应用LCM结合RNA-Seq技术研究减数分裂后奶牛精子细胞变形的基因表达机制

The studies on mechanism of spermiogenesis are the premises of promoting efficient and normal sperm production, which is the basis for improving the fecundity of male animals and plays important roles in increasing the efficiency of animal husbandry production. Although the spermatogenesis mechanism has been relatively clear, the molecular mechanism of spermiogensis is still a gap, which is not conductive to further improve the breeding efficiency of stud. The applicant's previous study about transcriptomics and proteomics in sperm showed that the expressions of genes are significantly different between X and Y sperm, which results in the significanat phenotypic difference beteween them. According to the previous studies, the applicant pointed out that the changes of morphology and physiology of haploid spermatids during spermiogenesis process are resulted from the differential gene expression in different stages. Therefore, the applicant will combine the techniques of laser capture microdissection (LCM) with RNA-Seq to capture the round spermatides, elongated spermatides, sperm and spermatogonia stem cells (SSCs) and to performe the transcriptome sequencing of all the captured cell samples using SSCs samples as diploid controls. The strategy breaks through the bottleneck of capturing high purity definite type of cell samples from complex cell population of seminiferous tubles in testis, and the molecular mechanism of spermiogenesis will be revealed through bioinformatics analysis. This study will fill the vacancy of the research on the spermiogensis mechanism of mammalian sperm, which will play important roles in improving the breeding efficiency, controlling the sex ratio and controlling the disease in dairy cattle and other livestocks production. It also has great value to the human male infertility, contraception and well bear and well rear.

精子变形机理研究是促进精子正常高效生产，充分挖掘种公畜繁殖力的基础，对提高畜牧业生产效率具有重要意义。虽然精子发生机理已较为清晰，但圆形精细胞通过漫长而复杂的变形过程发育成有功能精子的机理仍为空白，不利于繁殖效率进一步提高。申请人前期的转录组和蛋白组学研究表明，X、Y精子间基因表达存在显著差异，致使X、Y精子间产生表型差异。由此，申请人提出，精子间及单倍体精细胞变形期间的形态及生理变化是基因表达变化的结果。为此，申请人拟采用LCM结合RNA-Seq技术，突破睾丸曲细精管复杂细胞群体中高纯度特定细胞样本的获取瓶颈，以精原干细胞为对照，进行奶牛圆形精细胞、变形精细胞及成熟精子转录组测序，并通过生物信息学分析揭示奶牛精子形成期的分子机理。本研究将填补哺乳动物精子变形机理研究的空白，对奶牛及其他家畜高效繁殖、性别控制和疾病控制具有重要意义，对人类男性不育、避孕及优生优育也同样具有参考价值。

本项目以小鼠为模式动物，建立了精子变形期间基因表达差异分析技术路线后，采用冷冻切片和激光显微切割技术获取了奶牛精原干细胞、圆形精子细胞和长形精子细胞细胞样品，浮游法获得附睾尾精子，通过转录组测序分析，共发现18906个转录表达差异基因（P< 0.05）。其中，精原干细胞到圆形精子细胞表达差异基因3174个，圆形到长形精子的表达差异基因994个；长形到成熟精子表达差异基因4771个；还发现了转录本基因的可变剪接差异。通过 GO（Gene Ontology）功能分类将这些差异基因富集于三类GO条目：细胞过程、单一生物过程、生物调节等生物过程，细胞组成、细胞器、膜等细胞组分，催化、转运体、酶调节物等分子功能；通过通路富集发现，长形与圆形精子细胞差异基因仅富集于metabolicpathways，成熟精子与长形精子细胞差异基因主要富集于metabolic pathways、protein processing in endoplasmic reticulum等通路，圆形精子细胞和精原干细胞差异基因主要富集于metabolic pathways、protein processing in endoplasmic reticulum等通路。根据基因注释，将这些基因分为组蛋白、线粒体、微管、中心体等10类。通过差异基因的qPCR验证，证明了测序数据可用于后续的基因功能验证试验。