In mammals and birds, sex differences in brain function and disease are thought to derive exclusively from sex differences in gonadal hormone secretions. For example, testosterone in male mammals acts during fetal and neonatal life to cause masculine neural development. However, male and female brain cells also differ in genetic sex; thus, sex chromosome genes acting within cells could contribute to sex differences in cell function. We analyzed the sexual phenotype of the brain of a rare gynandromorphic finch in which the right half of the brain was genetically male and the left half genetically female. The neural song circuit on the right had a more masculine phenotype than that on the left. Because both halves of the brain were exposed to a common gonadal hormone environment, the lateral differences indicate that the genetic sex of brain cells contributes to the process of sexual differentiation. Because both sides of the song circuit were more masculine than that of females, diffusible factors such as hormones of gonadal or neural origin also likely played a role in sexual differentiation.T heories of sexual differentiation in birds and mammals postulate different mechanisms for sexual differentiation of gonadal and nongonadal tissues (1). Gonadal sex is determined by sex chromosome gene(s). In mammals, the Y-linked SRY gene is expressed within cells in the undifferentiated gonadal ridge to induce testicular development (2). In contrast, sexual differentiation of nongonadal (somatic) tissues, such as the brain, is caused by sex differences in early gonadal hormones (3, 4). A key question is whether somatic sexual differentiation also involves cell-autonomous action of sex chromosome genes as occurs in gonadal differentiation.An exclusively hormonal theory of brain sexual differentiation has been challenged by studies of zebra finches (Taeniopygia guttata). Males, but not females, sing a courtship song. The male's neural song nuclei are much larger and have larger neurons (5). Treatment of hatchling females with estradiol induces more masculine neural development, and they sing (6), suggesting that estrogens derived from gonadal secretions normally induce masculine song system development in males. However, treatments with gonadal hormones do not completely sex-reverse females, and blocking testicular hormones in males does not prevent masculine development (1). Moreover, testicular tissue induced to develop in genetic females does not masculinize the song system (7). One explanation for these results is that sex chromosome genes are expressed differently within brain cells of the two sexes and act in a cell-autonomous fashion to cause differences in song system development (8).The discovery of a rare bilateral gynandromorphic zebra finch presented us with an opportunity to test this hypothesis, because cells on one half of its brain and body were genetically male and cells on the other half were genetically female. Because both halves of the brain developed in the same gonadal hormonal environment, a complete...

The brains of males and females differ, not only in regions specialized for reproduction, but also in other regions (controlling cognition, for example) where sex differences are not necessarily expected. Moreover, males and females are differentially susceptible to neurological and psychiatric disease. What are the origins of these sex differences? Two major sources of sexually dimorphic information could lead to sex differences in brain function. Male and female brain cells carry a different complement of sex chromosome genes and are influenced throughout life by a different mix of gonadal hormones. Until recently all sex differences in the brain have been attributed to the differential action of gonadal hormones. Recent findings, however, suggest that brain cells that differ in their genetic sex are not equivalent, and that difference may contribute to sex differences in brain function. Here we discuss evidence for sex chromosome effects on both neural and nonneural systems, which together provide support for the idea that XX and XY cells differentiate even before they are influenced by gonadal hormones, and even if they are exposed to similar levels of gonadal steroids. Fortunately, new model systems for studying sex chromosome effects have recently been developed, and they should help in testing further the role of sex chromosome genes.

Previous research suggests that sex differences in the nigrostriatal system are created by direct effects of the sex chromosomes (XX vs. XY), independent of the action of gonadal hormones. Here we tested for sex chromosome effects on expression of three mRNAs in the striatum and nucleus accumbens of adult mice of the four core genotypes model (XX and XY gonadal males, XX and XY gonadal females). Mice were gonadectomized (GDX) at 47-51 days old to eliminate group differences in the levels of gonadal steroids. Three weeks later, mice were killed and brains collected for in situ hybridization of the striatum, or the striatum was dissected out for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Expression in XX and XY mice was measured by in situ hybridization using riboprobes encoding the dynorphin precursor Pdyn (prodynorphin), the substance P precursor Tac1 (preprotachykinin) or dopamine D2 receptor. XX mice had higher expression, relative to XY mice of the same gonadal sex, of Pdyn and Tac1 mRNA in specific striatal regions. Quantitative PCR confirmed that GDX XX mice have higher Pdyn expression in striatum than XY mice, regardless of their gonadal sex. XX had higher Pdyn expression than XY or XO mice, indicating that the sex chromosome effect is the result of XX vs. XY differences in the number of X chromosomes, probably because of sex differences in the expression of X gene(s) that escape inactivation. We detected no sex chromosome effect on D2 receptor mRNA.

