Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq

L Fraser; P Brym; C S Pareek; M Mogielnicka-Brzozowska; Ł Paukszto; J P Jastrzębski; K Wasilewska-Sakowska; A Mańkowska; P Sobiech; K Żukowski

doi:10.1016/j.theriogenology.2019.11.001

Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq

Theriogenology. 2020 Jan 15:142:400-413. doi: 10.1016/j.theriogenology.2019.11.001. Epub 2019 Nov 2.

Authors

L Fraser¹, P Brym², C S Pareek³, M Mogielnicka-Brzozowska⁴, Ł Paukszto⁵, J P Jastrzębski⁵, K Wasilewska-Sakowska⁴, A Mańkowska⁴, P Sobiech⁶, K Żukowski⁷

Affiliations

¹ Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, 10-719, Olsztyn, Poland. Electronic address: fraser@uwm.edu.pl.
² Department of Animal Genetics, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, 10-719, Olsztyn, Poland.
³ Centre of Veterinary Sciences, Inter-University Centre of Veterinary Medicine, Nicolaus Copernicus University, 87-100, Torun, Poland.
⁴ Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, 10-719, Olsztyn, Poland.
⁵ Department of Plant Physiology and Biotechnology, Faculty of Biology and Biotechnology, University in Olsztyn of Warmia and Mazury in Olsztyn, 10-719, Olsztyn, Poland.
⁶ Department of Clinical Sciences, Internal Disease Unit, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-719, Olsztyn, Poland.
⁷ Department of Cattle Breeding, National Research Institute of Animal Production, 32-083, Balice, Poland.

PMID: 31711689
DOI: 10.1016/j.theriogenology.2019.11.001

Abstract

Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen.

Keywords: Boars; RNA profiles; Spermatozoa; Transcriptome; Transcripts.

MeSH terms

Animals
Cryopreservation / methods
Cryopreservation / veterinary
Freezing* / adverse effects
Gene Expression Profiling* / methods
Gene Expression Profiling* / veterinary
Male
RNA-Seq* / veterinary
Semen Analysis / veterinary
Semen Preservation* / classification
Semen Preservation* / standards
Semen Preservation* / veterinary
Spermatozoa / chemistry
Spermatozoa / metabolism*
Swine / genetics*
Swine / metabolism
Transcriptome