Regulatory Mechanism About Proliferation Of Primordial Germ Cells From Embryonic Chickens

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 from the gonadal ridges, and then the differentiating potency was examined. In addition, the effect of different somatic cells and two flavonoids (daidzein and quercetin) on proliferation of cultured PGCs were evaluated, together with the underlining mechanisms.1. Establishment of chicken PGC primary culture and subculture modelsGenital ridges were collected by dissection of chicken embryos at 3.5-4 days with a fine glass needle under a microsurgery scope and then were dispersed into single cells in a solution of 0.25% trypsin:0.02% EDTA. PGCs and somatic cells were co-cultured with medium 199 (containing 5%FCS) at 39°C in a water-saturated atmosphere of 95% air and 5% CO₂. The results showed that 5%FCS displayed a better pro-proliferating effect in the primary culture. While in subculture, it was found that the supplementation of FCS, ITS (insulin 10μg/ml, transferring 5μg/ml and selenite3×10^-8 mol/L) and conditioner medium (CM) could maintain survival and promote conglomerates formation of PGC (P<0.05). Staining of alkaline phosphatase (AKP), periodic acid-Schiff regent (PAS), c-kit and stage-specific embryonic antigen-1 (SSEA-1) immunocytochemistry all confirmed the characteristics of cultured PGCs. Proliferating cell nuclear antigen (PCNA) labeling suggested that the subcultured PGCs after 60h manifested positive staining, which indicated that PGCs possessed intensive proliferating activity. In addition, PGCs could form different cells, such as neuronal cells, epithelial and skeleton muscle cells according to cell morphology. The immunological characteristic of the differentiated cells could be identified by neuron specific enolase (NSE) and keratin immunocytochemical staining. The above results indicated that the primary and subculture models of PGCs could be used for studies about regulation of PGC proliferation and differentiation.2. Supporting effect of somatic cells on PGC proliferationThree different somatic cell feeder layer, including chicken fibroblast feeder (CEF), skeletal myoblast cell feeder layer (SMF) and follicular granulosa cell feeder (GCF) were adopted to screening the optional sustaining system for PGC culture in vitro. At the same time the effects of different growth factors or cytokines on PGC proliferation were detected with the media supplemented with FCS, ITS and leukemia inhibitory factor (LIF). BrdU incorporation was determined for the proliferate activity of PGCs. The effects of FCS, LIF and ginsenoside (GS) on subcultured PGCs were also evaluated. Results showed that the effects of CEF and GCF were superior to SMF on PGC proliferation and the number of PGC colonies was more than the latter significantly(P<0.05), and BrdU labeling displayed more intensive staining in PGC subculture on CEF than on GCF. On all the three feeder layers, medium with 0.5%FCS+LIF (10ng/ml) manifested better pro-proliferating effect on PGC colonies formation than media with FCS and ITS. Active proliferation of PGCs was found after 5～6d in subculture and large colonies were formed especially in the group treated with GS (10μg/ml). The above results indicated that chicken embryonic fibroblast feeder represented an optional sustaining system for chicken PGC culture and the low concentration FCS, LIF and antioxidant nutrient GS could promote PGC proliferation significantly.3. Regulation of protein kinases A and C on PGC proliferationInvolvement of intracellular signaling systems in PGC proliferation were evaluated by respective activation or inhibition of protein kinases A and C with the PGC culture model. The cultured cells were challenged with cyclic adenosine monophosphate (cAMP) kinase activator forskolin (FRSK) or phorbol-12-myristate-13-acetate to activate protein kinases A and C, or in combinations with their respective inhibitor H₈₉ or H₇ in subculture. BrdU incorporation was used to detect cell proliferation. Results showed that FRSK (10^-7～10^-5M) significantly stimulated PGC proliferation in a dose-dependent manner (PO.05), while this effect could be inhibited by H₈₉ at 10^-5M; PMA (10^-8M) also significantly increased PGC proliferation (P<0.05), but it was inhibited by H₇ (10^-7～10^-5M). BrdU-labeling index increased after treatment of FRSK and PMA. These data suggested that activation of protein kinases A and C might accelerate proliferation of PGCs.4. Effects of flavonoids on PGC proliferationThe effects of two flavonoids daidzein (DAI) and quercetin (QUE) on PGC proliferation was evaluated with elucidation of the underlining mechanisms. Results showed that DAI (1μg/ml) and QUE (0.01～1μg/ml) promoted proliferation of the cultured PGCs in a dose-dependent manner. DAI and QUE displayed toxicity on PGCs at 10μg/ml. Reactive oxygen substances (ROS) produced from hypoxanthine/xanthine oxidase (HX/XO) system caused serious oxidative damage on PGCs and the number of PGCs was significantly reduced (.PO.05). However, combined treatment of DAI and QUE could significantly attenuate the oxidative damage induced by ROS and this effect was further confirmed by analysis of superoxide dismutase (SOD) activity and glutathione (GSH) levels (P<0.05). In addition, it was revealed that PKA signal transduction pathway was involved in DAI and QUE-stimulated PGC proliferation. Furthermore, estrogenic effect of DAI or QUE was not related to the proliferation of PGCs. So it was deduced that the pro-proliferating effect of DAI and QUE on PGCs was mediated by antioxidative action involving PKA signaling pathway.The above results indicated that the primary and subculture models of PGCs from 3.5⁴d chicken embryonic germinal ridge 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 immunocytochemistry. The pluri-potency of PGCs was also demonstrated. PKA and PKC signaling pathways were found involve in PGC proliferation and vegetal flavonoids DAI and QUE could promote PGC proliferation through antioxidant action. All these data indicated that the chicken PGC culture model could be use to explore the regulating mechanisms of exogenous agents including nutrients on proliferation and differentiation of PGCs. On the other hand, we may promote PGC proliferation by using vegetal bioactive materials, via intracellular signaling pathways, to provide theoretic guidance and experimental platform for improving poultry reproductive performance and preparation of chimeras and transgenic poultry.