Transcriptome analysis and identification of genes associated with chicken sperm storage duration

The sperm storage tubules located in the mucosal folds of the uterovaginal junction (UVJ) are the primary site of sperm storage in chicken hens after natural mating or artificial insemination (AI). The short-term sperm storage (24 h after mating or AI) in hens was highly associated with immunity and pH-related pathway genes. However, the underlying mechanism of longer duration of sperm storage in female birds remains largely unclear. In the present study, transcriptome analysis was applied to uncover the dynamic gene expression changes in chicken UVJ tissues at two time points (day 3 and day 9) after AI. A total of 574 differentially expressed genes (DEG) were enriched, including 266 upregulated and 308 downregulated DEG. The validation of 5 DEG using quantitative PCR showed a similar expression tendency with RNA sequencing results. The gene ontology terms of DEG were highly enriched in heparin binding (9 genes including COMP, CTGF, and IMPG2), glycosaminoglycan binding (10 genes including PCOLCE, POSTN, and RSPO3), and response to estradiol and ion transport (AREG, RAMP3, SFRP1, and SSTR1). Kyoto encyclopedia of genes and genomes pathway-enrichment analyses of DEG revealed 10 significant pathways (P < 0.05) represented by calcium signaling pathway (7 genes including CACNA1G, PDE1C, PDGFRB, and SLC8A1) and glycosaminoglycan biosynthesis (B3GNT7, CSGALNACT1, GLCE, and ST3GAL1). Protein-protein interaction network of DEG established the connection-regulating epithelial cell or cell-matrix adhesion and migration. The enriched pathways and genes were highly correlated with temporary sperm storage in and possibly sequential sperm release from chicken UVJ overtime after AI. Of these, HIP1, PDE1C, and calcium-related genes were the most interesting candidates associated with sperm storage duration. This report provided a global gene expression profile of the chicken UVJ regarding the capacity of sperm storage overtime after AI. The outcome of this study will contribute to further understanding of the long-term sperm maintenance in avian females and eventually improving the duration of fertile egg performance by selected chicken breeding.