The inhibition of the conversion of testosterone into 5α-dihydrotestosterone in the reproductive organs of the male rat

The inhibition of testosterone 5α-reductase activity by 3-oxo-4-androstene-17β-carboxylic acid in the male reproductive organs of the rat was demonstrated $\text{in vitro}$. The medium for incubation of caput epididymis showed the highest concentration of 5α-dihydrotestosterone (5α-DHT) whereas the highest concentration of testosterone (T) was recorded in medium for incubation of decapsulated testis after two hours of incubation. The 3-oxo-4-androstene-17β-carboxylic acid (1.58 × 10^?5M) inhibited the conversion of T to 5α-DHT in all the organs tested (testis, caput and cauda epididymis and ventral prostate) under identical incubation conditions.     

Identification by capillary gas chromatography-mass spectrometry of eleven non-ecdysteroid steroids in the haemolymph of larvae of Sarcophaga bullata

$\text{O-}\text{Pentafluorobenzyloxime}$ (OPFB)-heptafluorobutyrylester (HFB) derivatives and $\text{OPFB}\text{-O-}\text{methyloxime}$ (MO)-trimethylsilylether (TMS) derivatives of non-ecdysteroid steroids were prepared from haemolymph extracts of last instar larvae of the fleshfly Sarcophaga bullata. Using a negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS) technique the following steroids could be identified: progesterone, testosterone, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, androst-5-ene-3β,17β-diol, androstenedione, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, 17α-hydroxyprogesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxyprogesterone. Although the technique is very sensitive, estrogens could not be detected. These results suggest an active metabolism of progesterone and testosterone.     

Androgen receptor protein binding properties and tissue distribution of 2-selena-a-nor-5α-androstan-17β-ol in the rat

2-selena-A-nor-5α-androstan-17β-ol was studied $\text{in vitro}$ and $\text{in vivo}$ in the rat prostate gland. The data demonstrates the ability of this compound to selectively complex with the specific receptors of 5α-dihydrotestosfcerone (5α-DHT) in the cytosol and to be retained in the nuclei in an unaltered form. Studies with selenium-75 labeled material suggests that the uptake and localization is similar to endogenous 5α-dihydrotestosterone.     

Rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihyorotestosterone in the male rat anterior pituitary: in vivo and in vitro studies

The $\text{in vivo}$ and $\text{in vitro}$ metabolism of $\text{(}{}^3\text{H}\text{)-5α-}\text{androstane}\text{-α, 17β-}\text{diol}$ by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73 % of the recovered radioactivity were constituted by 5α-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5α-dihydrotestosterone was the main steroid recovered except from the 105,000 × g pellet. From $\text{in vivo}$ and $\text{in vitro}$ experiments it was concluded that the transformation of 5α-dihydrotestosterone into 5α-androstane-3α,17β-diol was a reversible process, and that this last steroid could exert its biological action mainly $\text{via}$ 5α-dihydrotestosterone.     

Effects of steroidal hormones on sexual, attack, and search behavior in the isolated male chick

Immature male chickens were treated with testosterone (1 mg/day), Δ₄-androstenedione (1 mg/day), 5α-dihydrotestosterone (5α-DHT; 1 mg/day), 5α-androstanedion (1 mg/day), or estradiol (100 μg/day) in order to assess the effects of these steroids on copulatory behavior, agonistic behavior, and attentional processes. Testosterone, estradiol, and 5α-DHT were most effective in stimulating male copulatory behavior above that of oil-treated controls; whereas Δ₄-androstenedione and 5α-androstanedione had less, but nevertheless significant, effects on this behavior. Testosterone and 5α-DHT facilitated agonistic behavior; however, estradiol, 5α-androstanedione, and Δ₄-androstenedione were ineffective in this capacity. The persistence of response to a given stimulus type was increased by testosterone and decreased by 5α-DHT: 5α-Androstanedione had no discernible effect on this behavior. These findings suggest that in the male chicken the neural structures regulating male copulatory and aggressive behavior as well as attentional processes are differentially sensitive to sex steroids. The effects of all these steroids on somatic structures were assessed.     

