Sexually dimorphic SCAMP1 expression in the forebrain motor pathway for song production of juvenile zebra finches

Abstract

Mechanisms regulating sexual differentiation of the zebra finch song system are not well understood. The present study was designed to more fully characterize secretory carrier membrane protein 1 (SCAMP1), which was identified in a cDNA microarray screen as showing increased expression in the forebrains of developing male compared with female zebra finches. We completed the sequence of the open reading frame and used in situ hybridization to compare mRNA in song control regions of juvenile (25-day-old) individuals. Expression was significantly greater in the HVC (used as a proper name) and robust nucleus of the arcopallium (RA) in males than in females. Immunohistochemistry revealed that SCAMP1 protein is also expressed in these two brain regions, and qualitatively appears greater in males. Western analysis confirmed that the protein is increased in the telencephalon of males when compared with females at 25 days of age. These results are consistent with the idea that SCAMP1 is involved in masculinization of these brain areas, perhaps facilitating the survival of cells within them.

Figure 1

Comparison of predicted amino acids in three mammalian species and chicken to the zebra finch. Those that differ from the zebra finch are indicated with lower case letters. The available sequence for the chicken does not cover the entire protein coding region.

Figure 2

SCAMP1 mRNA in the forebrain of 25-day-old zebra finches. Analyses of the percent area covered by silver grains were conducted for regions in which expression had been previously detected (see text for details), including the lateral magnocellular nucleus of the anterior nidopallium (lMAN), the portion of the medial striatum that in males contains Area X, HVC (used as a proper name), and the robust nucleus of the arcopallium (RA). As a control, silver grains were also assessed in a region of the arcopallium (A) outside of RA. Overall, expression was increased in males compared with females, and differed among brain regions only in males. * indicates significant difference between the sexes within brain area.

Figure 3

SCAMP1 mRNA in the song control nuclei HVC and RA. Darkfield images from each brain region and some surrounding tissue are depicted from representative males and females. In addition to the tissue labeled with the antisense probe, adjacent tissue sections incubated with the sense probe are shown to indicate the level of background. Scale bars: 500 μm for each brain region.

Figure 4

SCAMP1 protein expression in HVC and RA, as detected with immunohistochemistry. Arrows in each of the low magnification images indicate the boarders of the song nucleus. The small box includes a high magnification image of a cell from the male's HVC. Scale bars: 100 μm for the lower magnification photographs of HVC and RA, and 5 μm for the insert.

Figure 5

Quantification of protein expression in the forebrain of males and females from a Western blot. The optical densities of bands representing SCAMP1 and ZENK were obtained for each individual and a ratio was calculated. Values represent the mean + standard error of six birds of each sex; *p = 0.004. Representative bands for each protein are shown below the histogram.