Genetics and Genomics

Abstract

Primordial germ cells (PGCs), are an efficient approach for transgenic technology . Therefore, an improved expansion of PGCs is required . Although PGC migration is crucial for a genetic continuation to the next generation, the factors and mechanisms that control their proliferation remain largely unknown . In the present study the proliferative effect of transforming growth factor-β (TGF-β) was examined on quail PGCs, employing the number analysis and several factors related to PGC phenotype as an in vitro and the migratory ability of PGCs to gonad as in vivo model . The quail has been proven to be a good model in numerous fields of research, especially for transgenic technology due to short generation time . In this study, initially we attempted to isolate quail PGCs from gonad (at stage 28–30) and blood (at stage 13–15) . The isolated PGC were cultured on feeder cells derived from chicken embryonic fibroblast. The cultured gonadal PGC proliferated about 400times versus 100-times in blood PGC during the first 40–50 d. Upon in vitro exposure to an Activin or IDE1 small molecule, the number of PGCs increased significantly 26% and 64%, respectively. In contrast, the inhibition of TGF-β signaling pathway using SB0431542 resulted in a significant decrease of PGC number after 10 d of treatment. In addition, PGCs were found to be upregulated for phosphorylated SMAD2/3, suggesting that the potential involvement of TGF-β/ SMAD2/3 in the PGC proliferation . Moreover, phosphorylation of SMAD2/3 in IDE1 group was higher than Activin group . The identities of the PGC were confirmed using periodic acid-Schiff (PAS) staining or anti-SSEA1, β-Catenin, β-integrin, and Nanog immunofluorescence staining or reverse transcription-polymerase chain reaction for several factors related to PGC phenotype, DAZL, PRDM14, VASA and Sox2 . Exogenously IDE1 expanded PGCs migrated toward the embryonic gonads when they were transplanted into the heart of the recipient embryo at stage 13–15 Hamburger–Hamilton (HH) . Our results suggest that the application of small molecule IDE1 into quail PGCs represents a step toward achieving an efficient expansion of the PGCs.