Role of Membrane Estrogen Receptor Alpha on the Positive Feedback of Estrogens on Kisspeptin and GnRH Neurons.

Estrogens act through nuclear and membrane-initiated signaling. Estrogen receptor alpha (ERα) is critical for reproduction, but the relative contribution of its nuclear and membrane signaling to the central regulation of reproduction is unclear. To address this question, two complementary approaches were used: estetrol (E4) a natural estrogen acting as an agonist of nuclear ERs, but as an antagonist of their membrane fraction, and the C451A-ERα mouse lacking mERα. E4 dose- dependently blocks ovulation in female rats, but the central mechanism underlying this effect is unknown. To determine whether E4 acts centrally to control ovulation, its effect was tested on the positive feedback of estradiol (E2) on neural circuits underlying luteinizing hormone (LH) secretion. In ovariectomized females chronically exposed to a low dose of E2, estradiol benzoate (EB) alone or combined with progesterone (P) induced an increase in the number of kisspeptin (Kp) and gonadotropin-releasing hormone (GnRH) neurons coexpressing Fos, a marker of neuronal activation. E4 blocked these effects of EB, but not when combined to P. These results indicate that E4 blocked the central induction of the positive feedback in the absence of P, suggesting an antagonistic effect of E4 on mERα in the brain as shown in peripheral tissues. In parallel, as opposed to wild-type females, C451A-ERα females did not show the activation of Kp and GnRH neurons in response to EB unless they are treated with P. Together these effects support a role for membrane-initiated estrogen signaling in the activation of the circuit mediating the LH surge.

Exploring neuronal markers and early social environment influence in divergent quail lines selected for social motivation.

Many species, including humans exhibit a wide range of social behaviors that are crucial for the adaptation and survival of most species. Brain organization and function are shaped by genetic and environmental factors, although their precise contributions have been relatively understudied in the context of artificial selection. We used divergent lines of quail selected on their high versus low level of motivation to approach a group of conspecifics (S + and S-, respectively) to investigate the influence of genetic selection and early social environment on sociability. We observed distinct sex- and brain-region-specific expression patterns of three neuronal markers: mesotocin, and vasotocin, the avian homologues of mammalian oxytocin and vasopressin, as well as aromatase, the enzyme that converts androgens into estrogens. These markers displayed pronounced and neuroanatomically specific differences between S + and S- quail. Additionally, in a second experiment, we assessed the influence of early social environment on social skills in juvenile birds. Mixing S + and S- resulted in more S- males approaching the group without affecting the sociability of S + or other behaviors, suggesting that the early social environment may influence the results of genetic selection. In conclusion, the divergent quail lines offer a valuable model for unraveling the neuronal and behavioral mechanisms underlying social behaviors.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

Role of neuroestrogens in the regulation of social behaviors - From social recognition to mating.

In this mini-review, we summarize the brain distribution of aromatase, the enzyme catalyzing the synthesis of estrogens from androgens, and the mechanisms responsible for regulating estrogen production within the brain. Understanding this local synthesis of estrogens by neurons is pivotal as it profoundly influences various facets of social behavior. Neuroestrogen action spans from the initial processing of socially pertinent sensory cues to integrating this information with an individual's internal state, ultimately resulting in the manifestation of either pro-affiliative or - aggressive behaviors. We focus here in particular on aggressive and sexual behavior as the result of correct individual recognition of intruders and potential mates. The data summarized in this review clearly point out the crucial role of locally synthesized estrogens in facilitating rapid adaptation to the social environment in rodents and birds of both sexes. These observations not only shed light on the evolutionary significance but also indicate the potential implications of these findings in the realm of human health, suggesting a compelling avenue for further investigation.

Role of membrane estrogen receptor alpha (ERα) in the rapid regulation of male sexual behavior.

