Spatial Expression Pattern of Novel Undifferentiated Spermatogonial Markers, TCL1 and IRX1, in the Adult Mouse Testis

Spermatogonial stem cells (SSCs) are a population of adult germline stem cells in the testis responsible for the self-renewal and differentiation of daughter spermatogonia via spermatogenesis. Any dysregulation to the stem cell pool, may impair spermatogenesis and ultimately cause infertility. Yet, understanding the molecular mechanisms driving SSC maintenance in relation to their niche environment could advance assisted reproductive technology (ART) procedures. Single-cell mRNA analysis from adult mice carrying an Id4-eGFP transgene, revealed a panel of potential SSC genes, including Tcl1 and Irx1 in the ID4-EGFPbright transplantable fraction (Hermann et al., 2018; Suzuki et al., 2021). However, the spatial organization of SSCs within the testis is lost with tissue dissociation necessitated by single-cell gene expression profiling. To address this, I sought to spatially map cells expressing Tcl1 and Irx1 mRNAs as prototypical markers of SSCs using multiple spatial gene expression approaches to understand the spatial profile of labeled spermatogonia and determine whether they are preferentially positioned along the basement membrane of the seminiferous tubules. The spatially-resolved transcriptome results revealed Tcl1+ spots are randomly distributed within the testis with respect to the stage of the cycle of the seminiferous epithelium. Too few Irx1-containing spots were identified to draw definitive conclusions. Adult mouse testicular cryosections labeled using smFISH for Tcl1, Irx1, Kit, Gfra1, and Id4 mRNAs revealed Tcl1+ and Irx1+ spermatogonia localize near and outside the interstitial tissue and blood vessels. Taken together, these studies have provided spatial data features including topology and stem cell marker gene attributes of Tcl1+ and Irx1+ spermatogonia.