Estrogen and sex-dependent loss of the vocal learning system in female zebra finches

Abstract

Sex hormones alter the organization of the brain during early development and coordinate various behaviors throughout life. In zebra finches, song learning is limited to males, and the associated song learning brain pathway only matures in males and atrophies in females. This atrophy can be reversed by giving females exogenous estrogen during early post-hatch development, but whether normal male song system development requires estrogen is uncertain. For the first time in songbirds, we administered exemestane, a potent third generation estrogen synthesis inhibitor, from the day of hatching until adulthood. We examined the behavior, brain, and transcriptome of individual song nuclei of these pharmacologically manipulated animals. We found that males with long-term exemestane treatment had diminished male-specific plumage, impaired song learning, but retained normal song nuclei sizes and most, but not all, of their specialized transcriptome. Consistent with prior findings, females with long-term estrogen treatment retained a functional song system, and we further observed their song nuclei had specialized gene expression profiles similar, but not identical to males. We also observed that different song nuclei responded to estrogen manipulation differently, with Area X in the striatum being the most altered by estrogen modulation. These findings support the hypothesis that song learning is an ancestral trait in both sexes, which was subsequently suppressed in females of some species, and that estrogen has come to play a critical role in modulating this suppression as well as refinement of song learning.

Neurogenesis and the development of neural sex differences in vocal control regions of songbirds

“…Two young females had a visible Area X, as defined by NeuN‐ir, but in other birds, it was not distinguishable. Area X is typically impossible to discern in female zebra finches, though may be delineated by visualizing patterns of gene expression (Choe et al, 2021). LMAN displayed a different pattern and was measurable in young females but became undetectable by the motor phase (Figure 3(c)).…”

“…Additionally, this early exposure to estradiol significantly increased the amount of androgen‐accumulating cells in females' HVC, LMAN, and Area X (Kim et al, 2004; Nordeen et al, 1986). However, hormone manipulations such as this have never fully masculinized the brains of females and attempts to inhibit song learning and masculinization of the developing song control system in males through early castration (Adkins‐Regan & Ascenzi, 1990; Arnold, 1975) and exposure to aromatase inhibitors or anti‐estrogens (Balthazart et al, 1994; Choe et al, 2021; Mathews & Arnold, 1991; Wade & Arnold, 1994) have proven unsuccessful. Thus, hormones may be an important part of the behavioral and neural sex differentiation, but sex‐linked genes expressed differently in male and female brains contribute to the morphological and behavioral sex differences independent of gonadal hormones (Agate et al, 2003; Tomaszycki et al, 2009; Wade & Arnold, 2004).…”

Sex chromosome gene expression associated with vocal learning following hormonal manipulation in female zebra finches

“…We used RNA-Seq data from a previous study on the effects of E2 manipulation on the song system which was analyzed using a differential expression approach after mapping reads to an older genome lacking the W sex chromosome 26 . We first re-mapped RNAseq reads to the new assembly from the four major song nuclei (HVC, LMAN, RA, and Area X) and their adjacent non-vocal motor surrounds (DN, AN, LAI, and Str respectively; Fig.…”

“…1a ), taken from quiet males and females at 30 days old; treated with either E2 or a vehicle control since hatch ( Fig. 1b ) 26 . The non-vocal surrounds allow us to examine specialized up-or down-regulated gene expression patterns in the song control nuclei 12,13 .…”

“…Amazingly, female zebra finches treated with estrogen or a synthetic analog at an early age do not exhibit song system atrophy and instead form a functional neural circuit with all the anatomical components and connections seen in males [2][3][4]25 . This "masculinized" song system allows estrogen supplemented females to vocally imitate adult male conspecifics, though not with the same accuracy as males 2,4,26 . Interestingly, lesioning female HVC prevents estrogen dependent anatomical "masculinization" of its postsynaptic targets, RA and Area X 27 .…”