大菱鲆生殖细胞减数分裂和性腺分化调控机制初探

Females of turbot Scophthalmus maximus, a main species of the marine fish cultured in China and Europe, grow remarkably faster than males. However, the technology for the production of all-female turbot juveniles has not been established so far, owning to the uncertainty of its mechanisms of sex differentiation. Recently, several studies suggested that the metabolic regulation of retinoic acid (RA) can control the initiation of the meiosis of the germ cells in model animals of vertebrates, such as mice, chicken and zebrafish, and hence decides the direction of sex differentiation of their fetal gonads. Therefore, the aim of this project is to reveal the molecular mechanism behind the control of the germ cell meiosis and sex differentiation in turbot by the metabolic regulation of RA. This aim will be achieved by following measures: 1) to clone the coding sequences of the enzyme families involved in the metabolism of RA, and to identify the expressing types of these genes in the turbot gonad; 2) to compare the paradigms of the expression of genes of the metabolic enzymes of RA and the activities of the meiosis of the germ cells before, during and after the differentiation of the ovary/testis, on the base of our earlier results of the histological observation on the differentiation of the gonads and the cloning of the genetic sequence marking the meiosis in turbot. The results of this study will amplify our knowledge on the diversity and evolution of the mechanism of the control of the meiosis in the germ cells of vertebrates, and lay down the theoretical basis for the development of the all-female juvenile production of turbot and other similar flatfish.

大菱鲆雌鱼生长优势显著，其性别分化机制迄今尚不明确，是这一欧亚优良养殖鱼种的全雌制种技术研发徘徊不前的原因之一。新近研究认为，维甲酸代谢调节能调控小鼠、鸡和斑马鱼等模式脊椎动物生殖细胞减数分裂的启动时机，从而决定这些动物原始性腺的性别分化方向。因此，本项目拟获取大菱鲆维甲酸合成酶和降解酶的编码基因家族各成员序列，明确它们在性腺组织中表达的基因型；在大菱鲆性腺分化组织学观察和减数分裂标记基因序列克隆等前期研究基础上，采用PCR和原位杂交等多种技术手段，比较研究在卵巢和精巢分化的前、中、后3个关键时期维甲酸代谢酶编码基因表达时空模式和生殖细胞减数分裂活动规律，以揭示维甲酸代谢调控大菱鲆生殖细胞减数分裂和性别分化的分子机制。研究结果可以丰富对维甲酸代谢调控脊椎动物生殖细胞减数分裂机制的多样性及进化途径的认识，同时为大菱鲆乃至鲆鲽类单性苗种制种技术开发提供理论依据。

大菱鲆雌鱼生长优势显著，明确其性别分化的调控机制，有助于构建这一欧亚优良养殖鱼种稳定的全雌制种技术。已有研究证明，维甲酸是调控小鼠、鸡和斑马鱼等模式动物生殖细胞减数分裂启动的关键因素。本项目主要探讨维甲酸在大菱鲆生殖细胞减数分裂和性别分化中的调控作用。通过设计兼并引物扩增核心片段、RACE技术和End to End全长序列验证，获得大菱鲆维甲酸合成酶aldh1a2和降解酶cyp26a1、cyp26b1基因序列，利用生物信息学方法预测了蛋白的结果和功能，并进行了同源性序列多重比对和分子系统发生分析。采用染色体组操作技术，构建了大菱鲆全雌苗种家系，克服了其缺乏有效性别分子标记和早期性腺组织难以识别分离的困难，对3个基因在75-360日龄卵巢发育过程中的表达规律进行了研究，其中合成酶aldh1a2基因在75-95日龄卵巢中持续高表达，其后表达量下调维持较低的水平，仅140日龄短暂少量升高；降解酶cyp26b1基因在孵化后75-95日龄逐渐上调，随后则显著下调又呈上调趋势，140日龄达最高值，其后表达下调；降解酶cyp26a1基因在140日龄前均维持低表达，150日龄后表达水平快速增加；结合同期大菱鲆卵巢组织学切片结果和生殖细胞标记基因的表达规律，75-95日龄aldh1a2基因高表达，cyp26b1基因低表达，95日龄后减数分裂标记基因scp3和dmc1持续高表达，初级卵母细胞数量显著增加；140日龄cyp26b1瞬间高表达，对应的减数分裂标记基因scp3和dmc1则显著下调表达，生殖细胞转录因子figla上调表达，卵泡快速生长和发育。初步表明合成酶aldh1a2和降解酶cyp26b1协同作用调节卵巢组织内源性RA浓度，参与卵母细胞减数分裂的调控。相关研究结果，理论上丰富了维甲酸代谢调控脊椎动物生殖细胞减数分裂机制的多样性和进化途径的认识，实践上可通过外源性RA或降解酶抑制剂的添加促进大菱鲆生殖细胞减数分裂的启动和维持，从而抵制外界环境因素如温度对性别分化的影响，建立稳定的大菱鲆全雌苗种生产技术。