The activation of male sexual behavior depends on brain estrogen synthesis. Estrogens act through nuclear and membrane receptors producing effects within hours/days or seconds/minutes, respectively. In mice, estrogen receptor alpha (ERα) is the main estrogen receptor (ER) controlling the activation of male sexual behavior. Although neuroestrogens rapidly modulate mouse sexual behavior, it is not known whether these effects involve membrane ERα (mERα). This study combines two complementary approaches to address this question. C451A-ERα mice carry an ERα that cannot signal at the membrane, while estetrol (E4) is a natural estrogen acting as an agonist on nuclear ERα but as an antagonist on membrane ERα. In wild-type males, E4 decreased the number of mounts and intromissions after 10 min. In C451A-ERα males, E4 also altered sexual performance but after 30 min. E4 did not affect time spent near the female in both wild-type and C451A-ERα mice. However, regardless of genotype, the aromatase inhibitor 1,4,6-Androstatriene-3,17-dione (ATD) decreased both sexual performance and the time spent near the female after 10 and 30 min, confirming the key role of aromatization in the rapid control of sexual behavior and motivation. In conclusion, the shift in timing at which the effect of E4 is observed in mice lacking mERα suggests a role for mERα in the regulation of rapid effects of neuroestrogens on sexual performance, thus providing the first demonstration that E4 acts as an antagonist of a mER in the brain. The persisting effect of ATD on behavior in C451A-ERα mice also suggests the implication of another ER.

Contribution of birds to the study of sexual differentiation of brain and behavior.

Behavioral neuroendocrinology has largely relied on mammalian models to understand the relationship between hormones and behavior, even if this discipline has historically used a larger diversity of species than other fields. Recent advances revealed the potential of avian models in elucidating the neuroendocrine bases of behavior. This paper provides a review focused mainly on the contributions of our laboratory to the study of sexual differentiation in Japanese quail and songbirds. Quail studies have firmly established the role of embryonic estrogens in the sexual differentiation of male copulatory behavior. While most sexually differentiated features identified in brain structure and physiology result from the different endocrine milieu of adults, a few characteristics are organized by embryonic estrogens. Among them, a sex difference was identified in the number and morphology of microglia which is not associated with sex differences in the concentration/expression of neuroinflammatory molecules. The behavioral role of microglia and neuroinflammatory processes requires further investigations. Sexual differentiation of singing in zebra finches is not mediated by the same endocrine mechanisms as male copulatory behavior and "direct" genetic effect, i.e., not mediated by gonadal steroids have been identified. Epigenetic contributions have also been considered. Finally sex differences in specific aspects of singing behavior have been identified in canaries after treatment of adults with exogenous testosterone suggesting that these aspects of song are differentiated during ontogeny. Integration of quail and songbirds as alternative models has thus expanded understanding of the interplay between hormones and behavior in the control of sexual differentiation.

Sex differences in song syntax and syllable diversity in testosterone-induced songs of adult male and female canaries.

BACKGROUND: Behavioral sex differences are widespread in the animal world. These differences can be qualitative (i.e., behavior present in one sex but not the other, a true sex dimorphism) or quantitative (behavior is present at a higher rate or quality in one sex compared to the other). Singing in oscine songbirds is associated with both types of differences. In canaries, female rarely sing spontaneously but they can be induced to do so by treatments with steroids. Song in these females is, however, not fully masculinized and exhibits relatively subtle differences in quality as compared with male song. We analyzed here sex differences in syllable content and syllable use between singing male and female canaries. METHODS: Songs were recorded from three groups of castrated male and three groups of photoregressed female canaries that had received Silastic™ implants filled with testosterone (T), with T plus estradiol (E2), or left empty (control). After 6 weeks of hormone treatment, 30 songs were recorded from each of the 47 subjects. Songs were segmented and each syllable was annotated. Various metrics of syllable diversity were extracted and network analysis was employed to characterize syllable sequences. RESULTS: Male and female songs were characterized by marked sex differences related to syllable use. Compared to females, males had a larger syllable-type repertoire and their songs contained more syllable types. Network analysis of syllable sequences showed that males follow more fixed patterns of syllable transitions than females. Both sexes, however, produced song of the same duration containing the same number of syllables produced at similar rates (numbers per second). CONCLUSIONS: Under the influence of T, canaries of both sexes are able to produce generally similar vocalizations that nevertheless differ in specific ways. The development of song during ontogeny appears to be a very sophisticated process that is presumably based on genetic and endocrine mechanisms but also on specific learning processes. These data highlight the importance of detailed behavioral analyses to identify the many dimensions of a behavior that can differ between males and females.

