Genetically Triggered Sexual Differentiation of Brain and Behavior

The dominant theory of sexual differentiation of the brain holds that sex differences in brain anatomy and function arise because of the action of gonadal steroids during embryonic and neonatal life. In mammals, testicular steroids trigger masculine patterns of neural development, and feminine patterns of neural development occur in the absence of such testicular secretions. In contrast, gonadal differentiation in mammals is not initiated by hormonal mechanisms, but is regulated by the action of gene products such as SRY, a testis-determining gene on the Y chromosome. This paper argues that such genetic, nonhormonal signals may also trigger specific examples of sexual differentiation of the brain. This thesis is supported by two arguments. The first is that “direct genetic” (i.e., nonhormonal) control of sexual differentiation may be as likely to evolve as hormonal control. The second line of argument is that neural and nonneural dimorphisms have already been described that are not well explained by classical theories of steroid-dependent sexual differentiation and for which other factors need to be invoked.     

Early Administration of 17β-Estradiol Partially Masculinizes Song Control Regions and α2-Adrenergic Receptor Distribution in European Starlings (Sturnus vulgaris)

The vocal control system in many songbird species is a sexually dimorphic neural circuit that mediates learning and production of song. The mechanism by which this system is sexually differentiated has been investigated in only one species, the zebra finch (Taeniopygia guttata). Estradiol may be involved in the sexual differentiation of this system, as female zebra finches treated with estradiol as nestlings develop a male-like song system; however, blocking estradiol action in embryonic and nestling male zebra finches does not demasculinize the song system. Therefore, the role of estradiol in song system development is unclear. The role of estradiol in song system sexual differentiation was assessed in European starlings (Sturnus vulgaris). This species is of potential interest because it is less extreme in the degree of sexual dimorphism of the song system and song behavior than zebra finches. While in the field, starling nestlings were implanted with 500 μg of estradiol at 3 days of age. These birds were brought into the laboratory at Day 11 and hand-reared. In females, estradiol produces significant increases in the volumes of song control regions defined by Nissl stain, as well as by autoradiography for α₂-adrenergic receptors; however, these estradiol-treated females have song systems that more closely resemble those of control females than control males. Estradiol-treated males exhibit significant hypermasculinization at 210 days of age, but this effect is transient and hypermasculinization is no longer evident at Day 345. The role of estradiol in sexual differentiation of the neural circuit mediating song behavior remains enigmatic.     

Sexual differentiation of the zebra finch song system parallels genetic, not gonadal, sex.

Mechanisms regulating sexual differentiation of the zebra finch song system present an intriguing puzzle. Masculine development of brain regions and behavior can be induced in genetic females by posthatching estradiol treatment. That result is consistent with the hypothesis that estradiol, converted within the brain from testicular androgen via the aromatase enzyme, masculinizes neural structure and function. In contrast, treatment during specific stages of development with the aromatase inhibitor Fadrozole has not prevented masculine development, and the presence of testicular tissue in genetic females did not induce masculine organization of neuroanatomy or singing behavior. Fadrozole treatments in those previous studies were limited, however, and most genetic females had both ovarian and testicular tissue. The present experiments were designed to provide increased aromatase inhibition and to reliably produce genetic females with only testicular tissue. Eggs received a single injection at a later age or with higher doses of Fadrozole than had been used previously. Some embryos were exposed to Fadrozole more frequently by either injecting eggs on 2 days of development or dipping them for 10-12 days in Fadrozole. Finally, in some individuals from Fadrozole-treated eggs, the left gonad was removed, leaving each genetic male and female with a single right testis. None of these treatments significantly affected development of the song system compared to appropriate control groups. These results suggest that sexual differentiation of the zebra finch song system is not regulated by embryonic aromatase activity or by gonadal secretions and instead involves events that need not be mediated by steroid hormones.     