Biliary metabolites of testosterone and testosterone glucosiduronate in the rat

A mixture of testosterone-4-¹⁴C and testosterone-1,2-³H-17-glucosiduronate was intraperitoneally administered into male and female rats with bile fistulas. Biliary metabolites were separated and purififd by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the blue. 5β-Androstane-3α, 17β-diol was found principally in monoglucosiduronate fraction and was produced preferentially from the injected conjugate in both sexes. Very marked sex differences from the injected conjugate in both sexes. Very marked sex differences were observed in the following metabolites: Androsterone was present only in the female as monoglucosidironate, which was preferentially derived from testosterone. 5α-Androstane-3α,17β-diol was identified in both monoglucosiduronate and diconjugate fractions of the female, which was formed significanrly more from the conjugate than testosterone. These findings provide evidence that testosterone glucosiduronate could be converted directly into 5α-steroids as well as 5β-ones $\text{in}$$\text{vivo}$. In marked contrast, the major portion of testosterone was metabolized to polar steroids in the male.     

Effects of sex steroids on calling,locomotor activity,and sexual behavior in castrated male Japanese quail

Castrated male Japanese quail were implanted with Silastic capsules containing testosterone (T), estradiol-17β (E₂), 5β-dihydrotestosterone (5β-DHT), Δ₄-androstenedione (Δ₄) 5α-androstanedione (A), 5α-dihydrotestosterone (5α-DHT) or with empty capsules. Calling, monitored continuously and automatically, was induced significantly by T and Δ₄. Locomotor activity, also monitored continuously by floor deflection, was enhanced by T, Δ₄, and E₂. Additional data concerning heterosexual and homosexual behavior were obtained from castrated quails after implantation of T, Δ₄, E₂, or 5α-DHT. T and Δ₄ restored hetero- and homosexual behavior as did E₂ but to a lesser extent. 5α-DHT did not induce either sexual behavior. Growth of the cloacal protrusion was induced in birds implanted with T, Δ₄, A, and 5α-DHT but not with 5β-DHT and E₂. These results indicate that calling and locomotor activity enhancement (including sexual behavior) are two different components of reproductive behavior which require different androgens or their metabolites to be activated.     

5α-reduction of an anti-androgen TSAA-291, 16β-ethyl-17β-hydroxy-4-estren-3-one,by nuclear 5α-reductase in rat prostates

Inhibition of 5α-reduction of testosterone by an anti-androgen TSAA-291 (16β-ethyl-17β-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of ³H-TSAA-291 was examined both $\text{in vitro}$ and $\text{in vivo}$. In the $\text{in vitro}$ experiment using nuclear 5α-reductase of the prostate, 5α-dihydrotestosterone formation from ³H-testosterone was inhibited in a competitive manner by the anti-androgen. In the $\text{in vitro}$ experiment using ³H-TSAA-291, 5α-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 μCi/rat of ³H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5α-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency on the 5α-reductase activity of several other 16β-substituted androstane and estrane analogues was also examined.     

Stimulatory effects of 5 beta-dihydrotestosterone on the sexual behavior in the domestic chick

During in vitro incubations, brain tissues of chicks transform testosterone mainly into 5β-reduced androgens including 5β-dihydrotestosterone (β-DHT). Although β-DHT is generally believed to have no androgenic effects, we showed recently that it stimulates sexual behavior in young chicks during hand thrust tests. This result was confirmed during three experiments presented here and the possible interactions of β-DHT with the endogenous sex steroids have been analyzed. There is no synergism between β-DHT and estradiol in the induction of sexual behavior in the chicks. β-DHT is still partly active in chicks injected with the antiandrogen, cyproterone acetate in doses sufficient to block all action of the endogenous testosterone. β-DHT is also active in castrated chicks. The effects of β-DHT on the sexual behavior thus do not seem to depend on an interaction with other sex steroids and the reasons why it is active in chicks during hand thrust tests and not in other test situations in other birds are briefly discussed.     

