Table_10_Characterization of the Impact of Density Gradient Centrifugation on the Profile of the Pig Sperm Transcriptome by RNA-Seq.xlsx

RNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPureTM on porcine sperm. Four boar ejaculates were purified with BoviPureTM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high—although not complete—similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs.

针对人类精液的RNA测序（RNA-Seq）研究表明，若要探究精子转录组，需先从射精样本中去除携带有大量RNA的体细胞。在包括猪在内的其他物种中，为研究精子转录组也需开展精液纯化工作，该步骤通常采用密度梯度离心法，从新鲜射精样本或人工授精精液中获取具有活性的精子，但此方式会限制单次研究可处理的样本通量与样本可及范围。本研究旨在评估采用BoviPure™密度梯度离心法进行纯化对猪精子的影响。研究人员对4份公猪射精样本采用BoviPure™进行纯化，并通过RNA测序对其转录组进行测序，随后将其与对应未纯化样本的RNA测序图谱进行比对分析。共鉴定得到7519个蛋白质编码基因。相关性分析、聚类分析与主成分分析结果显示，纯化样本与对应未纯化射精样本的转录组虽不完全一致，但整体相似度较高。共有372个基因在纯化与未纯化处理间的RNA表达水平存在显著差异。此类差异基因大多在纯化后表达丰度降低，且主要富集于翻译、转录及代谢过程相关的功能通路。研究人员观察到，差异丰度基因中附睾起源基因的占比为1.3%，而未发生表达改变的基因集合中该占比仅为0.2%，二者存在显著差异。与之相反，未发生表达改变的基因集合中睾丸特异性基因的占比为4%，显著高于差异丰度基因集合的0%。在前列腺、白细胞、淋巴结、扁桃体、十二指肠、骨骼肌、肝脏及乳腺组织起源的基因中，未观察到类似的占比差异。综合上述结果可知，精液纯化过程仅会影响少数基因的RNA表达水平，其潜在机制大概率与去除了附睾上皮细胞、未成熟生殖细胞、未成熟或异常精子，以及携带有独特RNA载荷的精液外泌体有关。