CircRNA Role and circRNA-Dependent Network (ceRNET) in Asthenozoospermia

Abstract

The role of circRNA in reproduction is under investigation. CircRNAs are expressed in human testis, spermatozoa (SPZ), and seminal plasma. Their involvement in embryo development has also been suggested. Asthenozoospermia, a common cause of male infertility, is characterized by reduced or absent sperm motility in fresh ejaculate. While abnormal mitochondrial function, altered sperm tail, and genomic causes have been deeply investigated, the epigenetic signature of asthenozoospermic derived SPZ still remains unexplored. CircRNAs may take part in the repertoire of differentially expressed molecules in infertile men. Considering this background, we carried out a circRNA microarray, identifying a total of 9,138 transcripts, 22% of them novel based and 83.5% with an exonic structure. Using KEGG analysis, we evaluated the circRNA contribution in pathways related to mitochondrial function and sperm motility. In order to discriminate circRNAs with a differential expression in SPZ with differential morphological parameters, we separated sperm cells by Percoll gradient and analyzed their differential circRNA payload. A bioinformatic approach was then utilized to build a circRNA/miRNA/mRNA network. With the aim to demonstrate a dynamic contribution of circRNAs to the sperm epigenetic signature, we verified their modulation as a consequence of an oral amino acid supplementation, efficacious in improving SPZ motility.

Keywords: asthenozoospermia; circRNAs; epigenetic signature; infertility; mitochondria-dependent ceRNET.

Figure 1

Overview of circRNA expression in asthenozoospermic derived SPZ. (A) The proportion of circRNAs from circBASE (http://circbase.mdc-berlin.de) and other databases/literature on a total of 9,138 circRNAs identified. (B) The proportion of different types of circRNAs among all predicted circRNAs. (C) Chromosomal distribution of SPZ derived circRNAs, on strand + and strand –. (D) The distribution of up- and down-regulated circRNAs in fraction B compared to fraction A asthenozoospermic derived SPZ among all circRNAs. (E) Hierarchical clustering analysis of total circRNAs samples arranged into two groups, based on their expression levels; in detail, this analysis used different colors to represent the expression values of circRNAs detected in fraction A (n = 3, indicated as 1A, 2A, 3A) and B (n = 3, indicated as 1B, 21B, 3B) SPZ of asthenozoospermic patients.

Figure 2

Differential expression of circRNAs between fraction A and B asthenozoospermic derived SPZ. Differential expression of circRNAs between fraction A and B SPZ. (A) The distribution of up- and down-regulated DE-circRNAs in fraction B compared to fraction A SPZ. (B) The distribution of up- and down-regulated DE-circRNAs in the human genome, according to their host gene location. (C) Hierarchical clustering analysis of DE-circRNAs in A SPZ (samples 1A, 2A, 3A) and B SPZ (samples 1B, 2B, 3B). The expression values (Fold change ≥ 1.5, p ≤ 0.05) are represented in different colors, indicating expression levels above and below the median expression level across all samples. (D) The volcano plot was constructed using Fold-Change and p-values; in detail, the values on X and Y axes are log2 (FC = Fold-Change) and –log10 (p-values), respectively. Red points in the volcano plot represent the DE-circRNAs with statistical significance.

Figure 3

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of host genes. (A) The Top 15 KEGG signaling pathway annotations of circRNAs up-regulated in fraction A asthenozoospermic derived SPZ. (B) The Top 15 KEGG signaling pathway annotations of circRNAs down-regulated in fraction A asthenozoospermic derived SPZ.

Figure 4

(A) Validation of the circRNA-microarray results up-regulated in B compared to A asthenozoospermic derived SPZ; ^**p < 0.01. (B) Validation of the circRNA microarray results down-regulated in B compared to A asthenozoospermic derived SPZ; ^**p < 0.01. A significant high score of normalized intensity and related host genes involved in sperm physiology and embryonic development functions represent the “selection criterion” of the DE-circRNAs validated.

Figure 5

Functional clustering of DE-circRNA down-regulated in B compared to A asthenozoospermic derived SPZ. One circRNA, circUSP54, tethers a group of miRNAs as targets, all involved in mitochondria-dependent pathways. Networks were built using Cytoscape. Hexagonal and rectangular symbols represent circRNAs and miRNAs, respectively. The arrow indicates the tethering activity of circRNAs toward miRNAs, while the dotted arrow indicates the pathways upstream of the miRNAs.

Figure 6

(A) Expression of five circRNAs up regulated in A normozoospermic derived SPZ (N) compared to A asthenozoospermic derived SPZ from men pre- (pre-A) and post-treatment with oral amino acid supplement (post-A); ^**p < 0.01. (B) Expression of five circRNAs up-regulated in A asthenozoospermic derived SPZ from men pre-treatment with oral amino acids supplement (pre-A) compared to A normozoospermic derived SPZ (N) and to A asthenozoospermic derived SPZ from men post-treatment with oral amino acids supplement (post-A); ^**p < 0.01.