Neurosteroidogenic enzymes and their regulation in the early brain of the protogynous grouper Epinephelus coioides during gonadal sex differentiation

Abstract

The regulatory role of neurosteroids in the early brain during gonadal sex differentiation is unclear. The aim of this study was to investigate the expression and cellular localization of key steroidogenic enzymes in the early brain of the protogynous orange-spotted grouper Epinephelus coioides and the temporal expressions has been correlated with gonadal sex differentiation. In this study, we showed that peak neurosteroidogenesis occurs in the early brain during gonadal sex differentiation. The temporal expressions of key enzymes, cyp11a1 (cytochrome P450 side chain cleavage), hsd3b1 (3β-hydroxysteroid dehydrogenase) and cyp17a1 (cytochrome P450c17) were studied at different developmental ages (from 90- to 150-dah: days after hatching) using quantitative real-time PCR (q-PCR). q-PCR analysis indicated that the transcript expressions of cyp11a1, hsd3b1 and cyp17a1 were increased in the brain around the period of gonadal sex differentiation. Further, in situ hybridization (ISH) analysis showed that cyp11a1, hsd3b1 and cyp17a1 transcripts were widely expressed in several discrete brain regions, especially the intense expression in the forebrain, with an overall similar expression pattern. High density in the cyp19a1b/Cyp19a1b expression was detected in radial glial cells. Thus, the expression of grouper cyp19a1b/Cyp19a1b is restricted to radial glial cells, suggesting estrogens can modulate their activity. Next, by combining Cyp19a1b immunohistochemistry (IHC) with florescence ISH (FISH) of cyp11a1, hsd3b1 and cyp17a1, we showed that sub-cellular localization of cyp11a1, hsd3b1 and cyp17a1 transcripts, in partial, appeared to be in Cyp19a1b radial glial cell soma. Further, exogenous estradiol (E(2)) increased the expression of cyp17a1 and cyp19a1b/Cyp19a1b in the brain of grouper. Consequently, our results illustrated that the locally synthesized E(2) upregulated neurosteroidogenic enzymes in the early brain and suggest a role for these enzymes in the neurogenic process during gonadal sex differentiation.