The postnatal demasculinization of sexual behavior in the Japanese quail (Coturnix coturnix japonica)

Three experiments were performed to analyze the time course of demasculinization in the Japanese quail and to test the activating and organizing effects of estradiol (E2) in adult sexually active birds. In Experiment 1, males and females were castrated at the age of 1 day or 1, 2, 4, and 6 weeks and treated as adults with testosterone (T). The age of castration had no effect on behavior and morphology in males. Plasma gonadotrophins (LH and FSH) were, however, higher in males castrated at or before than in those castrated after 2 weeks of age. This suggests that postnatal testicular secretions have organizing effects on the pituitary activity. Females which were castrated before 1 week of age were less sensitive to the activating effects of T than males, but were not fully demasculinized. The demasculinization of different reproductive characteristics such as male sexual behavior, cloacal gland size, and weight of the syringeal muscles is achieved in females at different times posthatching. In Experiment 2, castration of male and female quail at the ages of 4 days or 4 weeks confirmed that postnatal ovarian secretions contribute to the full behavioral and morphological demasculinization of females. It is easier to elicit mounting in T-treated females when they are tested in their home cage instead of a test arena. This difference was not observed in males. During Experiment 3, it was impossible to demasculinize sexually active adult males or females by treatment with Silastic implants of E2. E2 did not maintain sexual behavior in ovariectomized females showing male sexual behavior when treated with T but maintained the behavior in males.     

Estrogen receptor 2b is the major determinant of sex-typical mating behavior and sexual preference in medaka

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

     

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.     

Changes in androgen receptor, estrogen receptor alpha, and sexual behavior with aging and testosterone in male rats

Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.     

Distribution of androgen receptor-immunoreactive cells in the quail forebrain and their relationship with aromatase immunoreactivity

The distribution of androgen receptor-like immunoreactive (AR-ir) cells in the quail brain was analyzed by immunocytochemistry with the use of the affinity-purified antibody PG-21-19A raised against a synthetic peptide representing the first 21 N-terminal amino acids of the rat and human AR. This antibody is known to bind to the receptor in the absence as well as in the presence of endogenous ligands, and it was therefore expected that a more complete and accurate characterization of AR-ir cells would be obtained in comparison with previous studies using an antibody that preferentially recognizes the occupied receptor. Selected sections were double labeled for aromatase (ARO) by a technique that uses alkaline phosphatase as the reporter enzyme and Fast blue as the chromogen. AR-ir material was detected in the nucleus of cells located in a variety of brain areas in the preoptic region and the hypothalamus including the medial preoptic (POM), the supraoptic, the paraventricular (PVN), and the ventromedial (VMN) nuclei, but also in the tuberculum olfactorium, the nucleus accumbens/ventral striatum, the nucleus taeniae, the tuberal hypothalamus, the substantia grisea centralis (GCt), and the locus ceruleus. Cells exhibiting a dense AR-ir label were also detected in the nucleus intercollicularis. Preincubation of the primary antibody with an excess of the synthetic peptide used for immunization completely eliminated this nuclear staining. A significant number of AR-ir cells in the POM, VMN, PVN, and tuberal hypothalamus also contained ARO-ir material in their cytoplasm. These data confirm and extend previous studies localizing AR in the avian brain, and raise questions about the possible regulation by androgens of the metabolizing enzyme aromatase. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 323–340, 1998     

Effect of estrogen and its antagonist on the expression of arginine vasotocin (AVT) and its oxytocic-like receptor VT3 in the shell gland of Japanese quail, Coturnix coturnix japonica

Avian neurohypophysial hormone arginine vasotocin (AVT) is known to regulate shell gland contractility during oviposition. While studying the role of estrogen in the expression and regulation of AVT and its oxytocic-like receptor VT3, using in situ hybridization and immunohistochemistry, it was observed that the expression of AVT and its receptor was not detected in the shell gland of sexually immature Japanese quail. However, administration of estrogen to these birds not only stimulates the growth and activity (as assessed by increased mucosal fold length, total protein content and alkaline phosphatase level) of the shell gland but also upregulates the expression of AVT and VT3. Further, administration of estrogen antagonist tamoxifen to sexually mature bird shows opposite results. On the other hand, localization of ir-AVT, observed in the ovary of sexually mature bird, was not detected in the estrogen treated sexually immature quail. It is concluded that estrogen not only affects the growth and differentiation of avian oviduct, but also regulates the expression of shell gland AVT and its receptor VT3. Present findings suggest that the locally synthesized AVT acts in a paracrine way to upregulate VT3 receptor and thus facilitates the endocrine function of neurohypophysial AVT during oviposition.     

