Development of novel markers for the characterization of chicken primordial germ cells

Abstract

This study was undertaken to develop novel markers for chicken primordial germ cells (PGCs), which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a Dulbeccos minimal essential medium and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 hours after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain, antibodies to stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins α6 and β1, several lectins (Solanum tuberosum agglutinin [STA], Dolichos biflorus agglutinin [DBA], concanavalin A agglutinin [ConA], and wheat germ agglutinin [WGA]), and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin α6, or integrin β1 and then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin α6, and integrin β1. The control mouse ES cells reacted with PAS, anti-SSEA-1, and anti-EMA-1 antibodies, as well as with antibodies to integrins α6 and β1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3 and SSEA-4 and integrins α6 and β1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.