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| This article will appear in a forthcoming issue of Trends in Neurosciences. | |
Abstract
A core belief of vertebrate behavioral neuroendocrinologists is that gonadal hormones stimulate sexually dimorphic growth of neural circuits. The complete hypothesis for birds and mammals states that sex chromosomal genes dictate gonadal differentiation and gonad specific hormones induce sex specific brain organization. In recent years, this principle has been challenged with evidence suggesting that some sexually dimorphic brain development occurs autonomously, independent of peripheral signals. Strong evidence supporting the latter hypothesis has been lacking, until now.
Carl Holloway, working with David Clayton, reported recently in Nat. Neurosci. [1] that a sexually dimorphic connection within the neural song system of songbirds might develop in males but not in females because the functional signalling molecule estradiol (E2) is synthesized de novo from cholesterol in greater amounts in the male brain compared with the female brain. The paper is worthy of our attention as the results bear profoundly on our ideas about the very basic mechanisms underlying brain sexual differentiation and the functional capacity for steroid synthesis in the brain.
The neural song system of passerine songbirds can be a striking and extensive sexually dimorphic circuit, especially in zebra finches in which adult males, but not females, sing. It has been the subject of considerable investigation into the hormonal and cellular events associated with differential neural growth. Surprisingly, in spite of much research, the mechanisms underlying sexually dimorphic development of the neural song system remain in dispute [2,3]. Treatment of females with E2 during a crucial, posthatching, developmental period induces growth of a substantially masculine song system and the capacity to sing as adults. The masculinizing effects of E2 are generally undisputed; they lead to the hypothesis that the song system in males develops naturally under the influence of estrogen. Because gonadal secretions are considered the determinants of brain sexual differentiation, testicular steroidogenesis presumably exceeds that of the ovaries during a crucial developmental period. The neurally active estrogens could be produced in the testes or in the brain from testicular androgens.
Paradoxically, neither experimental manipulations of the hormonal environment in males (blocking sex steroid synthesis or action) nor most measures of endocrine physiology in males and females (plasma hormone levels, expression or activity of steroidogenic enzymes) support the conclusion that males make more estrogen than females to induce a masculine song system [2,3]. Importantly, genetic females induced to develop with functional testes retain a feminine song system [4].
Sexual-differentiation in a Slice
To investigate this problem further, Holloway and Clayton developed an in vitro slice-culture preparation of the developing zebra finch brain. Using this preparation, they investigated the development of a major connection between two crucial telencephalic song control nuclei, HVC (high vocal center) and RA (robust nucleus of the archistriatum). In vivo, in males, but not females, axons arising in HVC innervate nucleus RA ∼30 days after hatching [5]. To label this developing connection in vitro, the authors placed crystals of DiI directly into the HVC of slices from 25-day-old males and females. Slices were survived for three weeks and were cultured alone or co-cultured (male or female slices with female slices). Slices were treated with E2, the estrogen receptor antagonist tamoxifen, or the estrogen synthesis inhibitor fadrozole. They were careful to monitor or remove steroids or estrogenic compounds from the media. They measured E2 levels in media removed from the slices at various times. Regardless of treatment or measure, all of their results led to the conclusion that male slices synthesized more E2 de novo compared with the female slices, and that this E2 caused the formation of the male HVC-RA connection.
Their results raise two fundamental questions. First, can we conclude that E2 is synthesized naturally at higher levels in the brains of developing males compared with developing females in vivo? Second, if more E2 is made in the male brain, can we conclude that masculine differentiation of this neural circuit occurs autonomously? In our view, the answer to the first question is probably yes, to the second question, perhaps.
