孕激素（DHP）调控鱼类雌性性别分化过程的机制研究

In teleosts, maturation inducing hormone (DHP) is a progestin to promote gametes maturation and ovulation (spermiation). During the critical period of sex determination/differentiation in tilapia, specific expression of steroidogenic enzymes (Cyp17a2 and 20β-HSD2) for DHP biosynthesis, and DHP nuclear receptor (PGR) was detected in the XX gonad, but not in the XY gonad. Moreover, we also found that treatment of XX fish by RU486 (a PGR antagonist) from 5 to 20 days after hatching (dah) led to the significant repression of cyp19a1a gene expression, decline of estrogen production, and sex reversal. Therefore, we propose, for the first time, that DHP might play a key role in female sex differentiation in fish. In this proposal, we intend to further investigate the expression level and cellular localization of Cyp17a2, 20β-HSD and PGR during the critical period of sex differentiation in fish gonads. Meanwhile, the plasma DHP level during these stages will also be measured. To clarify the molecular and epigenetic mechanisms of DHP on female sex differentiation in fish, a serials of investigations will be carried out. First of all, RU486 treatment of XX tilapia from 5 dah will be performed and sampled at different time points to see the morphological and molecular changes. High throughput sequencing will be carried out to investigate the changes of miRNA and LncRNA during RU486 treatment. Knockout of Cyp17a2, 20β-HSD and PGR gene by CRISPR/Cas9 based transcriptional silencing technology will be accomplished to check the effects on sex differentiation. Simultaneously, rescue assay will be applied to check whether estrogen or DHP is capable of restoring the effects of RU486 treatment or gene knockout on gonadal differentiation. The target genes of PGR will be screened and characterized by ChIP-seq and real-time PCR, and then the molecular network of DHP in the sex differentiation will be constructed. Our final goal is to clarify the molecular and epigenetic mechanisms that DHP regulates fish gonadal sex differentiation directly or indirectly via estrogen.

成熟诱导激素(DHP)是促进硬骨鱼类配子成熟和排卵（精）的孕激素。我们发现DHP合成酶和DHP核受体（PGR）在罗非鱼性别决定和分化早期（孵化后5-20天）的性腺呈现雌性特异表达。PGR拮抗剂(RU486)处理（孵化后5-20天），能够抑制雌激素合成酶基因的表达和雌激素合成，导致XX个体性腺性逆转为精巢。我们创新性地提出DHP在鱼类雌性性别分化过程中发挥重要作用的学术思想。本项目拟进一步证实DHP合成酶和PGR在罗非鱼卵巢分化早期的表达水平和细胞类型，检测血清DHP合成水平。通过RU486处理、激素回救、基因敲除和RNA-Seq揭示DHP影响雌激素通路和雌性性别分化的机制。通过PGR抗体的ChIP-Seq和real-time PCR筛选和鉴定PGR的靶基因，构建DHP调控性别分化的分子网络。最终阐明DHP直接，或者通过雌激素间接调控性腺分化过程的分子机制和表观遗传机制。

DHP（17α, 20β-dihydroxy-4-pregnen-3-one，17α, 20β-DP）作为鱼类孕激素，其合成酶（Cyp17a2）和核受体（Pgr）在罗非鱼性别决定与分化早期的性腺中呈现雌性特异表达，于是推测DHP信号通路可能在鱼类雌性性别分化、配子发生早期等过程中也发挥重要的作用。因此，在该项目实施的过程中，我们以罗非鱼（Orochromis niloticus）为实验材料，紧紧围绕DHP信号通路在鱼类性别分化、配子发生、成熟与排卵过程中的作用机制开展工作。研究结果概述如下：.1、从孵化后5天开始用pgr的拮抗剂（RU486）持续处理雌性罗非鱼，研究结果发现XX个体性腺发育为可育的精巢。RU486处理的同时添加雌激素E2能够恢复雌性性腺的发育，证明DHP在罗非鱼雌性性别决定和分化中可能发挥重要作用，并且雌激素和孕激素协同调控雌性发育。RU486处理雄鱼导致精子发生受到阻滞，但停止处理后精子发生过程得到恢复。在RU486处理的同时，添加DHP能够恢复精子发生过程，但11-KT不能回救由RU486处理导致的精子发生受阻的情况，表明DHP对于生殖细胞的减数分裂启动及雄性精子发生非常重要。.2、转录组和长非编码RNA测序分析发现，RU486处理之后XX个体性腺中mRNA和lncRNA的表达模式与对照雄鱼的表达模式基本一致，而与对照组雌鱼性腺的基因表达谱呈现显著差异，表明Pgr下游信号通路的阻断，导致XX个体雄性化，而非编码RNA在性别重塑过程中发挥重要作用。.3、采用CRISPR/Cas9基因编辑技术，在罗非鱼中成功实现DHP通路相关基因的敲除。 研究发现pgr和cyp17a2基因敲除，导致雌性和雄性性腺分化延迟和阻滞，严重影响生殖细胞减数分裂的启动过程。pgr和cyp17a2基因敲除导致雄性精子活力降低，育性降低。cyp17a2基因敲除敲除个体不能成熟和排卵，而pgr基因敲除导致雌性个体不能排卵。总之，本项目深入研究了DHP在硬骨鱼类雌性性别分化、配子成熟、排卵等过程中的作用和分子机制。.4、在项目执行期间，项目负责人申请获准国家自然科学基金面上项目1项；培养学生17人（博士2人，硕士15人）；发表SCI收录论文共17篇（标注该项目号的7篇）；获得重庆市自然科学基金三等奖。