Identification of the crucial circ-mi-mRNA interaction networks regulating testicular development and spermatogenesis in ganders

Semen quality has an important impact on the reproductive performance of ganders, and the quantity and quality of spermatozoa in semen are the determinants of semen quality. In our practical work, a small number of azoospermic ganders were observed in adult goose breeding populations, but the underlying regulatory mechanisms remain unknown. In the present study, we firstly compared the morphological and histological differences in the testes of ganders from normozoospermic group (NG) and azoospermic group (AG), and then analyzed the testicular expression patterns of circRNAs, miRNAs, and mRNAs between the two groups by using whole-transcriptome sequencing technology. Results from histomorphological analysis demonstrated that the body weight alone was not accountable for the occurrence of gander azoospermia, and the possible cause might be the observed testicular abnormalities. At the morphological level, the left, right, and bilateral testicular weights, the right and bilateral testicular organ indexes, and the long, short, and dorsoventral diameters of the left, right and bilateral testes were significantly lower in AG than in NG (P < 0.05). At the histological level, most testicular histological parameters, such as the testicular parenchymal area, the diameter of seminiferous tubules, and the number of germ cells, were significantly higher (P < 0.05) in NG than in AG. The RNA-seq results showed that a total of 683 differentially expressed circRNAs (DEcircRNAs), 24 differentially expressed miRNAs (DEmiRNAs), and 1,118 differentially expressed Genes (DEGs) were identified in the gander testes between NG and AG. Subsequent functional enrichment analysis revealed that most of the DEGs and the target genes of DEcircRNAs and DEmiRNAs were significantly enriched in either the biological processes related to male gonad development, spermatid development, and regulation of cell differentiation or the KEGG terms including the MAPK, TGF-beta, Wnt, and cell cycle signaling pathways. By constructing the core ceRNA regulatory networks, several key DEcircRNAs, including 1:98100313|98104995, 1:171413706|171419341, 6:3414226|3418193, and 2:115876735|115880760, were identified to regulate the expression of TGFB2 and BCL2 through interactions with specific miRNAs such as novel-miR-265 and novel-miR-266, and such interactions could play crucial roles in regulating the gander testicular cell apoptosis, proliferation, and spermatogenesis. This study provides novel insights into the function and molecular mechanisms of ceRNAs in regulating the gander testicular development and semen quality.