The Bovine Spermatozoal Transcriptome and Sire Fertility

Abstract

Ejaculated bovine spermatozoa retain a pool of RNAs that may have a function in early embryogenesis and be used as predictors of male fertility. The bovine spermatozoal transcript profile remains incomplete because previous studies have relied on hybridization-based techniques, which evaluate a limited pool of transcripts and cannot identify full-length transcripts. The goal of this study was to sequence the complete cryopreserved bovine spermatozoal transcript profile using Illumina RNA-Seq. Spermatozoal RNA was pooled from nine bulls with conception rate (CR) scores ranging from-2.9 to 3.5 and confirmed to exclude genomic DNA and somatic cell mRNA. After selective amplification of polyA + RNA and high-throughput sequencing, 6,166 transcripts were identified via alignment to the bovine genome (UMD 3.1/bosTau6). RNA-Seq transcript levels (n=9) were highly correlated with qPCR copy number (r 2 =0.9747). The bovine spermatozoal transcript profile is a heterogeneous population of degraded and full-length predominantly nuclear-encoded mRNAs. Highly abundant spermatozoal transcripts included PRM1, HMGB4 and mitochondrial-encoded transcripts. Full-length transcripts comprised 66% of the top 368 transcripts (FPKM>100) and amplification of the full-length transcript or 5' and 3' ends was confirmed for selected transcripts. In addition to the identification of transcripts not previously reported in spermatozoa, several known spermatozoal transcripts from various species were also found. Gene ontology analysis of the FPKM>100 spermatozoal transcripts revealed that translation was the most predominant biological process represented. This is the first report of the spermatozoal transcript profile in any species using high-throughput sequencing, supporting the presence of mRNA in spermatozoa for further studies.