Activity and localization of 3β‐hydroxysteroid dehydrogenase/ Δ⁵‐Δ⁴‐isomerase in the zebrafish central nervous system

Abstract

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 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3betaHSD), 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 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. By using Western immunoblotting with the polyclonal antiserum directed against purified bovine adrenal 3betaHSD, a 3betaHSD-like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3betaHSD-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). 3betaHSD-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 3betaHSD-like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3betaHSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3betaHSD expression in the fish CNS.

Biosynthesis and Biological Actions of Neurosteroids in Brain Neurons

“…Interestingly, in vitro studies using cultured cerebellar slices of newborn rats showed that progesterone promotes dendritic growth of the Purkinje neuron (Sakamoto et al, 2001b). A similar result was obtained by in vivo studies.…”

“…A similar result was obtained by in vivo studies. Electron microscopic analysis further revealed that progesterone induces an increase of the density of synapses on the Purkinje neuron (Sakamoto et al, 2001b). Furthermore, intranuclear receptors for progesterone were expressed in the Purkinje neuron (Sakamoto et al, 2001b).…”

“…Ukena and Tsutsui (2001) further demonstrated that the embryonic avian brain actively metabolizes progesterone to 5ß-dihydroprogesterone. In addition to these nonmammalian vertebrates, the expression of 3β-HSD and progesterone formation were also obtained in the brain of zebrafish (Sakamoto et al, 2001a). Thus, it is now established that de novo steroidogenesis from cholesterol occurs in vertebrate brains .…”

“…In contrast to mammalian studies, little has been known regarding de novo steroidogenesis in the brain of nonmammalian vertebrates. We therefore looked for steroids formed from cholesterol in the brain of birds Usui et al, 1995;Tsutsui et al, 1997aTsutsui et al, , 1997bUkena et al, 1999bUkena et al, , 2001Matsunaga et al, 2001;Tsutsui and Schlinger, 2001), amphibians and fish (Sakamoto et al, 2001a). Independently, other groups, such as Vaudry's laboratory (Mensah-Nyagan et al, 1994) and Schlinger's laboratory (Vanson et al, 1996), also contributed to this area.…”

Mode of Action and Functional Significance of Estrogen-Inducing Dendritic Growth, Spinogenesis, and Synaptogenesis in the Developing Purkinje Cell

“…The Purkinje cell is a major site for the formation of neurosteroids including estradiol in rats and other vertebrates (Usui et al, 1995;Ukena et al, 1998Ukena et al, , 1999Takase et al, 1999;Matsunaga et al, 2001;Sakamoto et al, 2001bSakamoto et al, , 2003aMatsunaga et al, 2004). We found previously that estradiol promotes dendritic growth of Purkinje cells in newborn rats and show now that this result generalizes to newborn mice.…”