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.     

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.     

Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats

The effect of antiestrogens (MER-25, ICI-46474, and cis-clomiphene) and aromatase inhibitors (5-α-androstanedione, metopirone, and aminoglutethimide) on androgen induced copulatory behavior was tested in sexually inexperienced castrated male tats. Daily injections of 1 mg testosterone (T) for 21 days induced sexual activity in most subjects (61% mounting). Daily pretreatment with MER-25 or cis-clomiphene at three dose levels did not block the behavioral response to T. ICI-46474 at the high dose level (1 mg/kg) elicited a significant depressory effect on the sexual behavior of the T treated castrated rats. A single injection of 6 mg testosterone propionate (TP) induced mounting behavior in 56% of the tested rats within 120 hr. Treatment with metopirone or 5 α-androstanedione (injections every 12 hr for 96 hr) did not inhibit the response to TP. By contrast, aminoglutethimide (5 or 15 mg every 12 hr for 96 hr) abolished the behavioral response to androgen.     

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.     

Effects of androgens on burst forming units (BFU-E) in normal rabbit bone marrows

The mechanism of action of androgenic steroids on erythropoiesis is not well understood. In order to assess whether the site of action of androgens is on the early erythroid committed stem cell compartment, the $\text{in}$$\text{vitro}$ effects of testosterone (T), 5α-dihydrotestosterone (5α-DHT) and 5β-dihydrotestosterone (5β-DHT) on the so-called erythropoietic burst forming unit (BFU-E) in normal rabbit bone marrows were studied. Even though all of the steroids studied increased the number of BFU-E in the presece of Ep, 5β-DHT was the most potent in stimulating BFU-E. Testosterone was moderately effective in increasing BFU-E. Even though 5α-DHT produced a significant increase in BFU-E, it was the least effective of the 3 steroids studied. Preincubation (2 hrs) of normal rabbit bone marrow cells with testosterone followed by removal of T from the culture system resulted in a significant increase in BFU-E when compared with that of non-treated marrow cells in the presence of Ep. These data suggest that testosterone and 5β-DHT and possibly 5α-DHT act on an early uncommitted stem cell, perhaps the CFU-S, to increase the numbers of erythroid committed stem cells to eventually cause an increase in erythropoiesis in combination with Ep.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Relevance of the selective oestrogen receptor modulators tamoxifen, toremifene and clomiphene in doping field: endogenous steroids urinary profile after multiple oral doses

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography–mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration.The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios.     

Effects of dihydrotestosterone and estradiol benzoate pretreatment upon testosterone-induced sexual behavior in the castrated male rat

Three groups of inexperienced castrated male rats were treated daily for 15 days with oil, estradiol benzoate (1 μg), or dihydrotestosterone (1 mg), and thereafter injected daily with testosterone (1 mg) for 21 days. Sexual behavior was tested every third day after the start of the pretreatment until day 36. Estradiol benzoate or dihydrotestosterone failed to elicit sexual behavior. Pretreatment with dihydrotestosterone, but not estradiol benzoate, significantly shortened the intervals to initiation of mounting and intromission in response to testosterone. The results suggest that fully developed genitals (penis and/or sexual accessories) facilitate initiation of copulatory behavior in response to testosterone administration.     

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.     

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Effects of estrone, estradiol and estriol combined with dihydrotestosterone on mounting and lordosis behavior in castrated male rats

Prepuberally castrated male rats were injected with estrone (1 or 5 μg), estradiol (1 or 5 μg) or estriol (1, 5, or 25 μg) either alone or in combination with dihydrotestosterone, (0.5 mg). Each of these steroids, when given alone, had no or only weak stimulatory effects on male sexual behavior. When combined with dihydrotestosterone all estrogens stimulated full copulatory behavior, the order of potency being estradiol, estrone, and estriol. Lordosis behavior in response to male mounting or manual stimulation was facilitated by all estrogens. All estrogens caused a slight weight increase of the seminal vesicles, ventral prostate and glans penis.     

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     

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.     

Aromatizable and 5alpha-reduced androgens: differentiation between central and peripheral effects on male rat sexual behavior.

