Progesterone modulates aggression in sex-role reversed female African black coucals

Testosterone is assumed to be the key hormone related to resource-defence aggression. While this role has been confirmed mostly in the context of reproduction in male vertebrates, the effect of testosterone on the expression of resource-defence aggression in female vertebrates is not so well established. Furthermore, laboratory work suggests that progesterone inhibits aggressive behaviour in females. In this study, we investigated the hormonal changes underlying territorial aggression in free-living female African black coucals, Centropus grillii (Aves; Cuculidae). Females of this sex-role reversed polyandrous bird species should be particularly prone to be affected by testosterone because they aggressively defend territories similar to males of other species. We show, however, that territorial aggression in female black coucals is modulated by progesterone. After aggressive territorial challenges female black coucals expressed lower levels of progesterone than unchallenged territorial females and females without territories, suggesting that progesterone may suppress territorial aggression and is downregulated during aggressive encounters. Indeed, females treated with physiological concentrations of progesterone were less aggressive than females with placebo implants. This is one of the first demonstrations of a corresponding hormone-behaviour interaction under challenged and experimental conditions in free-living females. We anticipate that our observation in a sex-role reversed species may provide a more general mechanism, by which progesterone--in interaction with testosterone--may regulate resource-defence aggression in female vertebrates.     

Polymorphic microsatellite loci in pheasant coucal (Centropus phasianinus)

Little is known about the effect of male parental care and behavioural sex‐role reversal on the mating system of birds because genetic markers for species with these characteristics are lacking. We developed primers for nine polymorphic microsatellite loci in pheasant coucals (Centropus phasianinus). Eight of the primers were also polymorphic in African black coucals (Centropus grillii). Pheasant coucals are of particular interest in the study of evolutionary and behavioural ecology, because their sex‐role reversal and extensive male parental care suggests low levels of extra‐pair fertilizations, yet they have large testes indicating sperm competition.     

Degree of sex reversal as related to plasma steroid levels in genetic female chickens (Gallus domesticus) treated with Fadrozole

The objectives of this work were to determine whether or not plasma levels of testosterone and estradiol reflect the various grades of sex reversal in genetic female chickens treated with Fadrozole (CGS 16949 A), a nonsteroidal aromatase inhibitor, and whether gonadal aromatase activity and plasma levels of testosterone and estradiol in treated females can or not be modified by post-hatch treatments with Fadrozole or Fadrozole + testosterone. Eggs were injected with 1 mg Fadrozole on day 4 of incubation. In females having developed sex-reversed gonads, endocrine parameters (estradiol and testosterone) at and after 13 weeks of age were indicative of the degree of sex reversal, with, for example, sex-reversed females with two testes having the highest levels of testosterone and the lowest levels of estradiol. Among these females, eight (from a total of 13) produced ejaculates with scarce and abnormal spermatozoa. Some motility was observable in the ejaculates from five of them. None of the post-hatch treatments had a significant effect on plasma levels of testosterone or estradiol (measured at 3-week intervals from week 4 to week 28 post-hatch) or on gonadal aromatase activity (measured at 12 and 28 weeks). In conclusion, these results indicate that plasma levels of testosterone and estradiol at and after 13 weeks of age are valuable indicators of the degree of sex reversal in female chickens treated with Fadrozole prior to gonadal sex differentiation. In pre-cited conditions, post-natal treatments with either Fadrozole or Fadrozole + testosterone had no apparent effect on the degree of sex reversal in these birds. Finally, the occurrence of ejaculates with motile although scarce and abnormal spermatozoa, revealed that epididymes and ducti deferens can develop and become functional in sex-reversed female chickens.     