Hormonal differentiation of sexual behavior in Japanese quail

The effect of hormones on the development of Japanese quail during the postembryonic period was examined. First, subcutaneous implants of estradiol monobenzoate (EB) and testosterone propionate (TP) were implanted 6–12 hr after hatching. EB and TP had no effect on the differentiation of sexual behavior in genetic males or females. However, EB had marked feminizing effects on plumage in genetic males. Second, the role of gonadal hormones during development was examined by gonadectomizing males and females 6–12 hr after hatching and treating them intramuscularly with EB or TP as adults. EB-treated adult females displayed sexual behavior typical of the genetic female and developed female plumage. A significant proportion of TP-treated females (57%) displayed male sexual behavior patterns. Cloacal gland development and male-type vocalizations were induced. EB-treated males displayed either male or female sexual patterns depending on the stimulus conditions. Third, to test whether bisexuality in gonadectomized males and females is maintained despite steroid treatment and expression of sexual behavior in adulthood, gonadectomized quail which were originally treated with EB received TP and vice versa. The results indicate that in the absence of gonadal hormones after hatching female quail remain bisexual until exposed to estrogen, whereas gonadectomized male quail retain behavioral bisexuality irrespective of prior estrogen or androgen exposure.     

Estrogen-Independent Ovary Formation in the Medaka Fish, Oryzias latipes

To investigate whether a female sex steroid, estrogen, acts as a natural inducer of female gonadal sex determination (or ovary formation) in the medaka fish, Oryzias latipes, the effects of an aromatase inhibitor and anti-estrogens on sexual differentiation of gonads were examined. We found that both drugs did not show any discernible effects on the genetically determined sex differentiation in both sexes. However, the aromatase inhibitor impaired the paradoxical effects of androgen (a male sex steroid), and the anti-estrogens inhibited the male-to-female sex reversal caused by estrogen. Treatments of the fertilized eggs with androgen disturbed the gonadal sex developments in both sexes, suggesting that sex steroid synthesis is detrimental to the gonadal sex developments in the medaka embryos. These results are consistent with the previous observation that sex steroids are not synthesized before the onset of gonadal sex differentiation, and suggest that ovary formation in the genetic females of the medaka fish is not dependent on estrogen.     

Estrogen accumulation in zebra finch song control nuclei: implications for sexual differentiation and adult activation of song behavior

In zebra finches the gonadal steroid estradiol (E2) directs the sexual differentiation of neural regions controlling song and synergizes with androgens to stimulate song in adulthood. To identify regions where E2 may act to exert these effects, steroid autoradiographic techniques were used to assess cellular accumulation of 3[H]-E2 or its metabolites within various nuclei of the zebra finch brain. In Experiment 1 we examined brains from juvenile females, still within the critical period for E2's effect on sexual differentiation. In Experiment 2 the pattern and extent of labeling in adult male brains was determined following injection of 3[H]-E2, 3[H]-testosterone, or 3[H]-dihydrotestosterone. The results suggest that, both during development and in adulthood, most song-control nuclei contain few E2-accumulating cells. In contrast, many cells densely labeled by 3[H]-E2 or its metabolites are present in the hypothalamus and in close proximity to one song-control region, the hyperstriatum ventralis pars caudalis (HVc). The distribution of these latter cells overlaps with cells that project to another song-related nucleus, Area X. Thus, in Experiment 3 fluorescent retrograde tracing and steroid autoradiographic techniques were combined to determine if E2-accumulating cells project to Area X in adult males. Although a few retrogradely labeled cells were lightly labeled by 3[H]-E2 or its metabolites, for the most part these appear to be two distinct populations of cells. The sparse accumulation of E2 in the zebra finch song system contrasts with that described in other song birds and has important implications as to the mechanism of E2 action on the developing and mature song system.     

