High-Throughput Sequencing Methods (RNA-Seq and PolyA-Seq) to Characterize Full-Length mRNAs in Transcriptionally-Silent Bull Sperm; Potential Non-Invasive Measures of Bull Fertility.

Sperm production is a highly regulated cell differentiation event that is characterized by spatio-temporal control of gene and protein expression. Extensive co- and post-transcriptional processing of male germ cell transcript 3’ untranslated regions (UTRs) occurs to coordinate the unique developmental events of spermatogenesis that impact sire fertility. An emerging co-transcriptional regulatory process of spermatogenesis is differential polyA site (PAS) selection in the 3’UTR, termed alternative polyadenylation. The messenger RNA (mRNA) population that remains in the terminally differentiated spermatozoa has the potential as a molecular marker for sire fertility because the characteristics of this mRNA population may reflect gene expression patterns that occurred during spermatogenesis. Furthermore, full-length mRNA may have a functional role in early embryonic development. The high-throughput sequencing methods RNA-Seq and PolyA-Seq can revel the full mRNA and 3’UTR use profiles in bull sperm as a fingerprint of spermatogenic gene expression regulation. From RNA-Seq analysis, the oligo-dT selected 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 these studies, we compared the oligo-dT selected spermatozoal transcript profiles of higher fertility (Conception Rate (CR) 1.8 to 3.5) and lower fertility (CR -2.9 to -0.4) sires using RNA-Seq. A total of 3,227 transcripts and 5,366 transcripts were identified in the higher and lower fertility populations, respectively. While common transcripts between the two populations were identified (2,422 transcripts), several transcripts were also unique to the fertility populations including 805 transcripts that were unique to the higher fertility population and 2,944 transcripts that were unique to the lower fertility population. From gene ontological analysis, the transcripts unique to each fertility population differed in Biological Processes (BP), including enrichment of regulatory transcripts for growth and protein kinase activity in the higher fertility bulls. Biological variation in transcript presence among individual sires was also found and transcripts with a correlation to sire fertility were identified. We are currently investigating 3’UTR use in bovine spermatozoa to investigate fertility-related patterns of alternative polyadenylation.