The post-natal differentiation of sexual behaviour in the Japanese quail (Coturnix coturnix japonica)

An experiment was carried out to study the contribution of endogenous steroids during postnatal life to the sexual differentiation in Japanese quail. Male and female quail were gonadectomized at one week of age and when adult received testosterone silastic capsules. Seventy seven percent of these females showed male-type sexual behaviour and 46 percent even copulated. All males showed sexual behaviour including cloacal contact movements. Males and females grew cloacal glands of nearly identical sizes in response to the testosterone treatment. It is concluded that sexual differentiation in quail is at least in part a postnatal process and more research is needed to elucidate its exact mechanisms and time course.     

Diurnal variations of sexual receptivity in the female Japanese quail (Coturnix coturnix japonica)

A series of experiments was performed to study the changes during the day of female receptivity in Japanese quail. In each experiment, the frequency of squatting and the percentage of male approaches which were followed by squatting increased at the end of the day, approximately 11 to 13 hr after lights on (in a photoperiod of 16L:8D). In some cases this increased receptivity was associated with a significant decrease of the long-avoid frequency. Analyses are presented which demonstrate that the increased receptivity at the end of the day is not directly caused by the oviposition and does not result directly from changes in the behavior of the male stimuli. This is strongly supported by the observation that the increase in receptivity was observed at the same time after lights on (but different clock times) in two groups of females which were raised in two different photoperiods shifted by 6 hr and tested with the same group of males raised in one of the two photoperiods. The increase in receptivity coincides with an increase in plasma estradiol and progesterone. Considering that this behavior is suppressed by ovariectomy, it is argued that the daily changes in receptivity could be controlled by the hormonal changes associated with the ovulatory cycle.     

Genotype/age interactions on aggressive behavior in gonadally intact estrogen receptor beta knockout (betaERKO) male mice

Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, alpha (ER-alpha) and beta (ER-beta), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-alpha gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-beta gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-beta gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-alpha whereas it may inhibit aggression via ER-beta. In the present study, we further investigated the role of ER-beta in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-beta gene knockout (betaERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident-intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, betaERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which betaERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of betaERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when betaERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-beta mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.     

Effects of the nonsteroidal inhibitor R76713 on testosterone-induced sexual behavior in the Japanese quail (Coturnix coturnix japonica)

A new triazole derivative, R76713 (6-[4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole), was recently shown to inhibit aromatase selectively without affecting other steroid-metabolizing enzymes and without interacting with estrogen, progestin, or androgen receptors. This compound was tested for its capacity to intefere with the induction of copulatory behavior by testosterone (T) in castrated Japanese quail (Coturnix coturnix japonica). In a first experiment, R76713 inhibited (range 0.01 to 1 mg/kg) the activation of sexual behavior by T silastic implants and hypothalamic aromatase activity in castrated male quail in a dose-dependent manner. The 5 alpha- and 5 beta- reductases of T were not systematically affected. Stereotaxic implantation of R76713 in the medial preoptic area similarly blocked the behavior activated by systemic treatment with T, demonstrating that central aromatization of androgen is implicated in the activation of behavior. These inhibiting effects of R76713 on behavior were observed when implants were placed in the medial part of the nucleus preopticus medialis, confirming the implication of this brain area in the control of male copulatory behavior. Finally, the behavioral inhibition produced by R76713 could be reversed by simultaneous treatment with a dose of estradiol, which was not behaviorally effective by itself. This suggests that the behavioral deficit induced by the inhibitor was specifically due to the suppression of estrogen production. This also shows that the activation of copulatory behavior probably results from the interaction of androgens and estrogens at the brain level, as the two treatments separately providing these hormonal stimuli (T with the aromatase inhibitor on one hand and a low dose of estradiol on the other hand) had almost no behavioral effects but they synergized to activate copulation when given concurrently. These data confirm the critical role of preoptic aromatase in the activation of reproductive behavior and demonstrate that R76713 is a useful tool for the in vivo study of estrogen-dependent processes.     

