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 CD71high 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 CD71high donor cells. Our findings collectively indicate that CD71high 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.