Sakata Natsuko, Tamori Yoichiro, Wakahara Masami
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Int J Dev Biol. 2005;49(4):417-25. doi: 10.1387/ijdb.041916ns.
Sex differentiation of gonads in amphibians is believed to be controlled genetically, but altered epigenetically or environmentally. When larvae of the salamander Hynobius retardatus were reared at defined temperatures from hatching to metamorphic stages, a high temperature (28 degrees C) induced exclusively female gonads (ovaries), whereas intermediate (20 and 23 degrees C) or lower (16 degrees C) temperatures produced a 1:1 sex ratio of the morphological gonads. The thermosensitive period was determined to be restricted from 15 to 30 days after hatching, just before or when sexual differentiation occurred. Hynobius P450 aromatase (P450arom) cDNA was isolated from adult gonads and the partial nucleotide or deduced amino acid sequences were determined, showing a high level of identity with various vertebrate species. The P450arom gene was expressed predominantly in the adult ovary and brain, weakly in testis, but not in other somatic organs. A typical sexual dimorphism in P450arom expression was detected in normally developing larvae by a quantitative competitive RT-PCR; strong expression in the female gonads but very weak in male gonads. The dimorphism was detected much earlier than the morphological sexual differentiation of the gonads. When larvae were reared at the female-producing temperature (28 degrees C), strong expression was detected in all the temperature-treated larvae, suggesting that P450arom was up-regulated, even in genetic males. Our results confirm the importance of the P450arom regulation in the sexual differentiation of gonads and demonstrate that an up-regulation of P450arom is involved in the process of temperature-sensitive sex reversal in this species.
两栖动物性腺的性别分化被认为受遗传控制，但可通过表观遗传或环境因素改变。当将日本林蛙的幼体从孵化到变态阶段饲养在特定温度下时，高温（28摄氏度）仅诱导产生雌性性腺（卵巢），而中等温度（20和23摄氏度）或低温（16摄氏度）则产生形态性腺1:1的性别比例。确定热敏期为孵化后15至30天，即性分化发生之前或之时。从成年性腺中分离出日本林蛙细胞色素P450芳香化酶（P450arom）的cDNA，并测定了部分核苷酸或推导的氨基酸序列，其与各种脊椎动物物种具有高度同源性。P450arom基因主要在成年卵巢和大脑中表达，在睾丸中表达较弱，而在其他体细胞器官中不表达。通过定量竞争性RT-PCR在正常发育的幼体中检测到P450arom表达存在典型的性别二态性；在雌性性腺中表达强烈，而在雄性性腺中非常弱。这种二态性比性腺的形态学性别分化要早得多被检测到。当幼体在产生雌性的温度（28摄氏度）下饲养时，在所有经该温度处理的幼体中均检测到强烈表达，这表明即使是遗传雄性，P450arom也会上调。我们的结果证实了P450arom调节在性腺性别分化中的重要性，并证明P450arom的上调参与了该物种温度敏感性性别逆转的过程。
