Regulation of cholesterogenic gene expression in Japanese medaka (Oryzias latipes) exposed to 17β-trenbolone

The anabolic androgen 17β-trenbolone (TB) can cause masculinization and reduce fecundity of fish. However, the underlying mechanisms of various biological pathways including metabolism, biosynthesis etc. are largely unknown. Here, we evaluated the effects of TB using the medaka DNA microarray representing 36,398 genes. Larval medaka, Oryzias latipes, (within 24 hrs posthatch) were exposed to TB at various concentrations (2, 6, 20, 60, 100, 200 ng/L) for up to 7 days. Dose-response relationships in gene expression levels of the categorized genes were analyzed using the cumulative chisquared method. Microarray analyses of the TB-exposed larvae showed that 117 and 32 genes were determined as up and down-regulated genes, respectively. The most significant GO term for biological process identified within this gene list was lipid metabolic process, which contained 26 genes in up-regulated genes. In this category, “cholesterol biosynthetic process” was highlighted as an important subcategory with 15 genes, including hydroxymethylglutaryl-CoA synthase cytoplasmic, squalene monooxygenase, lanosterol synthase etc. RT-PCR measurements in these genes were consistent with the microarray results in the direction and magnitude of these changes in gene expression. On the other hands, in the category of “sexual differentiation and development”, genes related to hypothalamic-pituitary-gonadal (HPG) axis were not affected by TB treatment except for one gene encoded to cytochrome P450 19A1. Genes related to oogenesis, such as choriogenins and vitellogenins were weakly down regulated at 2-200 ng/L of TB. Our findings demonstrate that genes encoding cholesterol synthesis pathway via the mevalonate pathway were controlled by TB in larval medaka. TB concentrations used were 0 (control), 2, 6, 20, 60, 100 and 200 ng/L for 7 days of exposure. Each TB treatment had 90 larval medaka for each chamber. At day 7 of the exposures, triplicate samples (30 larvae/sample) from each chamber respectively were collected, flash-frozen in liquid nitrogen, and stored in liquid nitrogen until RNA extraction.

合成代谢雄激素17β-群勃龙（17β-trenbolone, TB）可诱发鱼类雄性化并降低其繁殖能力。然而，包括代谢、生物合成在内的各类生物通路的潜在作用机制仍未得到充分阐明。本研究采用涵盖36398个基因的青鳉DNA微阵列（DNA microarray），评估了TB的生物学效应。选取孵化后24小时内的日本青鳉（Oryzias latipes）幼鱼，以2、6、20、60、100、200 ng/L的梯度浓度TB进行为期7天的暴露处理。采用累积卡方分析法，对分类基因的基因表达水平与剂量的响应关系进行了分析。对TB暴露幼鱼的微阵列分析结果显示，分别有117个和32个基因被鉴定为上调基因与下调基因。在该基因集合中，富集程度最高的生物过程基因本体（Gene Ontology, GO）术语为脂质代谢过程，其中上调基因包含26个相关基因。该脂质代谢过程类别中，“胆固醇生物合成过程”作为关键亚类尤为显著，涵盖15个基因，包括细胞质型羟甲基戊二酰辅酶A合酶、角鲨烯单加氧酶、羊毛甾醇合酶等。针对这些基因的逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）检测结果，在基因表达变化的方向与幅度上均与微阵列分析结果一致。另一方面，在“性别分化与发育”类别中，除编码细胞色素P450 19A1的单个基因外，下丘脑-垂体-性腺（hypothalamic-pituitary-gonadal, HPG）轴相关基因未受TB处理的影响。与卵子发生相关的基因（如卵壳蛋白原与卵黄蛋白原）在2~200 ng/L的TB浓度下仅呈现微弱的下调表达。本研究结果表明，在青鳉幼鱼体内，经由甲羟戊酸通路的胆固醇合成通路相关基因受TB调控。本研究设置的TB暴露浓度为0（对照组）、2、6、20、60、100及200 ng/L，暴露时长为7天。每个培养舱内每组处理接种90尾青鳉幼鱼。暴露第7天时，从每个培养舱中分别采集三份重复样本（每份样本含30尾幼鱼），经液氮快速冷冻后，于液氮中保存直至RNA提取。