结合蛋白质组学和RNA-seq技术分析海藻糖对绵羊冷冻精子的保护机制

As an important cryoprotectant, trehalose has been extensively used for cryopreservation of livestock semen. However, the action mechanism of trehalose still needs elucidation. Based on our previous study, both RNA-Seq and proteome are used to investigate the molecular base of cryoprotective effects of trehalose on frozen sperm. At first, flow cytometry and electron microscopy are used to analyze effects of trehalose on ultrastructure and organelles of frozen ram sperm. Simultaneously, effects of trehalose on subsequent embryonic development are also investigated in order to optimize the freezing procedure of ram sperm. Secondly, RNA-Seq was used to analyze the difference of gene expression of frozen or unfrozen ram sperm. Effects of trehalose on transcription profile of frozen ram spermatozoa are determined. Finally, two dimensional electrophoresis and iTRAQ are used to analyze the effects of cryopreservation on proteome of ram sperm. Simultaneously, the stabilizing effect of trehalose on proteome of frozen ram spermatozoa was also observed. This study firstly used proteome and RNA-Seq technologies to investigate effects of trehalose on structure and genetic stability of frozen mammalian spermatozoa, which has great significance for elucidation of long-term pending cryoprotective mechanism of trehalose in the cryobiology and reproductive biology field. Moreover, this study can provide certain theoretic and technique supports for improvement of quality of frozen livestock sperm.

作为一种重要的冷冻保护剂，海藻糖已经广泛应用于家畜精液的冷冻保存，但是对于其作用机制尚无定论。本课题组在前期工作基础上结合转录组和蛋白质组等技术探讨海藻糖对精子冷冻保护作用的分子基础。首先，采用流式细胞术和电镜技术分析海藻糖对绵羊冷冻精子超微结构和细胞器的影响，并进行胚胎发育实验，优化绵羊精子的冷冻保存程序；其次，采用RNA-Seq技术分析冷冻前后绵羊精子基因表达的差异，探讨海藻糖对冷冻精子RNA转录组的影响；最后，采用双向电泳和iTRAQ技术分析冷冻对绵羊精子蛋白质组的影响，同时观察海藻糖对相关蛋白质丰度的稳定效果。本研究首次采用蛋白质组学和RNA-Seq技术探讨海藻糖对家畜精子结构和遗传信息稳定性的影响，对于解决低温生物学和繁殖生物学研究领域长期悬而未决的海藻糖冷冻保护机制问题具有重要意义，同时为提高家畜精子冷冻保存质量提供一定的理论和技术支撑。

海藻糖已经广泛应用于家畜精液的冷冻保存，但是对于其作用机制尚无定论。本课题组常规细胞生物学手段、结合转录组和蛋白质组等技术探讨海藻糖对精子冷冻保护作用的分子基础。首先，流式结果表明，海藻糖可以有效抑制冷冻解冻过程引起的精子早期凋亡，并维持质膜蛋白的正常分布。另外，海藻糖可以维持冻精线粒体膜电位的稳定。TEM表明，冷冻过程严重损伤精子质膜和顶体完整性，而海藻糖可以缓解冷冻过程对精子超微结构造成的损伤。此外，新鲜精子、对照组和海藻糖冷冻组精子的囊胚发育率分别为38.2%、35.9%和38.7%，三个处理组之间差异不显著；其次，采用TRAQ技术分析海藻糖对精子相关蛋白质丰度的稳定效果。结果表明，和不添加海藻糖冷冻组精子相比，海藻糖冷冻组精子解冻后表达量下降的蛋白14个，上升的11个，GO聚类分析结果表明，差异蛋白主要参与氧化还原活性、离子结合以及结构分子活性等；最后，采用RNA-Seq技术探讨海藻糖对冷冻精子RNA转录组的影响，冷冻造成1208个基因表达量下降。然而，海藻糖并不影响冻精mRNA的表达，这暗示海藻糖对冻精的保护作用主要体现在细胞和蛋白水平。本研究联合采用蛋白质组学和RNA-Seq技术分析海藻糖对家畜精子结构和遗传信息稳定性的影响，对于解决低温生物学和繁殖生物学研究领域长期悬而未决的海藻糖冷冻保护机制问题具有重要意义，同时为改善家畜冻精质量提供必要的理论和技术支撑。