A spatial single-cell type multiplex map of human spermatogenesis

Mapping the spatial localization of proteins is essential for understanding their function. Human testis constitutes a complex organ with thousands of proteins activated or repressed during spermatogenesis. By combining single-cell RNA sequencing (scRNA-seq) with fluorescence-based multiplex immunohistochemistry (mIHC), we performed large-scale spatiotemporal mapping of specific testicular cell states within their native tissue context. Single-cell state expression patterns of almost 500 proteins were quantified and clustered according to differentiation stages, providing a framework for functional interpretation. Interestingly, comparative analysis of mRNA-to-protein expression dynamics revealed multiple instances of temporal discordance within defined cellular states. Notably, contradicting previous findings, PIWIL4 transcripts were detected in earlier germ cell states, whereas protein abundance peaked at later stages, coinciding with proliferative transition. These findings highlight the importance of assessing protein expression in single-cell mapping approaches. This workflow establishes a scalable framework for proteome-wide spatial tissue analysis applicable to studying cellular dynamics in both health and disease.