Expression of cytochrome P450 side-chain cleavage enzyme mRNA and production of pregnenolone in the brain of the red-bellied newt Cynops pyrrhogaster

It is becoming clear that the vertebrate brain has the capability of forming steroids de novo, the so-called " neurosteroids" To understand neurosteroidogenesis in the brain, it is essential to demonstrate the formation of pregnenolone, a main precursor of neurosteroids. In amphibians, the pregnenolone formation from cholesterol is still unclear, although the brain accumulates pregnenolone, pregnenolone sulfate and 7α-hydroxypregnenolone. This study was addressed to obtain basic information about pregnenolone formation in the newt brain. Firstly, we demonstrated that the newt brain produces pregnenolone from cholesterol. Subsequently, cDNA encoding cytochrome P450 side-chain cleavage enzyme (P450scc), a key steroidogenic enzyme catalyzing pregnenolone formation, was isolated from the newt. The sequence analysis showed that the isolated P450scc cDNA contained a putative coding region consisting of 1569. bp, which encoded 523 amino acids. The steroid- and heme-binding domains of P450scc were highly shared in amino acids among vertebrates. RT-PCR analysis amplified the authentic fragment corresponding to newt P450scc showed its transcription in the brain. However, the transcription level in the brain was lower than those of the gonad and the kidney including adrenals. The restricted cells in the four major regions of the newt brain, such as the telencephalon, diencephalon, mesencephalon, and rhombencephalon, were demonstrated to express P450scc transcripts by RT-PCR and in situ hybridization. Taken together, these results indicate that the newt brain expresses P450scc mRNA and produces pregnenolone from cholesterol.