雌激素诱导金钱鱼性分化的作用机制

Previous studies demonstrate that estrogens and cytochrome P450 aromatase (Cyp19a), the key enzyme responsible for estrogen synthesis, have critical roles in not only ovarian, but also testicular differentiation. In addition, sex differentiation of teleost fishes can be induced efficiently by exposing sexually undifferentiated fish to exogenous steroids. However, the mechanism of exogenous steroids on the sex differentiation in fishes is not quite clear. In order to elucidate the mechanism, we propose to select Scatophagus argus, the only species of Scatophagidae in China, as our experimental animal. We propose to study the following issues using histological, physiological and molecular biology techniques: 1. To clone the cDNAs of sex-related genes Cyp19, Dmrt1, Foxl2 and estrogen receptor genes (ERs) of Scatophagus argus, and to examine the histological signs of sex differentiation to determine the sampling points and the correct treatment timing; 2.To measure the growth performance, sex steroids levels (estradiol-17?, E2 and testosterone, T，or 11-ketotestosterone，11-KT) in tissues, as well as Cyp19, Dmrt1, Foxl2 and ER mRNA expression and location of Scatophagus argus during their period of gonadal sex differentiation under natural conditions, in order to find the interrelationships between the above indexes and the sex differentiation; 3. To feed (or immerse) undifferentiated Scatophagus argus with estradiol-17?, antiestrogens Tamoxinfen (Ta), and aromatase inhibitor (AI, Letrogen or Fadrogen), and measure gonadal morphology and sex ratios, as well as the above indexes, in the mean time compare to those under natural conditions, in order to find the key factors causing sex differentiation; 4.To interferer ER (RNAi) in the estradiol-17? treated Scatophagus argus, and check its effects on sex differentiation. The results of these studies could elucidate the mechanism of estrogens on inducing the sex differentiation in Scatophagus argus, and have wide application prospects in aquaculture.

雌激素和芳香化酶基因（Cyp19a）在卵巢和精巢分化中均起重要作用；在性腺分化的关键时期，外源激素可有效诱导鱼类性分化，但迄今其作用机制尚未明了。为此，本项目拟以中国境内金钱鱼科唯一物种金钱鱼为研究对象，采用组织学、生理学及分子生物学等手段，①探明性分化关键时期，以确定取样及处理时间；②检测自然性分化过程组织匀浆中性类固激素水平(雌二醇、睾酮、11-酮基睾酮)、性别相关基因(Cyp19a、Dmrt1、Foxl2)及雌激素受体基因（ERs）的表达及定位，探明各指标与性分化的内在联系；③以雌二醇、雌激素受体拮抗物和芳香化酶抑制剂诱导性分化，观察药物对性腺发育及上述指标的影响，探求引起性分化的主要因子；④探索雌二醇诱导性分化过程中，RNA干扰ERs对金钱鱼性分化的影响。预期成果不仅有助于阐明雌激素诱导金钱鱼性分化的作用机制，还具有广阔的应用前景。

采用雌二醇（17β-E2）（50，100和200 ug/L）浸泡处理金钱鱼（Scatophagus argus），探讨雌激素诱导其性分化的作用机制，结果表明：（1）金钱鱼仔鱼0.9 cm时出现原始卵巢腔雏形，判断其性分化时间最早为18d；（2）克隆获得Cyp19a1b（登陆号JQ366071）、Dmrt1（登陆号KJ000674 ）、Foxl2（登陆号 KF272907 ）基因，证实上述基因参与金钱鱼性分化过程；（3）雌激素处理20天，100和200 ug/L剂量组金钱鱼雌雄性比2∶1，而对照组和50 ug/L组为1∶1。此外，研究发现早春提高水温至23～26℃，或在饲料中填加6%鱼油可有效促进金钱鱼的卵巢发育；建立了金钱鱼雌雄判别模型，鉴别准确率达85%以上；探明影响金钱鱼雌雄生长差异与GH（登录号：JQ693976）表达、日摄饵量及饵料转化率有密切关系。研究结果丰富和完善了鱼类生长及生殖内分泌调控机制，为金钱鱼全人工繁殖及后续单性化苗种培育奠定基础，后续成果开发应用可望产生重大的经济效益和社会效益。