Small non coding RNA from frozen bull sperm cells: biomarkers of male fertility?

The sperm cell has been generally considered as a cab driver, carrying and protecting his genetic heritage, but a new vision of its functions has emerged recently. Indeed, sperm cells also carry small noncoding RNAs (sncRNAs) into oocytes during fertilization. Although their roles remain largely unclear, involvement of sperm-borne miRNAs in mouse epigenetic inheritance has been evidenced and paternal miRNAs and/or endo-siRNAs have been shown to be dispensable for fertilization but crucial for the transcriptomic homeostasis and the developmental of zygotes and 2 cells-embryos. This study aimed at unraveling the sncRNA content from bull frozen sperm cells and identifying miRNA or piRNAs associated with fertility. Total RNA has been produced from 40 ejaculates originating from Holstein and Montbeliard bulls with contrasting fertility. To achieve this task, a novel protocol has been developed to ensure good quality and reproducibility. Quality control was ensured by total RNA quantification using the RNA HS Qubit Assay kit, as well as miR125 quantitation by RTqPCR (miRCURY LNA Universal RT microRNA PCR kit; Exiqon). Starting from 77 million of frozen sperm cells on average, RNA yield was 51 ± 15ng, leading to miR125 Cycle Threshold values in a 19 - 20 range. Only small RNA (<200 nucleotides) were retained to construct NGS libraries, which were sequenced at modest depth (40 million 50 bp single reads, Illumina HiSeq; Exiqon). BWA mapping and annotation was performed, identifying about two thirds of the sequenced reads. Among identified sequences, 16% are annotated as miRNA, 13% as rRNA, 7.5% as tRNA, 7.2% as long non coding RNA, 6.5% as mitochondrial RNA and 17% as mRNA. By the use of miRDeep2 software, 3196 miRNA expressed within all samples have been identified, including 583 known and 2613 putative miRNAs. Interestingly, the remaining unknown sequences are consistent in terms of size with piRNA, which are known to be expressed spermatocytes, spermatids and sperm cells. Expression normalization and statistical analysis were carried out using the DESeq2 package, highlighting 47 differentially expressed miRNA between fertility groups.