Abstract
BACKGROUND: MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of 22-24 nucleotides that regulate gene expression. In the last decade, miRNAs have been described in sperm of several mammals, including cattle. It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans; however, little is known about the content, expression, and function of sperm-borne miRNAs in early bovine embryo. In this study, total sperm RNA was isolated from 29 cryopreserved sperm samples (each coming from a separate bull) using a RNeasy kit and treatment with DNase I. RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer. The expression of 10 candidate miRNAs in bovine sperm (bta-miR-10a, bta-miR-10b, bta-miR-138, bta-miR-146b, bta-miR-19b, bta-miR-26a, bta-miR-34a, bta-miR-449a, bta-miR-495 and bta-miR-7), previously identified in testis and/or epididymis, was evaluated with RT-qPCR. The cel-miR-39-3p was used as a spike-in exogenous control. Nonparametric Mann-Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility [HF; non-return rates (NRR) ranging from 39.5 to 43.5] and those with subfertility (SF; NRR ranging from 33.3 to 39.3). Several sperm functionality parameters (e.g., viability, membrane stability or oxygen consumption, among others) were measured by multiplexing flow cytometry and oxygen sensing technologies. RESULTS: RNA concentration and purity (260/280 nm ratio) (mean ± SD) from the 29 samples were 99.3 ± 84.6 ng/µL and 1.97 ± 0.72, respectively. Bioanalyzer results confirmed the lack of RNA from somatic cells. In terms of the presence or absence of miRNAs, and after applying the Livak method, 8 out of 10 miRNAs (bta-miR-10b, -138, -146b, -19b, -26a, -449a, -495, -7) were consistently detected in bovine sperm, whereas the other two (bta-miR-10a, and -34a) were absent. Interestingly, the relative expression of one miRNA (bta-miR-138) in sperm was significantly lower in the SF than in the HF group (P = 0.038). In addition to being associated to fertility potential, the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption. The expression of three other miRNAs (bta-miR-19b, bta-miR-26a and bta-miR-7) was also correlated with sperm function variables. CONCLUSIONS: In conclusion, although functional validation studies are required to confirm these results, this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond, and supports its use as a fertility biomarker in cattle.