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.     

Steroid biosynthesis by phalarope (Steganopus tricolor and Lobipes lobatus) adrenal tissue

Phalarope (Steganopus tricolor and Lobipes lobatus) adrenal tissue homogenates incubated with pregnenolone-4-¹⁴C formed primarily ¹⁴C-labeled progesterone, 11-deoxycorticosterone, and corticosterone. Formation of radioactive dehydroepiandrosterone, testosterone, or androstenedione was not detected. No differences with regard to the products formed or the time course of their formation were observed between adrenal tissue from male and female phalaropes.     

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 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.     

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.     

Biosynthesis of 11-deoxycorticosteroids and androgens by the ovary of the newt Triturus alpestris alpestris Laur

Steroidogenesis by the ripe ovarian tissue of Triturus alpestris alpestris was studied in vitro using both incubation and superfusion techniques. The following metabolites were identified by their isopolarity and isomorphicity with authentic compounds after the tissue was incubated or superfused with [4-¹⁴C]progesterone, 11-deoxycorticosterone, 11-deoxycortisol, 17α-hydroxyprogesterone, androstenedione, and testosterone. Product yield versus time curves approached a steady-state plateau in the superfusate and displayed a differently timed single peak in the incubation medium. In both cases, the integrated yields of 11-deoxycorticosterone and, to a minor extent, of 11-deoxycortisol were greater than those of androgens. The amounts of 11-deoxycorticosterone retained by the superfused and incubated tissues also exceeded those of the other identified compounds. No steroid hormone was isolated after incubation with sodium [1-¹⁴C]acetate, even in the presence of ovine LH or newt pituitary homogenate. On the other hand, 11-deoxycorticosterone was produced from endogenous precursors during superfusion of ovarian tissue with medium alone (0–180 min) or with medium containing HCG (10 IU/ml; 180–300 min). During the first period, 11-deoxycorticosterone output declined to a baseline of 2 ng/g/hr, from which it increased sixfold in the presence of HCG. The final 11-deoxycorticosterone concentration in the tissue was 46.7 ng/g. This steroid was measured by radioimmunoassay following a chromatographic separation from interfering compounds.     

Metabolism of radioactive pregnenolone and progesterone in the embryonic gonads of quail (Coturnix coturnix japonica) in organ culture

Gonads from quail embryos of 10 and 15 days of incubation were explanted for 24 hr on a gelified synthetic medium (Eagle) containing pregnenolone 16-³H and progesterone 4-¹⁴C in equimolecular quantities.The biochemical analyses of these media confirm earlier histoenzymological results. Embryonic gonads of quail, like those of chick, are able to synthesize sex steroids (testosterone by the testis, oestrogens by the ovaries) from the above radioactive precursors. However, in the quail, and mainly in the testis, a delayed appearance and a lower activity of the enzyme 3β HSDH Δ5-4 isomerase was found.It seems likely that for the synthesis of sex hormones, the quail embryonic gonads use preferentially the Δ5-3β hydroxysteroid pathway.     

Biosynthesis of 11-deoxycorticosterone by the ovary of the yucca night lizard, Xantusia vigilis

The steroidogenic capability of the fully grown ovaries of the lizard Xantusia vigilis was studied in vitro using pregnenolone-4-¹⁴C as a precursor. The following metabolites were identified in the incubate by their isopolarity and isomorphicity with standard compounds: predominantly progesterone and 11-deoxycorticosterone and smaller amounts of androstenedione. Testosterone was absent and 17α-hydroxylated intermediates could not be isolated. The enzyme steroid 21-hydroxylase, essential for the formation of 11-deoxycorticosterone, is reported for the first time in the ovary of a reptile.     

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.     

In vitro incubations of presumptive adrenocortical cells from the opisthonephros of the adult sea lamprey, Petromyzon marinus

Presumptive adrenocortical cells from the glomus region of the opisthonephros of the sea lamprey, Petromyzon marinus, were incubated with [4-¹⁴C] progesterone, cholesterol, 11-deoxycortisol, and 11-deoxycorticosterone in a buffered medium for 4 hr with an NADPH generating system. After extraction and chromatographic purification of the incubation media, a considerable amount of ¹⁴C radioactivity was observed in the cortisol, corticosterone, cortisone, and 17α-hydroxyprogesterone areas in several of the incubations. Following the addition of the appropriate authentic tritium labeled steroid and further purification by chromatography and recrystallization the formation of 17α-hydroxyprogesterone was confirmed from progesterone. The formation of progesterone from cholesterol may also have occurred but definitive evidence was lacking. Cortisol, cortisone, 11-deoxycortisol, and 11-deoxycorticosterone were not detected in these incubations.     

Leucopoietic Effect of Calusterone (7 β, 17 α-Dimethyltestosterone) in Women with Advanced Breast Cancer

Calusterone, a new anabolic, weakly androgenic testosterone derivative, found highly effective in women with advanced breast cancer, was studied for its peroral effect on white blood cell count (WBC). A significant rise in WBC occurred during treatment of 80 women, as compared both with a contol group of 21 untreated breast cancer patients and a similar group of 27 patients receiving Δ¹-estololactone 1, 000 mg daily. Δ¹-estololactone produced no significant change in WBC. The combination of 5-fluorouracil 500 mg i.v. once a week plus Δ¹-estololactone 1, 000 mg a day by mouth in 28 patients produced significant mild leucopenia. The effect of androgens on leucopoiesis and the potential value of calusterone in leucopenia, in addition to its efficacy in breast cancer, is discussed.     

