Androgen metabolism in skin and skeletal muscle of the rainbow trout (Salmo gairdnerii) and in accessory sexual organs of the spur dogfish (Squalus acanthias).

The in vitro metabolism of testosterone, 4-androstene-3,17-dione (androstenedione) and dehydroepiandrosterone by skin and muscle from the rainbow trout (Salmo gairdnerii), and by skin and accessory sexual tissues from the spur dogfish (Squalus acanthias) was studied. In trout skin, testosterone was transformed mainly into 5α-dihydrotestosterone together with smaller amounts of 5α-androstane-3α, 17β-diol, androstenedione, 5α-androstane-3,17-dione and androsterone. Androstenedione was transformed mainly into 5α-androstane-3,17-dione with smaller amounts of testosterone, 5α-dihydrotestosterone, androsterone and 5α-androstane-3α,17β-diol. Dehydroepiandrosterone was transformed to 5-androstene-3β,17β-diol with trace quantities of androstenedione and 5α-androstane-3,17-dione. Unidentified polar nonconjugated metabolites and traces of steroid glucuronides were formed from the three substrates. The patterns of steroid metabolism were similar in dorsal and ventral skin, and in dorsal skin from male and female, adult and immature fish. Most of the 5α-reductase activity in the skin was in the dermis, only a small fraction of the total activity being in the epidermis. The trout muscle converted testosterone into 5α-dihydrotestosterone but in much lower yields than did skin.The skin, clasper, sperm sac and vas deferens of an adult male spur dogfish converted testosterone to 5α-dihydrotestosterone and androstenedione, though in much lower yields than did trout skin. Androstenedione was converted into testosterone, 5α-androstane-3, 17-dione and androsterone, while dehydroepiandrosterone was converted into 5-androstene-3β,17β-diol. No metabolism of testosterone was detected in the skeletal muscle of the dogfish.     

Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses

The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC–MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC–MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC–MS is a valuable tool for the validation of immunoassays.     

DIFFERENT ASPECTS OF 5α-REDUCTASE DEFICIENCY IN MALE PSEUDOHERMAPHRODITISM AND HYPOTHYROIDISM

The 5α-reductase activity that mediates the transformation of testosterone to dihydrotestosterone in various anatomical sites of human beings, has been studied in different pathological conditions related to 5α-reductase deficiency. We have studied two patients with male pseudohermaphroditism due to 5α-reductase deficiency, four patients with the complete form of testicular feminization syndrome and four men with primary hypothyroidism. Results were compared with those obtained in seven normal men. In vivo: radioactive tracers of testosterone were administered to each subject by different routes: intravenous, oral and subcutaneous. The urinary metabolites of these labelled precursors were measured. The 5β: 5α ratios of 17-ketosteroids (aetiocholanolone: 5α-androsterone) and androstanediols (5β-androstane-3α, 17β-diol: 5α-androstane-3α, 17β-diol) were calculated in the urine recovered after each mode of administration of radioactive testosterone. When testosterone was administered subcutaneously these ratios were highly increased in one patient with male pseudohermaphroditism due to 5α-reductase deficiency. In all the other patients, the ratios were found to be in the normal range for men. After oral administration of radioactive testosterone, both 5β: 5α ratios were very high in hypothyroid and in 5α-reductase deficient patients. These results suggest that the defective 5α-reductase activity observed in hypothyroid patients is only localized in the hepatic compartment. Conversely, in male pseudohermaphroditism, the 5α-reductase defect might affect both hepatic and extra-hepatic compartments. In vitro: the diagnosis of 5α-reductase deficiency was confirmed in the two male pseudohermaphrodite patients after incubation with ³H testosterone of skin homogenates from the external genital area. No 5α-reduction of testosterone occurred in the two skin specimens studied. In contrast, 5α-reductase activity was normal in genital skin from hypothyroid and testicular feminization syndrome patients. In pubic skin, 5α-reductase activity was absent in patients with testicular feminization syndrome. It was in the normal range in homogenates from hypothyroid patients and varied in the 5α-reductase deficient patients. Based on these data, it may be postulated that the programming of hepatic and extrahepatic 5α-reductase enzymes is fundamentally different. In addition, the enzyme that mediates the appearance of secondary sex characteristics seems to be androgen dependent, while the 5α-reductases present in the external genital area and the liver are not androgen dependent.     