Inhibitor of heme synthesis in polycythemic rabbit serum

Sera from hypertransfused polycythemic rabbits were found to significantly inhibit ⁵⁹Fe incorporation into heme in erythroid cells in normal rabbit bone marrow cultures when compared with that of normal serum controls suggesting a higher concentration of this inhibitor in polycythemic serum. This serum inhibitor delayed the time of peak cumulative heme synthesis $\text{in}$$\text{vitro}$ and the delay in peak cumulative heme synthesis was increase with increasing concentrations of polycythemic serum. It is suggested from these studies that this serum inhibitor may be involved in a negative feedback system in the control of erythropoiesis and may act specifically on differentiated nucleated erythroid cells to delay their entry into the cell cycle, consequently inhibiting heme synthesis.     

Effects of diverse androgens on the sexual behavior and morphology of castrated male quail

Castrated male Japanese quail were injected for 15 days with 1 mg/day of testosterone propionate (TP), testosterone (T), androstenedione (AE), androsterone (AO), 5α-dihydrotestosterone benzoate (5α-DHTB), or 5β-dihydrotestosterone (5β-DHT), or with oil. Copulation was activated to a significant extent only by TP and T. Strutting was activated only by TP, T, and AE. Proctodeal (foam) glands were well-developed in birds injected with TP, T, AE, or 5α-DHTB. Additional data were obtained following implantation of pellets of crystalline T, AE, AO, or 5α-DHT. T pellets activated copulation, but AO and 5α-DHT pellets did not. Effects of AE require further study. These results suggest that conversion of androgen to estrogen is necessary for the activation of copulation in the male quail.     

Regulation of aromatase, 5α- and 5β-reductase in primary cell cultures of developing zebra finch telencephalon

Sex steroids act on the developing and adult telencephalon of songbirds to organize and activate the neural circuits required for the learning and production of song. Presumably, the availability of active androgens and estrogens to steroid-sensitive neural circuits controlling song is modulated by the local expression of androgen-metabolizing enzymes. Two enzymes, 5α- and 5β-reductase, are expressed widely in the songbird telencephalon, as they are in the telencephalons of other avian species. These enzymes convert circulating testosterone (T) into the active and inactive metabolites, 5α- and 5β-dihydrotestosterone (DHT), respectively. A third enzyme, aromatase, converts T into estradiol (E₂) and is expressed at unusually high levels in several regions of the songbird telencephalon. In many tissues, including the brain, the regulation of expression of one or more of these enzymes can be a critical feature of their ability to control the production of active sex steroids. We have used primary cell cultures to examine factors that might regulate the expression of these enzymes in developing zebra finch telencephalon. Cultures were treated for 0-72 h with sex steroids (T, E₂, 5α-DHT, and 5β-DHT) or with dibutyryl cAMP. Afterward, activities of aromatase, 5α-, and 5β-reductase were determined or total RNA was extracted for Northern analysis. Treatments with cAMP increased both aromatase activity and aromatase mRNA levels by 220%. E₂ significantly reduced aromatase activity by an average of 65%, whereas 5α- and 5β-DHT had no effect on aromatase activity. Compared to untreated controls, E₂ treatment decreased aromatase mRNA levels by 56%. None of these treatments consistently affected either 5α- or 5β-reductase activities. These results suggest that telencephalic E₂ may regulate its own synthesis by repression of aromatase expression, whereas factors that upregulate cAMP in the telencephalon can increase the local concentrations of E₂. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 30–40, 1998     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

The uptake and metabolism of labelled testosterone by the brain and pituitary of the male rhesus monkey (Macaca mulatta)

The ventricular systems of three male rhesus monkeys (one castrate) were infused over a one hour period with a small volume of labelled testosterone of high specific activity. The pituitary and various areas of the brain and samples of blood and spinal fluid were secured following infusion. Radioactive steroids were extracted from the tissues, separated chromatographically, and identified by recrystallization to constant specific activity. Radioactive testosterone (T), 5α-dihydrotestosterone (5α-DHT), 5α-androstane-3α, 17β-diol (5α-A-diol) and δ₄-androstenedione (δ₄-A) were found in all samples of pituitary and brain and at a much higher concentration per unit weight than that noted in blood. The spinal fluid samples contained primarily unchanged T. Uptake of labelled T appeared to be greater in the pituitary and hypothalamus than in other areas of the brain. It is concluded that (1) ventricular infusion of labelled testosterone under the conditions of these experiments provided a suitable means of supplying deep structures of monkey brain and pituitary with a high concentration of steroid with relatively little reaching the systemic circulation, and (2) steroidal 5α-reductase and 17β-dehydrogenase activity was present throughout the brain.     