Activation of sexual behavior by implantation of testosterone propionate and estradiol benzoate into the preoptic area of the male Japanese quail (Coturnix japonica)

Intracranial implantation of minute pellets of gonadal steroids was performed to determine neuroanatomical loci at which steroids activate sexual behavior in the Japanese quail (Coturnix japonica). In this species, systemic treatment of castrated males with either testosterone propionate (TP) or estradiol benzoate (EB) restores male-typical copulatory behavior (head grabbing, mounting, and cloacal contact movements). In addition, EB activates female-typical receptive behavior (crouching). Adult male castrated quail were implanted intracranially with 300-micrograms pellets containing TP, EB, or cholesterol (CHOL) and behavior was tested with intact males and females. Either TP or EB pellets in the preoptic area (POA) activated male-typical copulatory behavior. Mounting was specifically activated without concomitant activation of other steroid-sensitive sexual and courtship behaviors. TP and EB implants in adjacent nuclei containing receptors for these steroids and CHOL implants in POA had no effect on male-typical copulatory behavior. Eighteen percent of all males tested for female-typical receptivity crouched, but no specific effect of EB was seen at any site. The similarity of the POA sites for activation of mounting by TP and EB is consistent with the hypothesis that cells within the POA aromatize testosterone to estrogens, which directly stimulate the cellular processes leading to behavioral activation.     

Parallel evolution between aromatase and androgen receptor in the animal kingdom

There are now many known cases of orthologous or unrelated proteinsin different species that have undergone parallel evolutionto satisfy a similar function. However, there are no reportedcases of parallel evolution for proteins that bind a commonligand but have different functions. We focused on two proteinsthat have different functions in steroid hormone biosynthesisand action but bind a common ligand, androgen. The first protein,androgen receptor (AR), is a nuclear hormone receptor and thesecond one, aromatase (cytochrome P450 19 [CYP19]), convertsandrogen to estrogen. We hypothesized that binding of the androgenligand has exerted common selective pressure on both AR andCYP19, resulting in a signature of parallel evolution betweenthese two proteins, though they perform different functions.Consistent with this hypothesis, we found that rates of aminoacid change in AR and CYP19 are strongly correlated across themetazoan phylogeny, whereas no significant correlation was foundin the control set of proteins. Moreover, we inferred that genomictoolkits required for steroid biosynthesis and action were presentin a basal metazoan, cnidarians. The close similarities betweenvertebrate and sea anemone AR and CYP19 suggest a very ancientorigin of their endocrine functions at the base of metazoanevolution. Finally, we found evidence supporting the hypothesisthat the androgen-to-estrogen ratio determines the gonadal sexin all metazoans.     

The acute phase response in Japanese quail (Coturnix coturnix japonica)

Experiments were conducted to determine the effect of injection of lipopolysaccharide (LPS, from S. typhimurium) or muramyl dipeptide (MDP, N-acetylmuramyl-L-ala-isoglutamine) in Japanese quail. Doses of MDP between 0.3 and 10 mg/kg body wt. had no effect on body temperature. In contrast, doses of 1.0-22.5 mg LPS/kg body wt. caused significant increases in body temperature. None of the doses of LPS or MDP resulted in mortality. The febrile response to LPS was diminished following a second injection 48 h after the first, and was absent following a third injection. Plasma zinc, an indicator of the acute phase response, was significantly reduced by either LPS or MDP after the first injection (P<0.001), but not after the second or third injection. Splenic interleukin 1-beta (IL-1beta) mRNA expression was increased after the first and last injection of LPS (P<0.001), but only after the first injection of MDP (P<0.005). Hepatic IL-1beta mRNA expression was increased after the first, but not the third injection of LPS (P<0.001), while MDP had no effect. These data indicate that Japanese quail are less sensitive to MDP than LPS, and that quail demonstrate tolerance to LPS following repeated injections.     

