IMMUNOCYTOCHEMICAL LOCALIZATION AND BIOLOGICAL-ACTIVITY OF 3-BETA-HYDROXYSTEROID DEHYDROGENASE IN THE CENTRAL-NERVOUS-SYSTEM OF THE FROG

The enzyme 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isome rase (3 beta-HSD) catalyzes biosynthesis of progesterone (P) and all p recursors of glucocorticoids, mineralocorticoids, androgens, and estro gens. Despite the broad interest raised by neurosteroids, the cellular localization of 3 beta-HSD has never been investigated in the brain. We took advantage of the availability of an antiserum raised against h uman placental 3 beta-HSD to determine the distribution of 3 beta-HSD- immonoreactive structures in the brain of the frog pans ridibunda by t he indirect immunofluorescence technique. Three populations of 3 beta- HSD-immunoreactive cell bodies were observed in the hypothalamus, name ly, in the rostral region of the preoptic nucleus, the dorsal infundib ular nucleus, and the dorsal part of the ventral infundibular nucleus. A dense network of 3 beta-HSD-immunoreactive nerve fibers was visuali zed in the dorsal area of the diencephalon, that is, in the lateral ne uropil, the corpus geniculatus lateralis, and the nucleus posterolater alis thalami. Reversed-phase HPLC analysis of frog hypothalamic extrac ts combined with RIA detection showed the presence of substantial amou nts of immunoreactive steroids coeluting with P and 17-hydroxy-progest erone (17 OH-P). The synthesis of Delta(4)-3-keto-steroids in the frog hypothalamus was investigated using the pulse-chase technique with H- 3-pregnenolone (H-3-Delta(5)P) as a precursor. The formation of five t ritiated metabolites of H-3-Delta(5)P was observed, one of which coelu ted with 17OH-P. Conversion of H-3-Delta(5)P into this radioactive met abolite was significantly reduced by trilostane, a specific inhibitor of 3 beta-HSD. Immunodetection of newly synthesized steroids in HPLC f ractions of hypothalamic extracts, using 17OH-P antibodies, revealed t he existence of an immunoreactive steroid that exhibited the same rete ntion time as synthetic 17OH-P. The present study provides the first i mmunocytochemical mapping of 3 beta-HSD, a key enzyme of the steroid b iosynthetic pathway, in the CNS of a vertebrate. The data also demonst rate for the first time biosynthesis of neurosteroids in the brain of a nonmammalian vertebrate.