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To assess which hormones are capable of masculinizing the neural song system of zebra finch hatchlings, we implanted female hatchlings with estrogen (estradiol [E2], 75 micrograms, n = 9), testosterone (T, 75-88 micrograms, n = 13), androstenedione (AE, 75 micrograms, n = 7), progesterone (P, 117 micrograms, n = 10), or nothing (Blanks, n = 10) and compared these to unimplanted males (n = 7). Implants, consisting of a hormone and Silastic mixture encased in polyethylene tubing, were placed under the skin of the breast on the day of hatching. Birds were killed when they were sub-adult (58 to 68 days old). We measured volumes of area X, the higher vocal center (HVC), and the robust nucleus of the archistriatum (RA); measured soma sizes in the lateral magnocellular nucleus of the neostriatum (lMAN), HVC, and RA; and counted RA neurons. E2 masculinized all measures in the song system and nearly sex-reversed the size of RA neurons. T masculinized volumes of nuclei and soma sizes but not the number or spacing of RA neurons. E2 was always at least as effective as T in masculinizing measures of the song system and was usually more effective. AE and P did not significantly masculinize any measure. These data suggest that E2 is more potent than aromatizable androgens or P in masculinizing the female song system in development and that the action of E2 alone may be sufficient to masculinize the volume of song control nuclei and the size and number of neurons.

Previous studies have suggested that both major active metabolites of testosterone, estradiol (E2) and dihydrotestosterone (DHT), are needed for complete masculinization of the brain regions that control song in passerine birds. However, DHT treatment of hatchling female zebra finches has only small masculinizing effects on the song system. To assess whether E2 and DHT have a synergistic effect on the masculinization of the zebra finch song system, female zebra finches were given Silastic implants of E2 on the day of hatching (day 1) either without any additional hormone treatment or in combination with DHT on days 1, 14, or 70. At 105 to 110 days of age, we measured the volumes of Area X, higher vocal center (HVC), robust nucleus of the archistriatum (RA), soma sizes in HVC, RA, and the lateral magnocellular nucleus of the neostriatum (IMAN), and neuron density and number in RA. E2 masculinized all of the measures in the song system with the exception of the number of neurons in RA. DHT did not synergize with E2 to produce any additional masculinization of the attributes measured. These data demonstrate that the combination of E2 and DHT did not result in the complete masculinization of the song control nuclei and argue against the importance of androgen in sexual differentiation of the song system.

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Song behavior and the neural song system that serves it are sexually dimorphic in zebra finches. In this species, males sing and females normally do not. The sex differences in the song system include sex differences in the proportion of neurons that express androgen receptors, which is higher in specific brain regions of males. Estradiol (E2) administered in early development profoundly masculinizes the song system of females, including the proportion of neurons expressing androgen receptors. We examined whether or not the expression of these androgen receptors was causally related to the E2-induced masculinization of this system by co-administering Flutamide, which blocks androgen action at the receptor, along with E2 at hatching. E2 alone had its usual masculinizing effect on the female song system, measured in adulthood: increasing the size of song nuclei, the size of neurons in HVC, RA, and 1MAN, and the number of neurons in HVC. E2's masculinizing action, however, was significantly diminished on all measures by co-administering Flutamide. Indeed, females receiving both E2 and Flutamide were never significantly more masculine than controls on any measure. Flutamide alone had no effect. Our results strongly suggest that the activation of androgen receptors is necessary for the E2-induced masculinization of the song system in females.