Expression and Activity of 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4-Isomerase in the Rat Purkinje Neuron during Neonatal Life.

Recently, we demonstrated that cytochrome P450 side-chain cleavage enzyme (P450scc) occurs in the rat cerebellar Purkinje cell after differentiation and remains during neonatal development and into adulthood. 3b-Hydroxysteroid dehydrogenase/D-D-isomerase (3bHSD) is also an essential enzyme for progesterone biosynthesis not only in peripheral steroidogenic glands but also in the nervous system. In the present study, therefore, the expression of 3bHSD in the rat cerebellum was investigated during neonatal development and in the adult. RT-PCR analysis showed that the expression of 3bHSD messenger RNA (mRNA) in the cerebellum was higher at 7–14 days of age than at other times. Biochemical studies together with HPLC analysis revealed that cerebellar slices at 10 days of age converted pregnenolone to progesterone, suggesting enzymatic activity of 3bHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3bHSD. A specific RIA indicated that progesterone concentrations in the cerebellum were higher at 3 and 10 days of age than at 60 days of age. The progesterone level in the cerebellum was significantly higher than that in plasma at 10 days of age. In contrast, the concentrations in both cerebellum and plasma at 3 and 60 days of age were similar. In the present study, the site of 3bHSD mRNA expression in the cerebellum was further examined in neonatal and adult rats using in situ hybridization. The cerebellar expression of 3bHSD mRNA was obscure at 3 days of age, whereas intense expression occurred in Purkinje cells and external granule cells throughout the cerebellum at 10 days of age. 3bHSD mRNA was also expressed in Purkinje cells and granule cells at 60 days of age, but a restricted expression was observed along the cerebellar meninges. These results suggest that the steroidogenic enzyme 3bHSD as well as P450scc are expressed at least in the cerebellar Purkinje cell. The expression of 3bHSD, however, may increase for a limited period around 10 days of age, unlike P450scc. (Endocrinology 140: 805–813, 1999) N such as pregnenolone and dehydroepiandrosterone are synthesized de novo in the nervous system through mechanisms partly independent of peripheral steroidogenic glands, e.g. the adrenal and gonads (for a review, see Ref. 1). A number of studies in several species of mammals and birds have reported that the brain contains abundant quantities of pregnenolone, dehydroepiandrosterone, and their fatty acid or sulfate esters (2–11). It has also been demonstrated that certain structures in the brain possess the cytochrome P450 side-chain cleavage enzyme (P450scc), which cleaves cholesterol to form pregnenolone (12–19). In addition, progesterone and its metabolites, such as 3a-dehydroxyprogesterone and 3a,5atetrahydroxyprogesterone, are produced and accumulate in the nervous system as neurosteroids (20–26). Many investigators have reported that progesterone and its metabolites act through ion-gated channel receptors, such as g-aminobutyric acidA and glycine, to modulate interneuronal communication and excitability as well as through nuclear steroid receptors (27–36). Furthermore, progesterone has been shown to be involved in myelination of the peripheral ner-