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.     

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.     

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.     

Testicular steroidogenesis in the mature and immature baboon Papio anubis.

The following studies were undertaken to compare testicular steroidogenesis in the mature and immature baboon. Testicular fragments (50 mg) were incubated for 3 hr with [7-³H]pregnenolone, or with [7-³H]progesterone. The mature testis formed more testosterone (4.6%), androstenedione (1.6%), and progesterone (28.5%) from pregnenolone than did the immature testis (0.6, 0.5, and 26.1%). The immature testis formed more 17α-hydroxyprogesterone (34.7%) and 20α-dihydroprogesterone (23.2%) from pregnenolone than did the mature testis. Similar conversions were obtained in progesterone incubates. 5α-Androstanediol was identified only in mature incubates. These results suggest that the mature baboon testis has greater C₁₇-C₂₀ lyase, 17β-hydroxysteroid dehydrogenase, and 5α-reductase activities than the immature testis, while the immature testis has greater 20α-reductase activity.     

Steroid production in the ovary of the gray mullet (Mugil cephalus) during stages of egg ripening

Steroid production in the ovary of the mullet, Mugil cephalus, during several stages of gonadal development and following spawning, were examined. Pieces of ovarian tissue were incubated with labeled androstenedione and the steroid metabolites isolated by thin-layer and gas-liquid chromatographic methods and identified by derivative formation and recrystallization to constant specific activity. The results indicate that with increasing development of the ovary there occurs a reduction in the concentration of 5α-reduced steroids, 5α-androstane-3α, 17β-diol, and 3α-hydroxy-5α-androstan-17-one, and a concomitant increase in the δ-4, 3-oxosteroids; the ratio of 5α-reduced to 4-ene-3-oxosteroids declining to about one-seventh of the original value. This ratio is not changed following spawning. During all stages of gonadal development, an active 11β-hydroxylase is present in the ovary producing 11β-hydroxyandrost-4-ene-3, 17-dione, 11β-hydroxytestosterone, and 11-ketostestosterone. Only the concentration of 11-ketotestosterone increases with advancing gonadal development.     

Steroid 21-hydroxylase activity in the ovary of the snake Storeria dekayi during pregnancy

The steroidogenic profiles of the corpora lutea and the remaining ovarian tissue from the snake Storeria dekayi at early and midpregnancy were compared after incubation with [4-¹⁴C] pregnenolone. Both tissues produced the following metabolites identified by their isopolarity and isomorphicity with standard compounds: 17α-hydroxypregnenolone, progesterone, 11-deoxycorticosterone, androstenedione, and testosterone. The steroids 17α-hydroxyprogesterone and dehydroepiandrosterone were not detected. Integration of the yield-time curves showed that much more 11-deoxycorticosterone and progesterone were synthesized by the corpora lutea at both stages of pregnancy than by the remaining ovarian tissue, whereas the latter produced more androstenedione and testosterone. Steroid 21-hydroxylase activity was almost exclusively confined to the luteal tissue. The corpora lutea at midpregnancy were smaller but showed greater steroid-converting activity per unit weight of tissue than those at early pregnancy.It is suggested that 11-deoxycorticosterone secretion may be involved in the function of the corpus luteum which is, supposedly, essential for embryonic survival during early pregnancy in some viviparous snakes.     

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.     

PLASMA PREGNENOLONE, PROGESTERONE, 17-HYDROXYPROGESTERONE, TESTOSTERONE AND 5α-DIHYDROTESTOSTERONE IN DIFFERENT TYPES OF CONGENITAL ADRENAL HYPERPLASIA

Unconjugated pregnenolone, progesterone, 17-hydroxyprogesterone, testosterone and 5a-dihydrotestosterone were simultaneously measured by radioimmunoassay in plasma from children with congenital adrenal hyperplasia (CAH) due to a deficiency of 21-hydroxylase (21-OHase), 11 β-hydroxylase (11β-OHase) or 3β-hydroxysteroid dehydrogenase (3β-HSD). These steroids were also measured in a reference group of children of similar age. The following concentrations of these five steroids were observed in the prepubertal children aged 0-4-10-9 years: pregnenolone <0-2-0-85 ng/ml; progesterone 0-17-0-68 ng/ml; 17-hydroxyprogesterone 0-10-0-53 ng/ml; testosterone <0-05-0-14 ng/ml, and 5α-dihydrotestosterone <0-05 ng/ml (detection limit of the method). The concentrations were clearly elevated in the plasma of children with CAH. A very high plasma concentration of 17-hydroxyprogesterone differentiates a 21-OHase deficiency from the two other types: children with this defect had levels mostly in excess of 100-fold that of normal. Plasma progesterone concentrations in these patients were also high being in the range found during the luteal phase level in the adult. Their plasma testosterone concentrations showed a scatter from normal values to high adult male levels being mostly at the level of adult females. The concentrations of 5a-dihydrotestosterone were at or above those of adult males. A high plasma concentration of pregnenolone with at most slightly elevated progesterone and 17-hydroxyprogesterone levels differentiated the 3β-HSD defect from the other two forms of CAH. The plasma profile of the five steroids determined in a patient with an 11 β-OHase deficiency was close to normal, but slightly elevated pregnenolone, progesterone and 17-hydroxyprogesterone levels were found to be characteristic of this enzyme deficiency.     

