Expression and localization of cytochrome P450_11β,aldo mRNA in the frog brain

The present study is focused on biosynthesis of adrenal steroids in the frog brain. Employing RT-PCR method using total RNA from the adult Rana nigromaculata brain, we isolated a 419-bp fragment of cDNA encoding cytochrome P450_11β,aldo, which catalyzes the final step of biosynthesis of the frog adrenal steroids, corticosterone and aldosterone. The deduced amino acid sequence of R. nigromaculata brain cytochrome P450_11β,aldo shared a high homology (88.8%) with that of R. catesbeiana adrenal cytochrome P450_11β,aldo. Southern blot analysis of the RT-PCR product confirmed the P450_11β,aldo transcription in the frog brain without a clear-cut sex difference. Then, we analyzed the P450_11β,aldo mRNA expression in different brain regions of the adult frog by RT-PCR method. The P450_11β,aldo gene was transcribed in the telencephalon, diencephalon, midbrain, and cerebellum. The transcript level of the frog β-actin gene was relatively constant in all the frog samples examined. In situ hybridization analysis showed that the P450_11β,aldo gene was transcribed abundantly in the cells throughout the frog brain, such as the pallium mediale in the telencephalon, the nucleus preopticus in the diencephalon, the stratum griseum superficiale tecti in the midbrain, and Purkinje cells in the cerebellum. These results taken together suggest that the frog brain synthesizes adrenal steroids, such as corticosterone and aldosterone.