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. 2020 Feb 11;30(3):211–228. doi: 10.1038/s41422-020-0279-8

Fig. 6. Sperm-borne circRNAs are abundant, highly conserved across species and variable with aging and fertility statuses.

Fig. 6

a Levels of circRNAs in three types of spermatogenic cells (pachytene spermatocytes, round, and elongating spermatids), whole sperm and cytoplasmic droplets. CircRNA reads were identified from RNA-seq data in triplicates (biological replicates, n = 3 in whole sperm and cytoplasmic droplets), followed by normalization against aligned total RNA reads. b Enriched GO terms for cytoplasmic droplet-enriched and whole sperm-enriched circRNAs. c CircRNA abundance in sperm heads and whole sperm. CircRNA reads were identified from RNA-seq data in biological triplicates (n = 3), followed by normalization against aligned total RNA reads. d Dot plot showing circRNAs are more abundant in young than those in old mouse testes. Normalized FPKM values of circRNAs in old (2-year-old) mouse testes are plotted against those in young (4-month-old) mouse testes (biological replicates, n = 2). The regression line is indicated in blue with gray shading. e Representative semi-qPCR gel images showing levels of the circular and linear forms of two genes (Sept10 and Ttil5) in young and old mouse testes (Technical replicates n = 2, results are similar). f High fertility human sperms contain more circRNAs and less linear RNAs than low fertility human sperms. CircRNA reads were identified from RNA-seq data (biological replicates n = 8 after removing outlier) from whole sperm of healthy donors, followed by normalization against aligned total RNA reads (Upper panel). Normalized counts of circRNAs and their linear isoforms are used to calculate the ratios and to generate the accumulation curve (lower panel). g Venn diagram showing sperm circRNAs are conserved across species. Unique circRNAs in rat, human, and rabbit sperm were identified from RNA-seq data (biological replicates, n = 2–3) of whole sperm, followed by normalization against aligned total RNA reads. h Significantly enriched GO terms in differentially expressed circRNAs between high and low fertility human sperms. GO terms related to nuclear functions are in orange, whereas those involved in sperm motility are in gray. The Student’s t test was used for statistical analyses. *P < 0.1; **P < 0.05; ***P < 0.001.