热应激下公兔睾丸组织形态和精液转录组分析

doi:10.11843/j.issn.0366-6964.2023.11.020

摘要/Abstract

摘要： 旨在深入了解公兔在热应激环境中睾丸组织形态改变及相应基因谱,对揭示热应激下公兔精液质量下降的机理具有重要意义。本研究选取了6只具有相同饲养水平,体况和体重相近的8月龄新西兰种公兔,通过建立热应激模型得到热应激组(8月份,HS)和未热应激组(5月份,NHS)各3只,分别采集2组公兔的睾丸和精液。通过HE染色,比较不同条件下睾丸组织结构的差异,并利用Illumina Hiseq高通量测序技术对精子进行转录组测序分析,通过生物信息学方法对2组样本间的差异表达基因进行GO、KEGG富集分析,并通过RT-qPCR方法验证转录组测序数据。公兔睾丸组织切片结果显示,与NHS组相比,HS组的睾丸中央出现明显的空泡化和撕裂,生精上皮较薄,受损情况显著,精子细胞数量下降。进一步以log₂FoldChange>1和P<0.05为阈值,共筛选出与精子热应激响应相关的差异表达基因1 676个,包括ATP5MC1、MDH2、ALDH7A1、GAPDHS、PRPS1等,其中上调基因894个,下调基因782个。GO分析结果显示,差异表达基因富集在应激反应、蛋白质代谢过程、催化活性等相关条目上,KEGG结果显示,差异表达基因富集到MAPK、PI3K-AkT、Toll-like receptor、Ras等信号通路上。RT-qPCR验证了ATP5MC1、MDH2、PRPS1、ALDH7A1和GAPDHS等5个差异表达基因的表达水平,与转录组结果趋势一致。综上,热应激能够调控精子发生相关基因的表达,影响相关信号通路及生物学功能。本研究结果为精子发生的分子调控机制提供线索,并为高温环境下家兔的选育提供理论基础。

关键词: 热应激, 种公兔, 精子, RNA-Seq, 差异表达基因

Abstract: The study aimed to understand the morphological changes of testicular tissue and responding gene profile of male rabbits in heat stress environment to reveal the mechanism of heat stress of the decline of semen quality of male rabbits. In this study, six 8-month-old New Zealand male rabbits with the same feeding level and similar body condition and weight were selected. By establishing a heat stress model, 3 rabbits in the heat stress group (August, HS ) and 3 rabbits in the non-heat stress group (May, NHS ) were obtained. The testis and semen of the two groups were collected, respectively. HE staining was used to compare the differences in testicular tissue structure under different conditions, and Illumina Hiseq high-throughput sequencing technology was used to analyze the transcriptome sequencing of sperm. GO and KEGG enrichment analysis of differentially expressed genes between the two groups of samples were performed by bioinformatics methods, and the transcriptome sequencing data were verified by RT-qPCR method. The results of testicular tissue sections of male rabbits showed that, compared with the NHS group, the central part in testis of the HS group showed obvious vacuolization and tearing, the spermatogenic epithelium was thinner, the damage was significant, and the number of sperm cells decreased. With log₂FoldChange>1 and P<0.05 as the threshold, a total of 1 676 differentially expressed genes related to sperm heat stress response were screened, including ATP5MC1, MDH2, ALDH7A1, GAPDHS, PRPS1, etc. Among them, 894 genes were up-regulated and 782 genes were down-regulated. GO analysis result showed that differentially expressed genes was enriched in stress response, protein metabolic process, catalytic activity and other related items, and KEGG result showed that differentially expressed genes was enriched in MAPK, PI3K-AkT, Toll-like receptor, Ras and other signaling pathways. RT-qPCR verified the expression levels of ATP5MC1, MDH2, PRPS1, ALDH7A1 and GAPDHS, which were consistent with the trend of transcriptome results. In summary, heat stress can regulate the expression of spermatogenesis-related genes and affect related signaling pathways and biological functions. The results of this study provide clues for the molecular regulation mechanism of spermato-genesis and provide a theoretical basis for the breeding of rabbits under high temperature environment.

Key words: heat stress, male rabbit, sperm, RNA-Seq, differentially expressed genes

中图分类号:

S814
S829.1

蔡佳炜, 张琛, 靳荣帅, 鲍志远, 张希宇, 王璠, 翟频, 赵博昊, 陈阳, 汤先伟, 吴信生. 热应激下公兔睾丸组织形态和精液转录组分析[J]. 畜牧兽医学报, 2023, 54(11): 4653-4663.

CAI Jiawei, ZHANG Chen, JIN Rongshuai, BAO Zhiyuan, ZHANG Xiyu, WANG Fan, ZHAI Pin, ZHAO Bohao, CHEN Yang, TANG Xianwei, WU Xinsheng. Analysis of Testicular Tissue Morphology and Semen Transcriptome of Male Rabbits under Heat Stress[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4653-4663.

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