芳香化酶抑制剂来曲唑对胡子鲇性腺分化及相关基因表达的影响

Effects of aromatase inhibitor letrozole on sex differentiation and related gene expression in Clarias fuscus

采用组织学和荧光实时定量PCR方法,检测饲喂不同剂量芳香化酶抑制剂来曲唑(letrozole,50、100和200μg/g)对胡子鲇(Clarias fuscus)性腺组织学和性别比率的影响,以及200μg/g来曲唑对性分化前后脑型芳香化酶基因(Cyp19a1b)和翼状螺旋/叉头转录因子2(Foxl2)基因表达的影响,结果表明,50μg/g剂量的来曲唑对胡子鲇性腺分化无显著影响;但100μg/g和200μg/g剂量的来曲唑可促进胡子鲇精巢分化,抑制卵巢分化,卵巢腔最早出现时间和初级卵母细胞出现时间均分别推迟3 d和6 d,而初级精母细胞最早出现时间则分别提前2 d和5 d,且雄性率分别达65.8%和71.3%,显著高于50μg/L组和对照组(P<0.05)。胡子鲇性腺分化前(出膜后12 d),Cyp19a1b和Foxl2基因即开始表达,性腺分化前后Cyp19a1b相对表达量无显著差异(P>0.05),但Foxl2相对表达量则随性分化而逐渐降低,且来曲唑显著抑制性腺分化过程中Cyp19a1b和Foxl2的表达(P<0.05)。结果表明,100μg/g以上剂量的来曲唑可有效诱导胡子鲇分化为雄性;Cyp19a1b可能不直接参与胡子鲇性腺分化,但Foxl2直接参与此过程。

In this study, Clarias fuscus, a common freshwater fish in China, were selected to determine the effects of an aromatase inhibitor on sex differentiation and related gene expression. Two-day-old juvenile C. fuscus were fed different doses of the aromatase inhibitor, letrozole （50, 100, and 200 μg/g diets） for 30 days. The effects of letrozoleon sex ratio, gonad histology, and Foxl2 and Cyp19a1b expression were examined by morphological observation, histology, and real-time fluorescent quantitative PCR during the period of sex differentiation （12？30 d after hatching）. The results showed that letrozole doses of 100 μg/g and 200 μg/g produced more males （65.9% and 71.3%, respectively） than did a dose of 50 μg/g （64.7%） （P〈0.05）. However, no significant difference in sex ratio was observed between the 50 μg/L 17α-MT treated group and the control group （51.1%）. In addition, letrozole accelerated the occurrence of primary spermatocytes for 2 and 5 days, but deferred that of ovarian cavity and primary oocytes for 3 and 6 days, respectively. Both Cyp19a1b and Foxl2 were expressed prior to sex differentiation in C. fuscus. No difference was observed in the level of Cyp19a1b expression prior to and post sex differentiation （12？30 d after hatching）. However, Foxl2 expression decreased with gonadal differentiation. In addition, letrozole at dose of 200 μg/g inhibited the expression of both Cyp19a1b and Foxl2 （P〈0.05）. Our results suggest that letrozole doses above 100 μg/g could induce C. fuscus masculinization, and that Cyp19a1b is not directly involved in the process of gonadal differentiation but Foxl2 is.