Glucocorticoid metabolism in the in ovo environment modulates exposure to maternal corticosterone in Japanese quail embryos (Coturnix japonica)

Maternal effects have gained attention as a method by which mothers may alter the physiological condition and phenotype of their offspring based upon current environmental conditions. The physiological and phenotypic outcomes of glucocorticoid-mediated maternal effects have been extensively studied in a variety of vertebrates; however, the underlying mechanism is currently unclear. Here, we injected tritiated corticosterone into the yolks of freshly laid Japanese quail eggs (Coturnix japonica) and traced its movement and metabolism through the in ovo development period. We found that corticosterone was extensively conjugated throughout the egg by the end of development, and while minimal corticosterone was detected within the embryo during development, accumulation of a conjugated metabolite in the embryo started to occur on day 6 of development. Because no movement and metabolism of corticosterone occurred in infertile eggs, our findings suggest that embryos are not passive recipients of maternal steroids, but instead appear to possess extensive metabolic capabilities, which may modulate their exposure to maternal steroids.     

Dynamics of crowing development in the domestic Japanese quail (Coturnix coturnix japonica)

Species-specific behaviours gradually emerge, via incomplete patterns, to the final complete adult form. A classical example is birdsong, a learned behaviour ideally suited for studying the neural and molecular substrates of vocal learning. Young songbirds gradually transform primitive unstructured vocalizations (subsong, akin to human babbling) into complex, stereotyped sequences of syllables that constitute adult song. In comparison with birdsong, territorial and mating calls of vocal non-learner species are thought to exhibit little change during development. We revisited this issue using the crowing behaviour of domestic Japanese quail (Coturnix coturnix japonica). Crowing activity was continuously recorded in young males maintained in social isolation from the age of three weeks to four months. We observed developmental changes in crow structure, both the temporal and the spectral levels. Speed and trajectories of these developmental changes exhibited an unexpected high inter-individual variability. Mechanisms used by quails to transform sounds during ontogeny resemble those described in oscines during the sensorimotor phase of song learning. Studies on vocal non-learners could shed light on the specificity and evolution of vocal learning.     

Inter-individual variability during morning choruses in Japanese quail males (Coturnix coturnix japonica)

We compared the organization of dawn choruses in five groups of Japanese quail, Coturnix coturnix japonica, males (four groups of kin-related males and one group of unrelated males). We documented the structure of chorus groups: one male sang much more than the others during the dawn peak in four of the five groups, and this regardless of the number of males in the group. The dominant singers were then removed from two similar-sized groups, one of kin-related (K3) and the other of unrelated (NK) males. The quantity of song emitted by the remaining NK males was modified, but not that emitted by the K3 males.     

Short latency vestibular evoked potentials in the Japanese quail (Coturnix coturnix japonica)

Short-latency vestibular-evoked potentials to pulsed linear acceleration were characterized in the quail. Responses occurred within 8 ms following the onset of stimuli and were composed of a series of positive and negative peaks. The latencies and amplitudes of the first four peaks were quantitatively characterized. Mean latencies at 1.0 g ms⁻¹ ranged from 1265 ± 208 μs (P1, N = 18) to 4802 ± 441 μs (N4, N = 13). Amplitudes ranged from 3.72 ± 1.51 μV (P1/N1, N = 18) to 1.49 ± 0.77 μV (P3/N3, N = 16). Latency-intensity (LI) slopes ranged from −38.7 ± 7.3 μs dB⁻¹ (P1, N = 18) to −71.6 ± 21.9 μs dB⁻¹ (N3, N = 15) and amplitude-intensity (AI) slopes ranged from 0.20 ± 0.08 μV dB⁻¹ (P1/N1, N = 18) to 0.07 ± 0.04 μV dB⁻¹ (P3/N3, N = 11). The mean response threshold across all animals was −21.83 ± 3.34 dB re: 1.0 g ms⁻¹ (N = 18). Responses remained after cochlear extirpation showing that they could not depend critically on cochlear activity. Responses were eliminated by destruction of the vestibular end organs, thus showing that responses depended critically and specifically on the vestibular system. The results demonstrate that the responses are vestibular and the findings provide a scientific basis for using vestibular responses to evaluate vestibular function through ontogeny and senescence in the quail. Accepted: 18 January 1997     

