| Embryonic germ cells (Embryonic germ cells, EGCs) are derived from primordial germ cells (PGCs). During early embryonic development, PGCs can proliferate quickly and migrate to the genital ridge, and after entering the genital ridge, PGCs undergo epigenetic reprogramming and ultimately differentiate into gametes. But in appropriate culture condition, PGCs can withdraw from specialization and reprogram to EGCs, which possess multiple differentiation potential. Till now, the studies of the mechanism of EGC specialization mainly based on mice, and the studies on biological characteristics of porcine PGCs, particularly expression patterns of pluripotency and germline markers during porcine PGCs reprogramming rarely reported. However, characterizing porcine PGCs is favorable for the acquisition of porcine EGCs as well as elaborating developmental mechanism of porcine germ cells in vivo. In this study, we observed growth behaviors of primary porcine PGCs, expression patterns of pluripotency and germline markers during their reprogramming into EGCs and the role of bFGF on this process, expecting to cast a light on the mechanism of porcine PGC reprogramming in vitro and providing theoretical support for the derivation of porcine EGCs in vitro. The main findings are as follows:(1) By HE staining of tissue sections of embryos, we observed that the genital ridge was thin and close to the mesonephrost in24-day embryos; the genital ridge became thicker and began to separate from mesonephrost in26-day embryos; the genital ridge developed into spindle-shaped and completely separated from mesonephrost, and it started sexual differentiation in28-day embryos; the genital ridge developed into sexual gonads in30-day embryos.(2) In serum-free media supplemented with different growth factors, freshly isolated PGCs can form EGCs-like colons after5days culture in vitro. In spontaneous differentiation experiment, colons can differentiate into neural cell-like cells, fibroblast-like cells and epithelial cell-like cells; and can form EBs. By immunofluorescence test, we observed the positive expressions of Oct4, Sox2, Nanog and SSEA-3in EGCs-like colons, and by real-time PCR, we found that the expression levels of Oct4, Sox2, Nanog, C-myc and Klf4in EGCs-like colons were significantly higher than those in porcine fetal fibroblast cells. Besides, we observed the expression of Stella in EGCs-like coloning, but the expression of Blimpl was not detected. Real-time PCR showed that the level of Blimp1was significantly lower than that in porcine fetal fibroblast cells, while the level of Ifitm3was significantly higher than that in porcine fetal fibroblast cells, and the levels of Stella and Nanosl made no difference between EGCs-like colons and porcine fetal fibroblast cells.(3) Set three different media:the first group is without any growth factors; the second group contains Lif and SCF cytokines; the third group is the second one adding bFGF. Freshly isolated PGCs with the same density were cultured in the three media, and we found little clones in the first culture medium, a small amount of colons in second culture medium and a large number of colons in the third culture medium, and the average number of colons from a genital ridge is about26in the third group. Then we explored the role of bFGF on colony formation rate, and found that colony formation rate is dependent on the concentration of bFGF and adding time. With5ng/ml bFGF, the number of colons reached a plateau. In order to verify the time-dependent effect of bFGF, bFGF was withdrawed at different time, and the result showed that bFGF could also inhibit apoptosis of PGCs, and the later apoptosis assay verified this finding.In summary, this study on one hand characterized the biological characteristics of porcine PGCs, and on the other hand found bFGF could promote porcine PGCs reprogramming and inhibit apoptosis of PGCs in vitro. |
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