progesterone, 11-deoxycorticosterone, corticosterone, 11β-hydroxyprogesterone, and aldosterone were identified as metabolites of these radioactive precursors. The rate of conversion of pregnenolone to progesterone by the slices and progesterone to corticosterone by the mitochondrial fraction significantly increased by the addition of ACTH to the medium. The activity of Δ⁵-3β-hydroxysteroid dehydrogenase associated with Δ⁵-Δ⁴ isomerase upon pregnenolone and the activity of 21-hydroxylase upon progesterone were concentrated in the microsomal fraction, while the activity of 11β-hydroxylase upon 11-deoxycorticosterone was in the mitochondrial fraction. No 17α-hydroxylase activity was observed.The main pathway for steroidogenesis in the chicken adrenal gland is proposed to be: pregnenolone → progesterone → 11-deoxycorticosterone → corticosterone.     

Androgen metabolism in the brain and non-neural tissues of the bullfrog Rana catesbeiana.

By investigating steroid conversions in the bullfrog Rana catesbeiana, the present study tests the hypothesis that androgen metabolism by the central nervous system (CNS) is a primitive vertebrate characteristic. Parts of the brain containing the amygdala, preoptic area, and hypothalamus (AMY/HTH), remaining forebrain (RFB), midbrain and hindbrain (MB/HB), and also the ovary, testis, muscle, and fat were homogenized and incubated with [7α-³H]androstenedione and cofactors for 60 min at 25°. Products were isolated and characterized by thin-layer chromatography and recrystallized to constant specific activity; estrone (E₁) derived from brain incubates was also methylated. E₁, testosterone (T), and 5α-dihydrotestosterone (DHT) were the metabolic products in brain incubates. In addition to E₁, ovarian homogenates synthesized estradiol-17β (E₂-17β) and estradiol-17α (E₂-17α), but no aromatase activity was detectable in the testis, fat, or muscle. T was the primary metabolite in the testis. Radioimmunoassayable T, DHT, E₁, and E₂ were present in the plasma of male and female bullfrogs. Concentration of aromatase in the HTH/AMY of the bullfrog is consistent with a similar distribution of enzyme activity in the limbic system or its homologs in a reptile and mammals. Centers for the control of reproduction and cells which bind sex steroids have been identified in the same brain areas in other anuran species. The experiments reported here indicate that the conversion of androgen to estrogen and other neutral metabolites by the brain is a primitive tetrapod characteristic and suggest that metabolism is an integral component of brain-steroid interactions which has been conserved during the evolution of vertebrates.     

Effect of photoperiod on the testicular steroidogenesis of the bank vole (Clethrionomys glareolus, Schreber): an in vitro study

Testicular minces from juvenile bank voles subjected to short (6L:18D, light on 0800 to 1400, Group S) and long (18L:6D, 0600–2400, Group L) photoperiods for 6 to 9 weeks were incubated (1 hr) with [4-¹⁴C]pregnenolone (Δ⁵P) and [4-¹⁴C]dehydroepiandrosterone (DHA). The main metabolites formed were identified by thin-layer chromatography (TLC). In the presence of exogenous cofactors (NADPH/NADP 1.3 mmol/liter) [4-¹⁴C]Δ⁵P was almost totally metabolized in Group L testes and was mainly converted to androstenedione (Δ⁴A; 36.9%), testosterone (T; 29.5%), and DHA (6.7%). In similar incubations with Group S testes no C₁₉-steroids were formed from [4-¹⁴C]Δ⁵P which was predominantly converted to progesterone (60.7%). A substantial portion (32.1%) of the substrate was not metabolized. When [4-¹⁴C]DHA was incubated with exogenous cofactors it was mainly converted to Δ⁴A (41.4%, Group S and 53.5%, Group L), T (37.7%, Group S and 36.1%, Group L), and dihydrotestosterone (DHT) (2.5%, Group L4 only). In Group S testes the substrate (DHA) was not utilized as effectively as in Group L (15.5 and 2.3% unmetabolized substrate, respectively). When exogenous cofactors were not added there was a marked decrease in substrate utilization in both groups. The results indicate that at least in vitro the testes of voles subjected to short photoperiods have a drastically reduced ability to synthesize such potent androgens as Δ⁴A and T from exogenous [4-¹⁴C]Δ⁵P and that this incapability is largely due to a C₁₇-C₂₀-lyase and/or 17α-hydroxylase deficiency.     