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

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Social Reversal of Sex‐Biased Aggression and Dominance in a Biparental Cichlid Fish (Julidochromis marlieri)

In biparental species, aggression, dominance, and parental care are typically sexually dimorphic. While behavioral dimorphism is often strongly linked to gonadal sex, the environment—either social or ecological—may also influence sex‐biased behavior. In the biparental cichlid fish Julidochromis marlieri, the typical social environment for breeding pairs consists of large females paired with smaller males. While both sexes are capable of providing territory defense and parental care, the larger female provides the majority of defense for the pair, while the smaller male remains in the nest guarding their offspring. We examine the contributions of sex and relative mate size to these sex‐biased behaviors in monogamous J. marlieri pairs. Both female‐larger and male‐larger pairs were formed in the laboratory and were observed for territorial aggression (against conspecifics and heterospecifics), dominance, and parental care. In female‐larger pairs, territorial aggression and intra‐pair dominance were female‐biased, while in male‐larger pairs this bias was reversed. For both pairing types, the presence of an intruder amplified sex differences in territorial aggression, with the larger fish always attacking with greater frequency than its mate. Though less robust, there was evidence for plasticity of sex‐bias for some egg care related behaviors in the inverse direction. Our study suggests that relative mate size strongly influences the sex bias of aggression and dominance in J. marlieri and that this aspect of the social environment can override the influence of gonadal sex on an individual's behavior. The remarkable plasticity of this species makes Julidochromis an exciting model that could be used to address the relationship between proximate and ultimate mechanisms of behavioral plasticity.     

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.     

Androgen‐metabolizing enzymes show region‐specific changes across the breeding season in the brain of a wild songbird

The Lapland longspur (Calcarius lapponicus) is an arctic‐breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate‐zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen‐metabolizing enzymes in the longspur brain explain hormone‐behavior patterns in this species. We measured the activities of aromatase, 5α‐reductase and 5β‐reductase in free‐living longspur males. Aromatase and 5α‐reductase convert T into the active steroids 17β‐estradiol (E₂) and 5α‐dihydrotestosterone (5α‐DHT), respectively. 5β‐Reductase deactivates T via conversion to 5β‐DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5β‐Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region‐specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5β‐reductase do not explain the effects of plasma T on aggressive behavior. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 176–188, 1999     

Sex-role reversal of a monogamous pipefish without higher potential reproductive rate in females

In monogamous animals, males are usually the predominant competitors for mates. However, a strictly monogamous pipefish Corythoichthys haematopterus exceptionally exhibits a reversed sex role. To understand why its sex role is reversed, we measured the adult sex ratio and the potential reproductive rate (PRR), two principal factors influencing the operational sex ratio (OSR), in a natural population of southern Japan. The adult sex ratio was biased towards females throughout the breeding season, but the PRR, which increased with water temperature, did not show sexual difference. We found that an alternative index of the OSR (Sf/Sm: sex ratio of 'time in') calculated from the monthly data was consistently biased towards females. The female-biased OSR associated with sex-role reversal has been reported in some polyandrous or promiscuous pipefish, but factors biasing the OSR differed between these pipefish and C. haematopterus. We concluded that the similar PRR between the sexes in C. haematopterus does not confer reproductive benefit of polygamous mating on either sex, resulting in strict monogamous mating, and its female-biased adult sex ratio promotes female-female competition for a mate, resulting in sex-role reversal.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad‐intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad‐intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen‐responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor–immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28 ± 0.3 units) than in males (2.21 ± 0.2 units; p < .05). These sex differences in the number and staining intensity of androgen‐responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 359–370, 1999     

Within species support for the expensive tissue hypothesis: a negative association between brain size and visceral fat storage in females of the Pacific seaweed pipefish

The brain is one of the most energetically expensive organs in the vertebrate body. Consequently, the high cost of brain development and maintenance is predicted to constrain adaptive brain size evolution (the expensive tissue hypothesis, ETH). Here, we test the ETH in a teleost fish with predominant female mating competition (reversed sex roles) and male pregnancy, the pacific seaweed pipefish Syngnathus schlegeli. The relative size of the brain and other energetically expensive organs (kidney, liver, heart, gut, visceral fat, and ovary/testis) was compared among three groups: pregnant males, nonpregnant males and egg producing females. Brood size in pregnant males was unrelated to brain size or the size of any other organ, whereas positive relationships were found between ovary size, kidney size, and liver size in females. Moreover, we found that the size of energetically expensive organs (brain, heart, gut, kidney, and liver) as well as the amount of visceral fat did not differ between pregnant and nonpregnant males. However, we found marked differences in relative size of the expensive organs between sexes. Females had larger liver and kidney than males, whereas males stored more visceral fat than females. Furthermore, in females we found a negative correlation between brain size and the amount of visceral fat, whereas in males, a positive trend between brain size and both liver and heart size was found. These results suggest that, while the majority of variation in the size of various expensive organs in this species likely reflects that individuals in good condition can afford to allocate resources to several organs, the cost of the expensive brain was visible in the visceral fat content of females, possibly due to the high costs associated with female egg production.     

