Cyp19a1基因在中华鳖精巢发育与精子发生中的功能及转录调控研究

Chinese soft-shelled turtle （Pelodiscus sinensis） is an importantly commercial aquaculture reptile. In the breeding programs, improvement of the male turtle hatching rate is important for the better growth performance compared with female turtle. Aromatase which is encoded by Cyp19a1 is crucial for converting androgen into estrogen. The crucial role that Cyp19a1 in ovary differentiation and development had been proved in vertebrates. However, the exact function in testis of non-mammalian vertebrates remains to be elucidated. Our previous findings showed that the aromatase inhibitor (Letrozole) treatment result in decreasing spermatids counts in testis indicating that Cyp19a1 might play an important role in regulation of spermatogenesis. To clarify the function and transcriptional regulation mechanisms of Cyp19a1 in the turtle testis development, a series of investigations will be carried out. In vivo gene knockout of Cyp19a1 by CRISPR/Cas9 will be implemented. Simultaneously, rescue assay in Cyp19a1 knockout individuals by estrogen treatment will be investigated. The serum hormone levels, testis histological morphology and the expression of genes involved in testis development, spermatogenesis and cell apoptosis would be checked. The miRNA-Cyp19a1 mRNA-lncRNA network will be constructed by screening and characterizing the function of transcription factors, miRNAs and lncRNAs. The implementation of this proposal will provide data for the mono-sex breeding in the turtle and provide a theoretical basis for the function of Cyp19a1 in gonadal development of aquaculture animals.

中华鳖是我国名特养殖种类之一，雄鳖较雌鳖有显著的生长优势，因此培育“全雄鳖”是育种的重要目标。芳香化酶是雌激素合成的关键酶，由Cyp19a1基因编码。Cyp19a1基因在水产动物性别分化与卵巢功能维持中有关键作用，但在雄性生殖中的功能尚不明确。我们前期研究发现芳香化酶活性降低会影响中华鳖睾丸精子发生，在此基础上，本项目拟采用CRISPR/Cas9技术敲除Cyp19a1基因并进行雌激素回救，通过检测睾丸、附睾、输精管不同发育时期组织形态、血清性激素水平、精子发生基因表达，研究Cyp19a1在精巢发育与精子发生中的作用。通过分析转录因子调控作用、鉴定相关miRNAs和lncRNAs功能，建立精巢中miRNA-Cyp19a1 mRNA-lncRNA调控网络。通过上述内容，研究Cyp19a1基因在中华鳖精巢发育与精子发生中的作用并探讨其表达调控机制，为中华鳖单性育种与水产动物性腺发育调控提供参考。

Cyp19a1编码的芳香化酶可以将雄激素转化为雌激素，是动物性别分化和卵巢功能维持的关键。雌激素也是哺乳动物精子发生中有重要作用，但在水产动物精巢发育中的作用尚少。本项目围绕Cyp19a1及雌激素在中华鳖精巢中的作用开展了研究。从动物水平采用雌激素及其受体激动剂、拮抗剂腹腔注射雄性中华鳖，分析了雌激素在中华鳖精子发生中的作用；采用激动剂、拮抗剂和RNAi方法开展了Cyp19a1潜在转录因子作用分析；筛选了性腺差异表达miRNA、lncRNA，构建了ceRNA网络，筛选了Cyp19a1靶向miRNA。研究主要结果：（1）在雄性中华鳖中进行腹腔注射Letrozole，处理后精子数量显著降低，凋亡信号明显增强，表明内源性雌激素是中华鳖精子发生的重要因素；（2）在稚鳖中腹腔注射雌激素受体激动剂和抑制剂，采用苏木素-尹红（HE）染色法观察了卵巢与精巢组织形态变化，表明雌激素受体Esr1和Esr2在性腺发育中存在功能差异；（3）克隆Cyp19a1启动子序列，预测并克隆了潜在转录因子（Sf-1、RXRα/γ、Sirt1），采用RNAi分析了RXRα/γ、Sirt1在中华鳖性别分化中的作用，表明RXRα/γ、Sirt1参与了性腺分化过程，Sirt1敲降后会影响雌激素合成；（4）转录组测序获得了性腺差异表达mRNA、miRNA、lncRNA，构建了差异表达基因ceRNA网络，筛选了以Cyp19a1为靶基因的miRNA。本项目研究了雌激素及其受体在中华鳖精子发生中的作用，筛选了Cyp19a1基因的转录因子并分析了在性腺发育中的作用，为中华鳖性腺发育提供参考，丰富了水产动物性腺发育与分子调控理论。