Inhibition of lordosis in female rats by subcutaneous implants of testosterone, androstenedione or dihydrotestosterone in infancy

Female rats were implanted on the day of birth with Silastic capsules containing nonesterified testosterone, androstenedione, or dihydrotestosterone. The date of vaginal opening was assessed until sacrifice. The animals were ovariectomized, treated with estradiol benzoate and progesterone, and tested for the display of lordosis. The animals were then administered testosterone propionate and the size of the phallus was taken. Testosterone and dihydrotestosterone completely inhibited vaginal opening; androstenedione was partially effective. Testosterone almost completely inhibited lordosis behavior; androstenedione was partially effective and dihydrotestosterone was ineffective. All three androgens facilitated phallic development.     

Testosterone,androstenedione and dihydrotestosterone: Effects on mating behavior of male rats

The relative effectiveness of testosterone, androstenedione, and dihydrotestosterone in maintaining mating behavior following castration of male rats was studied. In Experiment 1 testosterone, but not dihydrotestosterone, was found to maintain mating. In Experiment 2 testosterone and androstenedione were found to be equally effective in maintaining mating. Dihydrotestosterone failed to maintain mating and was no more effective than no treatment at all. Testosterone, androstenedione, and dihydrotestosterone significantly enhanced seminal vesicle and penis weight. In Experiment 3 castrated male rats were administered radiolabeled testosterone, androstenedione, or dihydrotestosterone. Radioactivity was found in hypothalamic and seminal vesicle samples indicating that these steroids can be accumulated by brain as well as peripheral androgen-sensitive tissues. It was concluded that the peripherally active steroid dihydrotestosterone probably plays no role in the maintenance of sexual behavior.     

Seasonal changes in circulating testosterone and androstenedione concentration and their correlation with the anomalous reproductive pattern in the male indian sheath-tailed bat, Taphozous longimanus

The relationship between reproductive organs, circulating testosterone, and androstenedione concentration in the male sheath-tailed bat, Taphozous longimanus, was studied. The masses of testis, accessory sex gland (prostate, ampullary), and epididymides showed three peaks, one each in October, January, and April. Monthly changes in testosterone also peaked during October, January, and April and closely coincided with the peak spermatogenesis. Serum androstenedione concentration peaked during November and January. Testosterone showed a strong correlation with masses of testis and accessory sex glands, while androstenedione showed strong correlation with the body mass. Different threshold levels of testosterone may be required to trigger spermatogenesis, secretory activity of accessory sex glands and mating in Taphozous longimanus and may be responsible for reproductive asynchrony in this species. Higher circulating concentrations of testosterone and androstenedione throughout the year in this species, as compared with other mammalian species, may be responsible for prolonged retention of sperm in the epididymides.     

The behavioral responses in ovariectomized cattle to either estradiol,testosterone, androstenedione,or dihydrotestosterone

Steroid hormone effects on sexual behavior were measured in 15 sexually mature nulliparous cattle which were bilaterally ovariectomized. They were alloted at random to five groups of three animals each (sesame oil vehicle control, estradiol, testosterone, androstenedione, and dihydrotestosterone) in the fall of the year and reassigned at random to replicate the study the following spring. Each experiment was divided into three weekly trials. Animals within treated groups were reassigned each week to receive in random order one of three levels of a particular hormone (200, 400, and 800 μg of estradiol and up to 1000 times these doses of androgens).Estradiol, and to a lesser extent, testosterone were capable of increasing the frequencies of occurrence of most behavioral parameters studied. These were: (1) elicitation of vulval interest; (2) vulval sniffing; (3) agonistic interactions; (4) giving chin rests; (5) receiving chin rests; (6) attempted mounts; (7) successful mounts; and (8) standing when mounted. The mean interval from treatment to first standing to be mounted was 25.4 ± 0.8 and 33.3 ± 5.2 hr for the estradiol-treated and testosterone-treated heifers, respectively. Peak activity generally occurred the second day after initiation of hormone treatment and rapidly declined after the third day. Flehmen lip curl and bellowing were not stimulated by either hormone. Neither androstenedione nor dihydrotestosterone was capable of stimulating sexual behavior in these heifers, as measured by any of the parameters studied.     

Aggression, dominance, and affiliation: Their relationships with androgen levels and intelligence in 5-year-old children

This study explores the potential relationship between social behavior (aggression, dominance, and affiliation) and testosterone, androstenedione, and DHEA measurements in 5-year-old children while also analyzing the moderating effect of IQ on the hormone-behavior relationship. 129 healthy normal Iberian children (60 boys and 69 girls) were videotaped in free play interactions in the school playground. Their behavior was then evaluated with particular emphasis on aggression, government, and affiliation. Testosterone, androstenedione, and DHEA levels were measured using an enzyme immunoassay technique in saliva samples. A test (K-BIT) which provides an IQ measurement for children was also administered to subjects. The correlation analysis revealed a positive relationship between the behavioral factor of Provocation and androstenedione in boys, and a regression analysis indicated that this relationship was moderated in a positive direction by the subject's intelligence. In girls, we observed a positive relationship between testosterone and Affectivity, with this relationship being moderated in a negative direction by intelligence.     

