Organization and activation of behavior in quail: role of testosterone metabolism

In quail, the hypothalamus enzymatically transforms testosterone (T) into estradiol (E2), 5 alpha-dihydrotestosterone (5 alpha-DHT), and 5 beta-dihydrotestosterone (5 beta-DHT). During the embryonic life, the 5 beta-reductase activity is very high, which probably protects the brain of males from being behaviorally demasculinized by their endogenous T. 5 beta androstanes are inactive androgens. The decrease of 5 beta reductase with age during sexual maturation corresponds to a potentiation of the effects of T as shown by experiments that compared the effects of T and 5 alpha-DHT in adult and young quail. T metabolism is also involved in the activation of male behavior in the adult. T aromatization is probably essential for behavioral activation, but nonaromatizable androgens such as methyltrienolone, and to some extent 5 alpha-DHT, can also stimulate sexual behavior in castrates. These enzymatic activities show a clear neuroanatomical localization and are sexually dimorphic. Males produce more active metabolites (E2, 5 alpha-DHT) than females, which could explain the male's greater sensitivity to T treatments. It thus appears that T metabolism is involved in the differentiation and activation of behavior in quail.     

Altered brain metabolism of testosterone is correlated with reproductive decline in aging quail

In aging quail, an increasing proportion of males show no sexual behavior. A decrease in the mean size of the tests, cloacal gland, and sternotracheal muscles is also observed. In both sexually active and inactive males, plasma testosterone decreases with age but more so in inactive birds. The behavioral and morphological changes observed during aging are correlated with shifts in the intracellular testosterone metabolism resulting in a change in the ratio of active versus inactive metabolites. In the hypothalamus there is a steady decrease with age of 5 beta-reductase activity in all birds and an increase in 5 alpha-reductase activity only in the birds which remain sexually active. In the cloacal gland, the 5 beta-reductase activity markedly increases with age but more so in the birds which become sexually inactive. These data support the notion that the effects of testosterone are controlled by enzymatic shifts which could modulate the sensitivity to the hormone at the cellular level.     

Vasotocinergic innervation of areas containing aromatase-immunoreactive cells in the quail forebrain

In the male quail forebrain, aromatase-immunoreactive (ARO-ir) elements are clustered within the sexually dimorphic medial preoptic nucleus (POM), nucleus striae terminalis (nST), nucleus accumbens (nAc), and ventromedial and tuberal hypothalamus. These ARO-ir cells are sensitive to testosterone and its metabolites: Their number and size increase after exposure to these steroids. The POM and lateral septum are also characterized by a dense vasotocinergic innervation that is also sensitive to testosterone. We analyzed here the anatomical relationships between ARO-ir elements and VT-ir fibers in the quail prosencephalon. Sequential staining for vasotocin, aromatase, or vasotocin plus aromatase was performed on adjacent 30-μm-thick cryostat sections. High concentrations of thin VT-ir fibers were observed within the POM, nST, lateral septum, periventricular mesencephalic central gray, and ventromedial and tuberal hypothalamus. There was a close correspondence between the extension of the ARO-ir cells and of VT-ir fibers. In double-labeled sections, all clusters of ARO-ir cells with the exception of those located in the nAc were embedded in a dense network of VT-ir fibers. Many of the VT-ir terminals appeared to end in the neuropile surrounding ARO-ir elements rather than directly on their cell bodies. This study supports the idea that the testosterone-dependent aromatase system is directly innervated by a testosterone-dependent peptidergic system. Aromatase-containing cells could therefore be modulated by steroids both directly and indirectly through the vasotocin system. Alternatively, this neuroanatomical arrangement may mediate the control of vasotocin synthesis or release by steroids. Functional studies demonstrate that both aromatase and vasotocin affect reproductive behavior in quail, and the present data provide anatomical support for the integration of these effects. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 45–60, 1997     

Relationship of cloacal gland with testes, testosterone and fertility in different lines of male Japanese quail

An attempt has been made to investigate the relationship of cloacal gland with testes, testosterone and fertility in different lines of Japanese quail (Coturnix coturnix japonica). For this study, three lines of healthy adult male Japanese quails (<12 weeks) viz., heavy body weight (HB), white breasted (WB), and white egg shell (WES) were taken. They were housed in individual cages under uniform husbandry conditions and were provided with normal quail breeder ration and water ad libitum, with 14 h light/day. The experimental birds were selected from each of these three lines and categorized into different groups (15 birds/group) based on the increasing order of the area of cloacal gland. At the end of this experiment (24 weeks) the data indicated that size of the cloacal gland was directly proportional to foam discharge, foam weight, testicular weight, fertility and testosterone concentration in plasma. From this study it may be concluded that area of cloacal gland in Japanese quail is positively correlated with testicular weight, level of testosterone in plasma and fertility. Visual examination of cloacal gland and cloacal foam may provide a valuable non-invasive tool for predicting the fertilizing ability of individual male bird.     

