Spermatogenesis sculpts cell surface RNA landscapes and glycoRNA signatures in the mouse germline
Mammalian spermatogenesis entails extensive remodeling of both the germ cell RNA repertoire and the cell surface glycocalyx, yet whether these two molecular layers intersect during male germ cell differentiation remains unknown. Here, we show that mouse spermatogenesis is accompanied by dynamic remodeling of cell surface RNA (csRNA) landscapes and RNA-associated glycan signatures. Orthogonal cell surface labeling approaches revealed RNase-sensitive J2-reactive RNA signals on intact germ cells and glycan-proximal csRNAs on testicular germ cells. Stage-resolved profiling by in-situ amplification of outer membrane surface RNA (AMOUR) further revealed substantial remodeling of csRNA composition across spermatocytes (SPC), round spermatids (RS), and cauda sperm (CdS), with a markedly expanded and compositionally distinct csRNA repertoire in CdS. In parallel, aqueous identification of RNA elements (AquIRE) detected PNGase F- and O-glycosidase-sensitive glycoRNAs in purified testicular germ cell and sperm RNA preparations. Mass spectrometry (MS)-based glycan analysis identified RNA-associated N-glycans in small RNA fractions of SPC and RS and revealed pronounced remodeling between these stages. Together, these findings establish RNA as a dynamically remodeled component of the male germ cell surface and reveal developmental remodeling of RNA-associated N-glycan signatures during spermatogenesis. This convergence of RNA and glycocalyx remodeling uncovers a previously unrecognized molecular dimension of male germ cell differentiation and provides a foundation for investigating its roles in testicular germ cell-niche interactions and sperm function.