Shift with age in steady-state concentrations of androstenedione and testosterone in incubations of guinea-pig testis

Minces of testes from mature and immature guinea pigs were incubated with [(14)C]androstenedione. Other portions of the same testes were simultaneously incubated with [(14)C]testosterone. Under steady-state conditions, the predominant steroid was [(14)C]androstenedione in the presence of tissue from young animals and [(14)C]testosterone in that from mature animals, irrespective of the compound added initially. It is concluded that the steady-state equilibrium favours the weak androgen androstenedione in the presence of tissue from the young animals, but favours the potent testosterone in that from the mature animals. Portions of tissue were homogenized in phosphate buffer. The net reduction of [(14)C]androstenedione to [(14)C]testosterone was less by homogenates of testes from young animals than by those from mature animals. Doubling the concentration of the NADPH-generating system did not affect this difference between the homogenates.     

Testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3, 17-dione in the plasma of male and female grey squirrels (Sciurus carolinensis)

Extracts of squirrel plasma have been chromatographed on partition columns (using a hydrophilic stationary phase) at atmospheric pressure (Celite support) and a reversed phase system at high pressure (HPLC). Both methods effectively separated testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one (DHT) and 4-androstene-3,17-dione; they gave elution patterns that differed considerably. Chromatographic mobility of the three androgens on the two systems was identical with that of fractions of squirrel plasma extracts that gave responses measured by appropriate androgen radioimmunoassays; good evidence for the occurrence of these androgens in squirrel plasma is thus provided. Plasma testosterone levels were 300 pmol/l in juvenile males, 800-7000 pmol/l in sexually-active males but undetectable (less than 50 pmol/l) in sexually-regressed males. Plasma DHT levels were also high in sexually-active males, but undetectable in other males except for one regressed individual. Plasma androstenedione was higher in juvenile males than in adult males, in which it was similar whether or not they were sexually regressed. Plasma testosterone and DHT, unlike androstenedione, were totally dependent on the presence of the testes. In females testosterone and DHT were undetectable in plasma but androstenedione levels were high, especially at oestrus. Androstenedione was dependent on the presence of the ovaries.     

Microbial transformation of steroids--II. Transformations of progesterone, testosterone and androstenedione by Phycomyces blakesleeanus

Phycomyces blakesleeanus transformed progesterone, testosterone and androstenedione into mixtures of products. Five monohydroxylated metabolites were obtained in reasonable yields from the progesterone transformation. Only 7 alpha- and 15 beta-hydroxyprogesterone have been reported previously from this organism. We find that it gives these two metabolites and also 6 beta-, 14 alpha- and 15 alpha-hydroxyprogesterone as major products. Five compounds were also purified from testosterone transformation mixtures. Two of these were monohydroxylated, two were ring A dehydrogenation products, and two were oxidised at C-17. The products were identified as 6 beta-hydroxytestosterone, 7 alpha-hydroxytestosterone, androsta-1,4-diene-3,17-dione (1-dehydroandrostenedione), 17 beta-hydroxyandrosta-1,4-diene-3-one (1-dehydrotestosterone) and androstenedione. All five metabolites were produced in reasonable yields, although hydroxylation was the minor transformation in this case. Only two significant products were formed from androstenedione. Both were reduced at C-17; one was also monohydroxylated. They were testosterone and 14 alpha-hydroxytestosterone. The testosterone and androstenedione transformation products have not been reported previously for this organism. We also report for the first time the preparation of P. blakesleeanus cell-free extracts which transformed progesterone reasonably efficiently and faithfully in vitro, although the proportions of each product varied from one extract to another.     

Sexual behavior of male golden hamsters receiving diverse androgen treatments

Four androgens were compared for their effectiveness in maintaining the sexual behavior of castrated male golden hamsters. Sexually experienced males were divided into 4 experimental treatment groups which received 500 μg daily of testosterone, androstenedione, dihydrotestosterone or androsterone. Control castrates were given oil. All animals were tested for sexual behavior every 2 wk for 10 wk following the onset of experimental treatment. Testosterone and androstenedione were the only androgens that maintained intromissions above the oil control level. However, testosterone, androstenedione and androsterone, but not dihydrotestosterone were effective in maintaining mounting behavior above the oil control level. No differences were detected between these 4 androgens in their maintenance of penile papillae.     

