Exploring the Landscape of Circular RNAs in Testes of Dezhou Donkey During Distinct Developmental Stages

Genetic selection is crucial to increase the reproductive performance of stud donkeys. Testicular development and spermatogenesis involve numerous genes, including circular RNAs (circRNAs). However, circRNAs in the male reproductive system of donkeys remain unexplored. This study comprehensively analysed testicular samples from Dezhou donkeys at three different age stages for circRNA using transcriptome data. A total of nine Dezhou donkeys were divided into three groups based on their developmental stages: juvenile (two-month-old, n = 3), adolescent (12-month-old, n = 3) and adult (24-month-old, n = 3) donkeys. We identified 12,084 candidate circRNAs, with 6409 circRNAs specific to the juvenile stage, 6616 to the adolescent stage and 10,031 to the adult stage, respectively. Among these, 3908 circRNAs were expressed across all three age stages. Differential expression analysis revealed 2419 circRNAs exhibiting age-dependent expression changes. Eight circRNAs showed consistent differential expression across all age stages, while specific differential expression patterns were observed between different age pairs. Five representative differentially expressed circRNAs were validated by RT-qPCR, and their expression patterns were consistent with the RNA-sequencing results. Moreover, enrichment analyses of differentially expressed circRNA target genes demonstrated significant enrichment in pathways related to male reproductive processes, including sperm egg recognition and sperm binding to the zona pellucida. Additionally, a circRNA-miRNA-mRNA network, comprising 350 circRNAs, 29 miRNAs and 39 mRNAs, is providing insights into the regulatory mechanisms of non-coding RNAs in testes. In conclusion, circRNA expression patterns are involved in the reproductive biology of testicular development and spermatogenesis in Dezhou donkeys.