Charlotte Cornil

Estradiol rapidly activates male sexual behavior and affects brain monoamine levels in the quail brain.

Steroids are generally viewed as transcription factors binding to intracellular receptors and activating gene transcription. Rapid cellular effects mediated via non-genomic mechanisms have however been identified and one report showed that injections of estradiol rapidly stimulate chemoinvestigation and mounting behavior in castrated male rats. It is not known whether such effects take place in other species and what are the cellular underlying mechanisms. We show here that a single injection of estradiol (500 microg/kg) rapidly and transiently activates copulatory behavior in castrated male quail pre-treated with a dose of testosterone behaviorally ineffective by itself. The maximal behavioral effect was observed after 15 min. In a second experiment, the brain of all subjects was immediately collected after behavioral tests performed 15 min after injection. The preoptic area--hypothalamus (HPOA), hindbrain, telencephalon and cerebellum were isolated and monoamines measured by HPLC-ED. Estradiol increased levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA/serotonin ratios in the telencephalon and hindbrain independently of whether animals had mated or not. Estradiol also affected these measures in HPOA and cerebellum but this effect was correlated with the level of sexual activity so that significant effects of the treatment only appeared when sexual activity was used as a covariate. Interactions between estradiol effects and sexual activity were also observed for dopamine in the HPOA and for serotonin in the hindbrain and cerebellum. Together, these data demonstrate that a single estradiol injection rapidly activates male sexual behavior in quail and that this behavioral effect is correlated with changes in monoaminergic activity.

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.

Multiple mechanisms control brain aromatase activity at the genomic and non-genomic level

Evidence has recently accumulated indicating that aromatase activity in the preoptic area is modulated in parallel by both slow (hours to days) genomic and rapid (minutes to hours) non-genomic mechanisms. We review here these two types of control mechanisms and their potential contribution to various aspects of brain physiology in quail. High levels of aromatase mRNA, protein and activity (AA) are present in the preoptic area of this species where the transcription of aromatase is controlled mainly by steroids. Estrogens acting in synergy with androgens play a key role in this control and both androgen and estrogen receptors (ER; alpha and beta subtypes) are present in the preoptic area even if they are not necessarily co-localized in the same cells as aromatase. Steroids have more pronounced effects on aromatase transcription in males than in females and this sex difference could be caused, in part, by a sexually differentiated expression of the steroid receptor coactivator 1 in this area. The changes in aromatase concentration presumably control seasonal variations as well as sex differences in brain estrogen production. Aromatase activity in hypothalamic homogenates is also rapidly (within minutes) down-regulated by exposure to conditions that enhance protein phosphorylation such as the presence of high concentrations of calcium, magnesium and ATP. Similarly, pharmacological manipulations such as treatment with thapsigargin or stimulation of various neurotransmitter receptors (alpha-amino-3-hydroxy-methyl-4-isoxazole propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA)) leading to enhanced intracellular calcium concentrations depress within minutes the aromatase activity measured in quail preoptic explants. The effects of receptor stimulation are presumably direct: electrophysiological data confirm the presence of these receptors in the membrane of aromatase-expressing cells. Inhibitors of protein kinases interfere with these processes and Western blotting experiments on brain aromatase purified by immunoprecipitation confirm that the phosphorylations regulating aromatase activity directly affect the enzyme rather than another regulatory protein. Accordingly, several phosphorylation consensus sites are present on the deduced amino acid sequence of the recently cloned quail aromatase. Fast changes in the local availability of estrogens in the brain can thus be caused by aromatase phosphorylation so that estrogen could rapidly regulate neuronal physiology and behavior. The rapid as well as slower processes of local estrogen production in the brain thus match well with the genomic and non-genomic actions of steroids in the brain. These two processes potentially provide sufficient temporal variation in the bio-availability of estrogens to support the entire range of established effects for this steroid.

