Gene Expression Dynamics and Network Analysis Reveal Key Molecular Pathways in Spermatogenic Progression from Spermatogonial Stem Cells to Round Spermatids.

OBJECTIVE: Spermatogenesis is a tightly regulated process that involves an orchestrated transcriptional and translational event across the seminiferous epithelium. While individual regulators of this process have been studied, the network-level dynamics underlying the transition from spermatogonial stem cells (SSCs) to round spermatids (RSs) remain poorly understood. The dynamic molecular transitions between SSCs and RSs have not been fully characterised, particularly at the network level. Therefore, this study aimed to characterise the molecular and network-level dynamics underlying the transition from SSCs to RSs by integrating transcriptomic profiling, protein-protein interaction network analysis, functional enrichment, and immunohistochemical validation. MATERIALS AND METHODS: In this experimental study, we evaluated the gene expression dynamics from SSCs to RSs. We analysed publicly available microarray datasets (GEO) comprising four biological replicates for RSs and three biological replicates for SSCs. Differentially expressed genes (DEGs) were identified and mapped onto proteinprotein interaction (PPI) networks, followed by prioritisation of central regulators. Functional enrichment analysis was performed to characterise biological pathways. Immunohistochemistry (IHC) for SOX9, n-MYC, VASA, SOX2, and DAZL was conducted to examine spatial and developmental protein expressions across the seminiferous tubules. RESULTS: A set of 3598 DEGs was identified between SSCs and RSs. This analysis highlighted central genes, Actb, Kras, Pten, Jun, Cdc42, and Gart, that act as central nodes in spermatogenic networks. These genes were associated with pathways that govern cell cycle regulation, chromatin organisation, and signal transduction, consistent with their established roles in germline development. Protein-level localisation confirmed stage-specific expression patterns within the seminiferous tubules. SOX9 localised to Sertoli cells, n-MYC and VASA were activated in differentiating germ cells, DAZL was restricted to early progenitors, and SOX2 appeared only in late-stage subsets. CONCLUSION: This study used transcriptomic profiling with network analysis and molecular experiments to reveal novel regulatory connections that underlie the SSC-RS transition. These findings provide a systems-level framework for understanding male germline development and may facilitate future research on infertility and reproductive health.