Progesterone administered by percutaneous route: an antiandrogen locally useful (author's transl)]

A skin specimen has been obtained from 5 volunteered men submited to a percutaneous administration of progesterone. Plasma progesterone, testosterone and dihydrotestosterone were evaluated before and after this treatment. Progesterone percutaneously administered does not change plasma testosterone and dihydrotestosterone and only slightly increases plasma progesterone. On the contrary, in the skin samples obtained from subjects percutaneously treated by progesterone, radioactive testosterone was not converted to dihydrotestosterone by skin homogenates. In conclusion, progesterone when percutaneously administered inhibits testosterone 5alpha-reduction and may be considered as an antiandrogen locally useful.     

Changes in the testicular metabolism of dehydroepiandrosterone during the annual reproductive cycle of the hedgehog (Erinaceus europaeus L).

Testicular homogenates obtained from the hedgehog (Erinaceus europaeus L.) at different stages of its annual reproductive cycle were incubated with radioactive dehydroepian-drosterone as the substrate at 35 and 4.5° (hibernation temperature). Testosterone (T) and/or 5-androstene-3β,17β-diol (Δ⁵-A) were found as the main metabolites. In the incubations carried out in the absence of co-factors or in the presence of NADP the formation of Δ⁵-A dominated over that of T. In the presence of NADT was formed in high, and Δ⁵-A in negligible or undetectable amounts. The formation of both these steroids was clearly inhibited when the homogenates derived from hibernating animals were incubated at 4.5°. The nature of this cold-induced inhibition of steroidogenesis is discussed.     

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.     

In vitro biosynthesis of 11β-hydroxy- and 11-oxotestosterone by testes of the pike (Esox lucius) and the perch (Perca fluviatilis)

Testes from the pike, Esox lucius, and the perch, Perca fluviatilis, were incubated with radioactive pregnenolone, progesterone, and testosterone, and the major metabolites were identified by chromatography, chemical reaction, and crystallization to constant specific activity. 11-Oxotestosterone and 11β-hydroxytestosterone were the major products from all incubations of pike testes, testosterone being formed in lower yields. The two 11-oxygenated metabolites were also isolated from incubations of perch testes with testosterone, but could not be detected in incubations of this tissue with the other two substrates. Indications were found of the presence of testosterone glucuronide in all incubations with perch testes, but no conjugates could be isolated from incubations of the pike.     

Steroid synthesis in the adrenal gland of the sea snake Hydrophis cyanocinctus: The metabolism of exogenous precursors

Sea snake (Hydrophis cyanocinctus) adrenal glands (whole homogenates, preincubated minces, or mitochondrial preparations) were incubated in vitro with exogenous radioactive precursors. Hydrophis adrenal tissue was capable of synthesizing 17-deoxycorticosteroids from exogenous cholesterol, pregnenolone, progesterone, and DOC, but not from sodium [¹⁴C]-acetate. Products identified after incubation were pregnenolone, progesterone, 11β-hydroxyprogesterone, DOC, corticosterone, 18-hydroxycorticosterone, and aldosterone. The major product was corticosterone with lesser quantities also of 18-hydroxycorticosterone and aldosterone. In the case of the mitochondrial preparation 11β-hydroxyprogesterone predominated. No evidence for the biosynthesis of cortisol from cholesterol was found. Two types of kinetic incubation were employed: One sampled the incubation medium alone, while the other sampled both medium plus tissue. It was concluded that sampling the medium only did not allow the identification of the biosynthetic pathways operating in vitro. However, from sampling both the medium and the tissue it was concluded that both the C21-C11 and C11-C21 sequences of hydroxylation operated in the conversion of progesterone to corticosterone. The data contrast with those obtained from previous studies on cobra adrenal tissue, particularly with regard to the ability of sea snake adrenals in vitro to 18-oxygenate exogenous precursors.     

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.     

Longitudinal study of maternal plasma bioavailable testosterone and androstanediol glucuronide levels during pregnancy

OBJECTIVE The aim of this study to evaluate during normal pregnancy plasma bioavailable testosterone and androstanediol glucuronide levels. MEASUREMENTS Bioavailable testosterone, androstanediol glucuronide and SHBG levels were evaluated every 4 weeks from week 6 to week 38 in 10 normal pregnant women. We also measured plasma oestradiol, oestriol, Δ⁴-androstenedione, 17-hydroxyprogesterone, progesterone and testosterone. RESULTS The mean bioavailable testosterone levels were within the range of non-pregnant women but with an increasing trend until delivery. Androstanediol glucuronide had increased at weeks 6 and 8, decreased at week 14, remained low at week 30, and increased again at week 34. SHBG was significantly correlated with testosterone, oestradiol and oestriol. No correlation could be established between androstanediol glucuronide and any other parameter. DISCUSSION Bioavailable testosterone (non-SHBG bound testosterone) represents the sum of free testosterone plus albumin bound testosterone. The increase in testosterone concentrations with decreased albumin levels during pregnancy, could suggest reduced metabolic clearance of testosterone throughout pregnancy. No correlation was established between the decrease in androstanediol glucuronide and increase in progesterone, suggesting that the decrease in androstanediol glucuronide is not a consequence of the inhibitory effect of progesterone on 5α-reductase activity.     

Effects of C18, and C21 steroids in vitro maturation of oocytes of the catfish, Heteropneustes fossilis (Bloch)

Eleven steroidal compounds were tested for their ability to induce in vitro maturation of Heteropneustes fossilis oocytes. C₁₈ estrogenic steroids (estradiol-17α and -17β) were totally ineffective in inducing oocyte maturation, while androgenic steroids (19-nortestosterone and 11-ketotestosterone) were only marginally effective. Significant maturational ability was confined to C₂₁ steroids; the most potent among them were 11-deoxycortisol, 11-deoxycorticosterone, and 21-deoxycortisol.     

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.