Rapid control of male typical behaviors by brain-derived estrogens

Beside their genomic mode of action, estrogens also activate a variety of cellular signaling pathways through non-genomic mechanisms. Until recently, little was known regarding the functional significance of such actions in males and the mechanisms that control local estrogen concentration with a spatial and time resolution compatible with these non-genomic actions had rarely been examined. Here, we review evidence that estrogens rapidly modulate a variety of behaviors in male vertebrates. Then, we present in vitro work supporting the existence of a control mechanism of local brain estrogen synthesis by aromatase along with in vivo evidence that rapid changes in aromatase activity also occur in a region-specific manner in response to changes in the social or environmental context. Finally, we suggest that the brain estrogen provision may also play a significant role in females. Together these data bolster the hypothesis that brain-derived estrogens should be considered as neuromodulators.

Rapid changes in production and behavioral action of estrogens.

It is well established that sex steroid hormones bind to nuclear receptors, which then act as transcription factors to control brain sexual differentiation and the activation of sexual behaviors. Estrogens locally produced in the brain exert their behavioral effects in this way but mounting evidence indicates that estrogens also can influence brain functioning more rapidly via non-genomic mechanisms. We recently reported that, in Japanese quail, the activity of preoptic estrogen synthase (aromatase) can be modulated quite rapidly (within minutes) by non-genomic mechanisms, including calcium-dependent phosphorylations. Behavioral studies further demonstrated that rapid changes in estrogen bioavailability, resulting either from a single injection of a high dose of estradiol or from the acute inhibition of aromatase activity, significantly affect the expression of both appetitive and consummatory aspects of male sexual behavior with latencies ranging between 15 and 30 min. Together these data indicate that the bioavailability of estrogens in the brain can change on different time-scales (long- and short-term) that match well with the genomic and non-genomic actions of this steroid and underlie two complementary mechanisms through which estrogens modulate behavior. Estrogens produced locally in the brain should therefore be considered not only as neuroactive steroids but they also display many (if not all) functional characteristics of neuromodulators and perhaps neurotransmitters.

Human and Quail Aromatase Activity Is Rapidly and Reversibly Inhibited by Phosphorylating Conditions

Besides their slow genomic actions, estrogens also induce rapid physiological responses. To be functionally relevant, these effects must be associated with rapid changes in local concentrations of estrogens. Rapid changes in aromatase activity (AA) controlled by calcium-dependent phosphorylations of the enzyme can alter in a rapid manner local estrogen concentrations, but so far this mechanism was identified only in the avian (quail) brain. We show here that AA is also rapidly down-regulated by phosphorylating conditions in quail ovary homogenates and in various cell lines transfected with human aromatase (HEK 293, Neuro2A, and C6). Enzymatic activity was also rapidly inhibited after depolarization of aromatase-expressing HEK 293 cells with 100 mM KCl, and activity was fully restored when cells returned to control conditions. Western blot analysis demonstrated that the reduction of enzymatic activity is not due to protein degradation. We next investigated by site-directed mutagenesis the potential implication in the control of AA of specific aromatase residues identified by bioinformatic analysis. Mutation of the amino acids S118, S247, S267, T462, T493, or S497 to alanine, alone or in combination, did not block the rapid inhibition of enzymatic activity induced by phosphorylating conditions, but basal AA was markedly decreased in the S118A mutant. Altogether, these results demonstrate that the rapid inhibition of AA is a widespread and fully reversible process and that phosphorylation of specific residues modulate AA. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues.

Acute Stress Differentially Affects Aromatase Activity in Specific Brain Nuclei of Adult Male and Female Quail

The rapid and temporary suppression of reproductive behavior is often assumed to be an important feature of the adaptive acute stress response. However, how this suppression operates at the mechanistic level is poorly understood. The enzyme aromatase converts testosterone to estradiol in the brain to activate reproductive behavior in male Japanese quail (Coturnix japonica). The discovery of rapid and reversible modification of aromatase activity (AA) provides a potential mechanism for fast, stress-induced changes in behavior. We investigated the effects of acute stress on AA in both sexes by measuring enzyme activity in all aromatase-expressing brain nuclei before, during, and after 30 min of acute restraint stress. We show here that acute stress rapidly alters AA in the male and female brain and that these changes are specific to the brain nuclei and sex of the individual. Specifically, acute stress rapidly (5 min) increased AA in the male medial preoptic nucleus, a region controlling male reproductive behavior; in females, a similar increase was also observed, but it appeared delayed (15 min) and had smaller amplitude. In the ventromedial and tuberal hypothalamus, regions associated with female reproductive behavior, stress induced a quick and sustained decrease in AA in females, but in males, only a slight increase (ventromedial) or no change (tuberal) in AA was observed. Effects of acute stress on brain estrogen production, therefore, represent one potential way through which stress affects reproduction.

