Dynamics of microRNAs in bull spermatozoa

Abstract

Background: MicroRNAs are small non-coding RNAs that regulate gene expression and thus play important roles in mammalian development. However, the comprehensive lists of microRNAs, as well as, molecular mechanisms by which microRNAs regulate gene expression during gamete and embryo development are poorly defined. The objectives of this study were to determine microRNAs in bull sperm and predict their functions.

Methods: To accomplish our objectives we isolated miRNAs from sperm of high and low fertility bulls, conducted microRNA microarray experiments and validated expression of a panel of microRNAs using real time RT-PCR. Bioinformatic approaches were carried out to identify regulated targets.

Results: We demonstrated that an abundance of microRNAs were present in bovine spermatozoa, however, only seven were differentially expressed; hsa-aga-3155, -8197, -6727, -11796, -14189, -6125, -13659. The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa.

Conclusion: Identification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development.

Figure 1

Small molecular weight RNA transcripts as detected using Bioanalyzer. According to the gel image there is no contamination in the RNA and the miRNA will be at or below the green line on the gel, given that miRNAs range from 17-25 nt in length. Rhythm 1, Rhythm 2, Rhythm 3, and Rhythm 4 represent biological repeats for low fertility bull of -3.3 ± 3.3 fertility %. Devin 1, Devin 2, Devin 3, and Devin 4 represent biological repeats for high fertility bull of 4 ± 1.8 fertility %.

Figure 2

Heat map and the result of clustering to all MicroRNA probes detected. There are two specific groups. Column 1-3 represents bull R. Columns 4-6 represents bull D. There were differences between the two groups and some differences within the same groups.

Figure 3

Interactome of miRNA targets. Figures present predicted physical (protein-protein) interactions of a) TOB2 b) CHN2 c) BTBD2.

Figure 4

Expression dynamics of seven miRNAs in sperm from six bulls with varying fertility. Mean Ct values from the High fertility animals were used as reference points and Ct values for groups (high and low fertility) were used to calculate the fold change from the reference points for high fertility group using reference point Ct values according to 2-ΔΔCt method as described by Livak and Shimitgen [25]. Data were presented as mean value ± SEM for each miRNA and * indicates statistical significance at p < 0.05.