Erythroid colony formation and erythropoietin activity in mice treated with estradiol benzoate

In mice administered with estradiol benzoate (EB) at 0.2 or 5 μg dose levels, the number of erythroid colony-forming units (CFU-E) in marrow declines progressively starting from 12 through 48 hr. On the other hand hypoxic erythropoietin (Ep) production, although potentiated in animals primed with relatively large dosages of EB (5–25 μg), is significantly diminished after treatment with lower amounts of estrogen (0.2 μg). In mice primed with large amounts of EB, the enhancement of Ep activity apparently compensates the depleting influence on marrow CFU-E, thus leading to a nearnormal erythroid response to hypoxia. On the other hand, a sharp drop of this parameter is observed in animals primed with the lower dosage (0.2 μg), $\text{i.e.}$ the depleting influence on the CFU-E pool is not compensated here by potentiation of the Ep response.     

Identification and quantification of C21 and C19 steroids in the haemolymph of Leptinotarsa decemlineata,a phytophagous insect

o-Pentafluorobenzyloxime (OPFB)-heptafluorobutyrylester (HFB)-derivatives were prepared from extracts of haemolymph from last instar larvae of Leptinotarsa decemlineata and subjected to negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS). Ten C21 and C19 steroids could be positively identified: testosterone, dehydroepiandrosterone, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, androstenedione, progesterone, 17α-hydroxyprogesterone, pregnenolone and 17α,20β-dihydroprogesterone. No estrogens could be found in these larvae. Radioimmunoassay of chromatographed extracts of haemolymph taken from the larval and pupal stages showed fluctuations in testosterone (and 5α-dihydrotestosterone) titer.     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

De novo synthesis of steroids (from acetate) by isolated rat Sertoli cells.

Sertoli cells from 17 day old rats were shown to convert [¹⁴C]acetate to [¹⁴C]-labelled cholesterol, pregnenolone and 17α-hydroxypregnenolone$\text{in}$$\text{vitro}$. Identification was by several systems of thin layer and gas chromatography of the extracted steroids and their sylil and acetyl derivatives and by recrystallizations with authentic and acetylated unlabelled steroids. Several other steroids formed from acetate were tentatively identified. No androstenedione or testosterone were formed. That the Sertoli cell cultures were free of Leydig cells was established by the absence of histochemically detectable 3β-hydroxysteroid dehydrogenase activity and the inability of the cultures to oxidize the 3β-hydroxyl group of [¹⁴C]pregnenolone. This is the first direct evidence that Sertoli cells have the capacity to synthesize steroids $\text{de}$$\text{novo}$ from acetate.     

Behavioural and morphological dose-responses to testosterone and to 5α-dihydrotestosterone in the castrated male Japanese quail

Morphological and behavioural effects of testosterone (T) and of 5 α-dihydrotestosterone (5α-DHT) injected daily for a 3-week period at dosages of 0.5, 1, 5 and 10 mg for T and 1 and 5 mg for α-DHT were studied in the adult male castrated Japanese quail. Injections of 0.5 or 1.0 mg T produced only slight development of the cloacal gland while the other four treatments stimulated growth which reached maximal or submaximal values. Testosterone injections stimulated sexual activities; some such effects were also observed after treatment with 5α-DHT. Although both steroids elicited crowing, there were qualitative differences between quails given 5α-DHT and those given T and intacts. These differences were not due to the development of the sternotracheal (syringeal) muscles, the weights of which were increased and reached similar values in the 5α-DHT and T (5 and 10 mg) treated males. These results are discussed in the context of our present knowledge of the mechanisms of regulation of reproduction processes by testosterone and its metabolites in birds.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.