Male canaries normally sing complex songs at high rate while females only rarely sing very simple songs. Testosterone induces active singing in both male and female canaries, but female song is still not fully masculinized by these treatments even if song duration does not differ between the sexes. We analyzed the syllable repertoire and the sequence of use for different syllables in canaries of both sexes treated with testosterone or testosterone supplemented with estradiol. Compared to females, males had a larger syllable-type repertoire and their songs contained more syllable types. Syllable transitions were also more fixed in males. Sex differences in adult singing of canaries are thus a complex mixture of differences that result from the different endocrine condition of males and females (and are thus partially reversed by administration of exogenous testosterone) and of more stable differences that presumably develop during the ontogenetic process under the influence of endocrine and genetic differences and of differential learning processes. Canary song thus represents an outstanding model system to analyze the interaction between nature and nurture in the acquisition of a sophisticated learned behavior as well as the mechanisms controlling sex differences in vocal learning and production.

Does the syrinx, a peripheral structure, constrain effects of sex steroids on behavioral sex reversal in adult canaries?

We previously confirmed that effects of testosterone (T) on singing activity and on the volume of brain song control nuclei are sexually differentiated in adult canaries: females are limited in their ability to respond to T as males do. Here we expand on these results by focusing on sex differences in the production and performance of trills, i.e., rapid repetitions of song elements. We analyzed >42,000 trills recorded over a period of 6 weeks from 3 groups of castrated males and 3 groups of photoregressed females that received Silastic™ implants filled with T, T plus estradiol or left empty as control. Effects of T on the number of trills, trill duration and percent of time spent trilling were all stronger in males than females. Irrespective of endocrine treatment, trill performance assessed by vocal deviations from the trill rate versus trill bandwidth trade-off was also higher in males than in females. Finally, inter-individual differences in syrinx mass were positively correlated with specific features of trills in males but not in females. Given that T increases syrinx mass and syrinx fiber diameter in males but not in females, these data indicate that sex differences in trilling behavior are related to sex differences in syrinx mass and syrinx muscle fiber diameter that cannot be fully suppressed by sex steroids in adulthood. Sexual differentiation of behavior thus reflects organization not only of the brain but also of peripheral structures.

Effects of the depletion of neural progenitors by focal X-ray irradiation on song production and perception in canaries.

The song control nucleus HVC of songbirds has emerged as a widespread model system to study adult neurogenesis and the factors that modulate the incorporation of new neurons, including seasonal state, sex differences or sex steroid hormone concentrations. However, the specific function of these new neurons born in adulthood remains poorly understood. We implemented a new procedure based on focal X-ray irradiation to deplete neural progenitors in the ventricular zone adjacent to HVC and study the functional consequences. A 23 Gy dose depleted by more than 50 percent the incorporation of BrdU in neural progenitors, a depletion that was confirmed by a significant decrease in doublecortin positive neurons. This depletion of neurogenesis significantly increased the variability of testosterone-induced songs in females and decreased their bandwidth. Expression of the immediate early gene ZENK in secondary auditory areas of the telencephalon that respond to song was also inhibited. These data provide evidence that new neurons in HVC play a role in both song production and perception and that X-ray focal irradiation represents an excellent tool to advance our understanding of adult neurogenesis.

Does the syrinx, a peripheral structure, constrain effects of sex steroids on behavioral sex reversal in adult canaries?

We previously confirmed that effects of testosterone (T) on singing activity and on the volume of brain song control nuclei are sexually differentiated in adult canaries: females are limited in their ability to respond to T as males do. Here we expand on these results by focusing on sex differences in the production and performance of trills, i.e., rapid repetitions of song elements. We analyzed more than 42,000 trills recorded over a period of 6 weeks from 3 groups of castrated males and 3 groups of photoregressed females that received Silasticâ"¢ implants filled with T, T plus estradiol or left empty as control. Effects of T on the number of trills, trill duration and percent of time spent trilling were all stronger in males than females. Irrespective of endocrine treatment, trill performance assessed by vocal deviations from the trill rate versus trill bandwidth trade-off was also higher in males than in females. Finally, inter-individual differences in syrinx mass were positively correlated with trill production in males but not in females. Given that T increases syrinx mass and syrinx fiber diameter in males but not in females, these data indicate that sex differences in trilling behavior are related to sex differences in syrinx mass and syrinx muscle fiber diameter that cannot be fully reversed by sex steroids in adulthood. Sexual differentiation of behavior thus reflects organization not only of the brain but also of peripheral structures.

