Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes

Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle. Using samples from pre-pubertal calves, the CXCR4 protein was detected by immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1, OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell potential.