Usui M, Yamazaki T, Kominami S, Tsutsui K
Faculty of Integrated Arts and Sciences, Hiroshima University, Japan.
Brain Res. 1995 Apr 24;678(1-2):10-20. doi: 10.1016/0006-8993(95)00117-9.
We have recently demonstrated that the avian brain as well as the peripheral steroidogenic glands produces pregnenolone, the main precursor of steroid hormones, on the basis of biochemical studies. Therefore, the immunohistochemical and Western immunoblotting analyses with a polyclonal antibody directed against the purified bovine adrenal cytochrome P450scc, which is involved in pregnenolone formation, was undertaken to investigate the localization of a cytochrome P450scc-like substance in the brain of adult male Japanese quails. P450scc-like immunoreactive cells were distributed in several telencephalic, diencephalic and mesencephalic regions. An intense immunoreaction was observed in somata of Purkinje cells and in the dendrites extending through the cerebellar molecular layer. Clusters of immunoreactive cell bodies were also detected in the hyperstriatum accessorium (HA), the ventral portions of both the archistriatum and the corticoid area, the preoptic area (POA), the anterior hypothalamus (AHy) and the dorsolateral thalamus (DL). Preadsorbing the antibody with cytochrome P450scc resulted in a complete absence of P450scc-like immunoreactivity in all of positively stained cells. On Western immunoblotting with the P450scc antibody, a P450scc-like substance was present in homogenates of the several brain regions that contain immunohistochemically stained cells. Immunohistochemical experiments using both antibodies against P450scc and the glial fibrillary acidic protein (GFA), a specific marker of glial cells, indicated that many HA cells contained both P450scc-like and GFA-like immunoreactivities. However, no immunoreactivity for GFA was observed in Purkinje cells and the cells localized in the ventral portion of the corticoid area, despite the presence of P450scc-like immunoreactivity. These results confirm our previous findings of pregnenolone biosynthesis and suggest the presence of a P450scc-like substance in both neuronal and glial cells of the quail brain.
基于生化研究，我们最近证明，鸟类大脑以及外周类固醇生成腺体会产生孕烯醇酮，它是类固醇激素的主要前体。因此，我们使用针对纯化的牛肾上腺细胞色素P450scc（参与孕烯醇酮形成）的多克隆抗体进行免疫组织化学和蛋白质免疫印迹分析，以研究细胞色素P450scc样物质在成年雄性日本鹌鹑大脑中的定位。P450scc样免疫反应性细胞分布在几个端脑、间脑和中脑区域。在浦肯野细胞的胞体以及延伸穿过小脑分子层的树突中观察到强烈的免疫反应。在副新纹状体（HA）、旧纹状体和皮质oid区域的腹侧部分、视前区（POA）、下丘脑前部（AHy）和背外侧丘脑（DL）也检测到免疫反应性细胞体簇。用细胞色素P450scc预先吸附抗体导致所有阳性染色细胞中完全没有P450scc样免疫反应性。在用P450scc抗体进行蛋白质免疫印迹时，在含有免疫组织化学染色细胞的几个脑区的匀浆中存在P450scc样物质。使用针对P450scc和胶质纤维酸性蛋白（GFA，胶质细胞的特异性标志物）的抗体进行的免疫组织化学实验表明，许多HA细胞同时含有P450scc样和GFA样免疫反应性。然而，尽管存在P450scc样免疫反应性，但在浦肯野细胞和位于皮质oid区域腹侧部分的细胞中未观察到GFA的免疫反应性。这些结果证实了我们之前关于孕烯醇酮生物合成的发现，并表明鹌鹑大脑的神经元和胶质细胞中存在P450scc样物质。