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.     

Effects of testosterone on Reelin expression in the brain of male European starlings

Reelin, a large glycoprotein defective in reeler mice, is assumed to determine the final location of migrating neurons in the developing brain. We studied the expression of Reelin in the brain of adult male European starlings that had been treated or not with exogenous testosterone. Reelin-immunoreactive cells and fibers were widely distributed in the forebrain including areas in and around the song control nucleus, HVC. No labeling was detected in other song control nuclei with the exception of nucleus uvaeformis, which was delineated by a dense cluster of Reelin-immunoreactive perikarya. Reelin is thus expressed in areas incorporating new neurons in adulthood, such as HVC. Reelin expression was sharply decreased by testosterone in HVC, nucleus uvaeformis and dorsal thalamus but not in other brain regions. These results are consistent with the idea that seasonal changes in Reelin expression modulate the incorporation of neurons within HVC. The presence of Reelin in other brain areas that do not incorporate new neurons in adulthood indicates, however, that this protein must play other unrelated roles in the adult brain. Additional studies should now be carried out to determine the specific role played by this protein in the seasonal plasticity of the songbird brain.     

Effects of testosterone on ontogeny of urinary behavior in male and female dogs

Development of urinary behavior from birth to adulthood was observed in six groups of beagles including normal males (NM), normal females (NF), males castrated soon after birth (CM), males castrated soon after birth and treated with testosterone (T) for the next 90 days (CMT), females exposed to T in utero (FTU), and females exposed to T in utero and during infancy, i.e., the first 30-40 days postpartum (FTUI). Prenatal treatment with T had masculinizing effects on juvenile urinary behavior in FTU and FTUI. On the other hand normal development of fully adult masculine urinary patterns in males and females necessitated both prenatal and postnatal androgenic stimulation. It was not necessary that T be present at the time the overt behavior developed. For example, adult male behavior appeared in FTUI at the same time as in NM, i.e., 6-10 months, although the supply of exogenous androgen in FTUI had been exhausted within 30-40 days after birth. CMT showed precocious development of all components of the adult male pattern. Development of adult responses was markedly retarded in most CM, and their performance did not equal that of NM at 23 months. They were then injected with TP which promptly evoked completely normal male urinary behavior. It is tentatively concluded that T acting before birth and during the juvenile period "prepares" critical CNS mechanisms so that when general maturation reaches the appropriate point adult male behavior develops. Although the preparatory role of T is essential, the behavior is not dependent on T after it has developed.     

Specific activation of estrogen receptor alpha and beta enhances male sexual behavior and neuroplasticity in male Japanese quail

Two subtypes of estrogen receptors (ER), ERα and ERβ, have been identified in humans and numerous vertebrates, including the Japanese quail. We investigated in this species the specific role(s) of each receptor in the activation of male sexual behavior and the underlying estrogen-dependent neural plasticity. Castrated male Japanese quail received empty (CX) or testosterone-filled (T) implants or were daily injected with the ER general agonist diethylstilbestrol (DES), the ERα-specific agonist PPT, the ERβ-specific agonist DPN or the vehicle, propylene glycol. Three days after receiving the first treatment, subjects were alternatively tested for appetitive (rhythmic cloacal sphincter movements, RCSM) and consummatory aspects (copulatory behavior) of male sexual behavior. 24 hours after the last behavioral testing, brains were collected and analyzed for aromatase expression and vasotocinergic innervation in the medial preoptic nucleus. The expression of RCSM was activated by T and to a lesser extent by DES and PPT but not by the ERβagonist DPN. In parallel, T fully restored the complete sequence of copulation, DES was partially active and the specific activation of ERα or ERβ only resulted in a very low frequency of mount attempts in few subjects. T increased the volume of the medial preoptic nucleus as measured by the dense cluster of aromatase-immunoreactive cells and the density of the vasotocinergic innervation within this nucleus. DES had only a weak action on vasotocinergic fibers and the two specific ER agonists did not affect these neural responses. Simultaneous activation of both receptors or treatments with higher doses may be required to fully activate sexual behavior and the associated neurochemical events.     

