戊唑醇(TEB)是一种常用的杀菌剂,可以抑制芳香化酶Cyp19A并下调转录因子叉头盒L2(FoxL2),导致斑马鱼幼虫的男性偏向性别分化。然而,TEB暴露后FoxL2发挥功能的具体机制尚不清楚.在这项研究中,预测斑马鱼FoxL2蛋白(zFoxL2)的磷酸化位点和激酶特异性残基。随后,通过原核表达制备重组zFoxL2,并产生了多克隆兔抗zFoxL2抗体。将斑马鱼成纤维细胞(ZF4)细胞单独暴露于100μMTEB中8小时,之后,参与foxl2调控途径的基因表达发生变化(akt1,pi3k,cyp19a1b,检测到c/ebpb和sox9a)。当共同暴露于1μM雌二醇和100μMTEB时,这些关键基因的表达趋于恢复。有趣的是,TEB不影响foxl2基因或蛋白质的表达,但它显着抑制了FoxL2(pFoxL2)在丝氨酸238处的磷酸化(降低了43.64%,p=0.009)。免疫共沉淀试验表明,暴露于100μMTEB后,在抗zFoxL2抗体组和抗pFoxL2(ser238)抗体组中,ZF4细胞中的总沉淀蛋白分别减少了17.02%(p=0.029)和31.39%(p=0.027),分别,这表明TEB抑制了FoxL2蛋白通过抑制其自身磷酸化与其他蛋白结合的能力。下拉测定证实了这一结论。这项研究初步阐明了foxl2基因在TEB诱导的男性偏向性别分化过程中通过其编码蛋白的低磷酸化通过翻译后调节起作用。
Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (
FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which
FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish
FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-μM TEB alone for 8 h, after which changes in the expression of genes involved in the
foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-μM estradiol and 100-μM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of
FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-μM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.