Enhanced gene expression in the forebrain of hatchling and juvenile male zebra finches

The molecular mechanisms regulating sexual differentiation of the brain are largely unknown, although progress is being made, particularly in some mammalian systems. To uncover more of the key factors, a screen was conducted for genes involved in sexually dimorphic development of the neural song system in zebra finches. cDNA microarrays were initially used to compare gene expression in the telencephalons of hatchling and juvenile males and females. Then, real-time quantitative polymerase chain reaction (PCR) was employed to confirm sex differences, and the brain regions expressing the cDNAs of interest were localized using in situ hybridization. Several genes, including those likely to encode two ribosomal proteins (RPL17 and RPL37), SCAMP1, ZNF216, and a COBW-domain containing protein, showed enhanced expression in the telencephalon of males compared to females. In several cases, expression in the song control nuclei specifically was detected only in males. Interestingly, the sequences of some of these cDNAs shared substantial homology with regions of the chicken Z chromosome (male birds are ZZ, females ZW). Thus, we have identified genes likely to be involved in masculinization of the structure and/or function of the song circuit, some of which could be initial triggers for the sexual differentiation process.     

Independent Differentiation of Sexual and Social Traits

In the process of development in mammals and birds, an individual becomes either male or female. The dogma of sexual differentiation explains this development in terms of gonadal secretions, which direct the growth of an organism along masculine or feminine lines. However, this type of bimodality is not found in all traits that are displayed in reproductive contexts. For example, aggressive behaviors displayed during the breeding season can be expressed by both males and females. Furthermore, this reproductive aggression is highly variable between individuals, regardless of sex. The development of variable traits like reproductive aggression is therefore likely to be influenced by factors in addition to gonadal secretions. We propose that social traits associated with reproduction can differentiate along pathways that are distinct from the developmental pathways of primary sexual traits.     

Circuits,hormones, and learning: Vocal behavior in songbirds

Species-typical vocal patterns subserve species identification and communication for individual organisms. Only a few groups of organisms learn the sounds used for vocal communication, including songbirds, humans, and cetaceans. Vocal learning in songbirds has come to serve as a model system for the study of brain-behavior relationships and neural mechanisms of learning and memory. Songbirds learn specific vocal patterns during a sensitive period of development via a complex assortment of neurobehavioral mechanisms. In many species of songbirds, the production of vocal behavior by adult males is used to defend territories and attract females, and both males and females must perceive vocal patterns and respond to them. In both juveniles and adults, specific types of auditory experience are necessary for initial song learning as well as the maintenance of stable song patterns. External sources of experience such as acoustic cues must be integrated with internal regulatory factors such as hormones, neurotransmitters, and cytokines for vocal patterns to be learned and produced. Thus, vocal behavior in songbirds is a culturally acquired trait that is regulated by multiple intrinsic as well as extrinsic factors. Here, we focus on functional relationships between circuitry and behavior in male songbirds. In that context, we consider in particular the influence of sex hormones on vocal behavior and its underlying circuitry, as well as the regulatory and functional mechanisms suggested by morphologic changes in the neural substrate for song control. We describe new data on the architecture of the song system that suggests strong similarities between the songbird vocal control system and neural circuits for memory, cognition, and use-dependent plasticity in the mammalian brain. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 602–618, 1997     

Estrogenic contributions to sexual differentiation in the female guinea pig: influences of diethylstilbestrol and tamoxifen on neural, behavioral, and ovarian development

