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. 2017 Jan 4;18(1):14.
doi: 10.1186/s12864-016-3394-7.

Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations

E Capra  1 , F Turri  1 , B Lazzari  1   2 , P Cremonesi  1 , T M Gliozzi  1 , I Fojadelli  2 , A Stella  1   2 , F Pizzi  3
Affiliations

Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations

E Capra et al. BMC Genomics. .

Abstract

Background: Small RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed. To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized.

Results: Sperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT).

Conclusions: Small RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction.

Keywords: Cryopreserved; Sequencing; Sperm; miRNA; piRNA.

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Figures

Fig. 1
Fig. 1
Cluster analysis of the 83 differentially expressed DEmiRNAs (FDR <0.01) in High Motile (HM) and Low Motile (LM) fraction. In figure are shown the first 40 DEmiRNAs
Fig. 2
Fig. 2
Box plot showing the differentially expressed (DE) Bos taurus known miRNAs in high (gray bar) and low (white bar) motile fractions isolated from cryopreserved bovine semen. Central lines inside the boxes indicate median values; box width indicates 25 and 75% quartile ranges around the median; “T” indicates the maximum and minimum values, and black dots represent outliers. N = 12 for each treatment. In bold miRNA highly expressed in HM fraction
Fig. 3
Fig. 3
Canonical Pathway Chart of the (experimentally observed) genes targeted by 20 differentially expressed miRNA that found correspondence with human miRNA. Pathways analyses were calculated from: a) miRNAs up-regulated in the High Motile (HM) fraction; b) miRNAs up-regulated in the Low Motile (LM) fraction and c) Total of miRNAs differentially expressed between the (HM) and (LM) fractions. In squares, pathways that showed a positive or negative score and were shared between miRNAs up-regulated in the HM and LM fractions. The first 15 pathways are shown in the figure
Fig. 4
Fig. 4
Working hypothesis of the mechanism through which different miRNAs regulate the PTEN pathway, PI3K/AKT and STAT signalling in sperm. Arrows indicate miRNA expression and (+) activation or (−) inhibition of the related pathway. At the bottom of the figure other miRNAs and related target genes involved in the pathway regulation are reported
See this image and copyright information in PMC

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