Sexual behavior was observed in the 5 wk after castration in four separate experiments. In each, 50 male rats were allocated equally to the following treatment groups; intact controls; castrated controls; castrated + testosterone propionate, 75 μg/day; castrated + test steroid 1, 150 μg/day; castrated + test steroid 2, 150 μg/day. The following propionated compounds were used; dihydrotestosterone, androsterone, 3α- and 3β-androstanediols, androstenedione, androstanedione, 19-hydroxyandrostenedione, estradiol. With the exception of androstanedione, only aromatizable androgens or estrogen prevented post-castration increases in refractory period durations. However, aromatizable and 5α-reduced androgens stimulated penile spine growth. In each experiment there was a significant positive correlation between spine number and copulatory efficiency i.e., the ratio of intromission to mount frequencies.     

Conversion of testosterone to estradiol may not be necessary for the expression of mating behavior in male Syrian hamsters (Mesocricetus auratus)

Male sexual behavior is mediated in part by androgens, but in several species, mating is also influenced by estradiol formed locally in the brain by the aromatization of testosterone. The role of testosterone aromatization in the copulatory behavior of male Syrian hamsters is unclear because prior studies are equivocal. Therefore, the present study tested whether blocking the conversion of testosterone to estradiol would inhibit male hamster sexual behavior. Chronic systemic administration of the nonsteroidal aromatase inhibitor Fadrozole (2.0 mg/kg/day) for 5 or 8 weeks did not significantly increase mount latency or reduce mount frequency, intromission frequency, ejaculation frequency, or anogenital investigation relative to levels shown by surgical controls. However, Fadrozole effectively inhibited aromatase activity, as evidenced by the suppression of estrogen-dependent progesterone receptor immunoreactivity in the male hamster brain. The JZB39 anti-progesterone receptor antibody labeled significantly more neurons in brains of sham-treated hamsters than in brains of Fadrozole-treated hamsters. These data suggest that aromatization of testosterone to estradiol is not necessary for normal mating behavior in Syrian hamsters.     

Effects of ovarian hormones on the behavior of captive Macaca fascicularis

To examine the effects of ovarian hormones on the behavior of female Macaca fascicularis and their male partners, daily 1-hr behavior tests were conducted while ovariectomized females were (1) untreated, (2) given estradiol benzoate (EB) (5 μg subcutaneously [s.c.]/day), (3) given estradiol benzoate together with increasing doses of progesterone (P) (5 mg, 10 mg, and 20 mg. s.c./day), and (4) given testosterone propionate (TP) (0.25 mg s.c./day) (six pairs, 540 tests). Weekly blood samples were analyzed by radioimmunoassay for plasma hormone levels (81 samples). Estrogen treatment produced plasma estradiol levels similar to those of intact females during the late follicular phase of the menstrual cycle. Additional progesterone at the lowest dose produced plasma progesterone levels similar to or somewhat higher than those during the midluteal phase, while higher doses produced supraphysiological levels. Androgen treatment resulted in plasma levels well above the physiological range. Hormone treatments produced highly significant effects on the sexual, social, and aggressive interactions of the pairs. As in rhesus monkeys, estrogen increased male and female sexual activity, and increasing doses of additional progesterone reversed these effects. Unlike in rhesus monkeys, testosterone propionate increased both female sexual motivation (invitations) and also male sexual activity and ejaculatory performance. The direction of the hormone-dependent changes in grooming and aggressive interactions confirmed earlier results with intact females and indicated that aggressive interactions and male grooming times were highest, and female grooming times were lowest, when copulatory activity was at its height.     

Antiserum to 5alpha-dihydrotestosterone: production, characterization and use in radioimmunoassay

5α-Dihydrotestosterone (5α-DHT) was rendered antigenic by covalent attachment to bovine serum albumin (BSA) through position 1 of the steroid. Nucleophilic attack by β-mercaptopropionic acid on the 1,2-dehydro derivative of 5α-DHT yielded the corresponding 1α-thioether alkanoic acid which was coupled to bovine serum albumin by use of the carbodiimide reagent. The method should be generally applicable to 3-oxosteroids. Immunization of rabbits with 5α-DHT-1α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for 5α-DHT (K_a= 1.4 × 10⁹ 1/mol) that showed little cross reaction with 17β-hydroxy-5β-androstan-3-one (3%), and with a variety of 17-oxoandrostane compounds (≤0.5%). However the serum cross-reacted significantly with testosterone (10%) and with 5α-androstene-3α, 17β-diol (16%). A radioimmunoassay procedure for the determination of 5α-DHT in plasma is described. Chromatographic purification of the plasma extracts proved necessary for obtaining valid results. The plasma level of 5α-DHT(pg/ml; ean ± S.D.) was 364±79 (n = 7) in normal human adult males and 188 ± 62 (n = 5) in normal non-pregnant women.     