Metabolism of pregnenolone-4-14C and pregnenolone-7-alpha-3H sulfate by the Macaca mulatta fetal adrenal in vitro

These studies were initiated to ascertain the feasibility of utilizing the fetal monkey adrenal as a model for further studies on comparative steroid metabolism at various periods of gestation. Homogenates of midtrimester fetal monkey (Macaca mulatta) adrenals were incubated simultaneosly with pregnenolone-4-¹⁴C and pregnenolone-7α-³H sulfate. Conversion of both substrates to 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, 11β-hydroxyandrostenedione, and cortisol as well at 17α-hydroxypregnenolone sulfate and dehydroepiandrosterone sulfate was demonstrated. Conversion of pregnenolone to progesterone was also shown. Neither free nor conjugated 16α-hydroxypregnenolone or 16α-hydroxydehydroepiandrosterone were found.     

Sex differences in steroidogenesis by adrenal homogenates of adult possum (Trichosurus vulpecula) attributable to the steroids formed by the adrenocortical special zone of the female

A marked sex difference was found in the nature of steroid conversion products formed from [¹⁴C]progesterone by whole adrenal homogenates of adult possums. The major conversion products from adrenals of males were: cortisol in yields of 46 ± 2% and corticosterone, 25 ± 2% (mean ± SEM, n = 35). The total 5β-reduced products were <10%. With glands from females 5β-reduced products comprised >80% of the yield, the predominant steroids being 5β-pregnane-3α,17α-diol-20-one, 25 ± 4% and 5β-pregnane-3α-o1-20-one, 21 ± 3% (mean ± SEM, n = 39). The yields of cortisol was <8%. No significant difference was found in the yields of products from animals used on the day of capture from the wild, or taken from the animal house 1–12 months after capture. Studies were carried out on the separated adrenocortical special zone and cortex proper of females. Homogenized cortex proper incubated with [¹⁴C]progesterone formed mainly: cortisol, 28 ± 2%; corticosterone, 18 ± 3%; and 11β-hydroxyprogesterone, 24 ± 4% (mean ± SEM). From the same substrate the conversion products by the homogenized special zone were 5β-reduced pregnane derivates in yields >70% and reduced 5α- and 5β-androstan derivates, <15%. The products of major yield were: 5β-pregnan-3α-o1-20-one, 5β-pregnane-3α,17α-diol-20-one, and 5β-pregnane-3α,17α,20α-triol. Similarly from [17α-¹⁴C]hydroxyprogesterone and [11-¹⁴C]deoxycortisol substrates the conversion products by the special zone were mainly 5β-reduced pregnane derivates, the C19 steroids being <10%. Dissected medullary tissue had no steroidogenic activity. The addition of medullary tissue to cortex proper had no effect on the synthetic activity of the cortex. With mixtures of cortex proper and special zone the conversion products were similar to those obtained by whole adrenal homogenates. It is concluded that the dissimilarity in the adrenal steroid formation was due to the activity of the 5β-reductase, which prevailed in the female adrenal and was very low in the male adrenal. It was found that the location of the reducing enzymes was specific to the unique adrenocortical special zone which is present only in the female possum.     

