Devaleena S. Pradhan

Aggressive interactions rapidly increase androgen synthesis in the brain during the non-breeding season.

In male song sparrows (Melospiza melodia), territorial challenges during the breeding season can rapidly increase circulating levels of testosterone (T). During the non-breeding season, male song sparrows are highly aggressive, but the gonads are regressed and plasma T levels are non-detectable and unaffected by territorial challenges. The pro-hormone dehydroepiandrosterone (DHEA) is elevated in song sparrow plasma and brain during the non-breeding season and may be locally converted to sex steroids in the brain to regulate aggression. The enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) converts DHEA to androstenedione (AE) using the cofactor NAD(+), and this is a critical rate-limiting step. We predicted that brain 3beta-HSD activity varies seasonally and is rapidly modulated by aggressive challenges. In the first study, brain 3beta-HSD activity was highest in the non-breeding season in specific regions. In the second study, a simulated territorial challenge rapidly increased aggressive behavior in non-breeding song sparrows. Brain 3beta-HSD activity, when measured without exogenous NAD(+), increased by approximately 250 to 500% in telencephalic regions of challenged subjects. When brain 3beta-HSD activity was measured with exogenous NAD(+), these effects of territorial challenges were not observed. These data suggest that territorial challenges rapidly increase endogenous NAD(+) levels or increase 3beta-HSD activity specifically within a NAD-rich subcellular compartment. Together, these two studies suggest a shift from systemic to local sex steroid signaling in the non-breeding season. Local steroid signaling produces high spatial and temporal specificity of steroid signals and avoids the costs of high systemic T levels during the non-breeding season.

Dehydroepiandrosterone and Corticosterone Are Regulated by Season and Acute Stress in a Wild Songbird: Jugular Versus Brachial Plasma

Stress has well-known effects on adrenal glucocorticoid secretion, and chronic elevation of glucocorticoids can have detrimental effects on the brain. Dehydroepiandrosterone (DHEA), an androgen precursor synthesized in the adrenal glands or the brain itself, has anti-glucocorticoid properties, but little is known about the role of DHEA in the stress response, particularly in the brain. Here, we measured the effects of acute restraint on circulating corticosterone (CORT) and DHEA levels in wild song sparrows. Blood was collected from either the brachial or jugular vein. In songbirds, jugular plasma is enriched with neurally synthesized steroids, and therefore, jugular plasma is an indirect index of the neural steroidal milieu. Subjects were sampled during four times of year: breeding, molt, early nonbreeding, and mid-nonbreeding. Baseline CORT and DHEA levels showed similar seasonal changes; both steroids were elevated during the breeding season. Baseline CORT and DHEA levels were similar in jugular and brachial plasma. Acute stress had robust effects on CORT and DHEA that were season specific and vein specific. For CORT, during the molt, stress increased jugular CORT more than brachial CORT. For DHEA, during the breeding season, stress decreased jugular DHEA but not brachial DHEA. During the molt, stress increased jugular DHEA but not brachial DHEA. Acute stress did not affect brachial DHEA. These data suggest that acute stress specifically affects the balance between DHEA synthesis and metabolism in the brain. Furthermore, these results suggest that CORT and DHEA are locally synthesized in the brain during molt, when systemic levels of CORT and DHEA are low.