Excretion of non-conjugated androstenedione and testosterone in human urine.

A method for the measurement of unconjugated testosterone and androstenedione in human urine is described. The method uses chromatographic separation followed by radioimmunoassay and has been examined for reliability. The mean 24-hour excretion of androstenedione by adult male subjects was 2.5 mug and of testosterone was 0.8 mug. For women, the mean excretion was 2.9 mug of androstenedione and 0.25 mug of testosterone. In pregnancy, androstenedione excretion was occasionally elevated above the normal range, but testosterone excretion was quite commonly increased. Some hirsute subjects exhibited an increase in androstenedione excretion, which was decreased by administration of dexamethasone. The results suggest that the amount of unconjugated testosterone in urine is not a direct reflection of the plasma free testosterone, but urinary androstenedione may be a useful reflection of plasma androstenedione levels.     

Conversion of androstenedione to testosterone and other androgens in guinea-pig alveolar macrophages

Alveolar macrophages obtained by bronchoalveolar lavage of lungs of male and female guinea pigs were incubated with tritium-labelled androstenedione to evaluate the steroid metabolizing enzymes in these cells. The radiolabeled metabolites were isolated and thereafter characterized as testosterone, 5 alpha-androstanedione, 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol. Thus, the following androstenedione metabolizing enzymes are present in guinea-pig alveolar macrophages: 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase. The predominant androstenedione metabolizing enzyme activity present in alveolar macrophages was 17 beta-hydroxysteroid dehydrogenase. The rate of testosterone formation increased with incubation time up to 4 h, and with macrophage number up to 1.6 X 10(7) cells per ml. Androstenedione metabolism was similar in alveolar macrophages obtained both from male and female guinea pigs. These results suggest that alveolar macrophages may be a site of peripheral transformation of blood-borne androstenedione to biologically potent androgens in vivo and, therefore, these cells may contribute to the plasma levels of testosterone in the guinea pig.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

Kinetic evidence for the involvement of a common enzyme in the microsomal reduction of retinal and androstenedione in rat liver

Androst-4-ene-3,17-dione (androstenedione) was found to be a potent competitive inhibitor of the NADH-supported reduction of retinal in rat hepatic microsomes (K_i 42 μM, K_m/K_i ratio 1.1). Similarly, the NADH-mediated reduction of androstenedione was inhibited in mixed fashion by retinal (K_i 12 μM, K_m/K_i ratio 0.34). In subsequent experiments the cofactor NADH exhibited an identical K_m (8 μM) in the microsomal reductions of both substrates. Acidic pH markedly stimulated the microsomal reduction of androstenedione to testosterone and was also found to enhance retinal reduction to retinol, although the latter reaction exhibited a district pH optimum between 6.0 and 6.5. These results suggest that a common enzyme may participate in the reduction of both substrates but at least one other enzyme probably participates in hepatic microsomal testosterone production.     

Comparative studies of androgen metabolism in theca and granulosa cells of human follicles in vitro

In eight separate experiments, theca and granulosa were isolated from human follicles (5–25 mm in diameter), and their capacities to metabolize radiolabelled testosterone in 24 hour cultures were assessed. Theca metabolized testosterone primarily to androstenedione, however significant aromatization to estradiol-17β and to estrone was also observed. Granulosa metabolized testosterone primarily to estradiol-17β and estrone, while smaller quantities were converted to androstenedione. In seven of these experiments, the intermediate of aromatization, 19-hydroxytestosterone, was identified. In six of these experiments, theca, when compared to granulosa, produced more androstenedione but less estradiol-17β and estrone. 5α-Reduced androgens were non-detectable or produced in small quantities. In a single experiment, metabolism of androstenedione was compared to metabolism of testosterone by both theca and granulosa. Theca metabolized androstenedione to testosterone in smaller quantities than testosterone to androstenedione. Granulosa metabolized androstenedione to testosterone in higher quantities than testosterone to androstenedione. Both theca and granulosa aromatized androstenedione more readily than testosterone.     

Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22 h post-administration. However, IRMS could provide positive determinations for up to 55 h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36 h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.     

