Effect Of Retinoic Acid On Proliferation And Adhesion Of Chicken Primordial Germ Cells

PGCs, as precursor cells developing into ova and spermatozoa in embryonic stage, are model type cells for genome research and transgenic application in a variety of vertebrates. Recent years, the limit of great number and stability of cultured embryonic PGCs made the research of avian PGCs attenuated. Coordinating the proliferation and aggregation of cultured PGCs is a crucial method to get enough and survival cells for various studies.Intercellular connection plays an important role in male stem spermatogonium, spermatogonium, spermatocytes, and the female oogonia. In the mouse, PGCs connect to each other in the process of migrating to genital ridge until forming cell colonies. Proliferation and cellular aggregation were both crucial features on survival and self renewal of primordial germ cells (PGC). Adhesive proteins play pivotal roles in cell-cell adhesion and signal exchanges under the influence of many cytokines, growth factors and bioactive metabolites such as retinoic acid (RA). RA is a metabolite of vitamin A with essential biological activity. For many types of cells, RA plays an important role in regulating cell proliferation and differentiation. RA synthesis and signaling play an important role during early organogenesis.In this study, chicken PGC culture system was adopted to evaluate the effect of RA on germ cell aggregation by demonstration of the changes in expression of E-cadherin andα/βcatenins mRNAs. At the same time, the change in cell proliferation was also detected by the colony number and area of PGC aggregates. The results will facilitate to elucidate the regulation of PGCs proliferation by cell-cell adhesion.PGCs were isolated from the genital ridge of 4-day chicken embryos and cultured on chicken embryonic fibroblast feeder. Cells were primary seeded at 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS) and 10 ng/ml LIF in 6-well culture plates. Cells were cultured at 39℃in a water-saturated atmosphere of 5% CO2. After 48h culture, PGC colonies were picked up and dispersed, and then subcultured on CEF in medium supplemented with 0.5% FCS. Subcultured PGCs were treated with RA (10-7-10"5 M) along and in combinations with PKC inhibitor H7 (10-7-10-5 M) for 24 h, respectively.After culture, the PGCs proliferated from single cells to double or more cells. These cells colonized together to become cell aggregates, finally form compact PGCs colonies. PGCs were detected by SSEA-1 immunofluorescence and the nuclei were stained with DAPI Cultured PGCs colonies were also identified by SSEA-1/3 immunocytochemical staining with brown color. Slices of genital ridge from 5.5d chicken embryo were stained by SSEA-1 immunofluorescence method, with significant cell-surface red fluorescence for chicken PGCs clusters. In addition, expression of several pluripotency-associated genes including POU5F1, NANOG and SOX2 mRNA in PGCs were examined in cultures PGCs after treatment with 10-6 M RA for 24h. There was no significant difference in the mRNA abundance between the treated cells and the control.Adhesion index and expression of E-cadherin, a-catenin andβ-catenin of PGCs were determined to detected impact of RA on PGC adhesion. RA (10-7 M) reduced the adhesion index from 0.67 to 0.60 with 10% decrease, while in 10-6 M RA-treated cells, the adhesion index was reduced to 0.37 with 45% decrease with significant difference. No further decrease in the adhesion index was achieved after higher RA treatment (10-5 M). At the same time, the mRNA expression of relative adhesion proteins was altered. Compared with the control group, after treatment with RA at 10-7,10-6 and 10-5 M, the increase of E-cadherin mRNA expression were increase at 5%(P> 0.05), 18% and 21%(P< 0.05). The a-catenin mRNA was increased at 8%,22% and 24%, andβ-catenin was increased at 7%,25% and 28.5%(P< 0.05).Though the PKC inhibitor H7 at 10-7 M imposed no significant effect on RA-stimulated reduction of adhesion index of PGCs, higher H7 at 10-6 and 10-5M significantly attenuated the RA-elicited PGC aggregation. Simultaneous treatment of H7 at 10-6 and 10"5M remarkably attenuated the effect of RA on mRNA expression of adhesive proteins E-cadherin,α-catenin andβ-catenin. After treatment with RA at 10-7-10-5M for 24 h, the number and area of PGCs colonies was increased in a dose-dependent manner. However, the RA-elicited PGC proliferation was reduced by the combined treatment of H7. Meanwhile, the three-dimensional cell morphology of PGC colonies was weakened by H7.To further confirm the effect of RA on PGC proliferation, we examined the effect of RA on mRNA expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2 which are considered to be critical factors in G1-S progression in cell cycle.. Treatment with RA at 10"6 M increased the mRNA expression of all four genes significantly. (P<0.05). This effect was hindered by combined H7 treatment.In conclusion, proliferating effect of RA was revealed in cultured chicken PGCs. This stimulating action involves the increased expression of the adhesive proteins E-cadherin andα/βcatenins. RA-elicited effect was attenuated by PKC inhibition. Increased cell proliferation is accompanied with elevated mRNA expression of cyclins CCND1 and CCNE1, CDKs 6 and 2. These results indicated that RA promoted PGCs proliferation and strengthened aggregation of PGCs via E-cadherin andα/βcatenins expression through PKC signaling pathway in chicken PGCs.