We administered the synthetic estrogen, diethylstilbestrol (DES), or the antiestrogen, tamoxifen, to pregnant guinea pigs and observed the consequences for sexual differentiation of their female offspring. Hormones were administered during the period when treatment of fetuses with testosterone influences the development of sex-related traits (approximately Days 30 to 65 of gestation). Ovarian function, masculine and feminine sexual behavior, and the structure of a sexually dimorphic neural region in the preoptic area were assessed in adulthood in hormone-exposed animals and in oil-treated and untreated controls. Prenatal exposure to DES dipropionate (DESDP) caused masculinization and defeminization. DESDP-treated females mounted more than control females, both without hormonal stimulation and when given testosterone propionate (TP) as adults. The sexually dimorphic neural region was also masculinized in these females. In regard to defeminization, they showed delayed vaginal opening, impaired progesterone (P) production, an absence of corpora lutea, and impaired lordosis and mounting responses to estradiol benzoate (EB) and P. Prenatal treatment with tamoxifen produced a complicated pattern of results. Tamoxifen-exposed females evidenced less masculine-typical behavior, showing diminished mounting without hormonal stimulation and in response to TP. However, they also showed delayed vaginal opening, enhanced P production, and impaired mounting in response to EB and P. Their lordosis behavior and the volume of the sexually dimorphic neural region were unaffected. These results suggest that estrogens play a substantial role in sexual differentiation in the guinea pig. High levels of estrogen promote masculine-typical development, and unusually low levels may impair some aspects of both masculine-typical and feminine-typical development.     

Inhibition of adult social behaviour in ducks induced by juvenile administration of gonadal hormones

This experiment was designed to study the long-term behavioural effects of gonadal hormone injections into domestic ducklings. Ten male and four female ducklings were injected daily from their 4th day onwards: the males with testosterone propionate and the females with oestradiol benzoate. Five males were injected for 40 days, the other ducklings (5♀ + 4♂) for 80 days. Behavioural effects of these treatments were assessed when the birds were six months old. Social displays were strongly inhibited in the injected males as compared with control birds but no general significant effect was observed for the aggressive and sexual behaviour. The inhibition of social displays is consistent with the view that these behaviour patterns are regulated by the pituitary-gonadal axis and the different levels at which the functional inhibition could have occured are discussed. Far fewer social displays were performed by the males presented with injected females which, on the other hand, showed less sexual behaviour than the control females. This supports the idea that the female plays an important part in the social display of ducks.     

Activity pattern, reproductive behaviors and gonadal hormones in the raccoon dog (Nyctereutes procyonoides)

The objectives of this study were to (1) assess year-round behaviors and activity patterns of captive raccoon dogs (Nyctereutes procyonoides) and (2) characterize the species' reproductive endocrinology. Behaviors and activity patterns of 12 (5.7) animals were recorded over a 1-year period. During that time, fecal samples were collected 2-7 times/week from 16 (7.9) individuals (six of these were included in the behavioral study) for the analysis of testosterone, progesterone and estrogen metabolite concentrations. Activity pattern and excretion of gonadal steroids followed a seasonal pattern. Specifically, dogs were cathemeral in summer, and primarily nocturnal in winter. In the males, testosterone concentrations were at baseline from April through September, began to rise in October and reached peak concentrations in February (P<0.05). In the females, elevated estrogen (P<0.05) was observed in March followed by an increase in progestagen concentrations from March through May (P<0.05) in both pregnant and pseudopregnant animals. Gender significantly influenced monthly testosterone/estrogen ratio (P<0.01); values were higher in males than in females throughout the year with overall percentage of overlapping values between males and females being 28%. In summary, this study characterized cirannual fluctuations in behaviors and gonadal steroid metabolites in the raccoon dog maintained in captivity. Because there is no obvious sexual dimorphism, the differences in testosterone/estrogen ratio may be useful for gender differentiation (72% accuracy), especially among individuals living in the wild.     

