采用组织学和分子生物学方法,研究了投喂芳香化酶抑制剂来曲唑(LE)后暗纹东方(Takifugu obscures)初鲀孵仔鱼CYP19A、DMRT1基因表达以及性腺的组织学变化,以期进一步了解P450芳香化酶(P450arom)在鱼类早期性别分化过程中的作用.RT-PCR 结果显示,对照组样品 CYP19A 和 DMRT1表达显示性二态,雌性表达 CYP19A基因,雄性表达 DMRT1基因.LE 处理组在性别分化期间,雄性样品单一表达 DMRT1,雌性样品则同时表达CYP19A 和 DMRT1.qRT-PCR 结果显示： LE 处理组雌性仔鱼 CYP19A 基因表达被显著抑；虽然在仔鱼出膜后22 d(dph)的表达水平高于9 dph,但仅为同日对照组的2.11%.LE处理组雌性样品22 dph时DMRT1基因表达量上调,至150 dph时达对照组雄性水平.55 dph的性腺组织学结果表明, LE处理可导致暗纹东方稚鱼原始卵巢退化,并鲀向功能性精巢发育.150 dph的LE处理组性腺均为精巢,并与对照组精巢发育同步.结论认为,暗纹东方性腺鲀分化期间P450arom是卵巢形成和维持发育所必须的,抑制P450arom活性可导致雌性暗纹东方发生雄性化逆转.鲀%As in all other lower vertebrates, sex differentiation in fish is susceptible to environmental and steroi-dogenic stimulation or inhibition. Alteration of the sex differentiation process is possible in many fishes through the manipulation of the environment and steroid function. Some chemicals, which have androgenic or estrogenic function, are known to alter the gonadal sex in fish from female to male or vice versa if administered during the period of gonadal sex differentiation. Other than direct androgenic or estrogenic effects, there are chemicals which are capable of disrupting the enzymatic pathway of natural androgenic-estrogenic balance in fish. Among the non-steroidal aromatase inhibitors (AI), letrozole is typically used as an effective human drug in the treatment of estrogen-dependent disease, including breast cancer, and was found to be effective in suppression of all estrogens after oral administration. To assess the involvement of aromatase during gonadal sex differentiation in Takifugu obscures, we fed fry of this species with letrozole [1.25g/(kg·d)]. Gonadal development was observed histologically using a microscope, and the expression of CYP19A and DMRT1 was measured during the period of sex differentiation. The results of RT-PCR revealed that there was a sexually dimorphic expression pattern of CYP19A and DMRT1 in the T. ob-scures fry during gonad differentiation. CYP19A was expressed in female, and DMRT1 was expressed in male control fish. In fry treated with LE, both of CYP19A gene and DMRT1 gene expression were observed in female, but only the DMRT1 gene was expressed in male fry. A quantitative real-time PCR analysis revealed that the expres-sion of the CYP19A gene in female was significantly inhibited after treatment with LE. Though expression of CYP19A was higher at 22 days post hatch (dph) than at 9 dph, it was only 2.11% the level of the control female. DMRT1 gene expression was up-regulated gradually in LE treated female fry during sex differentiation, and reached the same level as control males at 150 dph. In LE treated female fry, degeneration of the ovarian cavity and primary spermatocytes were observed in sections of the gonad. The functional testes were developed at 150 dph. Our research demonstrates that aromatase activity is necessary for ovarian formation and development during sex differentiation in T. obscures. Masculinization can be induced by LE treatment in T. obscures during sex differentiation.
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