Testosterone treatment reveals marked sex differences in song diversity and syllable syntax in adult canaries.

Background. Behavioral sex differences are widespread in the animal world. These differences can be qualitative (i.e., behavior present in one sex but not the other, a true sex dimorphism) or quantitative (behavior is present at a higher rate or quality in one sex compared to the other). Singing in oscine songbirds is associated with both types of differences. In canaries, female rarely sing spontaneously but they can be induced to do so by treatments with steroids. Song in these females is however not fully masculinized and exhibits relatively subtle differences in quality as compared with male song. We analyzed here sex differences in syllable content and syllable use between singing male and female canaries. Methods. Songs were recorded from 3 groups of castrated male and 3 groups of photoregressed female canaries that had received Silasticâ"¢ implants filled with testosterone (T), with T plus estradiol (E2), or left empty (control). After 6 weeks of hormone treatment, 30 songs were recorded from each of the 47 subjects. Songs were segmented and each syllable was annotated. Various metrics of syllable diversity were extracted and network analysis was employed to characterize syllable sequences. Results. Male and female songs were characterized by marked sex differences related to syllable use. Compared to females, males had a larger syllable type repertoire and their songs contained more syllable types. Network analysis of syllable sequences showed that males follow more fixed patterns of syllable transitions than females. Both sexes however produced song of the same duration containing the same number of syllables produced at similar rates (numbers per second). Conclusions. Under the influence of T canaries of both sexes are able to produce generally similar vocalizations that nevertheless differ in specific ways. The development of song during ontogeny appears to be a very sophisticated process that is presumably based on genetic and endocrine mechanisms but also on specific learning processes. These data highlight the importance of detailed behavioral analyses in order to identify the many dimensions of a behavior that can differ between males and females.

Loss of function of the maternal membrane oestrogen receptor ERα alters expansion of trophoblast cells and impacts mouse fertility.

The binding of 17ß-oestradiol to oestrogen receptor alpha (ERα) plays a crucial role in the control of reproduction, acting through both nuclear and membrane-initiated signalling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss of function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do become pregnant, allowing the investigation of the role of ERα during pregnancy for the first time. All neonatal deaths of the mutant offspring mice resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibited partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality resulted from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells into the utero-placental unit, which is associated with an imbalance in expression of angiogenic factors. Together, these processes control the trophoblast-mediated spiral arterial remodelling. Hence, loss of membrane ERα within maternal tissues clearly alters the activity of invasive trophoblast cells during placentogenesis. This previously unreported function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.

Photoperiodic control of singing behavior and reproductive physiology in male Fife fancy canaries.

Temperate-zone birds display marked seasonal changes in reproductive behaviors and the underlying hormonal and neural mechanisms. These changes were extensively studied in canaries (Serinus canaria) but differ between strains. Fife fancy male canaries change their reproductive physiology in response to variations in day length but it remains unclear whether they become photorefractory (PR) when exposed to long days and what the consequences are for gonadal activity, singing behavior and the associated neural plasticity. Photosensitive (PS) male birds that had become reproductively competent (high song output, large testes) after being maintained on short days (SD, 8 L:16D) for 6 months were divided into two groups: control birds remained on SD (SD-PS group) and experimental birds were switched to long days (16 L:8D) and progressively developed photorefractoriness (LD-PR group). During the following 12 weeks, singing behavior (quantitatively analyzed for 3 × 2 hours every week) and gonadal size (repeatedly measured by CT X-ray scans) remained similar in both groups but there was an increase in plasma testosterone and trill numbers in the LD-PR group. Day length was then decreased back to 8 L:16D for LD-PR birds, which immediately induced a cessation of song, a decrease in plasma testosterone concentration, in the volume of song control nuclei (HVC, RA and Area X), in HVC neurogenesis and in aromatase expression in the medial preoptic area. These data demonstrate that Fife fancy canaries readily respond to changes in photoperiod and display a pattern of photorefractoriness following exposure to long days that is associated with marked changes in brain and behavior.

Treatment with androgens plus estrogens cannot reverse sex differences in song and the song control nuclei in adult canaries.

