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Characterization of the impact of density gradient centrifugation on the profile of the pig sperm transcriptome by RNA-Seq
Frontiers in Veterinary Science ( IF 3.2 ) Pub Date : 2021-06-22 , DOI: 10.3389/fvets.2021.668158
Yu Lian 1 , Marta Gòdia 1 , Anna Castello 1, 2 , Joan Enric Rodriguez-Gil 3 , Sam Balasch 4 , Armand Sanchez 1, 2 , Alex Clop 1, 5
Affiliation  

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. 7,519 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 catalogue 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.

中文翻译:

通过 RNA-Seq 表征密度梯度离心对猪精子转录组的影响

来自人类精液的 RNA-Seq 数据表明,精子转录组的研究需要事先从携带大量 RNA 的体细胞射精中消除。精液纯化也用于研究包括猪在内的其他物种的精子转录组,通常通过密度梯度离心从新鲜射精或人工授精剂量中获得活精子,从而限制了可在一项研究。这项工作的目的是评估 BoviPureTM 密度梯度离心纯化对猪精子的影响。用 BoviPureTM 纯化了四只公猪精液,并将它们通过 RNA-Seq 测序的转录组与其配对的未纯化样品的 RNA-Seq 谱进行了比较。7、鉴定了519个蛋白质编码基因。相关性、聚类和主成分分析表明纯化的和成对的未纯化的精液之间具有高度(尽管不完全)相似性。372 个基因在处理之间显示出不同丰富的 RNA 水平。大多数这些基因在纯化后的丰度较低,主要与翻译、转录和代谢过程有关。与未改变基因的目录 (0.2%) 相比,我们检测到差异丰度基因 (1.3%) 中附睾来源的基因比例发生了显着变化。相比之下,与差异丰富的基因列表 (0%) 相比,未改变基因组 (4%) 中睾丸特异性基因的比例更高。前列腺、白血、淋巴结、扁桃体、十二指肠、骨骼肌、肝脏和乳腺。总之,这些结果表明纯化对少数基因的 RNA 水平有影响,这很可能是由于去除附睾上皮细胞以及早熟的生殖细胞、未成熟或异常的精子或具有不同 RNA 负载的精液外泌体引起的.
更新日期:2021-06-22
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