Aromatase inhibition abolishes courtship behaviours in the ring dove (Streptopelia risoria) and reduces androgen and progesterone receptors in the hypothalamus and anterior pituitary gland

The aim of this study was to determine in the ring dove, the effects of aromatase inhibition on the expression of aggressive courtship and nest-soliciting behaviours in relation to the distribution of cells containing immunoreactive androgen (AR) and progesterone (PR) receptor in the hypothalamus and pituitary gland. Isolated sexually experienced ring doves were transferred in opposite sex pairs to individual breeding cages, and then injected with the aromatase inhibitor, fadrozole (four males and four females), or saline vehicle (four males and four females) for 3 days at 12 hourly intervals. Saline-injected control males displayed aggressive courtship behaviours (bow-cooing and hop-charging) and nest-soliciting throughout the study, and control females displayed nest-soliciting. By day 3, fadrozole treatment resulted in the disappearance of all these behaviours and in a decrease or disappearance of AR and PR in the anterior pituitary gland, and in the nucleus preopticus paraventricularis magnocellularis (PPM), nucleus preopticus medialis (POM), nucleus hypothalami lateralis posterioris (PLH), and ventral, lateral and dorsal nucleus tuberalis in the hypothalamus (VTu, LTu, DTu). In the nucleus preopticus anterior (POA), fadrozole treatment decreased AR in both sexes and decreased PR in females but not in males. Cells containing co-localized nuclear AR and PR were found in all hypothalamic areas examined, and in the anterior pituitary gland. Fadrozole is suggested to reduce the local availability of estrogen required indirectly for the induction of AR, and except in cells containing PR in the male POA, for the direct induction of PR. It is suggested that aggressive courtship behaviour is terminated by “cross talk” between aromatase-independent PR and aromatase-dependent AR co-localized in neurons in the POA. Aromatase-independent PR may increase in the male POA in response to visual cues provided by a partner. Aromatase-dependent PR in the POM, and basal hypothalamus may play a role in the facilitatory effect of progesterone on estrogen-induced nest-orientated behaviours. (Mol Cell Biochem 276: 193–204, 2005)     

Establishment of estrogen receptor 1 (ESR1)‐knockout medaka: ESR1 is dispensable for sexual development and reproduction in medaka,Oryzias latipes

Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss‐of‐function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen‐induced vitellogenin gene expression, an estrogen‐responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish.     

Female-specific target sites for both oestrogen and androgen in the teleost brain

To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout life, we examined sex differences in neural expression of all subtypes of nuclear oestrogen and androgen receptors (ER and AR) in medaka. All receptors were differentially expressed between the sexes in specific nuclei in the forebrain. The most pronounced sex differences were found in several nuclei in the ventral telencephalic and preoptic areas, where ER and AR expression were prominent in females but almost completely absent in males, indicating that these nuclei represent female-specific target sites for both oestrogen and androgen in the brain. Subsequent analyses revealed that the female-specific expression of ER and AR is not under the direct control of sex-linked genes but is instead regulated positively by oestrogen and negatively by androgen in a transient and reversible manner. Taken together, the present study demonstrates that sex-specific target sites for both oestrogen and androgen occur in the brain as a result of the activational effects of gonadal steroids. The consequent sex-specific but reversible steroid sensitivity of the adult brain probably contributes substantially to the process of sexual differentiation and the persistent sexual plasticity of the teleost brain.     

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex‐linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex‐reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress‐induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North‐Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female‐to‐male sex‐reversed adults had similar body mass as normal males. We recorded no events of male‐to‐female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human‐induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex‐reversed individuals surviving to adulthood may participate in breeding.