Transcriptional Regulation Analysis Provides Insight into the Function of GSK3β Gene in Diannan Small-Ear Pig Spermatogenesis

Glycogen synthase kinase-3β (GSK3β) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3β expression patterns in adult Diannan small-ear pig (DSE) testes. We identified the most important transcript ENSSSCT00000039364 of GSK3β, obtaining its full-length coding sequence (CDS) spanning 1263 bp. Gene structure analysis located GSK3β on pig chromosome 13 with 12 exons. Protein structure analysis reflected that GSK3β consisted of 420 amino acids containing PKc-like conserved domains. Phylogenetic analysis underscored the evolutionary conservation and homology of GSK3β across different mammalian species. The evaluation of the protein interaction network, KEGG, and GO pathways implied that GSK3β interacted with 50 proteins, predominantly involved in the Wnt signaling pathway, papillomavirus infection, hippo signaling pathway, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, endometrial cancer, basal cell carcinoma, and Alzheimer’s disease. Functional annotation identified that GSK3β was involved in thirteen GOs, including six molecular functions and seven biological processes. ceRNA network analysis suggested that DSE GSK3β was regulated by 11 miRNA targets. Furthermore, qPCR expression analysis across 15 tissues highlighted that GSK3β was highly expressed in the testis. Subcellular localization analysis indicated that the majority of the GSK3β protein was located in the cytoplasm of ST (swine testis) cells, with a small amount detected in the nucleus. Overall, our findings shed new light on GSK3β’s role in DSE reproduction, providing a foundation for further functional studies of GSK3β function.