Song post exposure, song features, and predation risk

Male birds use song to attract mates and deter other males,but in doing so, they also attract the attention of predatorsand parasites. Such viability costs are inherent in reliablesignals, potentially causing females to prefer mates that displayfrom the most exposed sites. However, viability costs of sexualsignals may be ameliorated by affecting the choice of microhabitat,which in turn may affect the design of song features that aremost efficiently transmitted in this microhabitat. We estimatedthe exposure of song posts (microsites used by males when singing)used by passerine birds in relation to prey selection by thesparrowhawk Accipiter nisus, by calculating the proportion ofmales that sang from song posts that were at the maximum levelof the vegetation, in an attempt to quantify the costs of sexualselection. We quantified prey susceptibility to predation asthe difference between the log-transformed observed number ofprey minus the log-transformed expected number of prey in theenvironment. This prey susceptibility index increased with increasingsong post exposure similarly in sexually dichromatic and monochromaticspecies, although the prey susceptibility index was relatedto sexual dichromatism. Song post exposure was dependent onhabitat, but comparative models controlling for the potentiallyconfounding effects of habitat, sexual dichromatism, hole nesting,coloniality, body mass, cognitive capacities, and flying abilitiesindicated that the relationship between the prey susceptibilityindex and song post exposure is strong. Path analyses of therelationship between song post exposure, sexual dichromatism,and prey susceptibility index revealed that selection actingon sexual dichromatism and song post exposure has secondaryimpact on prey susceptibility index. The opposite causal mechanismsby which predation affects sexual traits are less likely. Thesemodels suggest that female preference for high song posts ordichromatic plumage increases predation risk on an evolutionarytime scale.     

Human aldosterone synthase: recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level

Recent experiments from our laboratory are consistent with the idea that hypothalamic astrocytes are critical components of the central nervous system (CNS) mediated estrogen positive feedback mechanism. The "astrocrine hypothesis" maintains that ovarian estradiol rapidly increases free cytoplasmic calcium concentrations ([Ca(2+)](i)) that facilitate progesterone synthesis in astrocytes. This hypothalamic neuroprogesterone along with the elevated estrogen from the ovaries allows for the surge release of gonadotropin-releasing hormone (GnRH) that triggers the pituitary luteinizing hormone (LH) surge. A narrow range of estradiol stimulated progesterone production supports an "off-on-off" mechanism regulating the transition from estrogen negative feedback to estrogen positive feedback, and back again. The rapidity of the [Ca(2+)](i) response and progesterone synthesis support a non-genomic, membrane-initiated signaling mechanism. In hypothalamic astrocytes, membrane-associated estrogen receptors (mERs) signal through transactivation of the metabotropic glutamate receptor type 1a (mGluR1a), implying that astrocytic function is influenced by surrounding glutamatergic nerve terminals. Although other putative mERs, such as mERβ, STX-activated mER-Gα(q), and G protein-coupled receptor 30 (GPR30), are present and participate in membrane-mediated signaling, their influence in reproduction is still obscure since female reproduction be it estrogen positive feedback or lordosis behavior requires mERα. The astrocrine hypothesis is also consistent with the well-known sexual dimorphism of estrogen positive feedback. In rodents, only post-pubertal females exhibit this positive feedback. Hypothalamic astrocytes cultured from females, but not males, responded to estradiol by increasing progesterone synthesis. Estrogen autoregulates its own signaling by regulating levels of mERα in the plasma membrane of female astrocytes. In male astrocytes, the estradiol-induced increase in mERα was attenuated, suggesting that membrane-initiated estradiol signaling (MIES) would also be blunted. Indeed, estradiol induced [Ca(2+)](i) release in male astrocytes, but not to levels required to stimulate progesterone synthesis. Investigation of this sexual differentiation was performed using hypothalamic astrocytes from post-pubertal four core genotype (FCG) mice. In this model, genetic sex is uncoupled from gonadal sex. We demonstrated that animals that developed testes (XYM and XXM) lacked estrogen positive feedback, strongly suggesting that the sexual differentiation of progesterone synthesis is driven by the sex steroid environment during early development. This article is part of a Special Issue entitled 'Neurosteroids'.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.     

