The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined.

To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. A _dark and A _pale spermatogonia).

FACS analysis identified a THY-1 ⁺ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFRα1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFRα1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1 ⁺ , PLZF ⁺ , GFRα1 ⁺ , NGN3 ^+/− , cKIT ⁻ ) comprises most A _dark and A _pale and is considerably larger in primates than in rodents. It is noteworthy that the majority of A _dark and A _pale share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from A _dark , but is expressed by some A _pale and may mark the transition from undifferentiated (cKIT ⁻ ) to differentiating (cKIT ⁺ ) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF ⁺ , GFRα1 ⁺ , NGN3 ⁺ , cKIT ^+/− ) is smaller in primates than in rodents.

These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.