Biosynthesis of testicular steroids in the immature, adult and senescent guinea-pig

The potential biosynthetic capacity of testicular hormones was studied in immature, pubertal and aging guinea-pig. In their sexual development towards puberty, changes in the relationship of the steroids involved in the steroidogenic pathways were observed. The testosterone/androstenedione ratio changes markedly, showing an important increase with pubertal proximity. The testosterone in equilibrium androstenedione sequence, reversibly catalyzed by 17 beta-hydroxysteroid oxidoreductase (17 beta-oxido-reductase), clearly shifted towards androstenedione in immature animals irrespective of the precursor utilized. Post-pubertal animals showed a greater enzymatic activity in the 5-ene and 4-ene testicular synthesis pathways, testosterone production being greatest. In the aging animal, hormonal biosynthetic capacity falls. Reversion of the 17 beta-oxido-reductase activity could be one of the mechanisms responsible for the decrease in testosterone, as in immature guinea-pigs. In order to investigate the in vitro steroidogenic capacity of glands at different ages, minces of testicular tissue were incubated with labelled precursors. The studies were conducted in triplicate at 35 degrees C. For equal quantities of incubated tissue the non-metabolized amount of [3H]pregnenolone and [14C]progesterone, utilized as precursors, was different in post-pubertal and senescent animals: 55.7 +/- 3 vs 59.3 +/- 2.3% (P less than 0.01) for pregnenolone, and 50.1 +/- 3.3 vs 56.3 +/- 2.9% (P less than 0.01) for progesterone, respectively. Testosterone production was 12 +/- 2% in adult and 6.7 +/- 2.7% in senescent animals (P less than 0.01). The testosterone/androstenedione ratio was not significantly different in post-pubertal and senescent animals: 2.8 +/- 0.5 vs 2.4 +/- 0.4, but consistently higher than found in immature animals: 0.3 +/- 0.1. The lesser potential capacity of the aging tissue to synthesize testosterone could be explained by a decline in the glands capacity to metabolize the hormonal precursors.     

Androgen dynamics in vitro in the human prostate gland. Effect of cyproterone and cyproterone acetate

Hyperplastic and adenocarcinomatous human prostatic tissue was superfused in vitro with radioactively labelled androst-4-ene-3,17-dione, testosterone and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one), with and without addition of the anti-androgens cyproterone and cyproterone acetate. Cyproterone competitively inhibited the entry of the androgens into the majority of the tissues, whereas cyproterone acetate increased this entry. These findings indicated that transport of androstenedione, testosterone and 5alpha-dihydrotestosterone into prostatic tissue is performed by a specific mechanism, possibly involving a carrier situated in the cell membrane. The extent of metabolism of the three androgens was also modified: formation of 5alpha-dihydrotestosterone from testosterone, and of the latter from androstenedione, was decreased by cyproterone and increased by the acetate. Acetate was more effective than cyproterone in decreasing the ;uptake' of the perfused androgens by the tissue; at the same time, it increased the androgen clearance from the tissue. As cyproterone acetate is the more potent of the two anti-androgens, the possibility that these findings in vitro are related to the different anti-androgenic potency exhibited by the two compounds in vivo is discussed. ;Uptake' of the two anti-androgens and the response to their action on androgen dynamics were similar in adenocarcinomatous and hyperplastic glands.     

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.     

Substrate specificity of the placental microsomal aromatase

Using an accurate and sensitive assay for the human placental aromatase we have found apparent Km values for androstenedione (4-androstene-3,17-dione) and testosterone to be 14 ± 4.0 nM and 41 ± 12 nM respectively. These values were significantly different (p < 0.001). Analyses at substrate concentrations 5–10 fold above and below the Km values did not indicate any anomalous kinetic behavior. Mixed substrate experiments were consistent with a single enzyme metabolizing both steroids: each competitively inhibited the aromatization of the other, and the “Ki” values were the same as their apparent Km values. Sodium chloride (1.2M) significantly increased the rate of testosterone aromatization by decreasing its Km value and had no significant effect on the aromatization of androstenedione. However, in the presence of this salt testosterone still inhibited the aromatization of androstenedione competitively with a “Ki” equal to its apparent Km. Our data is therefore consistent with the proposal that human placental microsomes contain a single “high affinity” site for the aromatization of androstenedione and testosterone.     

Deleterious effects of androstenedione on growth and cell morphology of Schizosaccharomyces pombe

Androgens (androstenedione and testosterone) belong to the most important compounds in human steroidogenesis. The 17-hydroxysteroid dehydrogenase responsible for interconversion of the oxygenic group on C-17 of androgens ring is involved in steroid hormone synthesis. The fission yeast Schizosaccharomyces pombe 972 h^- was found to contain constitutive 17-hydroxysteroid dehydrogenase that was able to reduce androstenedione to testosterone and oxidize testosterone to androstenedione. The reductive pathway was found to be predominant while the oxidative one was carried out with much lower activity. Exogenous androstenedione, contrary to testosterone, inhibited S. pombe growth and stimulated the formation of aberrant swollen cells with slighter cell wall sensitivity to the action of the lytic enzyme Novozym. It is postulated that the 17hydroxysteroid dehydrogenase prevents the deleterious effects of androstenedione on the morphology and growth of the yeast's cells by androstedione reduction to testosterone.