Induction of sexual behavior in ovariectomized rhesus females with 19-hydroxytestosterone

Eight sexually experienced long-term ovariectomized female rhesus monkeys were given tests of sexual behavior following treatment with 19-hydroxytestosterone (19-OH-T, 1 mg/day for 13 days), and their performance was compared with that following treatment with estradiol benzoate (EB, 10 μg/day for 13 days). Each female was tested for 10 min with each of nine adult males. Blood samples were taken on the last day of treatment with EB, at the end of the intervening 3-month period of no treatment, and on the last day of treatment with 19-OH-T. Blood levels of testosterone and estradiol were quantified by radioimmunoassay. Mean rate of presenting at a distance (proceptive behavior) was significantly higher (P < 0.05) when they were treated with 19-OH-T, but the ratio of presents to male contacts (receptive behavior) was significantly higher (P <0.05) when they were treated with EB. All other components of female sexual behavior were the same. Males displayed fewer annoyance responses (rejecting jerk, P < 0.05) when the females were treated with 19-OH-T than when they were treated with EB. All other male responses occurred with the same frequencies under the two female treatment conditions. Injection of 19-OH-T and EB both resulted in plasma testosterone and estradiol levels higher than those found in the untreated condition. Testosterone levels did not differ under the two treatments (P > 0.05), but estradiol levels were higher under EB treatment than under 19-OH-T (P < 0.05). This study suggests that both testosterone and estradiol are essential for maximum sexual performance and that various components of sexual behavior may be differentially influenced by the ratio of testosterone to estradiol in plasma.     

Castration, sex steroids, and heterosexual behavior in adult male laboratory-housed stumptailed macaques (Macaca arctoides)

The sociosexual behaviors of six stable male-female pairs of stumptailed monkeys were studied in half-hour pair tests. Their performance before and after castration of the males was compared. The effects of replacement therapy with sex steroids on male-female interaction were studied. Also the effects of new females as sexual partners were investigated. Castration caused a significant decrease in sexual behavior. Individual males could display ejaculatory behavior up to about 1 year postcastration. Dihydrotestosterone propionate (75 mg/week/male) alone or in combination with estradiol benzoate (0.9 or 3 mg/week/male) was not effective in restoring sexual behavior to precastration levels in the three castrated males tested. Replacement therapy with testosterone propionate (75 or 10 mg/week/male) was effective in restoring copulatory behavior in half of the castrated males. In some males the introduction of a new female caused an increase in sexual activity, usually when sexual activity with their familiar partner was low. This occurred both in the castration condition and in the steroid treatment period, suggesting, that low activity was caused by low "motivation" and not by the inability to perform.     

Androgen- and estrogen-induced copulatory behavior and inhibition of luteinizing hormone (LH) secretion in the male rat

The purpose of the present investigation was to determine if estrogen, aromatizable androgen or nonaromatizable androgen is capable of (1) inducing copulatory behavior and (2) inhibiting the postcastration rise in plasma LH. Castrate male rats were injected daily with either 1 mg testosterone (T), androstenedione (A), dihydrotestosterone (DHT), or 25 μg estradiol benzoate (EB) or oil and tested weekly for masculine behavior and for lordosis behavior after 38 days of steroid treatment. On day 40 blood was collected for radioimmunoassay of plasma LH. At least 89% of the males treated with T, A, or EB and 55% of those treated with DHT displayed ejaculatory behavior whereas none of the oil-treated males showed male copulatory behavior. Only estrogen-treated males displayed lordosis behavior. T and to a lesser extent A treatment reduced high levels of plasma LH; however, DHT and EB further reduced plasma LH to undectable levels. The relative potency of the steroid effect in stimulating accessory sex tissues followed the order: DHT > T > A > EB = oil. Significant dissociation was observed between the effects of these steroids on peripheral morphology, negative feedback, and mating behavior. These results indicate that masculine behavior is facilitated to the greatest extent, although not exclusively, by centrally acting aromatizable androgen or estrogen, whereas under the present conditions only estrogen stimulates feminine behavior.