Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells

Many growth factors or cytokines regulate cell proliferation via different intracellular signaling pathways. The mechanisms remained quite unclear in avian primordial germ cells (PGCs). In the present study, two major protein kinases, PKA and PKC, were investigated to be involved in signal transduction of PGC proliferation. PGCs were isolated from genital ridge of 3.5-day chicken embryos and primary culture was performed with 5% fetal calf serum (FCS)-supplemented medium 199. After culture for 24 h, PGCs were subcultured on chicken embryonic fibroblast feeder (CEF) and the cells were characterized by histochemical stainings of alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) reagent as well as immunocytochermical stainings of c-kit and stage-specific embryonic antigen-1 (SSEA-1). In addition, cells were challenged with adenylate cyclase activator forskolin (FRSK) and PKC activator phorbol-12-myristate-13-acetate (PMA) alone or in combinations with PKA inhibitor H-89 and PKC inhibitor H-7, respectively. Results showed that subcultured PGCs on CEF displayed positive histochemical and immunocytochemical stainings for ALP, PAS, c-kit and SSEA-I and manifested intensive proliferating activity by colony formation. Downstream activation of PKA by FRSK (10(-7) to 10(-5) M) significantly promoted the proliferation of PGCs by increasing colony number (ALP-stained) in a dose-dependant manner. PMA (10(-8) M) also increased PGC colony number (P < 0.05). However, the proliferating effects elicited by FRSK or PMA could be inhibited by the respective protein kinase inhibitor H-89 or H-7. Therefore, the above results suggest that activation of intracellular protein kinases A and C by external factors may promote proliferation of cultured PGCs and PKA represents the most likely mediator of PGC proliferation in embryonic chickens. (c) 2006 Elsevier B.V. All rights reserved.