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 isomerase (3 beta-HSD) catalyzes biosynthesis of progesterone (P) and all precursors of glucocorticoids, mineralocorticoids, androgens, and estrogens. 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 human placental 3 beta-HSD to determine the distribution of 3 beta-HSD-immunoreactive structures in the brain of the frog Rana ridibunda by the indirect immunofluorescence technique. Three populations of 3 beta-HSD-immunoreactive cell bodies were observed in the hypothalamus, namely, in the rostral region of the preoptic nucleus, the dorsal infundibular nucleus, and the dorsal part of the ventral infundibular nucleus. A dense network of 3 beta-HSD-immunoreactive nerve fibers was visualized in the dorsal area of the diencephalon, that is, in the lateral neuropil, the corpus geniculatus lateralis, and the nucleus posterolateralis thalami. Reversed-phase HPLC analysis of frog hypothalamic extracts combined with RIA detection showed the presence of substantial amounts of immunoreactive steroids coeluting with P and 17-hydroxyprogesterone (17OH-P). The synthesis of delta 4-3-keto-steroids in the frog hypothalamus was investigated using the pulse-chase technique with 3H-pregnenolone (3H-delta 5P) as a precursor. The formation of five tritiated metabolites of 3H-delta 5P was observed, one of which coeluted with 17OH-P. Conversion of 3H-delta 5P into this radioactive metabolite was significantly reduced by trilostane, a specific inhibitor of 3 beta-HSD. Immunodetection of newly synthesized steroids in HPLC fractions of hypothalamic extracts, using 17OH-P antibodies, revealed the existence of an immunoreactive steroid that exhibited the same retention time as synthetic 17OH-P. The present study provides the first immunocytochemical mapping of 3 beta-HSD, a key enzyme of the steroid biosynthetic pathway, in the CNS of a vertebrate. The data also demonstrate for the first time biosynthesis of neurosteroids in the brain of a nonmammalian vertebrate.
Mensah-Nyagan
A G
AG
European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, University of Rouen, Mont-Saint-Aignan, France.
Feuilloley
M
M
Dupont
E
E
Do-Rego
J L
JL
Leboulenger
F
F
Pelletier
G
G
Vaudry
H
H
D019326:17-alpha-Hydroxyprogesterone; D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000818:Animals; D002490:Central Nervous System / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005455:Fluorescent Antibody Technique; D006908:Hydroxyprogesterones / Q000378:metabolism; D008297:Male; D011374:Progesterone / Q000378:metabolism; D011863:Radioimmunoassay; D011895:Rana ridibunda / Q000378:metabolism*; D014018:Tissue Distribution
D006908:Hydroxyprogesterones; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D015096:3-Hydroxysteroid Dehydrogenases