Characterization of urinary and fecal metabolites of testosterone and their measurement for assessing gonadal endocrine function in male nonhuman primates

The aims of the present study were (i) to provide basic comparative data on the time course, route, and characteristics of excreted [14C]testosterone (T) metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macaca fascicularis) and the chimpanzee (Pan troglodytes) and (ii) to use this information to help validate the measurement of urinary and fecal testosterone metabolites for assessing androgen status in Anthropoid primates. Radiolabeled 14C-T (10-30 microCi) was injected intravenously into one adult male of each species and the excreta collected over the next 5 days. Peak radioactivity in urine was detected within 2h and accounted for 67% (Mf), 80% (Cj) and 91% (Pt) of the total radioactivity recovered. The time course of excretion of radioactivity in feces showed a higher variation between species (4-26 h to peak values). In all three species, the majority (>90%) of urinary metabolites were excreted as conjugates whereas the proportion of conjugated metabolites in feces was substantially lower and more variable. High pressure liquid chromatography (HPLC) analysis of urinary and fecal extracts revealed multiple peaks of radioactivity in all three individuals, but each with a distinctive pattern. Native T was excreted in only small amounts into the urine, whereas it was virtually absent in the feces of all three individuals. Three C17 group-specific enzymeimmunoassays using antisera against testosterone, 5alpha-androstane-17alpha-ol-3-one and androsterone were evaluated for their ability to discriminate immunoreactive androgen levels between intact males, castrated males and females based on measurements in urine and feces. In the marmoset, all assays (except for T in feces) clearly discriminated between test groups; in the chimpanzee significantly higher levels of androgen immunoreactivity in intact versus castrated males were measured in urine, but not feces. In the macaque, only the 5alpha-androstanolone measurement in feces discriminated between groups. Data on the results of a radiometabolism study using 3H-DHEA (a weak adrenal androgen) in a long-tailed macaque suggested that co-measurement of metabolites derived from T and DHEA in the assays tested might explain the difficulties in discriminating gonadal status in the two Old World primate species. Collectively, the data show that T metabolism in primates is highly complex and that no single method for noninvasive assessment of androgen status can be used for application across species. The importance of a proper validation of the methodology for each species is emphasised.     

In vitro metabolism of testosterone in hepatic tissue of a hagfish, Eptatretus burgeri

The metabolism of testosterone in hepatic tissue (combined microsomal and cytosol fractions) of a hagfish, Eptatretus burgeri, was examined in the presence of NADPH and in aerobic or CO-saturated atmosphere. The metabolites formed in CO atmosphere were identified as 4-androstene-3,17-dione, 5α-dihydrotestosterone, 3β-hydroxy-5α-androstan-17-one, and 5α-androstane-3β,17β-diol. From formation of these metabolites of testosterone, the activities of 5α-reductase, 3β- and 17β-hydroxysteroid dehydrogenases were indicated. Under aerobic condition, 7α-hydroxytestosterone, 7α-hydroxy-5α-dihydrotestosterone, 3β,7α-dihydroxy-5α-androstan-17-one and 5α-androstane-3β,7α,17β-triol were formed, in addition to 4-androstene-3,17-dione. These results demonstrate the presence of 5α-reductase, 3β- and 17β-hydroxysteroid dehydrogenases and 7α-hydroxylase in the hagfish liver. Since the hagfish is regarded as a very primitive vertebrate, it may be suggested that these enzymes are phylogenetically old and that the 7α-hydroxylase represents one of the first forms of hepatic cytochrome P-450 appearing during development.     

Steroid biosynthesis by the ovary of the hagfish Myxine glutinosa

Ovaries of the hagfish Myxine glutinosa were incubated with [³H]progesterone and [³H]testosterone and the metabolites identified by chromatography, chemical reaction, and crystallisation to constant specific activity. 5α-Pregnanedione was the sole metabolite of progesterone, but testosterone was converted into dihydrotestosterone (32% yield), 5α-androstane-3β, 17β-diol (11%), and 6β-hydroxytestosterone (3.6%). A more polar metabolite (24.6% yield) was also obtained which had the same chromatographic and chemical properties as 5α-androstane-3β,7α,17β-triol. Formation of conjugated metabolites was not significant.     

CONTROL OF GONADOTROPHIN SECRETION BY STEROID HORMONES IN CASTRATED MALE TRANSSEXUALS. II. EFFECTS OF ANDROGENS ALONE AND IN COMBINATION WITH OESTRADIOL ON THE SECRETIONS OF FSH AND LH

