The xenoestrogen, 4-nonylphenol, impaired steroidogenesis in previtellogenic oocyte culture of Atlantic cod (Gadus morhua) by targeting the StAR protein and P450scc expressions
The xenoestrogen, 4-nonylphenol, impaired steroidogenesis in previtellogenic oocyte culture of Atlantic cod (Gadus morhua) by targeting the StAR protein and P450scc expressions
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DOI：
10.1016/j.ygcen.2006.10.008
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发表时间：
2007-02-01
影响因子：
2.7
通讯作者：
Arukwe, Augustine
中科院分区：
文献类型：
--
作者：
Kortner, Trond M.;Arukwe, Augustine
类固醇生成急性调节（星星）蛋白和细胞色素P450介导的胆固醇侧链裂解（P450 SCC）已定位于大多数类固醇生成器官，并迅速合成响应急性促激素刺激。本研究建立了鳕鱼卵母细胞体外培养体系，并采用组织学和分子生物学方法研究了壬基酚（NP）等内分泌干扰物对鳕鱼卵母细胞类固醇激素水平、星星蛋白和P450 SCC的影响。此外，由于细胞周期蛋白B作为鱼类卵母细胞生长的指示物的作用，研究了细胞周期蛋白-B的表达模式。卵母细胞体外培养技术是基于琼脂糖漂浮法。将组织在10 ℃的加湿培养箱中培养4、7、14和21天，并将不同浓度的壬基酚（0（对照）、1、10、50和100 μ M）溶解在乙醇（0.3%）中。使用经验证的实时聚合酶链反应（PCR）与特异性引物检测基因表达。星星蛋白和P450 sec的免疫组织化学使用针对两种蛋白的合成肽制备的抗血清进行。使用酶免疫测定法估计雌二醇-17 β（E2）和11-酮睾酮（11-KT）组织水平。我们的数据表明，壬基酚产生了独特的和一致的浓度特异性模式的调制的星星蛋白，P450 sce和细胞周期蛋白-B基因的表达在第14天后曝光。这种模式通常被描述为从0（对照）增加到1和10 μ M，并在50和100 μ M时降低。观察到的星星蛋白，P450 SCC和细胞周期蛋白-B水平的变化与暴露后第14天11-KT水平的变化有直接关系。星星和P450 SCC的细胞定位是特定的卵泡细胞的pretellogogenic卵母细胞，但没有显色强度的差异。在暴露组之间未观察到卵母细胞直径的显著变化。我们的数据揭示了一些新的方面的壬基酚的成熟和卵母细胞生长的硬骨鱼，表明受损的类固醇激素和激素失衡的卵黄发生过程和明显的繁殖力的潜在后果。(c)2006年爱思唯尔公司All rights reserved.
The steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated cholesterol side-chain cleavage (P450scc) have been localized in most steroidogenic organs and are rapidly synthesized in response to acute tropic hormone stimulation. In this study, we present the development of cod previtellogenic oocyte in vitro culture system, histological and molecular methods for evaluating the effects of endocrine disruptors such as nonylphenol (NP) on steroid hormone levels, the StAR protein and P450scc. In addition, expression pattern of cyclin-B was studied, because of cyclin B's role as an indicator of oocyte growth in fish. The in vitro previtellogenic oocyte culture technique was based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 4, 7, 14 and 21 d with different concentrations of nonylphenol (0 (control), 1, 10, 50 and 100 mu M) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time polymerase chain reaction (PCR) with specific primers. Immunohistochemistry of the StAR protein and P450sec were performed using antisera prepared against synthetic peptide for both proteins. Estradiol-17 beta (E2) and 11-ketotestosterone (11-KT) tissue levels were estimated using enzyme immunoassay. Our data show that nonylphenol produced a unique and consistent concentration-specific pattern of modulation for the StAR protein, P450sce and cyclin-B gene expression at day 14 after exposure. This pattern is generally described as increasing from 0 (control) to 1 and 10 mu M, and decreased at 50 and 100 mu M. The observed changes in the StAR protein, P450scc and cyclin-B levels showed a direct relationship with changes in 11-KT levels at day 14 after exposure. Cellular localization of StAR and P450scc were specific to the follicular cells of previtellogenic oocytes, but with no differences in staining intensities. No significant change in oocyte diameter was observed between the exposure groups. Our data reveal some novel aspects of nonylphenol effects on maturation and oocyte growth in teleosts, suggesting impaired steroidogenesis and hormonal imbalance with potential consequences for the vitellogenic process and overt fecundity. (c) 2006 Elsevier Inc. All rights reserved.