Steroids have profound effects on the brain. In the zebra finch (Taeniopygia guttata), regulation of the estrogenic steroid estradiol (E2) within the zebra finch brain may have functional consequences for song behavior. In zebra finches, males sing but females do not. Males also have a larger and more interconnected set of brain areas, or nuclei, specialized to learn and produce song than females . Early in development, E2 masculinizes the song circuit required for song production and in adulthood, the function of song areas can be altered by changes in E2 after song experience [2–5].
The gonads do not supply the steroids that direct masculinization of the song circuit, but the brain may be an essential source of E2 both during development and in adulthood [6–24]. It is still unclear, however, how steroids produced in the brain act on the song system [17, 24–28]. Therefore, two non-exclusive theories have been proposed to explain the origin of sex differences in this system: sex differences are caused by local differences in steroid synthesis in or near the song control circuit [6, 7, 11–13, 17], and sex differences are caused by the differential effect of genes localized to sex chromosomes [29, 30].
Evidence exists to support both theories. Many of the major steroid -producing and -transducing factors that can act on the song circuit are present in the zebra finch brain [6, 7, 10, 11, 18, 19, 31–37]. For example, early developmental E2 administration to females acts in the brain to induce a masculine song circuit and the closer the source of steroids is to a song nucleus the more potent its effect, cultured brain slices produce E2 and manipulations of E2 levels and estrogen receptor action in these slices prevent full masculinization of song circuitry, and the E2-synthetic enzyme aromatase is present in presynaptic terminals within major song nuclei; in fact, in adult male zebra finches, the brain is the major source of E2 [2, 8, 9, 15, 16, 38–42]. On the other hand, in vivo manipulations of E2 synthesis and action in males does not consistently significantly de-masculinize the song system, the song circuit in genetic females is not masculinized by the presence of functional steroid-secreting testes, and the song nuclei within the male hemisphere of a naturally-occurring gynandromorph (one half genetically male, one half genetically female) are larger than those in the genetically female hemisphere [22, 25, 28, 42–46]. Although in some cases, these findings are partially consistent with the neural steroid synthesis hypothesis, they primarily suggest that genetic differences between males and females contribute to the masculine development of the song system.
Indeed, in mammals and in zebra finches, data is accumulating that male brain cells are fundamentally different than female cells by virtue of their different complement of sex chromosomes: XX/XY in mammalian females and males, and ZW/ZZ in avian females and males, respectively. There is evidence that genes on the sex chromosomes might influence sexual differentiation of the zebra finch song system [25, 30, 47–50]. The potential for sex chromosome genes to display sex differences in expression levels is especially high in the zebra finch because sex chromosome dosage compensation is largely ineffective in birds at the mRNA level [51–53].
Here, we examine 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), a steroidogenic enzyme that converts E2 to a less potent estrogen, estrone (E1). Inasmuch as HSD17B4 is present and active in particular brain regions, it could regulate the local concentrations of E2. This could impact the organization and function of the song circuit [12, 13, 24, 50, 54–56]. Previous experiments have shown evidence that HSD17B4 is localized to the Z chromosome and has a male-biased sex difference in brain expression levels across development [50, 57]. HSD17B4 therefore represents a convergence of the steroidal and genetic theories of song system sexual differentiation. All of this also suggests, however, that E2 might be less available to the male brain, a prediction contrary to findings that E2 masculinizes the song system.
We therefore used several techniques to examine HSD17B4 DNA, mRNA, and enzyme activity in male and female zebra finch brains. We used posthatch day 5 (P5) and adult birds because at both ages, E2 meaningfully impacts the song system. P5 is within the period when steroid administration has the greatest masculinizing effect on song nuclei, other steroid-synthesizing genes are expressed in the brain at this early age, and it is just prior to the earliest emergence of identifiable song control nuclei [6, 11, 38, 58, 59]. Thus expression of HSD17B4 at this early age could alter the local steroid environment that contributes to the initial organization of song nuclei. In adulthood, measures in song nuclei show the potential for rapid and synaptic regulation of estradiol synthesis, which could have functional consequences for song production and perception [2–5]. Our results show that HSD17B4 is sexually dimorphic and could impact both developing and mature song nuclei, consistent with a previous report, and that dosage compensation of HSD17B4 gene may act at a post-transcriptional level . It will require further study to understand how these dosage compensation mechanisms affect neural steroid production, and therefore organization and function of the sexually dimorphic song control circuit.