Energetic trade-offs between immunity and reproduction in male Japanese quail (Coturnix coturnix)

We investigated a postulated trade-off between reproduction and immune function by comparing the energetic costs of an immune response with phytohemagglutinin challenge (or injection) in castrated (low testosterone [T]) and intact (high T) Japanese Quail (Coturnix coturnix). Intact birds had higher resting metabolic rate (RMR) and significantly lower immune response than castrates. RMR of intact birds did not change in response to an immune challenge, suggesting that maintenance of reproductive tissues and associated high T is both immunosuppressive and energetically costly. Despite having a greater immune response than intact quail, castrates had a lower pre-challenge RMR than intact birds and paradoxically tended to decrease RMR during an immune challenge. This paradox may be because of pro-inflammatory cytokines that are released during immune responses. Cytokines promote energy conservation through malaise and soporific behaviors, possibly explaining the co-occurrence of a relatively strong immune response and a decrease in nocturnal RMR in castrates. The lower immune response in intact birds may not elicit as great a response of pro-inflammatory cytokines owing to an already elevated RMR from reproductive state, thus reducing any effect on RMR. The suppressed immune response and elevated RMR in intact birds may be because of T; however, we cannot separate the effects of T per se from the metabolic requirements of reproductive tissues.     

雌激素受体α和β免疫反应在雄性和雌性棕色田鼠脑区分布的比较

单配制和多配制动物社会行为有差异,这些差异可能与雌激素受体类型有关（ERs）。虽然多配制大鼠和小鼠中枢神经雌激素受体α（ERα）和β（ERβ）免疫反应在大脑的分布已有报道,单配制雄性草原田鼠中枢神经ERα的分布也有报道,但单配制田鼠ERα和（或）ERβ在雌性和雄性分布差异未见报道。本研究对雄性和雌性棕色田鼠前脑区域ERα和ERβ免疫反应（IR）细胞数量进行比较。研究结果表明：（1）免疫反应主要分布在细胞核中。 （2）ERα-IR和ERβ-IR细胞广泛分布于整个雌性和雄性前脑区域,在许多脑区表达有重叠。然而,不同受体在雌雄不同脑核中的分布数量是不同的。（3）ERα 和ERβ的分布存在性别差异。例如,雌性ERα在视前核中部（MPN）,终纹床和（BNST）和杏仁内侧核（MeA）比雄性多,相反雄性ERβ在MPN和BNST比雌性多。这些研究结果可能为我们理解如何通过ERα和ERβ调节动物的社会行为,及雌性和雄性社会行为的差异提供一个重要的神经解剖学基础。     

Maternal presence influences vocal development in the Japanese quail (Coturnix c. japonica)

Social influences on vocal development of young birds have been widely studied in oscine songbirds who learn to sing by vocal imitation of conspecifics, mainly male adults. In contrast, vocal development of non-vocal learners such as Galliformes is considered as being under strong genetic influence and independent of the social environment. In this study, we investigated the role of the mother on the vocal development of young Japanese quail (Coturnix coturnix japonica). We compared the vocal development of mothered and non-mothered chicks during the first 21 days of life. We analysed the structural changes of two vocalisations: a) the rally call, emitted during long-distance communication and in stressful situations, b) the contact call, emitted during short-distance communication when chicks are in visual and/or auditory contact with congeners. We showed that temporal and spectral structures of the two types of calls changed during development and differed between mothered and non-mothered chicks. These results demonstrate that maternal presence influences the vocal development of the young in the Japanese quail. Even if the adaptive value of such changes was not assessed, these results highlight that plasticity of vocalisations in species considered as non-vocal learners has been underestimated.