Effects of testosterone metabolites on copulation and medial preoptic dopamine release in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. The recent presence of testosterone (T) may be necessary for this precopulatory increase in release. Previously, the postcastration loss of copulatory ability mirrored the loss of the DA response to an estrous female, and the restoration of copulation with exogenous T was concurrent with the reemergence of this DA response. The present study investigated the effectiveness of the two major metabolites of T in maintaining copulation and basal and female-stimulated DA levels. Adult male rats were castrated and received daily injections of estradiol benzoate (EB), dihydrotestosterone benzoate (DHTB), EB + DHTB, testosterone propionate (TP), or oil vehicle for 3 weeks. Microdialysis samples were collected from the MPOA during baseline conditions, exposure to an estrous female behind a barrier, and copulation testing. EB + DHTB- and TP-treated animals had normal basal DA levels and showed a precopulatory DA response, and most copulated normally. EB-treated castrates had high basal DA levels, but failed to show a female-stimulated increase; most intromitted, but none ejaculated. DHTB- and oil-treated groups had low basal levels of extracellular DA that did not increase during copulation testing; most failed to mount and none ejaculated. These results suggest that E maintains normal basal levels of extracellular DA in the MPOA, which are sufficient for suboptimal copulation, but that androgen is required for the female-stimulated increase in DA release and for facilitation of ejaculation.     

Effects of egg testosterone on female mate choice and male sexual behavior in the pheasant

Evidence is accumulating that sex steroids in the eggs, besides affecting progeny phenotype and behavior in the short term, also have enduring effects until adulthood, when they may translate into differences in reproductive strategies and success. Maternal steroids transfer may therefore affect both agonistic behavior and mate choice decisions, either through the promotion of body size and condition or through a priming effect on the neuroendocrine system. However, owing to the prevalence of a short-term perspective, relevance of maternal transfer of sex steroids to sexual selection processes has been seldom studied. Here we investigate the effects of an experimental increase in egg testosterone on male dominance and copulation success in the ring-necked pheasant, Phasianus colchicus, a polygynous galliform with multiple male ornamental traits, in captivity. We found that females from testosterone (T) injected eggs copulated less than control females. Males from T-injected eggs obtained more copulations than control males, specifically with control females. The effect of male ‘ordinary’ and secondary sexual traits on either dominance or copulation frequency did not depend on early exposure to T, nor did T treatment affect male dominance. Present results demonstrate that variation in the early hormonal environment set up by mothers affects sexual behavior of the offspring, which might translate into fitness differences.     

Effects of hemicastration and castration on foam production and its relationship with fertility in male Japanese quail

Healthy heavy body weight strain of adult male Japanese quail (Coturnix coturnix Japonica) of the same age were used in this study to observe the effect of hemicastration and castration on the frequency of foam discharges from cloacal gland and other related parameters. The quails were housed in individual cages and divided into four groups: control (intact birds), sham-operated control (intact birds with incision), hemicastrated, and castrated groups of birds. Hemicastration and castration were carried out surgically at 10 and 13 weeks of age, respectively. Subsequently, 3 weeks after castration birds were examined for different parameters. Hemicastration caused a significant (P < 0.05) drop in the foam discharge frequency, weight of the foam, and the level of testosterone in the plasma, whereas in castrated group these variables were observed nil or negligible as compared to both of the controls. A suppressive effect of hemicastration was also noticed on the body weight, area of the cloacal gland, as well as percent fertility. Castration induced the drastic regression of the cloacal gland and a significant reduction (P < 0.05) of the body weight was also noted as compared to other groups. Frequencies of foam discharges were twice the number in daytime (06:00-18:00 h) than night, irrespective of the groups. In another study, the effect of characteristics of foam or foam glands on fertility was examined in 77 male birds paired with females. Several characteristics of foam and foam glands were examined for infertility, such as light yellow foam, dark yellow foam, smaller area of foam gland (below 225 mm2), hardness of foam gland and certain unknown factors that contributed 0.23, 0.68, 5.23, 2.27 and 3.64% infertility, respectively. An overall 12.1% birds were found to be infertile. This data indicated that smaller sizes of foam glands might reflect the poorest fertilizing ability of the male birds. From this study, it may be concluded that the cloacal gland may be considered as an external indicator of testicular function of the birds. Characteristics of the cloacal gland or foam or both may be used as a simple and effective tool to predict the fertilizing ability of an individual male in view of the very small semen ejaculate that is very difficult to collect and evaluate for fertility.     

