Co-localization of sorting nexin 2 and androgen receptor in the song system of juvenile zebra finches

Abstract

Mechanisms regulating sexual differentiation of the zebra finch song system appear to include both genetic and hormonal factors. Sorting Nexin 2 (SNX2), which is involved in trafficking proteins between cellular membranes, and androgen receptor (AR) mRNA are both increased in song control nuclei of juvenile males compared to females. Here, in situ hybridization for SNX2 and immunohistochemistry for AR were used to evaluate these sexual dimorphisms in more detail. Estimates of the total number of HVC cells expressing SNX2 and AR, individually as well as together, were greater in 25-day-old males compared to females. The densities of these types of cells were generally also increased in males compared to females in HVC and Area X (or the equivalent portion of the medial striatum in females). On average, more than half of the AR+ cells co-expressed SNX2 in both brain regions. The potential, therefore, exists for both AR and SNX2 to be involved in masculinization of these two brain regions. One possibility is that they, either separately or in conjunction, enhance the action of trophic factors within the brain.

Figure 1

Double-labeling of SNX (blue) and AR (reddish brown) in the HVC of male (A) and female (B) zebra finches. In A and B, arrows indicate the ventral and lateral borders of the brain region. Panel C depicts double-labeling in the male at higher power. Panels D and E show single-labeld in situ hybridization for SNX2 and immunohistochemistry for AR, respectively, from a different male. Note that labeling, especially for SNX2, tended to be lighter in females. Scale bars = 200 μm for the upper and 25 μm for the bottom panels.

Figure 2

Stereologic analysis of AR-, SNX2- and AR+SNX2-labeled cell numbers in HVC. Estimates of these total cell numbers were all greater in males than females (***p < 0.0001).

Figure 3

Densities of AR-, SNX2- and AR+SNX2-labeled cells in HVC (A) and Area X (B). *p=0.004, **p=0.002, ***p<0.0001.

Figure 4

Photomicrographs of representative sections of the medial striatum of male (A) and female (B) juvenile zebra finches. The inset depicts a small portion from the male image in more detail; it includes a few double-labeled cells, as well as some single-labeled for AR (nuclei). Area X, which is ventromedial to the lamina pallio-subpallialis (LPS), is visible in the male, but cannot be detected from background in the female. While small numbers of positive cells were present in the medial striatum of females, as in HVC the labeling generally appeared lighter than in males. Scale bar = 200μm.