DEAD-box polypeptide 4 (DDX4) is an evolutionally conserved ATP-dependent RNA helicase that is exclusively expressed in germ cell lineage. Although DDX4 is believed to reside and function in the cytoplasm, recent studies in mice and humans suggest that its epitope is expressed on the cell surface of a small subpopulation in the ovary, putative oogonial stem cells. No study has examined whether such cell-surface DDX4(+) cells exist in the testes of any species. In this study, we explored cell-surface DDX4(+) cells in postnatal porcine testes before the onset of spermatogenesis, where gonocytes, which are the precursors of spermatogonial stem cells, are the only germ cell population. Transfection experiments demonstrated that recombinant porcine DDX4 can be expressed on the cell surface, and cell-surface DDX4-immunoreactive cells were identified in the testis by flow cytometry. Although the DDX4-expressing cells identified in the testis were indeed gonocytes, the cell-surface DDX4-immunoreactive cells expressed negligible DDX4 mRNA and protein levels. Furthermore, they did not express other germ cell markers, such as ZBTB16, NANOS2, and DAZL, but prominently expressed early primordial germ cell markers, such as PRDM1, IFITM3, and EPCAM. Nonetheless, the cell-surface DDX4-immunoreactive cells generated neither germ cell colonies nor teratomas following transplantation into immunocompromised mouse testes. Taken together, these results demonstrate that testicular cell-surface DDX4-immunoreactive cells are not germ cells and constitute a distinct subpopulation that is different from gonocytes. Moreover, the subpopulation in porcine testes might be species specific because no DDX4-immunoreactive cells were found in postnatal mouse testes.
Keywords: male germline stem cells; prespermatogenesis; prospermatogonia; testis.