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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Biosynthesis of 15-hydroxylated steroids by gonads of the river lamprey, Lampetra fluviatilis,in vitro

Ovaries and testes of the river lamprey, Lampetra fluviatilis, were incubated with [³H]testosterone and [³H]progesterone and the major metabolites identified by chromatography, chemical reaction, and gas chromatography-mass spectrometry. The major metabolite of progesterone with gonads of both sexes was 15α-hydroxyprogesterone (26.6% yield in ovary, 50.0% in testis); a small amount of 15β-hydroxyprogesterone was also found in the testis incubation. With testosterone as substrate, the major metabolite was 15β-hydroxytesterone (84.8% in testis, 65.2% in ovary). The formation of androstenedione, but not of testosterone, from progesterone was also demonstrated in both sexes.     

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.     

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.     

STEROID 17, 20-DESMOLASE DEFICIENCY: A NEW CAUSE OF MALE PSEUDOHERMAPHRODITISM

In a child with male pseudohermaphroditism (ambiguous external genitalia, XY sex chromosomal constitution and normal adrenocortical function), incubations of testicular tissue with pregnenolone/progesterone, 17α-hydroxy-pregnenolone/17α-hydroxyprogesterone and androstenedione/dehydroepiandrosterone showed that testosterone could be formed from androstenedione and dehydroepiandrosterone only, but not from other substrates. In urine, testosterone did not increase after HCG, but small amounts of pregnanetriolone were found, which increased after HCG and ACTH. There was no DHA increment after ACTH. It is concluded that this patient, as well as a first cousin and a gonadectomized maternal 'aunt' with the same clinical and urinary steroid findings have testicular and adrenal steroid 17,20-desmolase deficiency, causing a defect of androgen biosynthesis, which has not previously been described. The heredity of this condition seems to be autosomal or X-chromosomal.     

In VitroBiosynthesis of Androgens in the Australian Lungfish,Neoceratodus forsteri

The serum concentration of testosterone was estimated from a population of wild lungfish over 6–7 years of sampling. Male lungfish were found to have high circulating levels of testosterone (∼50 ng/ml) which varied seasonally and could be correlated with spermatogenesis as judged by testis histology. Incubation of testis tissue slices with [³H]progesterone, [³H]17-hydroxyprogesterone, or [³H]testosterone confirmed that testosterone is the major androgen inNeoceratodus.Not even trace amounts of 11-keto- or 11β-hydroxytestosterone or 5α-dihydrotestosterone could be identified by TLC separations. There was little or no conjugation of steroids by the testes, except during the spawning season, when glucuronides of androstenedione and testosterone were produced.     

Further evidence that serotonin may be a physiological melanocyte-stimulating hormone-releasing factor in the lizard Anolis carolinensis

Testes of the hagfish Myxine glutinosa were incubated with [³H]progesterone and [³H]-testosterone and their metabolites identified by chromatography and either chemical reaction followed by crystallisation to constant isotope ratio or by gas chromatography-mass spectrometry. The following metabolites of testosterone were identified: androstenedione (23% yield), 6β-hydroxytestosterone (6.3%), 5α-androstane-3β,7α,17β-triol (4.5%), 5α-androstane-3β,6β,17β-triol (trace), and a solvolysable conjugate of testosterone (0.7%). Testosterone was identified as a metabolite of progesterone (3% yield).     

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.     

Pathways of testosterone synthesis in decapsulated testes of mice.

In order to study the temporal relations in the biogenesis of testosterone, decapsulated testes of adult mice were incubated with carbon-14-labelled sodium acetate and attempts were made to isolate the most likely intermediates. Considerable quantities of radiochemically homogeneous squalene, lanosterol, cholesterol, testosterone and androstenedione, but no pregnenolone, progesterone, 17-hydroxypregnenoline, 17-hydroxyprogesterone, dehydroepiandrosterone, pregnenolone sulphate or dehydroepiandrosterone sulphate were isolated. The same pattern of incorporation was found when gradually increasing amounts of non-labelled pregnenolone, progesterone, 17-hydroxypregenenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate or testosterone were added to the system as "trapping agents" or when Leydig cell preparations rather than decapsulated testes were used. The presence of 10 mIU of HCG greatly enhanced the de novo formation of testosterone, androstenedione and 5alpha-dihydrotestosterone but did not change the pattern of acetate incorporation. Radioimmunoassays of the incubation medium with or without added HCG, and carried out at different periods of time indicated the presence of gradually increasing amounts of testosterone and androstenedione together with some 5alpha-dihydrotestosterone, whereas only trace amounts of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and dehydroepiandrosterone were present. An analysis of the incubated testes revealed that the addition of HCG significantly enhanced the content of testosterone, androstenedione and 5alpha-dihydrotestosterone. Little or no increase was observed as far as pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone or dehydroepiandrosterone were concerned. It is concluded that decapsulated testes of mice synthesize de novo testosterone from sodium acetate under conditions in which the formation of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnenolone sulphate and 17-hydroxypregnenolone sulphate cannot be demonstrated.     

Androgen biosynthesis in vitro by testes from amphibia.

Testes from three species of anuran Amphibia, Bufo marinus, Rana catesbeiana, and Rana esculenta, were incubated with radioactive pregnenolone, progesterone, and testosterone. In all incubations the major metabolite was dihydrotestosterone, which accounted for 30–47% of the initial radioactivity after a 3-hr incubation. In addition, 5α-androstanedione and 5α-androstane-3β,17β-diol were formed from all three substrates with testes of Bufo marinus and Rana catesbeiana. Testes of Rana esculenta however converted only testosterone into 5α-androstanedione and 5α-androstanediol, pregnenolone and progesterone being transformed to 5α-pregnane-3β,17α,20ξ-triol. Since in both R. catesbeiana and B. marinus significantly higher yields of 5α-androgens were obtained from pregnenolone and progesterone than from testosterone, it is possible that at least some of these compounds arise from a biosynthetic pathway not involving testosterone.     

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.     

Steroid metabolism in the testes of the breeding and nonbreeding three-spined stickleback, Gasterosteus aculeatus

Steroid metabolism in the testes of sticklebacks was studied in vitro by tissue incubations with [3H]pregnenolone or [3H]androstenedione as precursors. In males in full reproductive condition (nesting), [3H]pregnenolone was mainly converted via progesterone and 17 alpha-hydroxyprogesterone into androstenedione, 11 beta-hydroxyandrostenedione, and 11-ketoandrostenedione. The latter was the largest product formed. The main products from the [3H]androstenedione incubation, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione, confirm these findings. The rate of androgen synthesis, especially of 11-ketoandrostenedione, was much lower after the end of the breeding season.