Abstract：Primordial germ cells (PGCs) are kind of cells which have the potential to differentiate into germ cells. To investigate the effect of activin A on the survival and proliferation of chicken (Gallus gallus) embryonic blood PGCs cultured in vitro, PGCs were isolated from stage 13~15 chicken embryonic blood, and then the cultured PGCs were identified using PGCs marker antibodies and marker genes. After addition of activin A (0 and 100 ng/mL) into complete medium for 48 h, the cell number, morphology and cell cycle were detected. To study the regulative mechanism of activin A in PGCs proliferation, the DNA methylation level of deleted in azoospermia-like (Dazl) gene was measured. The protein expression and phosphorylation of transforming growth factor-β (TGF-β) and SMAD2/3 were tested by Western blot. The results showed that PGCs isolated from chicken embryonic blood was distinct from blood cells with big nuclear and rich in glycogen particles. The purity of PGCs rapidly rised by treatment with red blood cell lysis buffer (ACK), from (0.026±0.005)% to (69.2±4.6)%. The high purity and excellent growing trend of PGCs was obtained by subculture. After subculture for 3 passages, PGCs was positive with stage specific embryonic antigen 1 (SSEA-1), chicken vasa homologue (CVH) and periodic acid Schiff reaction (PAS). The expression of pluripotent related genes, PouV, Nanog and sex determining region Y (SRY)-box 2 (Sox2), suggested that the biology characterist theics were well maintained in PGCs cultured in vitro. After treated with activin A, numbers of PGCs were significantly increased and the morphology of PGCs was better than that of control group, with clear boundaries and cubic morphology. And the mRNA expression of cyclin-dependent kinases (CDK2)/Cyclin D1 and CDK6/Cyclin E were obviously up-regulated. Flow cytometry analysis confirmed that PGCs populations treated with activin A displayed a significant increase in the proportion of S and G2 phase cells, which meant that there were more PGCs which entered into mitosis process than that of control group. The survival and proliferation ability of PGCs was enhanced by addition of 100 ng/mL activin A. Moreover, the ratio of Dazl gene mythelation was decreased from 94.7% in control group to 52.6% in activin A treated group. However, the mRNA expression of Dazl was significantly increased. The results indicated that activin A promoted PGCs proliferation by reducing the mythelation of Dazl gene. The protein expressions of TGF-β and SMAD2/3 were up-regulated and the phosphorylation of SMAD2/3 was activated when PGCs were treated with activin A. Collectively, these results suggested that activin A significantly promoted the survival and proliferation of PGCs cultured in vitro via TGF-β/SMAD signaling pathway and Dazl mythelation. These data provides basic information for the mechanism of PGCs proliferation regulated by activin A, and provides theoretical foundation to regulate germ cell development by using PGCs culture model.
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