The interaction of testosterone and breeding phase on the reproductive behavior and use of space of male zebra finches

It is well known that androgens play a critical role in mediating the reproductive behavior of males. However, many laboratory experiments that examined the effects of testosterone in male songbirds typically limited their investigations to the early phase of breeding. We sought to determine the influence of testosterone on social behavior, pair bonding, nesting, and use of space in captive zebra finch (Taeniopygia guttata) males as a function of breeding stage (pre-laying, incubation, and nestling phases). Fourteen males were released into an aviary with 14 females and allowed to breed for 7 weeks. Half of the males were given testosterone implants and half were given control implants. During the pre-laying phase, testosterone-implanted males spent significantly more time in nesting activities than control birds and more time elapsed from starting to build a nest to when their mates initiated egg-laying. During the incubation phase, testosterone-implanted subjects spent significantly more time in female-directed and undirected singing. Use of space varied between hormone conditions depending upon breeding phase: there was no difference during pre-laying, but during the incubation and nestling phases, testosterone-implanted subjects used significantly more space. This significant increase in "home range" during the latter phase of the breeding cycle coincides with results from field studies on other species. These results underscore the importance of considering breeding phase in assessing the behavioral sensitivity to hormones.     

Rapid effects of aromatase inhibition on male reproductive behaviors in Japanese quail

Non-genomic effects of steroid hormones on cell physiology have been reported in the brain. However, relatively little is known about the behavioral significance of these actions. Male sexual behavior is activated by testosterone partly through its conversion to estradiol via the enzyme aromatase in the preoptic area (POA). Brain aromatase activity (AA) changes rapidly which might in turn be important for the rapid regulation of behavior. Here, acute effects of Vorozole, an aromatase inhibitor, injected IP at different doses and times before testing (between 15 and 60 min), were assessed on male sexual behavior in quail. To limit the risk of committing both types of statistical errors (I and II), data of all experiments were entered into a meta-analysis. Vorozole significantly inhibited mount attempts (P < 0.05, size effect [g] = 0.527) and increased the latency to first copulation (P < 0.05, g = 0.251). The treatment had no effect on the other measures of copulatory behavior. Vorozole also inhibited appetitive sexual behavior measured by the social proximity response (P < 0.05, g = 0.534) or rhythmic cloacal sphincter movements (P < 0.001, g = 0.408). Behavioral inhibitions always reached a maximum at 30 min. Another aromatase inhibitor, androstatrienedione, induced a similar rapid inhibition of sphincter movements. Radioenzyme assays demonstrated that within 30 min Vorozole had reached the POA and completely blocked AA measured in homogenates. When added to the extracellular milieu, Vorozole also blocked within 5 min the AA in POA explants maintained in vitro. Together, these data demonstrate that aromatase inhibition rapidly decreases both consummatory and appetitive aspects of male sexual behavior.     

Effects of neonatal septal lesions on reproductive behavior of male and female rats

The purpose of this study was to examine the effects of neonatally placed septal lesions (SL) in male, female, and androgenized female rats on reproductive behavior. Animals were castrated as adults and tested for both feminine and masculine sexual behavior. After treatment with estradiol benzoate (EB) alone (2 μg daily for 3 days), only the females with SL which had not been given testosterone propionate (TP) neonatally showed a facilitation of lordosis behavior. Following EB (2 μg for 3 days) plus 0.5 mg progesterone (P), both the lesioned and the sham-operated female groups showed an increase in the display of lordosis in either hormonal condition. All animals were given a pretest for masculine sexual behavior and tested on Days 4, 7, 11, and 15 of daily TP treatment (150 μg/day). There was no effect of the neonatally placed SL on masculine sexual behavior in female rats or in female rats androgenized with 30 μg TP. However, lesioned females treated neonatally with 1 mg TP showed a marginal enhancement of masculine sexual behavior. Male rats given SL neonatally showed a marked enhancement of masculine sexual behavior compared to that of controls. These results suggest that, depending on the neonatal hormone environment, SL selectively increase behavioral sensitivity to hormones. Although neonatally lesioned females show behavioral responses similar to females given SL as adults, male rats given SL neonatally are unique in that they show enhanced masculine sexual behavior whereas males lesioned as adults do not.     