Twenty-nine infusions in twenty castrated male transsexual volunteers were carried out over a period of 7 h with subjects lying in the supine position. The effects of different doses of testosterone and its Sα-reduced metabolites as well as the effect of testosterone in combination with oestradiol on gonadotrophin secretion were evaluated. Different and varying degrees of suppression of plasma levels of FSH and LH were observed. The infusions of 2·4 mg testosterone, 5α-androstan-3α-17β-diol (3α-diol), 5α-androstan-3β17β-diol (3β-diol) but not dihydrotestosterone (DHT) caused significant suppression of LH. FSH, on the the other hand was not significantly inhibited by the androgens at this rate. At higher doses all four androgens suppressed LH secretion significantly. FSH was similarly suppressed by the androgens except by DHT. A differential effect on FSH and LH secretions was noted with the combined regime of testosterone and oestradiol. The combined regime did not cause a significantly higher degree of FSH suppression compared with either 200 μg of oestradiol or 12 mg of testosterone infused alone. The level of LH, however, was suppressed to a greater extent than either of the hormones when given alone. The inhibitory effect of testosterone demonstrated in this study could be due to the parent hormone or its 5α-reduced metabolites. Pharmacological doses of testosterone could exert a greater degree of LH suppression through its conversion to oestradiol. It is likely that oestradiol and testosterone act on gonadotrophin secretion through different mechanisms and that they have an additive suppressive effect on the secretion of LH but not FSH. The potencies for the androgens to suppress gonadotrophin secretion can be ranked as: 3α-diol = 3β-diol > testosterone > dihydrotestosterone.     

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.     

Effects of in vivo corticosterone treatment on the in vitro metabolism of testosterone in the comb and brain of the young male chicken

Male chickens kept in constant light were injected from their second to their 12th day of life daily either with 2.5 mg of corticosterone or with a control solution. In both groups of birds, the in vivo metabolism of labeled testosterone by the comb and by brain tissues (hyperstriatum, hypothalamus, pituitary gland) was then studied.The experimental treatment blocked the growth of the comb observed in control birds. In this tissue, it also reduced the production from testosterone of 5α-dihydrotestosterone and of 5α-androstane-3α,17β-diol, but it increased the production of 5β-androstane-3α,17β-diol. These modifications did not appear in any brain tissue; they were not accompanied by changes either of LH or of testosterone circulating levels or of the testes' weights.     

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.     

Diurnal variation in plasma concentrations of testosterone, 5α-dihydrotestosterone,and corticosteroids in the Australian brush-tailed possum, Trichosurus vulpecula (Kerr)

Plasma concentrations of testosterone, 5α-dihydrotestosterone, and total adrenal corticosteroids were measured in intact and castrated males and female possums over a 24-h period. In males, testosterone levels varied diurnally, being significantly higher in the morning than in the evening, with an overall mean concentration of 3.30 ± 0.43 (SEM) ng/ml. Levels of 5α-dihydrotestosterone showed a similar variation but of lower magnitude and the overall mean plasma concentration was 0.63 ± 0.10 ng/ml. Total adrenal corticosteroid concentrations in males also appeared to cycle, but inversely to the androgens with a peak in the early evening, and the overall mean concentration was 0.61 ± 0.08 μg/100 ml. Testosterone concentrations averaged 0.46 ± 0.02 and 0.35 ± 0.01 ng/ml in castrated males and females, respectively, and the corresponding values for 5α-dihydrotestosterone were 0.20 ± 0.03 and 0.21 ± 0.03 ng/ml. These measurements indicate that plasma testosterone levels of the marsupial possum fall within the range reported for eutherian species.     

Endocrine characterization of the designer steroid methyl-1-testosterone: investigations on tissue-specific anabolic-androgenic potency, side effects, and metabolism

Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17β-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17β-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17β-diol and 17α-methyl-5α-androstane-3α,17β-diol. Additionally, the corresponding 17-epimers as well as 17β-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.     

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).     

Small doses of 5α-dihydrotestosterone mimic the effects of 5α-androstane-3β,17β-diol on acid phosphatase activity in the adult rat prostate gland

These studies were designed to further investigate whether 5α-androstane-3β,17β-diol was exerting unique effects on rat prostate acid phosphatase activity or could possibly be exerting its actions by a small peripheral conversion to 5α-dihydrotestosterone. Intraperitoneal administration of 5α-dihydrotestosterone in doses of 1 mg, 100 μg or 50 μg per day starting 7 days after castration led to the restoration of normal characteristics of acid phosphatase activity. However, when 5α-dihydrotestosterone was given in a dose of only 25 μg per day starting 7 days after castration, the changes in acid phosphatase activity were indistinguishable from those found when 5α-androstane-3β,17β-diol was administered in a dose of 2 mg per day. This suggests that the effects of 5α-androstane-3β,17β-diol can be explained by its conversion to small amounts of 5α-dihydrotestosterone.     

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.     