Neurosteroidogenesis
The idea that the male brain might synthesize more estrogen de novo than the female brain explains a great deal and is compatible with previous research. First, even though E2 is considered the functional masculinizing molecule, the pattern of steroidogenic enzyme expression in peripheral tissues does not support a mechanism for greater production of androgens or E2 in post-hatching males [6]. Direct brain synthesis resolves this paradox. Second, there have been concerns that the concentration of exogenous E2 required to masculinize the brains of females is higher than would be expected to occur systemically (see, for example, reference 7). High E2 levels, however, could be achieved by local neural synthesis. Finally, developmentally, more E2 can be found in the jugular blood of males compared with females [8], but not when birds are bled elsewhere [9,10]. The estrogen-synthetic enzyme aromatase is expressed in high amounts in the young songbird telencephalon [11], and estrogens made in brain can be released into the jugular when androgenic substrate is provided exogenously [12]. If androgen is directly synthesized in the brain, then jugular estrogens would be prominent, especially in males. The overall conclusion is that the steroidogenic enzymes required to synthesize estrogen from cholesterol are present in the zebra finch brain with higher expression levels of these enzymes in males.
It is not unreasonable to expect complete synthesis of active sex steroids in the brain. A substantial body of research indicates that steroids can be synthesized de novo in the brain. All five enzymes required to catalyze reactions that lead from cholesterol to E2 have been detected in the vertebrate brain [13,14]. Nevertheless, linking neural expression of these enzymes with a conspicuous neural function has been elusive. Holloway and Clayton have provided us with the first demonstration of neurosteroidogenesis that is associated with the formation of a prominent and behaviorally important neural circuit. Aromatase, the enzyme that converts androgens into estrogens, does not differ between the sexes in the developing zebra finch telencephalon [10,11]. Therefore, we assume that one or more enzymes leading to the synthesis of androgens must be present in greater amounts in the male brain, possibly the androgen-synthetic enzyme CYP17 [15]. A larger role for neurosteroids and their synthetic enzymes in vertebrate in neural development now requires serious consideration.
There are questions, however, that must be resolved. Are there steroidal substrates or factors in the medium, particularly in males, that induce or intensify steroid synthesis by the slice? Steroidogenesis in vitro might differ from in vivo conditions [16]. Does estrogen synthesis from cholesterol really occur in the brain in vivo? Why are anti-estrogens and aromatase inhibitors largely ineffective at blocking masculine song system development in vivo [3]? Perhaps neurosteroids are synthesized in greater amounts in songbirds than in other species, and these steroids influence hypothalamic-pituitary-gonadal function. In response, songbirds might have evolved compensatory endocrine mechanisms that make in vivo experiments more challenging.
Brain-autonomous Sexual Differentiation
Do these results imply that sexual differentiation of the song system occurs autonomously or do the gonads have a role? Can Holloway and Clayton's data be reconciled with hypotheses that neural expression of sex-chromosomal genes directly controls some sexually dimorphic brain development? First, it is well established that ovarian estrogens act in birds early in development (in ovo in chicks) to demasculinize or feminize neural circuits controlling copulatory behavior [2,17,18]. It is likely that ovarian estrogens have a similar role in songbirds, so male and female zebra finch brains are probably exposed to a different sex steroid environment long before the HVC-RA circuit is formed, perhaps even pre-hatching. Differential gonadal hormone exposure in ovo could account for all neural sex differences observed posthatching [19], including the dimorphic expression of the androgen synthetic enzymes.
Recent studies have documented neural expression of sex chromosomal genes in several species, including songbirds [3,20]. Theoretically, these genes could induce all sexually dimorphic neural development, independent of the gonads. However, steroids could be members of a genetically controlled neural signaling pathway. For example, E2 is known to be a key initiation signal for ovarian differentiation in birds [17] implying that steroidogenic enzymes in the avian genital ridge are controlled by sex-chromosomal gene products. A similar pathway is possibly used by the brain to induce masculine song system development, with E2 again as the functional paracrine signal. How sex-chromosomes might operate developmentally to upregulate this pathway in the female genital ridge and also in the male telencephalon is unknown.
The experiments of Holloway and Clayton are elegant and comprehensive in their design. Their results stimulate a shift in our thinking about the potential impact of steroid synthesis by the brain. They fuel even greater speculation that at least some brain sexual development results from a local signal, in this case a neurosteroid.