Effects of testosterone metabolites on copulation, medial preoptic dopamine, and NOS-immunoreactivity in castrated male rats

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. In a previous study, we identified 17beta-estradiol (E(2)) as the metabolite of testosterone (T) that maintains MPOA basal extracellular DA levels. However, the presence of dihydrotestosterone (DHT), an androgenic metabolite of T, is required for the female-induced increase in MPOA DA observed during copulation. Recently, we reported that assays of MPOA tissue DA content showed that castrates actually had more stored DA than did gonadally intact males. Therefore, the reduction in extracellular levels in castrates was not due to decreased availability of DA; most likely it was due to decreased release. Furthermore, T upregulates neuronal nitric oxide synthase (nNOS) in the MPOA. NO has been implicated in the regulation of DA release in the MPOA. It is not known, however, which metabolite(s) of T regulate(s) tissue stores of DA and/or nNOS in the MPOA of male rats. The present experiments were designed to test the following: (1) whether E(2), DHT, or the combination of the two influences MPOA DA tissue levels, an indication of stored DA, in male rat castrates; and (2) whether E(2), DHT, or the combination of the two influences NOS-ir in the MPOA of castrated male rats. The results indicate that E(2) up-regulates nNOS-ir in the MPOA and maintains tissue content of DA at levels similar to those in T-treated rats. DHT did not influence nNOS-ir, while attenuating the effect of castration on tissue DA content.     

Medroxyprogesterone acetate and the nuclear uptake of testosterone and its metabolites by brain, pituitary gland and genital tract in male cynomolgus monkeys.

The synthetic progestin, medroxyprogesterone acetate (MPA), is used to treat male sex offenders, and it is also suppresses sexual activity in male monkeys. To examine the possibility that MPA may act as an anti-androgen in the primate brain, 4 intact male cynomolgus monkeys were given MPA (40 mg i.m.) once a week for 16 weeks, while 4 control males received i.m. injections of vehicle. All males were then castrated and 3 days later were given 3 mCi [3H]testosterone ([3H]T) i.v.; 1 h after injection males were killed, and radioactivity in nuclear pellets obtained from the hypothalamus (HYP), preoptic area (POA), amygdala (AMG), septum, pituitary gland and genital tract was analyzed by HPLC. Concentrations of [3H]T and [3H]dihydrotestosterone in nuclear pellets were 65-96% lower in MPA-treated males than in controls (P less than 0.001), but the aromatized metabolite, [3H]estradiol, which was the major form of radioactivity present in nuclear pellets from HYP, POA and AMG, was unchanged. There were no differences in concentrations of [3H]T in supernatants from the tissues of MPA-treated and control males. Because the reduced nuclear uptake of androgen in brain occurred in males whose androgen-dependent behavior had been suppressed by MPA treatments, it is proposed that MPA may have anti-androgenic effects at the level of the cell nucleus in brain regions that control behavior.     

Daily patterns of courtship and mating behavior in the male Japanese quail

Crowing behavior was monitored constantly in male Japanese quail housed singly over 30 successive days. The photoperiod was 16h of light and 8 h of dark. A daily pattern in crowing was observed in which the frequencies were elevated in the afternoon and at the beginning of darkness. However, peak crowing occured 2 h prior to the onset of light. These rhythms were highly correlated among individuals and extremely repeatable over the sequential days of observation.In a second experiment, males which were paired with females were observed for frequencies of crowing, courtship, and mating behavior during the lighted portion of the day. In this experiment, the same photoperiod (16L:8D) was maintained. Paired males exhibited a daily pattern in crowing similar to that observed in the singly housed males. The frequency of mating was the highest between 1200 and 1300 h and lowest at 1400 h. Mating success was highest at midday, as were the number of males exhibiting mating behavior. These diurnal patterns in sexual behavior may depend on environmental cues such as photoperiod, which, in turn, may stimulate endocrine triggers.     

Effects of marijuana on testosterone in male subjects

Clinical studies have given contradictory reports on the effect of smoking marijuana on the plasma levels of testosterone in males. A reanalysis of existing data established that testosterone levels are depressed both after smoking one marijuana cigarette and after intravenous infusion of delta-9-tetrahydrocannabinol, a pharmacologically active component of marijuana. Simulation of the marijuana interaction, under the assumption that delta-9-tetrahydrocannabinol inhibits testosterone production or secretion, suggests a minimum of 24 hours are required for testosterone to return to pre-smoking levels. A series of clinical studies are specified to clarify the nature of the interaction.     

Effects of cyproterone acetate in the male Japanese quail

Male Japanese Quail were injected with oil or with one of four dosages of cyproterone acetate for 8 days. Copulatory behavior and cloacal gland size were measured daily. Cyproterone acetate reduced cloacal gland size at all dosages and in two experiments reduced copulation in eight out of 17 males at the two highest dosages. Thus, cyproterone acetate results in reproductive changes in at least two classes of vertebrates, and has behavioral as well as morphological effects. The morphological effects are more striking, however.