CD71 defines functionally active spermatogonial stem cells with enhanced transplantation potential in mouse testes

Spermatogonial stem cells (SSCs) are responsible for lifelong spermatogenesis in adult males; however, their scarcity and inherent heterogeneity, coupled with the lack of robust SSC-specific surface markers, continue to impede isolation and characterization. In the present study, we found CD71, which corresponds to transferrin receptor (TfR1; encoded by Tfrc), a candidate marker capable of enriching for SSC populations from mouse testes. The immunohistochemistry detected co-localization of CD71 with the undifferentiated spermatogonia marker glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1) on the seminiferous basement membrane, with approximately 81% co-localization. Consistent with this finding, the expression of Tfrc was up-regulated in SSC-enriched germ cell populations relative to mouse germ cell lines (GC-1 and GC-2) and somatic cell lines from testis (TM3 and TM4). Fluorescence-activated cell sorting (FACS) analysis further showed that GFRα1⁺ cells exhibited approximately 1.4-fold higher Tfrc mRNA expression than GFRα1^- cells. Similarly, CD71⁺ cells exhibited significantly higher expression of the undifferentiated spermatogonia markers Id4, Lhx1, Gfrα1, Zbtb16, and Etv5. Functional transplantation assays further demonstrate that CD71^high cells give rise to approximately 5.5-fold more colonies than freshy isolated, FACS-unsorted donor cells. Moreover, peanut agglutinin (PNA) lectin staining confirmed the normal spermatogenic differentiation within colonies derived from CD71^high donor cells. Our findings collectively indicate that CD71^high cells represent an SSC-enriched population with enhanced spermatogenic regenerative capacity and support the use of CD71 as a complementary marker for SSC enrichment and fertility restoration.