肌醇类SIB维持绵羊冷冻精子细胞结构和遗传信息稳定性的机制

Yunnan semi-fine wool sheep is the first breed of semi-fine wool sheep with 48-50 branches independently bred by our country. Cryopreservation of their semen has great significance on preservation of germplasm resources and acceleration of breeding and improvement. In the previous study, we have found the protective effect of cyclohexanhexol-derived SIB on frozen ram spermatozoa. The purpose of this study is to investigate the cryoprotective mechanism of SIBs on frozen ram spermatozoa based on investigation of molecular cryoinhuries of ram spermatozoa. The procedure of the slow freezing method controlled by program or the quick freezing method is optimized. The protective effect of SIBs on structure of frozen ram spermatozoa is studied. The effect of SIBs on calorie and morphology of spermatozoa or ice crystal during cryopreservation are observed by the thermophysical technology; The RNA-Seq was used to analyze the transcription profile of unfrozen or frozen ram spermatozoa. According to the results of RNA-Seq, the differentially expressing gene groups of frozen ram spermatozoa are determined. The effect of cryopreservation on proteome of ram spermatozoa is investigated by the two-dimensional electrophoresis and mass chromatographic analysis. The proteins significantly influenced by freezing are determined. Simultaneously, the stabilizing effect of SIBs on genetic information of frozen ram spermatozoa was also analyzed. This study firstly investigates the effect of SIBs on structure and genetic stability of frozen mammalian spermatozoa, which has great significance on the study of the cryoinjury mechanism of tissues and cells. Moreover, this study can provide certain theoretic and technique supports for improvement of quality of frozen livestock spermatozoa.

云南半细毛羊是我国自主培育的第一个48-50支半细毛羊品种，开展其精液冷冻保存对于种质资源保护和加快育种改良速度具有重要意义。本课题组前期发现肌醇类SIB可以改善云南半细毛羊精子的冷冻保存质量。本课题在研究冷冻对精子分子损伤机制的基础上，探讨肌醇类SIB对绵羊精子的冷冻保护机制。拟优化绵羊精子的程控降温慢速或快速冷冻程序，并探讨SIB对精子结构的保护作用；拟采用工程热物理技术观察SIB对冷冻体系热量、精子和冰晶形成的影响；拟采用RNA-Seq分析冷冻前后精子转录组的变化，确定差异表达的基因群；拟采用双向电泳和质谱分析冷冻对精子蛋白质组的影响，确定受冷冻影响明显的蛋白质种类，同时观察肌醇类SIB对精子遗传信息的稳定效果。本研究首次探讨了肌醇类SIB对家畜精子结构和遗传信息稳定性的影响，对组织细胞冷冻损伤机制研究具有重要意义，同时为提高家畜精子冷冻保存质量提供理论和技术支撑。

云南半细毛羊是我国自主培育的第一个半细毛羊品种，主要分布于云南省昭通地区，其对于该地区畜牧业发展和农民脱贫具有重要意义。然而，近年来由于绵羊市场逐渐转向以肉用为主，其饲养数量显著下降，因此开展云南半细毛羊精液冷冻保存对于其种质保存和育种改良具有重要意义。本项目采用流式细胞术和电镜技术观察肌醇类冰晶抑制剂（SIB）对绵羊精子结构功能的影响，并通过将解冻后精液进行人工授精和体外受精来观察SIB对绵羊冷冻精子受精能力的影响。在此基础上进一步优化绵羊精液冷冻保存程序以降低冷冻过程引起的渗透和化学毒性损伤。采用RNA-Seq分析冷冻前后精子转录组的变化，确定差异表达的基因；采用双向电泳和质谱技术分析冷冻解冻过程对精子蛋白质组的影响，确定冷冻前后差异表达显著的蛋白质种类，同时观察SIB对精子遗传信息的稳定效果。结果表明，50-100mM的环己二醇可以有效保护精子结构和功能，而且环己二醇可以抑制精子细胞凋亡。但是，肌醇类SIB并不能显著提高体外受精胚胎发育率和人工授精受胎率。蛋白质组学分析结果表明，冷冻前后差异显著蛋白点52个，差异蛋白主要参与精子结构、运动能力、应激反应、细胞代谢、分子功能和酶催化活性等方面。RNA-Seq结果表明，冷冻前后差异表达显著的mRNA共计1351个，其中下降1327个，可见冷冻导致大多数精子mRNA表达量的下降。聚类分析结果表明，差异表达mRNA主要参与结合、酶催化、分子传导、分子功能调节、酶调节等。此外，肌醇类SIB并不影响绵羊冻精mRNA的表达，这表明其对精子的保护作用主要体现在细胞水平。本项目首次探讨了肌醇类SIB对家畜精子结构和遗传信息稳定性的影响，对组织细胞冷冻损伤机制研究具有重要意义，同时为提高家畜冻精质量提供必要的理论和技术支撑。