3β-HSD ACTIVATES DHEA IN THE SONGBIRD BRAIN

Dehydroepiandrosterone (DHEA) is an abundant circulating prohormone in humans, with a variety of reported actions on central and peripheral tissues. Despite its abundance, the functions of DHEA are relatively unknown because common animal models (laboratory rats and mice) have very low DHEA levels in the blood. Over the past decade, we have obtained considerable evidence from avian studies demonstrating that (1) DHEA is an important circulating prohormone in songbirds and (2) the enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD), responsible for converting DHEA into a more active androgen, is expressed at high levels in the songbird brain. Here, we first review biochemical and molecular studies demonstrating the widespread activity and expression of 3β-HSD in the adult and developing songbird brain. Studies examining neural 3β-HSD activity show effects of sex, stress, and season that are region-specific. Second, we review studies showing seasonal and stress-related changes in circulating DHEA in captive and wild songbird species. Third, we describe evidence that DHEA treatment can stimulate song behavior and the growth of neural circuits controlling song behavior. Importantly, brain 3β-HSD and aromatase can work in concert to locally metabolize DHEA into active androgens and estrogens, which are critical for controlling behavior and robust adult neuroplasticity in songbirds. DHEA is likely secreted by the avian gonads and/or adrenals, as is the case in humans, but DHEA may also be synthesized de novo in the songbird brain from cholesterol or other precursors. Irrespective of its source, DHEA seems to be an important neurohormone in songbirds, and 3β-HSD is a key enzyme in the songbird brain.

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain.

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (3β‐HSD). Thus, brain 3β‐HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3β‐HSD activity in a sex‐specific manner. To elucidate endocrine regulation of brain 3β‐HSD, we asked whether 17β‐estradiol (E2) regulates DHEA metabolism in adult zebra finch (Taeniopygia guttata) and whether there are sex‐specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [3H]DHEA and radioinert E2in vitro. Within only 10 min, E2 significantly reduced 3β‐HSD activity in both male and female brain. Interestingly, the rapid effects of E2 were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.

A mechanism for rapid neurosteroidal regulation of parenting behaviour

While systemic steroid hormones are known to regulate reproductive behaviour, the actual mechanisms of steroidal regulation remain largely unknown. Steroidogenic enzyme activity can rapidly modulate social behaviour by influencing neurosteroid production. In fish, the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) synthesizes 11-ketotestosterone (KT, a potent teleost androgen) and deactivates cortisol (the primary teleost glucocorticoid), and both of these steroid hormones can regulate behaviour. Here, we investigated the role of neurosteroidogenesis in regulating parenting in a haremic bidirectionally hermaphroditic fish, Lythrypnus dalli, where males provide all requisite parental care. Using an in vitro assay, we found that an 11β-HSD inhibitor, carbenoxolone (CBX), reduced brain and testicular KT synthesis by 90% or more. We modulated neurosteroid levels in parenting males via intracerebroventricular injection of CBX. Within only 20 min, CBX transiently eliminated parenting behaviour, but not other social behaviour, suggesting an enzymatic mechanism for rapid neurosteroidal regulation of parenting. Consistent with our proposed mechanism, elevating KT levels rescued parenting when paired with CBX, while cortisol alone did not affect parenting. Females paired with the experimental males opportunistically consumed unattended eggs, which reduced male reproductive success by 15%, but some females also exhibited parenting behaviour and these females had elevated brain KT. Brain KT levels appear to regulate the expression of parenting behaviour as a result of changes in neural 11β-HSD activity.

3β-HSD in songbird brain: subcellular localization and rapid regulation by estradiol.

J. Neurochem. (2010) 115, 667–675.

Contextual modulation of androgen effects on agonistic interactions

Seasonal changes in steroid hormones are known to have a major impact on social behavior, but often are quite sensitive to environmental context. In the bi-directionally sex changing fish, Lythrypnus dalli, stable haremic groups exhibit baseline levels of interaction. Status instability follows immediately after male removal, causing transiently elevated agonistic interactions and increase in brain and systemic levels of a potent fish androgen, 11-ketotestosterone (KT). Coupling KT implants with a socially inhibitory environment for protogynous sex change induces rapid transition to male morphology, but no significant change in social behavior and status, which could result from systemically administered steroids not effectively penetrating into brain or other tissues. Here, we first determined the degree to which exogenously administered steroids affect the steroid load within tissues. Second, we examined whether coupling a social environment permissive to sex change would influence KT effects on agonistic behavior. We implanted cholesterol (Chol, control) or KT in the dominant individual (alpha) undergoing sex change (on d0) and determined the effects on behavior and the degree to which administered steroids altered the steroid load within tissues. During the period of social instability, there were rapid (within 2 h), but transient effects of KT on agonistic behavior in alphas, and secondary effects on betas. On d3 and d5, all KT, but no Chol, treated females had male typical genital papillae. Despite elevated brain and systemic KT 5 days after implant, overall rates of aggressive behavior remained unaffected. These data highlight the importance of social context in mediating complex hormone-behavior relationships.

