miR-34b/c在鲟鱼性别分化中的调控作用及其分子机制

Fish gonadal sex differentiation is regulated by the hypothalamo-pituitary-gonad (HPG) axis. Sturgeon aquaculture needs more female populations and reproduction-related regulation techniques, but the mechanism of sex determination and differentiation in sturgeon is still unclear. Recent studies indicate that post-transcriptional regulation of sex differentiation related genes expression mediated by microRNAs (miRNAs) is the important regulatory factors in animals. In previous studies, we have found many sturgeon miRNAs with sex-dimorphic expression pattern between ovary and testis by high-through sequencing and microarray. We concluded that miRNAs may also play a key role in sex differentiation regulation of sturgeonfrom functional prediction.Among them, miR-34b/c were the especially important candidate genes for sex differentiation, which may regulate the biological process by targeted AR gene expression. Therefore, in this study, based on our previous studies in Amur sturgeon (Acipenser schrenckii), which is an important economic sturgeon species in China, we plan to find the spatial-temporal expression profile of miR-34b/c in brain and gonad tissues during early sex differentiation, and characterizations of early differentiation of gonad by immunohistochemistry using SCP3 antibody. Then, comprehensive regulation relationships will be verified by luciferase assays at the cellular level between miR-34b/c and 3’UTR of targeted AR gene. Moreover, miR-34b/c overexpression and inhibition experiment in vitro and in vivo will be carried out to explore its effect on sex differentiation, respectively. Our research will clarify regulation roles of miR-34b/c by targeted AR gene expression in early sex differentiation process and expand the molecular mechanism of sex determination and differentiation regulated by miRNAs in sturgeon. The results of miRNAs involved in sex determination and differentiation can provide us with novel genes and techniques for sturgeon breeding, and enrich the theory of fish reproductive regulation.

鱼类性腺发育和性别分化受下丘脑-垂体-性腺轴（HPG）的调控。目前鲟鱼养殖产业急需性别调控技术，但阐明鲟鱼性别决定与分化机制是首要解决的关键科学问题。新近研究表明microRNA介导靶基因的转录后表达是动物性别决定与分化的重要调控机制。我们首次报道了鲟鱼物种特异microRNA，从幼龄鲟鱼microRNA的性二态表达特性和功能分析中发现microRNA参与其性别分化调控，并鉴定miR-34b/c是鲟鱼性别分化中的重要候选基因。本项目拟以施氏鲟早期性别分化事件为研究主线，以miR-34b/c为切入点，采用SCP3免疫组化等技术揭示其性腺早期分化特征，明确miR-34b/c在脑和性腺的时空表达谱；利用microRNA过表达/干扰技术从体外到体内深入研究miR-34b/c对鲟鱼性别分化的影响，并解析其作用的分子机制，将为鲟鱼养殖产业性别调控提供新的基因资源和技术依据，亦可丰富鱼类生殖调控理论。

多倍体鲟鱼不但是软骨硬鳞鱼类的重要代表类群，是发育和进化研究的理想模型，还是重要经济养殖鱼类，鲟鱼卵制成的鱼籽酱被誉为“黑色黄金”。目前鲟鱼养殖产业急需性别调控技术，但阐明鲟鱼性别决定与分化机制是首要解决的关键科学问题。新近研究表明microRNA介导靶基因的转录后表达是动物性别决定与分化的重要调控机制。利用高通量测序技术鉴定鲟鱼性腺特异miRNA，miRNA呈性二态表达特征结合功能分析暗示其重要参与鲟鱼性别分化调控，并鉴定miR-34b/c是鲟鱼性别分化中的重要候选基因。本项目在前期工作基础上，以miR-34b/c为研究切入点，从组织学水平揭示施氏鲟早期性别分化主要特征；从细胞学水平采用miR-34b/c过表达/干扰技术研究对施氏鲟性腺生殖细胞的影响；体外构建双荧光素酶报告基因验证miR-34b/c与雄激素受体(AR)基因的靶向关系；从动物水平采用miR-34b/c激动剂和抑制剂性腺注射方法揭示对施氏鲟性别分化方向影响的调控作用。研究主要结果如下：采用苏木素-尹红(HE)染色法系统观察了施氏鲟不同性腺发育阶段(5月龄~36月龄)的性腺分化特征(组织形态学特征和生殖细胞发育特征)。采用最新全长转录组测序并结合二代测序技术获得164,618条平均全长达到2,782 bp高质量非冗余的性腺全长转录本数据集。利用Real-time RT-PCR技术检测miR-34b和miR-34c在施氏鲟早期性别分化过程的时空表达谱，发现在12月龄表达量最高且呈性二态表达模式；利用原位杂交技术发现miR-34c主要在卵巢卵原细胞的细胞质中表达，而在原始生长卵泡中几乎不表达，在精巢中miR-34c在精原细胞的细胞核中表达，而在体细胞中不表达，如Sertoli细胞和leydig细胞。首次建立了施氏鲟未分化性腺原代生殖细胞体外增殖培养体系，在细胞水平，miR-34b和miR-34c对性腺生殖细胞的增殖和促抑制效果明显；在动物水平，miR-34b和miR-34c联合抑制显著增加了施氏鲟早期性别向雄性方向分化的比例。本项目揭示非编码miR-34b/c对鲟鱼早期性别分化的调控作用，为鲟鱼性腺发育和性别调控提供新的靶标和技术手段，亦丰富了鱼类性别决定与分子机制的理论。