Microarray and Single-Cell RNA Sequencing Reveals G-Protein Gene Expression Signatures of Spermatogonia Stem Cell

Proper testicular development is essential for spermatogenesis, a complex biological process that depends on the continuous proliferation and differentiation of spermatogonial stem cells (SSCs). These processes are tightly regulated by the SSC niche. Understanding the developmental mechanisms of SSCs is therefore critical for elucidating the basis of male fertility. Recent studies have shown that members of the G-protein-coupled receptor (GPCR) superfamily play key roles in ion and water balance in the epididymis, development of efferent ductules, blood-epididymal barrier formation, and sperm maturation. To investigate SSC development in humans, we performed microarray analysis to examine G-protein gene expression in single cells from six human testes. Our analysis revealed that genes such as LEPROT, LRRC15, LPAR1, SSR1, BMPR2, TNFRSF11B, TNFRSF10D, DDR2, SSR3, SIGMAR1, GRIA3, OGFRL1, GRIK2, TMEM87A, GPR108, TNFRSF1A, S1PR2, and VASN were down-regulated, while FLT1, ADGRG6, CSF1R, IL7R, ADGRL3, OR4N4, MMD, SIRPB1, OR5I1, PTGDR, MPL, and GPR107 were up-regulated. Single-cell transcriptomic and bioinformatic analyses were used to validate SSC-specific gene expression and assist in SSC isolation and sorting. Additionally, immunofluorescence labeling at different developmental stages provided insights into the spatial and temporal dynamics of spermatogonia. Our findings offer new insights into the molecular mechanisms governing human SSC development and provide a valuable foundation for advancing SSC-based fertility research and therapeutic applications.