Oshima Yuki, Kato Takuma, Wang Deshou, Murakami Takahiro, Matsuda Yoichi, Nagahama Yoshitaka, Nakamura Masahisa
Department of Biology, Faculty of Education and Integrated Arts and Sciences, Waseda University, Tokyo 169-8050, Japan.
Zoolog Sci. 2006 Jan;23(1):79-85. doi: 10.2108/zsj.23.79.
Sex is determined genetically in amphibians, but is reversed occasionally by steroid hormones. The phenotypic sex of some amphibian species can be reversed from male to female by estrogens. Estrogens, which are synthesized from testosterone irreversibly by the enzyme P450 aromatase (CYP19), are essential for ovarian development in vertebrates. CYP19 expression is reportedly regulated by steroidogenic factor-1 (SF-1), also designated as Ad4BP, in fish and mammals. However, it is unknown if this is also the case in amphibians. Thus, to elucidate the role of SF-1 in CYP19 gene expression in the gonad of amphibians, it is necessary to isolate and characterize the promoter region of the CYP19 gene of amphibians. For this purpose, we first cloned the promoter region of CYP19 from genomic DNA fragments of the frog Rana rugosa. As a result, a potential binding site of SF-1 was found in the region. When a luciferase promoter assay in HEK 293 cells was carried out to examine the ability of SF-1 as a transcriptional regulator, we found that R. rugosa SF-1 stimulated the expression of the CYP19 gene of the tilapia Oreochromis niloticus, but not that of the frogs R. rugosa and Xenopus laevis. RT-PCR analysis revealed that CYP19 mRNA was expressed at a higher level in the indifferent gonads of females than in those of males. This was also true to SF-1 mRNA In addition, FISH analysis showed that the CYP19 gene was located on chromosome 3 of R. rugosa. Taken together, our data suggest that CYP19, an autosomal gene, is expressed in the undifferentiated gonads to an extent greater in females than in males, but its expression probably is not regulated by SF-1 alone. Another factor(s) may be required if SF-1 promotes the expression of the CYP19 gene in R. rugosa as it does in fish and mammals.
两栖动物的性别由基因决定，但偶尔会被类固醇激素逆转。一些两栖动物物种的表型性别可通过雌激素从雄性逆转为雌性。雌激素由睾酮通过P450芳香化酶（CYP19）不可逆地合成，是脊椎动物卵巢发育所必需的。据报道，在鱼类和哺乳动物中，CYP19的表达受类固醇生成因子-1（SF-1，也称为Ad4BP）调控。然而，在两栖动物中情况是否如此尚不清楚。因此，为了阐明SF-1在两栖动物性腺中CYP19基因表达中的作用，有必要分离并鉴定两栖动物CYP19基因的启动子区域。为此，我们首先从皱皮蛙的基因组DNA片段中克隆了CYP19的启动子区域。结果，在该区域发现了一个潜在的SF-1结合位点。当在HEK 293细胞中进行荧光素酶启动子分析以检测SF-1作为转录调节因子的能力时，我们发现皱皮蛙的SF-1刺激了尼罗罗非鱼CYP19基因的表达，但未刺激皱皮蛙和非洲爪蟾的CYP19基因表达。RT-PCR分析显示，CYP19 mRNA在雌性未分化性腺中的表达水平高于雄性。SF-1 mRNA也是如此。此外，荧光原位杂交分析表明，CYP19基因位于皱皮蛙的3号染色体上。综上所述，我们的数据表明，CYP19作为一个常染色体基因，在未分化性腺中的表达在雌性中比在雄性中更高，但它的表达可能并非仅受SF-1调控。如果SF-1像在鱼类和哺乳动物中那样促进皱皮蛙CYP19基因的表达，可能还需要其他因素。