Biochemical, histochemical, and ultrastructural analyses of presumed steroid-producing tissues in the sexually mature sea lamprey, Petromyzon marinus L

In vitro incubations of testicular, ovarian, and presumed adrenocortical tissues (PAT) from the mature sea lamprey, Petromyzon marinus, with [1,2-³H]cholesterol failed to form cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, pregnenolone, and progesterone. “Isopolarity” and “isomorphicity” were establidhed for testosterone and androstenedione from the PAT incubation, but subsequent attempts at derivative formation indicated that no testosterone or androstenedione was formed. The interstitial cells of the testes and the cells within the ovarian follicles of P. marinus have a fine structure similar to those in other species of lamprey. The appearance of these tissues and of PAT remains unaltered after 4 hr of incubation. Histochemical procedures did not provide evidence for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity in ovary and PAT, and only a weak 3β-HSD activity was observed over the interstitial tissue of the testes. Spectrophotometric evidence for 3β-HSD activity was obtained from PAT and testicular tissue homogenates. No 3β-HSD activity was observed in mature ovarian tissue homogenates.     

Synthesis of 20 alpha-hydroxylated steroids by ovaries of the dab (Limanda limanda)

Dab (Limanda limanda) ovarian fragments were incubated in vitro with either [4,7-3H]pregnenolone or 17 alpha-hydroxy[1,2,6,7-3H]progesterone to investigate the pattern of steroidogenesis. A major enzyme found in the dab ovary was 20 alpha-hydroxysteroid dehydrogenase. Among the steroids that were tentatively identified in ovarian incubates were 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P). 17 alpha,20 alpha-dihydroxy-5 beta-pregnan-3-one, 3 beta, 17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta), and 3 alpha,17 alpha,20 alpha-trihydroxy-5 beta-pregnane. The presence of these steroids in plasma of mature female and male dabs was studied by radioimmunoassay. The antiserum was raised against 17,20 alpha-P. The 17,20 alpha-[3H]P label was produced by incubating place milt with 17 alpha-hydroxy [3H]progesterone. The radioimmunoassay was shown to have a high cross-reaction with the 5 beta-reduced analogues of 17,20 alpha-P and was therefore used, in conjunction with thin-layer chromatography, to measure the steroids. High concentrations of both 17,20 alpha-P and 3 beta,17,20 alpha-P-5 beta found in female and male dab plasma. The possible role of these steroids is discussed.     

Evidence of the in vitro formation of cortisol by the adrenal gland of embryonic and young chickens (Gallus domesticus)

The aim of this study is to elucidate the ontogenetic aspect of corticosteroidogenesis in the chicken. The adrenal gland of embryonic and very young chicks contains an enzyme system which converts progesterone to 17α-hydroxyprogesterone. 4-¹⁴C-Labeled cholesterol, pregnenolone, progesterone, and 17α-hydroxyprogesterone were incubated with the homogenates of adrenal gland from 17- and 21-day-old chick embryos and chickens. The metabolic products were identified by their mobilities on a thin-layer chromatogram and recrystallization to constant ³H:¹⁴C ratio after adding the corresponding ³H-labeled steroid. Cholesterol was metabolized to pregnenolone in the tissue homogenates from chick embryos and chickens at all ages. Pregnenolone was metabolized to progesterone, 11-deoxycorticosterone, and other minor metabolites, but not to 17α-hydroxyprogesterone. The major products from progesterone were 11-deoxycorticosterone and 17α-hydroxyprogesterone. The yield of 17α-hydroxyprogesterone decreased with advancing age and became zero at 7 days posthatching. 11-Deoxycortisol and cortisol were produced from progesterone by the homogenates from 17- and 21-day-old embryos and 3-day-old chicks, but neither was produced by those from 7-day-old chicks or those from 150-day-old hens. Radioactive 17α-hydroxyprogesterone was converted to 11-deoxycortisol and cortisol in large amounts and to cortisone in small amounts. Androstenedione and testosterone were detected in the adrenal homogenate from 17 days of incubation to 7 days posthatching, but not in the tissue from 14 days posthatching. The activity of 17α-hydroxylase was high at 17 days of incubation, decreasing with advancing age, and disappeared between 7 and 14 days posthatching. These results represent definite evidence of cortisol and testosterone formation in vitro by embryonic and very young chick adrenals.