Takase M, Ukena K, Yamazaki T, Kominami S, Tsutsui K
Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Higashi-Hiroshima, Japan.
Endocrinology. 1999 Apr;140(4):1936-44. doi: 10.1210/endo.140.4.6641.
To clarify whether the amphibian brain synthesizes de novo neurosteroids, we examined pregnenolone, pregnenolone sulfate ester, and cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc), an enzyme converting cholesterol to pregnenolone, using amphibians. Pregnenolone and its sulfate ester in the brain, gonad, and plasma of Xenopus laevis were measured by a specific pregnenolone RIA. The concentrations of these two steroids in the female brain were significantly larger than those in the ovary and plasma. A similar tendency was evident in the male. In both sexes, pregnenolone and its sulfate ester were concentrated more highly in the cerebellum than in the telencephalon, diencephalon, or midbrain. An immunoreactive protein band of electrophoretic mobility in the proximity of bovine adrenal P450scc was detected in the Xenopus brain as well as the testis by Western blot analysis. Immunohistochemical analysis indicated that Purkinje cells in the Xenopus cerebellum were specifically immunostained with the P450scc antibody. P450scc-like immunoreactive cells were further found in several telencephalic and diencephalic regions, such as the pallium mediale and nucleus preopticus, in the Xenopus brain. A similar localization of P450scc-like immunoreactive cells was evident in Rana nigromaculata, a seasonally breeding amphibian. In the present study, seasonal changes in pregnenolone and its sulfate ester were further examined as a possible physiological change using R. nigromaculata. In both sexes, pregnenolone concentrations in the brain were almost constant during the seasonally breeding cycle. In contrast, the pregnenolone sulfate concentration in the brain was significantly lower in the hibernating quiescent phase and higher in the breeding and postbreeding active phases, independent of the plasma steroid level. These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.
为了阐明两栖动物的大脑是否能从头合成神经甾体，我们以两栖动物为研究对象，检测了孕烯醇酮、硫酸孕烯醇酮酯以及细胞色素P450侧链裂解酶（细胞色素P450scc，一种将胆固醇转化为孕烯醇酮的酶）。利用特异性孕烯醇酮放射免疫分析法测定了非洲爪蟾大脑、性腺和血浆中的孕烯醇酮及其硫酸酯。雌性大脑中这两种甾体的浓度显著高于卵巢和血浆中的浓度。雄性也有类似趋势。在两性中，孕烯醇酮及其硫酸酯在小脑中的浓度都比端脑、间脑或中脑中的浓度更高。通过蛋白质免疫印迹分析在非洲爪蟾的大脑以及睾丸中检测到了一条电泳迁移率与牛肾上腺细胞色素P450scc相近的免疫反应性蛋白条带。免疫组织化学分析表明，非洲爪蟾小脑中的浦肯野细胞被细胞色素P450scc抗体特异性免疫染色。在非洲爪蟾大脑的几个端脑和间脑区域，如内侧皮质和视前核中还进一步发现了细胞色素P450scc样免疫反应性细胞。在季节性繁殖的黑斑蛙中也明显存在类似的细胞色素P450scc样免疫反应性细胞定位。在本研究中，还以黑斑蛙为对象进一步检测了孕烯醇酮及其硫酸酯的季节性变化，将其作为一种可能的生理变化。在两性中，大脑中孕烯醇酮的浓度在季节性繁殖周期中几乎保持恒定。相比之下，大脑中硫酸孕烯醇酮的浓度在冬眠静止期显著降低，而在繁殖期和繁殖后期活跃期则升高，且与血浆甾体水平无关。综合这些结果表明，两栖动物的大脑拥有甾体生成酶细胞色素P450scc，并能产生孕烯醇酮及其硫酸酯。硫酸孕烯醇酮可能在季节性繁殖动物一年中的繁殖期和繁殖后期活跃期发挥重要作用。
