Regional expression of tyrosinase in central catecholaminergic systems of colored mice

Abstract

A relationship between coat color and behavioral characteristics has been reported for numerous species. We previously indicated that particular behavioral traits contributing to the genotype at the agouti locus manifest only when possessing a wild-type allele at the albino (i.e., tyrosinase: Tyr) locus. The present study was performed to investigate tyrosinase expression with marked activity in central nervous systems. The whole brain of male B10 and B10-c mice, a B10 congenic strain of the albino locus from BALB/c, at 8 to 9 weeks of age was removed, and obtained several regions of brain, especially catecholaminergic. Comparatively large amounts of Tyr mRNA and its translation products of approximately 68 kDa were found in the regions obtained, and definitely possessed the enzyme activity for the oxidation of L-tyrosine. The present results indicate the possibility that the amount of catecholamines produced in albino mice is higher than that of colored mice due to the deficit in tyrosinase heritably.

Keywords: albino; central nervous system; striatum; tyrosinase.

Fig. 1.

(A) Diagram showing the tyrosinase gene (Tyr) locus, and annealing sites of primers for PCR-amplification of tyrosinase mRNA. Each primer pair was designed in the exons stepped over an exon to check for the presence of splice variants. (B) The expression of Tyr mRNA in specific brain regions of B10 mice. PCR products indicated by white arrows (a, b, c, and d) were sequenced to confirm the specific amplification by each primer pair (See DNA sequencing data in Fig. 2). M: DNA size standards with 100-1,000 bp in 100-bp increments. FC: frontal cortex, Str: striatum, Hip: hippocampus, SN: substantia nigra, Hypo: hypothalamus, LC: locus ceruleus, Non: non-template control.

Fig. 2.

Nucleotide sequences of the PCR products amplified by primers for the mouse tyrosinase gene (a–d in Fig. 1).

Fig. 3.

RT-qPCR analysis of Tyr mRNA from specific brain regions of B10 mice. Non-brain areas with biosynthesis pathways leading to the amino acid tyrosine were also examined regarding the expression and compared with the brain. Data are expressed as the mean ± SEM. Different lowercase letters indicate a significant difference between brain regions (n=3, P<0.05). AG: adrenal gland, TG: thyroid gland, FC: frontal cortex, Str: striatum, Hip: hippocampus, SN: substantia nigra, Hypo: hypothalamus, LC: locus ceruleus.

Fig. 4.

Western blot analysis of tyrosinase protein from specific brain regions of B10 mice. A single band of tyrosinase (A: approximately 68 kDa) and GAPDH (B) proteins were detected in each case by ImageJ software. Data analyzed are expressed as the mean ± SEM. Different lowercase letters indicate a significant difference between brain regions (n=3, P<0.05). FC: frontal cortex, Str: striatum, Hip: hippocampus, SN: substantia nigra, Hypo: hypothalamus, LC: locus ceruleus.

Fig. 5.

DOPA (3,4-dihydroxyphenylalanine) reactions in specimens of the striatum from B10 (A) and B10-c (B) mice (Bar, 100 µm), and Immunohistochemical staining with anti-tyrosinase antibody after DOPA reaction in the striatum from B10 mice (C: DOPA reaction, D: anti-tyrosinase antibody, E: Merged C and D). Arrowheads indicate a typical cell with colocalization of melanin pigments produced by the DOPA reaction and tyrosinase protein (Bar, 50 µm).