Nanos2 promotes differentiation of chicken (Gallus gallus) embryonic stem cells to male germ cells

Nanos2 is an evolutionarily conserved RNA-binding protein containing 2 CCHC-type zinc finger motives. Here, we report that Nanos2 is strongly expressed in the testis compared to other tissues in chicken (Gallus gallus). Overexpression and knockout plasmid vectors were constructed, and in-vitro Cas9/gRNA digestion and T7 endonuclease I (T7E1) assay indicated that Nanos2-g1 possessed the highest knockout activity. In vitro and in vivo, Nanos2 overexpression accelerated the production of embryoid bodies (EBs) and SSC-like cells and promoted cvh, c-kit, and integrin 6 expression. Immunofluorescence staining, periodic acid schiff (PAS) and flow cytometry (FCM) assay showed that primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) formation were significantly promoted. On the contrary, Nanos2 knockout delayed the production of EBs and SSC-like cells and correspondingly reduced cvh, c-kit, and integrin 6 expression. Simultaneously, the quantity of PGCs and SSCs was blocked. Collectively, these results uncovered a novel function of Nanos2 involved in chicken male germ cell differentiation, where it acts as a facilitator. To clarify the roles of Nanos2 on chicken male germ cell formation, combined with the RA induction system and chicken embryo injection system, we adopted overexpression and knockout in vitro and in vivo whereby biological function of Nanos2 was explored. The results indicate that Nanos2 is required for chicken male germ cells formation, which contributes to perfect chicken ESCs in vitro induction system and improvement of induction efficiency of differentiation of chicken ESCs into male germ cells.