93 MICRORNA SEQUENCES OF BULL SPERMATOZOA

Abstract

MicroRNA sequences (miRNA) are small RNAs 19 to 22 nucleotides in length that play a crucial role during mammalian development and disease. They are found in plants and animals and regulate the expression of protein-coding genes. Mechanisms regulating gene expression during gamete and embryo development are critical for setting the stage in later development; however, very little is known about the roles of miRNAs in male gametes (spermatozoa). Therefore, the objective of this study was to profile miRNA populations in spermatozoa collected from high and low fertility bulls. Frozen–thawed straws containing spermatozoa from one low and one high fertility bulls were purified using a Percoll gradient (which removes somatic cells) followed by 3 washing steps in PBS. Total RNA were isolated from pelleted spermatozoa using Trizol (3 independent isolations for each bull, total of 6 samples). RNA samples were quantified (NanoDrop Spectrophotometer, Thermo Scientific, Wilmington, DE) and verified for potential somatic cells (Agilent BioAnalyzer, Agilent, Foster City, CA) and DNA (No-RT-PCR) contaminations. Samples devoid of any contaminations went through an enrichment process of miRNA, consisting of dephosphorylation (CIP) and labeling (pCp-Biotin) of the 3′-end of miRNA, followed by their purification (BioSpin6, Bio-Rad, Hercules, CA). The purified miRNAs were analyzed by a miRNA microarray containing approximately 30 000 probes from human, rat, mouse, and other organisms (Ambion/Affymetrix DiscovArray, Asuragen, Austin, TX). The results showed that roughly 48% of total miRNA probe sets (14 215) were successfully analyzed (P < 0.05); of which only 0.5% (7) were significantly differentially expressed between the 2 bulls (fold change >2 and t-test P < 0.001). The roles of these miRNAs are not yet identified because the annotations are unavailable. Nevertheless, our preliminary results suggest that bull spermatozoa are rich in miRNAs, which could play important roles during fertilization and embryo development. Future studies will be aimed at identifying potential molecular mechanisms by which these 7 miRNAs might regulate bovine embryonic development.

This study was funded by the Mississippi Agricultural and Forestry Experiment Station and Alta Genetics Inc.