Functional profiling of microRNAs in stallions
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MicroRNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional regulators of gene expression in eukaryotic genomes and are thought to be critically involved in many biological processes. While the functions of sperm miRNAs in equine biology are yet to be determined, studies in mouse and humans suggest that sperm miRNAs regulate gene expression in the zygote and can indicate the status of male fertility. The aim of this study was to characterize the expression profiles of selected sperm miRNA in equine tissues and compare their expression levels in the sperm and testes of fertile/sexually mature and subfertile/sexually immature stallions. From sperm RNA-seq data, we selected 6 highly expressed miRNAs: miR-34b, -34c, -191, -223, -1248 and -1905c. Total RNA enriched with miRNAs was extracted from 10 adult tissues, sperm of 3 fertile and 3 subfertile stallions, and testes of five 1-year old and five 3-year old stallions. The RNA was polyadenylated, reverse transcribed into srcDNA, and examined through RT-PCR and qRT-PCR. Reverse transcriptase PCR on a panel of adult male tissues revealed ubiquitous expression of the 6 miRNAs, whereas transcription of miR-34c, -223, and -1905c was elevated in testes and sperm. Additionally, we showed that stallion sperm and testes contain transcripts of mature sperm-enriched tRNA-derived 2 small RNAs (mse-tsRNAs), which is a novel finding for the horse. A pilot study was conducted to quantify the expression of miR-34c and miR-1905c in the sperm of fertile and subfertile stallions. While the expression levels varied between individuals and the two fertility phenotypes, a significantly (p=0.04) elevated expression of miR-34c was observed in the subfertile group. Finally, due to the overall high expression of miR-1905c in sperm, its expression was qualified and quantified in the testes of 1-year old and 3-year old stallions. miR-1905c was expressed in all testes samples and no significant differences in expression level were observed between immature and maturing testes. Because the number of stallions was limited, the current results remain preliminary and further experimentation will be required. Nevertheless, the discovery of miRNAs in stallion sperm might lead to a new direction in the search of biomarkers for stallion fertility.
Wang, Aaron Stephen (2013). Functional profiling of microRNAs in stallions. Honors and Undergraduate Research. Available electronically from