精子三种RNA全基因组转录调控分析揭示鸡弱精症的发生机理

It is revealed from several studies and investigations that 12%-18% breeder roosters of indigenous breeds have the problem of low sperm motility (Asthenospermia), which increases the cost of keeping extra birds. The sperm RNA sequencing in human and other mammals showed that the presence of coding and non-coding RNA in the sperm, playing vital roles in the sperm motility function, capacitation, and embryo development. It indicates that multiple sperm RNA may also play important roles in chicken sperm motility regulation. Starting from the difference of semen motility, this study will focus on (1) The relationship between sperm motility and semen physiological and biochemical indices, (2) Constructing the mRNA, miRNA and lncRNA profiling of chicken sperm RNAs using RNA-seq, and revealing the tissue-specific expression profile in sperm and testis, (3) Identifying the differently expressed mRNA, miRNA, and lncRNA in the high and low motility sperm related with sperm motility, and (4) Identifying and verifying the co-expression regulatory networking of gene-miRNA-lncRNA related to sperm motility. This research will provide important clues for revealing the molecular mechanism of sperm motility regulation, and offer a theoretical basis for breeding of high fecundity chicken by molecular techniques.

研究调查发现，我国地方种公鸡中12-18%患有弱精症，其低精子活力导致种鸡养殖成本增加。人及哺乳动物精子转录组测序结果表明，精子中存在编码和非编码RNA，在精子运动、获能及胚胎发育等方面发挥重要作用，提示鸡精子中也存在不同种类的RNA参与调控精子活力。本研究拟以鸡精子活力差异为研究切入点，重点研究：（1）精子活力与精液生理生化指标的关系；（2）通过RNA测序构建鸡精子mRNA, miRNA和lncRNA表达谱，比较与睾丸基因表达谱差异；（3）比较高、低活力精子RNA的表达差异，结合前期蛋白组研究结果，筛选与精子活力相关的候选基因、miRNA和lncRNA；（4）在细胞水平验证多种候选RNA之间的调控关系，构建基因-miRNA-lncRNA互作网络。本研究可为揭示鸡精子RNA对精子活力的调控机理提供重要线索，为开展以提高繁殖性能为目标的分子育种奠定基础。

弱精症表现为精子活力低下，从而影响公鸡的种用效率，提高制种成本。本项目以北京油鸡为试验对象，针对精子活力调控机制展开研究并取得了显著的成果。利用计算机辅助精子分析系统结合多元统计分析策略，发现北京油鸡精子数量高，但精子运动速率不高、直线性较差，利用logic模型拟合北京油鸡公鸡受精率，进一步证实弱精症个体的持续受精能力显著降低；建立1套成熟鸡精子纯化与RNA提取方案，利用poly(A)非依赖的RNA测序（SUPeR-seq）技术探究鸡精子转录本，首次报告鸡成熟精子转录组RNA组成和功能，丰富了对成熟精子转录本的表达情况和功能的认识，并指出精子中AHI1、CUL3和DAW1的mRNA含量与精子活力相关；利用非标蛋白质组学技术分析精浆蛋白质组成，发现精浆中精子骨架蛋白、线粒体蛋白和胞质蛋白丰度的提高可以指示精子结构和功能损伤，并进一步揭示氧化应激是引起精子顶体、质膜和线粒体损伤，并导致弱精症的重要因素；利用高通量测序技术首次解析鸡睾丸RNA表达谱与新功能，在分子和细胞水平上验证了1ncRNA-MSTRG.15568.9靶向调控SPAG4参与精子的发生和活力调控。本项目丰富了对睾丸和成熟精子转录本的表达情况和功能的认识，鉴定了鸡精子发生与成熟过程中调控精子活力的关键候选基因和通路，对进一步挖掘鸡弱精症的分子调控机制和开展以高繁殖性能为目标的分子育种奠定了基础。