Regulation Of The Chicken Primordial Germ Cell Development

Poultry species has always been used as animal model for the research in developmental biology for profound elucidation of the mechanisms underlining poultry development, especially the reproductive system. The reproductive performance of poultry could be greatly improved by external manipulation based on endogenous regulating mechanisms. In this study primordial germ cells (PGCs) were isolated from Avian chicken embryo, and then subcultured on feed layer after initial primary culture with somatic cells. In addition, the effect of EGF, E2 and Ginsenosides on proliferation of cultured PGCs were evaluated, together with the underlining mechanisms. Meanwhile, we also investigate the effect of RA on the initiation of meiosis in chicken PGCs. These studies will provide theoretic guidance and experimental platform for improving poultry reproductive performance and preparation of transgenic poultry.1. Isolation, culture and identification of chicken PGCsPGCs were collected from stage 14 chicken embryonic blood, stage 19 embryonic genital ridges or stage 27 embryonic gonads with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGCs and somatic cells was seeded onto gelatin-treated 35 mm culture plates at a density of 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS), 10ng/ml leukemia inhibitory factor (LIF),10ng/ml human basic fibroblast growth factor (bFGF), 0.1mmol/L MEM nonessential amino acids, 0.1mmol/L 2-mercaptoethanol, 2mmol/L L-glutamine (GIn), 100U/ml penicillin and 100μg/ml streptomycin. The seeded cells were then maintained at 38.5℃in 5% CO2/95% air with 60%-70% relative humidity until the PGCs colonized as a primary culture. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle, dissociated with 0.25% trypsin-EDTA 5 days after plating and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and treated with 0.25% trypsin-EDTA to achieve single cell suspension and reseeded onto 6-well dishes. After three passages, staining of periodic acid-Schiff regent (PAS), stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) immunocytochemistry, and the expression of the pluripotency-associated genes cPouV, cNanog and Sox2; germ cell-specific genes c-kit, Vasa, Dazl, Stra8 and Scyp3 analysis all confirmed the characteristics of cultured PGC. The above results indicated that the primary and subculture models of PGCs could be used for studies about regulation of PGC proliferation.2. Effects of EGF on proliferation of chicken PGCsIn the present study, the effects of EGF and the EGF-related signaling pathway on proliferation of chicken primordial germ cells (PGC) were investigated. Results showed that EGF (10-100 ng/ml) increased the number and area of PGC colonies in a time and dose-dependent manner. EGF also activated PKC, a process that was inhibited by AG1478 (an EGFR tyrosine kinase inhibitor) and EGTA (an intracellular Ca2+ chelator). In addition, the degradation of IκBαand nuclear factor NF-κB (p65) translocation were observed after EGF treatment, which were significantly blocked by pretreatment with AG1478, EGTA, H7 or SN50 (NF-κB-specific inhibitor). Furthermore, we found that EGF-induced cell proliferation was significantly attenuated by AG1478, EGTA, H7 and SN50, respectively. On the other hand, inhibition of EGFR, Ca2+/PKC or NF-kB abolished the EGF-stimulated increase in the expression of cyclins CCND1 and CCNEI, cyclin-dependent kinase 6 (CDK6), CDK2 and BCL2, and restored the EGF-induced inhibition of BAX expression and caspase3/9 activity, indicating that EGFR, PKC and NF-kB signaling cascades were involved in EGF-stimulated DNA synthesis and anti-apoptosis action. In conclusion, EGF stimulated proliferation of chicken PGCs via activation of Ca2+/PKC involving NF-κB signaling pathway. These observations suggest that EGF signaling is important in regulating germ cell proliferation in the chicken embryonic gonad.3. Involvement of GPR30 on E2-induced proliferation of chicken PGCsIn the present study, The effect of 17β-Estradiol (E2) on chicken primordial germ cells (cPGC) was evaluated and the involvement of GPR30 as well as the underlying signaling pathway were investigated. Results showed that after injection of 100μg/ml E2 into dayO eggs, the percentage of SSEA-1 positive cells in day5.0 embryonic gonad was increased. Using