Abstract
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.
Highlights
Despite being a matter of debate for many years, the presence and role of sperm RNA is beginning to be elucidated
All the minus reverse transcription of the control samples showed no amplification of PRM1 and PTPRC and the qPCR on the intergenic region was undetectable in all P and not purified (NP) samples, thereby indicating no DNA contamination (Supplementary Table 2)
The NP aliquot of sample B2 showed more PTPRC signal ( CqPTPRC−PRM1) than any other of the eight samples thereby suggesting a degree of somatic cell presence in this sample
Summary
Despite being a matter of debate for many years, the presence and role of sperm RNA is beginning to be elucidated. Transcriptomic evaluations of the ejaculated spermatozoa are often preceded by a purification step to remove somatic and prokaryotic cells often by using a commercial colloidal silica suspension (e.g., PureSperm R , BoviPureTM, Nidacon, Sweden) in order to prepare density gradients and purify the sperm cells [11,12,13,14]. Our group has always purified the porcine ejaculates with BoviPureTM when characterizing the boar sperm transcriptome [5, 6, 8, 15] to be on the safe side since the potential impact of an even tiny presence of somatic cells on the semen transcriptome profile is unknown The objective of this pilot study was to assess the changes on the porcine ejaculate’s transcriptome after BoviPureTM purification and discuss the potential underlying causes. We compared the transcriptomes of purified vs. paired non-purified ejaculates from four male pigs
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