BULL SPERM MIRNAS PROFILING IN MOTILE AND LOW MOTILE CELL POPULATIONS

Mature spermatozoa contain coding and noncoding RNAs, some of them involved in the regulation of many physiological pathways including the control of sperm motility. The presence of miRNA transcripts in sperm is now well acknowledged, but little is known of the function of novel miRNAs in sperm cells or their potential involvement in spermatogenesis. The aim of this study was to characterize miRNAs expression in motile (M) and low motile (LM) sperm populations. Twelve frozen semen doses from 4 bulls were simultaneously thawed at 37°C and pooled. Each pool was overlaid on a dual-layer (90-45%) discontinuous Percoll gradient and centrifuged at 700 g for 30 minutes at 20°C. The two populations obtained (M and LM) from each tube were washed in TALP at 700 g for 10 minutes at 20°C and evaluated for sperm kinetic parameters by CASA and sperm viability and acrosomal status by flow citometry. Aliquots were kept at -80°C un6l RNA extraction. miRNAs were extracted and small RNA libraries were generated using the Illumina Truseq Small RNA Preparation kit according to manufacturer's instructions and sequenced on a single lane of Illumina Hiseq 2000, obtaining about 4.6 millions of reads from each sample. Using the mirDeep algorithm, a total of 811 miRNAs were classified as known in Bos taurus (481) or novel (330). Sperm quality parameters and miRNAs distribution in the two popula6tions were analysed by General Linear Model Procedure (SAS). According to the statistical analysis sperm cells were successfully fractionated in M and LM populations (Total motility: M = 48.4% vs LM = 4.8%; P<=0.0001). We found 37 known and 19 novel miRNAs that were differentially expressed in M and LM populations (P<=0.0001). Furthermore 92.8 % of miRNAs were upregulated in the M population. These results firstly report an integrated approach to evaluate miRNA expression between high and low motility sperm populations using deep sequencing.