Adult treatments with testosterone (T) do not activate singing behavior nor promote growth of song control nuclei to the same extent in male and female canaries (Serinus canaria). Because T acts in part via aromatization into an estrogen and brain aromatase activity is lower in females than in males in many vertebrates, we hypothesized that this enzymatic difference might explain the sex differences seen even after exposure to the same amount of T. Three groups of castrated males and 3 groups of photoregressed females (i.e., with quiescent ovaries following exposure to short days) received either 2 empty 10 mm silastic implants, one empty implant and one implant filled with T or one implant filled with T plus one with estradiol (E2). Songs were recorded for 3 h each week for 6 weeks before brains were collected and song control nuclei volumes were measured in Nissl-stained sections. Multiple measures of song were still different in males and females following treatment with T. Co-administration of E2 did not improve these measures and even tended to inhibit some measures such as song rate and song duration. The volume of forebrain song control nuclei (HVC, RA, Area X) and the rate of neurogenesis in HVC was increased by the two steroid treatments, but remained significantly smaller in females than in males irrespective of the endocrine condition. These sex differences are thus not caused by a lower aromatization of the steroid; sex differences in canaries are probably organized either by early steroid action or by sex-specific gene regulation directly in the brain.

Role of aromatase in distinct brain nuclei of the social behaviour network in the expression of sexual behaviour in male Japanese quail.

In male Japanese quail, brain aromatase is crucial for the hormonal activation of sexual behaviour, but the sites producing neuro-oestrogens that are critical for these behaviours have not been completely identified. This study examined the function of aromatase expressed in several nuclei of the social behaviour network on a measure of sexual motivation known as the frequency of rhythmic cloacal sphincter movements (RCSM) and on copulatory behaviour. Sexually experienced castrated males chronically treated with testosterone were stereotaxically implanted with the aromatase inhibitor vorozole (VOR), or cholesterol as control, and tested for sexual behaviour. In experiment 1, males were implanted in the medial preoptic nucleus (POM) with VOR, a manipulation known to reduce the expression of copulatory behaviour. This experiment served as positive control, but also showed that VOR implanted in the dorsomedial or lateral portions of the POM similarly inhibits male copulatory behaviour compared to control implants. In experiments 2 to 4, males received stereotaxic implants of VOR in the periaqueductal gray (PAG), the nucleus taeniae of the amygdala (TnA) and the ventromedial nucleus of the hypothalamus (VMN), respectively. Sexual behaviour was affected only in individuals where VOR was implanted in the PAG: these males displayed significantly lower frequencies of cloacal contact movements, the last step of the copulatory sequence. Inhibition of aromatase in the TnA and VMN did not alter copulatory ability. Overall, RCSM frequency remained unaffected by VOR regardless of implantation site. Together, these results suggest that neuro-oestrogens produced in the POM contribute the most to the control of male copulatory behaviour, while aromatase expressed in the PAG might also participate to premotor aspects of male copulatory behaviour.

Rapid changes in brain estrogen concentration during male sexual behavior are site and stimulus specific.

Classically, estrogens regulate male sexual behavior through effects initiated in the nucleus. However, neuroestrogens, i.e., estrogens locally produced in the brain, can act within minutes via membrane-initiated events. In male quail, rapid changes in brain aromatase activity occur after exposure to sexual stimuli. We report here that local extracellular estrogen concentrations measured by in vivo microdialysis increase during sexual interactions in a brain site- and stimulus-specific manner. Indeed, estrogen concentrations rose within 10 min of the initiation of sexual interaction with a female in the medial preoptic nucleus only, while visual access to a female led to an increase in estrogen concentrations only in the bed nucleus of the stria terminalis. These are the fastest fluctuations in local estrogen concentrations ever observed in the vertebrate brain. Their site and stimulus specificity strongly confirm the neuromodulatory function of neuroestrogens on behavior.

Perineuronal nets in HVC and plasticity in male canary song.