Aromatase inhibition affects testosterone‐induced masculinization of song and the neural song system in female canaries

Songbirds have a specialized steroid‐sensitive network of brain nuclei, the song system, for controlling song. Most nuclei of the song system express androgen receptors, and the sensory‐motor integration nucleus High Vocal Center (HVC) alone also expresses estrogen receptors. Apart from expressing estrogen receptors in the vocal control system, songbirds are unique among birds because they have high concentrations of the estrogen‐synthesizing enzyme aromatase in the neostriatum surrounding HVC. However, the role of estrogen in controlling the development of the song structure has been scarcely investigated. In this work, we show that blocking the production of estrogen during testosterone‐induced song motor development in adult female canaries alters the song pattern compared to control females treated with testosterone only. These effects were correlated with inhibition of the expression of estrogen‐sensitive genes, such as brain‐derived nerve growth factor, in HVC. The expression of the ATP‐synthase gene, an indicator of cell activity, in HVC, and the size of HVC, were not affected by the treatment. Our results provide the first example of estrogen‐sensitive mechanisms controlling the structural features of adult birdsong. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 370–379, 2003     

Castration and antisteroid treatment impair vocal learning in male zebra finches.

Both song behavior and its neural substrate are hormone sensitive: castrated adult male zebra finches need replacement of gonadal steroids in order to restore normal levels of song production, and sex steroids are necessary to establish male-typical neural song-control circuits during early development. This pattern of results suggests that hormones may be required for normal development of learned song behavior, but evidence that steroids are necessary for normal neural and behavioral development during song learning has been lacking. We addressed this question by attempting to eliminate the effects of gonadal steroids in juvenile male zebra finches between the time of initial song production and adulthood. Males were castrated at 20 days of age and received systemic implants of either an antiandrogen (flutamide), an antiestrogen (tamoxifen), or both drugs. The songs of both flutamide- and tamoxifen-treated birds were extremely disrupted relative to normal controls in terms of the stereotypy and acoustic quality of individual note production, as well as stereotypy of the temporal structure of the song phrase. We did not discern any differences in the pattern of behavioral disruption between birds that were treated with either flutamide, tamoxifen, or a combination of both drugs. Flutamide treatment resulted in a reduced size of two forebrain nuclei that are known to play some role unique to early phases of song learning [lateral magnocellular nucleus of the anterior neostriatum (IMAN) and area X (X)], but did not affect the size of two song-control nuclei that are necessary for normal song production in adult birds [caudal nucleus of the ventral hyperstriatum (HVc) and robust nucleus of the archistriatum (RA)]. In contrast, treatment with tamoxifen did not result in any changes in the size of song-control nuclei relative to normal controls, and it blocked the effects of flutamide on the neural song-control system in birds that were treated with both drugs. Castration and antisteroid treatment exerted no deleterious effects on the quality of song behavior in adult birds, indicating that gonadal hormones are necessary for the development of normal song behavior during a sensitive period.     

Intracranial administration of the G‐protein coupled estrogen receptor 1 antagonist,G‐15, selectively affects dimorphic characteristics of the song system in zebra finches (Taeniopygia guttata)

In zebra finches (Taeniopygia guttata), estradiol contributes to sexual differentiation of the song system but the receptor(s) underlying its action are not exactly known. Whereas mRNA and/or protein for nuclear estrogen receptors ERα and ERβ are minimally expressed, G‐protein coupled estrogen receptor 1 (GPER1) has a much greater distribution within neural song regions and the syrinx. At present, however, it is unclear if this receptor contributes to dimorphic development of the song system. To test this, the specific GPER1 antagonist, G‐15, was intracranially administered to zebra finches for 25 days beginning on the day of hatching. In males, G‐15 significantly decreased nuclear volumes of HVC and Area X. It also decreased the muscle fiber sizes of ventralis and dorsalis in the syrinx. In females, G‐15 had no effect on measures within the brain, but did increase fiber sizes of both muscle groups. In sum, these data suggest that GPER1 can have selective and opposing influences on dimorphisms within the song system, but since not all features were affected additional factors are likely involved. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018