性腺体细胞在牙鲆性腺分化过程中的作用及机制

Somatic cells are a main part of the gonad. The important role, possibly leading role of these cells during gonadal differentiation has caused concern. However, the knowledge about them is limited and few fish species have been studied. Their function in sex determination and differentiation is still unclear. Olive flounder Paralichthys olivaceus is an important maricultured fish and the females grow much faster than males. This project will study the function of the somatic cells in flounder gonadal differentiation. Initially, the style and rule of proliferation and differentiation of somatic cell during this process will be studied by using histological section and molecular marker. The function points of somatic cell and germ cell for initial time of gonadal differentiation will be analyzed combining with analysis of the sex steroid dynamics, and the interaction of these two cell styles will be presented with busulfan treatment and RNAi interference or knock-down method. The relationship between differential expression patterns and epigenetic modifications of key genes of somatic cells will be explored according to the data of single-cell transcriptome and genome wide DNA methylation sequencing. In the meantime, the regulation of signal pathways such as Wnt and TGF-β pathways are researched at the levels of cell and individual by analyzing the differential expression levels of key genes of these two signal pathways under activator or inhibitor condition. Finally, the function and mechanism of somatic cells during flounder gonadal differentiation process will be stated. The achievements will provide the basis for comprehensive analysis of mechanism of fish gonad differentiation and the production of unisex fish.

体细胞是性腺的主要组成部分，在鱼类性腺分化中的重要作用甚至主导作用已引起关注，但相关研究仅限少数鱼种，也缺乏作用机制的系统分析。本项目以具有雌雄生长差异的重要海水养殖鱼类牙鲆Paralichthys olivaceus为研究对象，拟首次从性腺体细胞角度，借助组织细胞学观察和分子标记对雌雄性腺分化过程中的增殖分化方式、规律进行了解；结合性激素水平来明确性腺体细胞与生殖细胞的作用时序。同时，在细胞和个体水平上通过抑制生殖细胞增殖或者敲降/干扰生殖细胞和体细胞主效基因等方法探讨两者的相互作用。对比单细胞转录组及表观组数据，分析关键基因差异表达和DNA甲基化表观修饰的关联，并通过激活剂/抑制剂厘清信号通路如Wnt和TGF-β对性腺体细胞分化的调控作用，最终系统阐述牙鲆性腺体细胞在性腺分化中的作用及机制。所获成果将为全面解析鱼类性腺分化机制提供理论基础，为鱼类性别控制等生产应用提供科学依据。

本项目以我国重要海水养殖鱼类-牙鲆为研究对象，解析性腺体细胞在性腺分化中的作用。首先通过组织学观察明确了性腺分化中生殖细胞、体细胞的增殖分化规律。随后分析精巢Sertoli细胞标记基因amh和卵巢颗粒细胞关键基因foxl2的表达图示，它们分别在精巢分化前和卵巢分化过程中高表达，显示其参与了精巢或卵巢的分化。进而amh的RNAi敲降导致性腺分化和发育滞后与萎缩，而性腺分化期鱼苗注射重组蛋白Foxl2后分别显著上调和下调分化早期的cyp19a和nr5a2表达、dmrt1和sox9a表达，并提升了分化后期E2水平，表明体细胞在牙鲆性腺分化中的关键作用。通过转录组测序，筛选到卵巢和精巢生殖细胞与体细胞的差异表达基因，将用于后续研究；也正在开展amh的CRISPR/Cas9基因编辑，获得了F0代突变成体。研究显示，减数分裂标记基因scp3和相关基因dazl在精巢和卵巢分化后高表达，表明性腺开始分化后生殖细胞才启动减数分裂。性激素主要由性腺体细胞合成，而雌雄激素水平在性腺分化启动前和分化过程中都升高，进一步显示体细胞对性腺分化方向发挥重要作用。白消安抑制了牙鲆成体性腺中的生殖细胞，利用其抑制分化期生殖细胞的增殖，性腺的正常分化受到了影响，表明生殖细胞在性腺分化中是不可或缺的。基因表达调控研究显示，foxl2差异表达与其启动子区DNA甲基化水平关联，雌雄性腺的RNA修饰、DNA/组蛋白甲基化等表观修饰差异也暗示它们在性腺分化中起重要作用。还利用TGF-β通路激活剂和抑制剂处理，研究了该通路对牙鲆性腺分化的影响。综上，初步解析了性腺体细胞在牙鲆性腺分化中的作用及其机制。相关成果共发表标注论文19篇，其中SCI论文16篇（第一标注7篇）；申请并获受理国家发明专利3件。这些成果将为鱼类性腺分化机制全面揭示和性别控制等生产应用提供理论基础。