牙鲆脑性别分化对性腺分化的作用机制

Fish has been regarded as an ideal animal model to investigate the mechanism of sex determination and differentiation in vertebrates due to the diversity of sex determination and differentiation. Most studies have focused on gonad differentiation. In recent years brain has been considered as the most important sexual organ in mammals, however the roles of brain sexual differentiation in fish gonadal differentiation keep unclear. This project will detect the density of glial cells or neurons by the Nissl staining and morphological differences in neuronal dendrites and dendritic spines using FD Rapid GolgiStainTM Kit between male and female brain before and during the gonadal differentiation period in olive flounder. The expression patterns of the sex dimorphic genes obtained from finished transcriptome analysis and the potential brain sex differentiation marker genes (th which encodes tyrosine hydroxylase and tph which encodes tryptophan hydroxylase) will be examined during the early brain differentiation period. The levels of serotonin and catecholamine will also be measured using HPLC method. We are supposed to obtain the brain sex differentiation marker genes of olive flounder. By investigating the expression pattern of brain sex differentiation marker genes, we could know when the brain sex differentiation begins in olive flounder. To explore the mechanism of flounder brain sex differentiation, we will compare the time when estrogen begin to be synthetised in gonad and brain by qPCR and HPLC. We will also investigate the expression of brain marker gene after inhibition the synthesis of estrogen or inhibition the binding of estrogen and estrogen receptor. The transcriptome and epigenomics analysis after inhibiting the DNA methyltransferase (DNMT) will be performed to explore mechanism of methylation in the brain sex differentiation. By inhibiting the brain sex differentiation with inhibitor of marker genes or DNMT, then analyzing the expression of key genes during the gonad differentiation and the sex of the gonad, we would know the influence of brain sex differentiation on the gonad differentiation. Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in China. Females grow much faster than males, so it is a proposed way to increase production by controlling the sex ratio.This project will throw light on the mechanism of the sex differentiation and be helpful to control the sex ratio.

摘要：鱼类性别分化机制的阐明有重要的理论和实践意义。目前对鱼类性别分化的研究侧重于对性腺的研究。已有文献报道，脑是最重要的性别器官。本项目拟以牙鲆为研究对象，研究脑性别分化在性腺分化中的作用及机制。结合牙鲆雌、雄性腺分化时期，根据脑神经元树突棘密度和数量、脑性别分化候选标记基因的表达及脑性别分化候选信号分子的含量，获得脑性别分化起始时期及标记基因；比较脑和性腺中雌二醇合成起始时间，分析添加雌二醇及雌二醇合成被抑制后脑性别分化的变化，同时借助全基因组甲基化测序及转录组学分析脑的视前区DNA甲基转移酶被抑制后甲基化调控的基因，从而阐述牙鲆脑性别分化机制；根据脑性别分化被抑制后性腺分化关键基因表达、性腺分化过程及性别表型的变化，解析脑性别分化对性腺分化的作用。牙鲆是我国重要海水养殖鱼类，雌雄生长差异大，其性别控制研究可丰富鱼类性别决定和分化理论，提高生产效益。

本项目以我国重要海水养殖鱼类-牙鲆为研究对象，从脑性别分化角度研究性腺性别分化。首先通过高尔基染色、尼氏染色及透射电镜分析了雌雄牙鲆脑神经元的形态学差异，发现在性腺分化期，雌鱼下丘脑神经元的树突长度从卵巢开始分化时即显著高于雄鱼，且雌鱼下丘脑神经元的树突棘密度在性腺分化后期显著高于雄鱼。利用ELISA检测了雌雄牙鲆脑中激素和神经递质的含量，结果显示脑中雌二醇和儿茶酚胺含量在性腺分化前已有极显著差异。通过定量PCR及转录组分析，发现在卵巢分化之前雌雄牙鲆的脑中已有cyp19b、tph等差异表达基因，差异基因富集在生物节律等11个信号通路。其次，分析了HPG轴基因的表达，发现脑中gnrh3在性腺分化前有雌雄二态性表达，垂体中的fsh及lh及性腺中的fshr及lhr在性腺分化前也均已开始表达，提示脑可通过HPG轴作用于性腺分化。利用GnRH受体拮抗剂处理性腺分化期的牙鲆鱼苗，虽然HPG轴基因表达显著下降，但性比并没有显著变化，显示除了HPG轴，脑可能还通过其他途径作用于性腺分化。最后，分析了DNA甲基化水平，发现雌二醇在性腺分化前提高了雌鱼脑的整体甲基化水平，并降低了cyp19b启动子甲基化水平，且雌二醇处理的牙鲆雌核发育鱼苗和孵育的雌鱼下丘脑组织中均显示，雌二醇可显著上调cyp19b表达。这些结果表明雌二醇可以通过DNA甲基化调控脑中基因表达，从而影响脑的性别分化。用DNA甲基转移酶抑制剂处理性腺分化期的牙鲆鱼苗，处理组雄性率显著提高，显示DNA甲基化在性别分化中发挥作用。此外，还发现雌雄脑部分差异表达基因受差异表达的miRNA调控，以及性腺分化的重要转录因子Foxl2调控HPG轴相关基因的表达。.本研究获得了牙鲆脑性别分化的标记基因，明确了牙鲆脑性别分化与性腺性别分化的先后，阐述了雌二醇对牙鲆脑性别分化影响及其机制，探讨了牙鲆脑性别分化对性腺分化的作用，获得的结果不仅对全面解析脑性别分化的机制及其对性腺分化的作用提供基础，也可指导鱼类性别控制。