the cPGC culture in vitro system, we first demonstrated that cPGCs express GPR30, however, both ERa and ERβwere not detected. Treatment with (1-100 nM) E2 significantly increased the area and number of cPGC colonies in a time-and dose-dependent manner. E2 also activated Akt, a process that was inhibited by AG1478 (EGFR inhibitor), LY294002 (PI3K inhibitor) or silencing GPR30 expression. However, ER inhibitor ICI182780 had no obvious inhibitory effect on this response. In addition, the phosphorylation of GSK3P and P-catenin translocation were observed after E2 treatment, which were significantly blocked by pretreatment with AG1478, LY294002, KP372-1(Akt inhibitor) and was enhanced by GSK3 inhibitor BIO. Furthermore, we found that E2-induced cell proliferation was significantly attenuated by AG1478, LY294002, KP372-1 or silencing GPR30 and was accelerated by BIO or GPR30 agonist G-1. On the other hand, E2 increased expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2 and protooncogenes (c-fos and c-myc). Increases of these cell cycle regulators and protooncogenes were abolished by inhibition of GPR30, EGFR, PI3K/Akt, or GSK3β/β-catenin. In conclusion, E2 stimulated proliferation of chicken PGCs via GPR30, EGFR, PI3K/Akt, and GSK3β/β-catenin signaling cascades. These observations suggest that E2/GPR30 signaling might play an important role in regulating germ cell development in the chicken embryonic gonad of early stage.4. Effects of ginsenosides on PGC in vitro expansionThe effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of NF-κB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in DMEM supplemented with 5%FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H7 or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-κB and degradation level of IκBαwas investigated by Western blotting analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100μg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H7(10-6-10-8M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10μg/ml stimulated the translocation of NF-κB (p65). However, the NF-κB translocation and the degradation of IκBαwere significantly blocked by combined treatment with H7 (10-6M). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-κB signaling pathway.5. Effect of RA on meiosis initiation of chicken PGCsHere we used the successful long-term culture of chicken PGCs, to investigate if retinoid acid (RA) play a conserved role in regulating entry into meiosis, and does RA act directly on PGCs or indirectly. Results showed that, using organ culture in vitro, addition of RA in stage 27 chicken gonads of both sexes siginificantly increase the mRNA expression of premeiotic gene Stra8, as well as meiotic markers Sycp3 and Dmcl. Using purified chicken PGCs culture with or without feeder layer, RA dramatically upregulated the Stra8 in both male and female PGCs, in parallel to similar increases in expressions of Dmcl and Sycp3. Flow cytometry analysis showed that, after 4-day RA treatment,29.5% male PGCs and 58.37% female PGCs were at sub G1 phase, indicating cells had entered meiosis. Statistatical analysis of giemsa stained cells showed the difference between female and male PGCs in the capability to enter into meiosis induced by RA and reach the zygotene/pachytene stage, male PGCs appeared lower capability to progress beyond zygotene/pachytene. In conclusion, we reported here for the first time that RA could induced chicken PGCs of both sexes to enter meiosis, and female PGCs respond more intensively to RA.The above results indicated that the primary and subculture models of PGCs from chicken embryos of different stages could be used to study the effects of endogenous and exogenous factors on proliferation and differentiation of cultured PGCs. PGCs were characterized by staining of AKP, PAS, c-kit and SSEA-1,3,4 immunocytochemistry. The pluripotency of PGCs was also demonstrated. EGF and GS were found to promote PGC proliferation through PKC-NF-κB signaling pathway. We first reported the involvement of the novel estrogenic receptor,GPR30 in the E2-induced proliferation of chicken PGC. We also first demonstrated the onset of meiosis in chicken PGC induced by RA. These findings provide theoretic guidance and experimental platform for improving poultry reproductive performance and preparation of chimeras and transgenic poultry.