The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells.

In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF-β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26-28 Hamburger-Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder-free substrates. The results showed that TGF-β signaling agonists (IDE1 and Activin-A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF-β, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency-associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.