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.     

Steroid formation in the larval and parasitic adult sea lamprey, Petromyzon marinus L.

Presumptive adrenocortical tissue (PAT) from all known sites in parasitic adults or larval sea lamprey, Petromyzon marinus L., were incubated with [4-¹⁴C]progesterone in a buffered medium with an NADPH generating system. Testicular tissue from parasitic adults was similarly incubated. PAT from both parasitic adults and larvae formed 11-deoxycortisol (S), 17α-hydroxyprogesterone (17αOHP), and androstene dione (AD) but no cortisol (F) cortisone (E), corticosterone (B), 11-deoxycorticosterone (DOC), or testosterone (T) were formed. Therefore PAT may be lacking in 11β-hydroxylase activity but does contain 17α- and 21-hydroxylase and 20-desmolase activities. Testicular tissue failed to produce F, E, B, S, T, 17αOHP, or AD. However, testicular tissue did form DOC indicating the presence of 21-hydroxylase activity. After the 4-hr incubations, histological examination indicated that the PAT and testicular tissue were normal in appearance.     

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.     

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.     

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.     

ADRENAL STEROIDOGENESIS IN HETEROZYGOTES FOR 21-HYDROXYLASE DEFICIENCY

Adrenal steroidogenesis has been studied in vivo in eight obligate heterozygotes (six men and two women) for 21-hydroxylase deficiency and the results compared with twenty-one normal subjects. Serum levels of nine steroids on the biosynthetic pathway (the Δ⁵3β-hydroxysteroids, pregnenolone (Pe), 17α-hyd-roxypregnenolone (17Pe), dehydroepiandrosterone (DHEA), androstenediol (Adiol), and their Δ⁴ 3-keto counterparts, progesterone (Po), 17α-hydroxypro-gesterone (17Po), androstenedione (Adione), testosterone (T)) as well as corti-sol were measured following ACTH stimulation from a dexamethasone-sup-pressed state. There was evidence of reduced 21-hydroxylase activity in the heterozygotes when the results were compared with the usual pattern of response seen in nineteen of twenty-two normal subjects. Thus the increment at 1 h for 17α-hydroxyprogesterone was greater in all the heterozygotes and three of them (one man, two women) also had a greater increment for progesterone. 17,20 desmolase activity may also be reduced in the heterozygotes for they had a lower increment than normal of dehydroepiandrosterone and androstenediol. The results were complicated by the finding that three additional subjects in the normal population tested have much greater 17Po and Po responses than the majority and clearly form a separate population. Our data suggest that there is a common variant within the normal population which may result from the heterozygous state of 21-hydroxylase deficiency; alternatively other forms of 21-hydroxylase may exist with reduced activity that even in the homozygous state may not produce clinical disease.     

Steroid synthesizing cellular sites in the ovary of catfish, Mystus cavasius: A histochemical study

The distribution of Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSDH), 17β-hydroxysteroid dehydrogenase (17β-HSDH), 11β-hydroxysteroid dehydrogenase (11 β-HSDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities has been studied by histochemistry in the ovary of the catfish, Mystus cavasius. The enzyme activities occurred in the thecal cells of the ovary. In addition, a weak enzyme activity was observed in the hypertrophied granulosa cells of early atretic follicles. In later stages these phagocytic granulosa cells degenerated and only thecal cells remained in the connective tissue, surrounded by ovarian stroma. These thecal cells transformed into interstitial gland cells and an intense Δ⁵-3β-HSDH, 17β-HSDH, 11β-HSDH and G-6-PDH enzyme activity occurred in them. It is suggested that the thecal cells of mature follicles and the interstitial gland cells form important sites of steroidogenesis in the ovary of M. cavasius.     

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.     

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.     

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.