Contextual modulation of social and endocrine correlates of fitness: insights from the life history of a sex changing fish

Steroid hormones are critical regulators of reproductive life history, and the steroid sensitive traits (morphology, behavior, physiology) associated with particular life history stages can have substantial fitness consequences for an organism. Hormones, behavior and fitness are reciprocally associated and can be used in an integrative fashion to understand how the environment impacts organismal function. To address the fitness component, we highlight the importance of using reliable proxies of reproductive success when studying proximate regulation of reproductive phenotypes. To understand the mechanisms by which the endocrine system regulates phenotype, we discuss the use of particular endocrine proxies and the need for appropriate functional interpretation of each. Lastly, in any experimental paradigm, the responses of animals vary based on the subtle differences in environmental and social context and this must also be considered. We explore these different levels of analyses by focusing on the fascinating life history transitions exhibited by the bi-directionally hermaphroditic fish, Lythrypnus dalli. Sex changing fish are excellent models for providing a deeper understanding of the fitness consequences associated with behavioral and endocrine variation. We close by proposing that local regulation of steroids is one potential mechanism that allows for the expression of novel phenotypes that can be characteristic of specific life history stages. A comparative species approach will facilitate progress in understanding the diversity of mechanisms underlying the contextual regulation of phenotypes and their associated fitness correlates.

Female, but not male, agonistic behaviour is associated with male reproductive success in stable bluebanded goby (Lythrypnus dalli) hierarchies

In many social species, there are important connections between social behaviour and reproduction that provide critical insights into the evolution of sociality. In this study, we describe associations between agonistic behaviour and male reproductive success in stable social groups of bluebanded gobies (Lythrypnus dalli). This highly social, sex-changing species forms linear hierarchies of a dominant male and multiple subordinate females. Males reproduce with each female in the harem and care for the eggs. Since aggression tends to be associated with reduced reproduction in social hierarchies, we hypothesized that males in groups with high rates of aggression would fertilise fewer eggs. We also hypothesized that a male’s agonistic behaviour would be associated with his reproductive success. Dominants often exert substantial control over their harem, including control over subordinate reproduction. To address these hypotheses, we quantified egg laying/fertilisation over 13 days and observed agonistic behaviour. We show that there was a significant, negative association between male reproductive success and the total rate agonistic interactions by a group. While no male behaviours were associated with the quantity of eggs fertilised, female agonistic behaviour may be central to male reproductive success. We identified a set of models approximating male reproductive success that included three female behaviours: aggression by the highest-ranking female and approaches by the lowest-ranking female were negatively associated with the quantity of eggs fertilised by males in their groups, but the efficiency with which the middle-ranking female displaced others was positively associated with this measure. These data provide a first step in elucidating the behavioural mechanisms that are associated with L. dalli reproductive success.

Simultaneous courtship and parenting in males and sex role reversal in females of the haremic bluebanded goby, Lythrypnus dalli

While males typically compete for females, species with female biased sex ratios and/or large male investment in offspring care often exhibit reversed sex roles. Here we investigated, in a haremic fish species, the bluebanded goby, Lythrypnus dalli, the impact of male and female courtship behaviour on male reproductive success, measured as the total number of eggs in the nest and total number of developed eggs. Reproductive success was not associated with rates of male behaviour, such as parenting, approaching and courtship, but was associated with rates of female courtship. Consistent with predictions for a role-reversed reproductive strategy, only males demonstrated nest care and females exhibited high rates of courtship and intrasexual competition, such that alpha females interrupted courtship solicitations by beta females. Overall, these data are consistent with sex role reversal in L. dalliand show that the expression of male courtship behaviour does not interfere with paternal care.