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Journal of Neuroscience, Vol 14, 7306-7318, Copyright © 1994 by Society for Neuroscience
Immunocytochemical localization and biological activity of 3 beta- hydroxysteroid dehydrogenase in the central nervous system of the frog
AG Mensah-Nyagan, M Feuilloley, E Dupont, JL Do-Rego, F Leboulenger, G Pelletier and H Vaudry
European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, University of Rouen, Mont-Saint-Aignan, France.
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.
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