Songbirds learn their vocalizations during developmental sensitive periods of song memorization and sensorimotor learning. Some seasonal songbirds, called open-ended learners, recapitulate transitions from sensorimotor learning and song crystallization on a seasonal basis during adulthood. In adult male canaries, sensorimotor learning occurs each year in autumn and leads to modifications of the syllable repertoire during successive breeding seasons. We previously showed that perineuronal nets (PNN) expression in song control nuclei decreases during this sensorimotor learning period. Here we explored the causal link between PNN expression in adult canaries and song modification by enzymatically degrading PNN in HVC, a key song control system nucleus. Three independent experiments identified limited effects of the PNN degradation in HVC on the song structure of male canaries. They clearly establish that presence of PNN in HVC is not required to maintain general features of crystallized song. Some suggestion was collected that PNN are implicated in the stability of song repertoires but this evidence is too preliminary to draw firm conclusions and additional investigations should consider producing PNN degradations at specified time points of the seasonal cycle. It also remains possible that once song has been crystallized at the beginning of the first breeding season, PNN no longer play a key role in determining song structure; this could be tested by treatments with chondroitinase ABC at key steps in ontogeny. It would in this context be important to develop multiple stereotaxic procedures allowing the simultaneous bilateral degradation of PNN in several song control nuclei for extended periods.

Effect of cyclo­oxygenase inhibition on embryonic microglia and the sexual differentiation of the brain and behavior of Japanese quail (Coturnix japonica).

Enduring sex differences in the brain are established during a developmental process known as brain sexual differentiation and are mainly driven by estrogens during a critical period. In rodents, the masculinization of the preoptic area by estrogens derived from the central aromatization of testosterone depends in part on the interaction between microglia and prostaglandin E2 (PGE2), a pro-inflammatory hormone of the prostanoid subclass. In contrast, in birds, estrogens produced by females induce a demasculinization, but whether an interaction with the neuro-immune system is involved in this process is unknown. This study addressed this question by testing the effects of blockade of cyclo­oxygenases (COX), the rate-limiting enzymes for prostanoid synthesis, on embryonic microglia and the sexual differentiation of brain and behavior using the Japanese quail as an animal model. The results show that COX inhibition does not affect the behavior of females, but impairs male sexual behavior and suppresses the sex difference in microglial profiles at embryonic day 12 (E12) in the medial preoptic nucleus by increasing the number of microglia in males only. However, neither prostanoid concentrations nor PGE2 receptors differed between sexes in the hypothalamus and preoptic area (HPOA) during development. Overall, these results uncovered a potential role of prostanoids in the demasculinization of Japanese quail. Moreover, the parallel effect of COX inhibition on behavior and microglia suggests an interaction between prostanoids and microglia in brain demasculinization, thus fueling the hypothesis of a conserved role of the neuroimmune system in the organization of the brain by estrogens.

DNA Methylation Regulates Transcription Factor-Specific Neurodevelopmental but Not Sexually Dimorphic Gene Expression Dynamics in Zebra Finch Telencephalon.

Song learning in zebra finches (Taeniopygia guttata) is a prototypical example of a complex learned behavior, yet knowledge of the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA-sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age-dependent gene expression was found for various transcription factors (i.e., OTX2, AR, and FOS) involved in neurodevelopment. Incomplete dosage compensation, independent of DNA methylation, was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon.

Effect of chronic intracerebroventricular administration of an aromatase inhibitor on the expression of socio-sexual behaviors in male Japanese quail.

Aromatase converts androgens into estrogens in the brain of vertebrates including humans. This enzyme is also expressed in other tissues where its action may result in negative effects on human health (e.g., promotion of tumor growth). To prevent these effects, aromatase inhibitors were developed and are currently used to block human estrogen-dependent tumors. In vertebrates including quail, aromatase is expressed in a highly conserved set of interconnected brain nuclei known as the social behavior network. This network is directly implicated in the expression of a large range of social behaviors. The primary goal of this study was to characterize in Japanese quail the potential impact of brain aromatase on sexual behavior, aggressiveness and social motivation (i.e., tendency to approach and stay close to conspecifics). An additional goal was to test the feasibility and effectiveness of long-term delivery of an aromatase inhibitor directly into the third ventricle via Alzet™ osmotic minipumps using male sexual behavior as the aromatase dependent measure. We demonstrate that this mode of administration results in the strongest inhibition of both copulatory behavior and sexual motivation ever observed in this species, while other social behaviors were variably affected. Sexual motivation and the tendency to approach a group of conspecifics including females clearly seem to depend on brain aromatase, but the effects of central estrogen production on aggressive behavior and on the motivation to approach males remain less clear.