Rapid Behavioural Effects of Oestrogens and Fast Regulation of Their Local Synthesis by Brain Aromatase

Besides their genomic effects, oestrogens, 17β‐oestradiol in particular, also activate cellular effects that may be too rapid (seconds to minutes) to result from de novo protein synthesis. Although the existence of such nongenomic actions has been extensively demonstrated in vitro, the understanding of their behavioural significance is only emerging. Recent findings provide evidence that acute oestrogen treatments significantly affect a variety of behavioural processes, including sexual behaviour, social communication and cognition. One question arising from these results concerns the source of the oestrogens mediating nongenomic effects in vivo. In this review, data collected in vitro and in vivo are presented supporting the notion that fast modulations of local testosterone aromatisation can rapidly control the local oestrogen concentration in a time frame compatible with their rapid actions. Taken together, these data provide compelling evidence of how rapid changes in the local production and action of oestrogens can shape complex behaviours.

Presence of aromatase and estrogen receptor alpha in the inner ear of zebra finches.

Sex differences in song behavior and in the neural system controlling song in songbirds are well documented but relatively little is known about sex differences in hearing. We recently demonstrated the existence of sex differences in auditory brainstem responses in a songbird species, the zebra finch (Taeniopygia guttata). Many sex differences are regulated by sex steroid hormone action either during ontogeny or in adulthood. As a first step to test the possible implication of sex steroids in the control of sex differences in the zebra finch auditory system, we evaluated via immunocytochemistry whether estrogens are produced and act in the zebra finch inner ear. Specifically we examined the distribution of aromatase, the enzyme converting testosterone into an estrogen, and of estrogen receptors of the alpha subtype (ERalpha) in adult zebra finch inner ears. The anatomy of the basilar papillae was visualized by fluorescein-phalloidin, which delineated the actin structure of hair cells and supporting cells at their apical surface. Whole mount preparations of basilar papillae stained by immunocytochemistry revealed in both males and females an abundant aromatase distribution in the cytoplasm of hair cells, while ERalpha was identified in the nuclei of hair cells and of underlying supporting cells. Double-labeled preparations confirmed the extensive co-localization of aromatase and ERalpha in the vast majority of the hair cells. These results are consistent with studies on non-avian species, suggesting a role for estrogens in auditory function. These findings are also consistent with the notion that estrogens may contribute to a sex difference in hearing. To our knowledge, this is the first demonstration of the presence of aromatase and of the co-localization of aromatase and ERalpha in the sensory epithelium of the inner ear in any animal model.

Is sexual motivational state linked to dopamine release in the medial preoptic area

The medial preoptic area (mPOA) is a key site for the dopaminergic enhancement of male sexual behavior. Dopamine release increases in the rat mPOA with mating, supporting the critical stimulatory role played by preoptic dopamine on male sexual behavior. However, it has been questioned whether dopamine is specifically related to the occurrence of male sexual behavior and not simply involved in general arousal. To address this question, we asked whether dopamine release in the mPOA is linked to the production of male sexual behavior in Japanese quail (Coturnix japonica), a species that exhibits a much shorter temporal pattern of copulation than rats and does not have an intermittent organ, resulting in a very different topography of their sexual response. Extracellular samples from the mPOA of adult sexually experienced male quail were collected every 6 min before, during, and after exposure to a female using in vivo microdialysis and analyzed using high-performance liquid chromatography with electrochemical detection. Extracellular dopamine significantly increased in the presence of a female and returned to baseline after removal of the female. However, quail that failed to copulate did not display this increased release. These findings indicate that it is not solely the presence of a female that drives dopamine release in males, but how a male responds to her. Furthermore, in quail that copulated, dopamine release did not change in samples collected during periods of no copulation. Together, these findings support the hypothesis that dopamine action in the mPOA is specifically linked to sexual motivation and not only to copulatory behavior or physical arousal.