Activity and localization of 3β-hydroxysteroid dehydrogenase/δ⁵-δ⁴-isomerase in the zebrafish central nervous system

Little information is available for neurosteroidogenesis in the central nervous system (CNS) of lower vertebrates. Therefore, in the present study, we examined the enzymatic activity and localization of 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase (3βHSD), a key steroidogenic enzyme, in the CNS of adult male zebrafish to clarify central progesterone biosynthesis. Biochemical studies together with HPLC analysis revealed that the zebrafish brain converted pregnenolone to progesterone, suggesting the enzymatic activity of 3βHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3βHSD. By using Western immunoblotting with the polyclonal antiserum directed against purified bovine adrenal 3βHSD, a 3βHSD-like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3βHSD-like substance in the zebrafish brain and spinal cord. Clusters of immunoreactive cell bodies were localized in the dorsal telencephalic areas (D), central posterior thalamic nucleus (CP), preoptic nuclei (NPO), posterior tuberal nucleus (PTN), paraventricular organ (PVO), and nucleus of medial longitudinal fascicle (NMLF). 3βHSD-like immunoreactivity was also observed in somata of cerebellar Purkinje neurons. A widespread distribution of immunoreactive fibers was found throughout the brain and spinal cord. In addition, positively stained cells were restricted to other organs, such as the pituitary and retina. Preabsorbing the antiserum with purified bovine adrenal microsome resulted in a complete absence of 3βHSD-like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3βHSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3βHSD expression in the fish CNS.