Seasonal changes in testosterone metabolism in the pituitary gland and central nervous system of the European starling (Sturnus vulgaris)

The metabolism of testosterone in the pituitary gland, the hypothalamus, and the hyperstriatum of the male European starling was studied in the breeding season (May) and at the beginning and the end of the photorefractory period (July and November). In the pituitary gland the percentage conversion of testosterone to androstenedione and to 5α-DHT did not show seasonal variation, while the conversion of testosterone to 5β-DHT and to 5β-3α-diol was increased two- to threefold when the birds became photorefractory (July and November). In the hypothalamus, the formation of the 5α-reduced metabolites did not show seasonal variation, while the formation of androstenedione was significantly greater in November than in July. In contrast, the formation of 5β-reduced metabolites in the hypothalamus was greater in May and July than in November. In the hyperstriatum, the formation of 5β-reduced metabolites was also greater in July and November than in May. These observations show that seasonal changes in the metabolism of testosterone in the pituitary gland and central nervous system of the starling are mainly characterized by changes in the formation of 5β-reduced metabolites and androstenedione.     

The effect of temperature on the hepatic catabolism of testosterone in the rainbow trout (Salmo gairdneri) and the goldfish (Carassius auratus)

Liver of the rainbow trout Salmo gairdneri and the goldfish Carassius auratus has been incubated with [³H]testosterone at a range of temperatures from 1 to 46°. With trout liver the main metabolites were identified as androstenedione, testosterone glucuronide, 5β-dihydrotestosterone glucuronide, 5β-androstane-3α, 17β-diol glucuronide, and 6β-hydroxytestosterone glucoronide. With goldfish liver the main products were testosterone glucuronide and androstenedione. With both species the effect of temperature on testosterone glucuronide formation was very similar to that found in the testis, maximum yields being obtained at 31 and 41° for the trout and the goldfish, respectively. An inverse pattern was observed for the recovery of unconjugated testosterone, indicating a more rapid removal of free androgen at higher temperatures. It is suggested that the hepatic glucuronyl transferase may act in conjunction with the testicular enzyme to regulate free plasma androgen levels so that reproductive development takes place at the environmentally most favourable temperature.     

Gonadal source of testosterone metabolites in urine of male cotton-top tamarin monkeys (Saguinus oedipus)

Examining gonadal function in the small excitable cotton-top tamarin monkey (Saguinus oedipus) requires noninvasive sampling techniques. Two studies were performed to identify the quantifiable urinary metabolites of testosterone in cotton-top tamarins and which of the measurable metabolites would best reflect a gonadal source of testosterone secretion. In the first study, we injected unlabeled testosterone i.m. in males at either 500-ng or 1-microg levels. Urine samples were analyzed for androgens and estrogens. Testosterone and dihydrotestosterone (DHT) increased significantly following the injections in test males but not in control males. No significant increases in androstenedione occurred. Mean levels of estradiol and estrone did not consistently increase during the 5 days following injection. In the second study, a gonadotropin-releasing hormone antagonist, Antide, was used to block LH stimulation of gonadal steroidogenesis. Males given Antide at either a 6 mg/kg dose or an 18 mg/kg dose showed significantly lower levels of urinary LH than controls. At the higher Antide dose, testosterone levels were significantly reduced during weeks 1 and 2 posttreatment, whereas DHT levels significantly declined during the 2nd week posttreatment. Estradiol levels were highly variable prior to treatment but decreased significantly following treatment, whereas estrone levels remained variable throughout. These results indicate that measurement of urinary testosterone and possibly DHT reflect gonadal function in male cotton-top tamarins. Other sources of urinary estrogens may occur for the male cotton-top tamarin, but these data suggest that a substantial part of urinary estradiol is from gonadal sources, whereas urinary estrone appears to be mainly from extragonadal sources.     

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.     

Effects of sex steroids on aggressive behavior of adult male Japanese quail

We found that the order of aggressiveness of adult male Japanese quail determined by paired fighting was not correlated with plasma testosterone level, plasma LH level, size of cloacal protrusion, testicular weight, nor body weight. Injections of testosterone into lower-ranked individuals did not elevate their ranks of aggressiveness. Aggressive behavior was lost after castration. Injections of testosterone, androstenedione, and estradiol-17β restored aggressive behavior in castrated males. The order of aggressiveness of these hormone-injected castrated birds was identical to the order observed before castration. Administration of individually different doses of testosterone did not change the order. Injections with 5α- and 5β-dihydrotestosterone did not restore aggressive behavior in castrated males. These results are consistent with the hypothesis that aggressive behavior in adult male Japanese quail, as well as their sexual behavior, is induced by estradiol-17β converted by aromatase in the brain from testosterone. However, no correlation seems to exist between the endogenous or exogenous testosterone level and the order of aggressiveness.