Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis

Gonocytes are primitive germ cells that are present in the neonatal testis and are committed to male germline development. Gonocytes differentiate to spermatogonia, which establish and maintain spermatogenesis in the postnatal testis. However, it is unknown whether large animal species have pluripotency-specific proteins in the testis. Nanog and Pou5f1 (Oct3/4) have been identified as transcription factors essential for maintaining pluripotency of embryonic stem cells in mice. Here, we show that NANOG protein was expressed in the germ cells of neonatal pig testes, but was progressively lost with age. NANOG was expressed in most of the lectin Dolichos biflorus agglutinin- and ZBTB16-positive gonocytes, which are known gonocyte-specific markers in pigs. NANOG was also expressed in Sertoli and interstitial cells of neonatal testes. Interestingly, POU5F1 expression was not detected at either the transcript or the protein level in neonatal pig testis. In the prepubertal testis, NANOG and POU5F1 proteins were primarily detected in differentiated germ cells, such as spermatocytes and spermatids, and rarely in undifferentiated spermatogonia. By using a testis transplantation assay, we found that germ cells from 2- to 4-day-old pigs could colonize and proliferate in the testes of the recipient mice, suggesting that primitive germ cells from neonatal pig testes have stem cell potential.