Mobilization of shell calcium by the chick chorioallantoic membrane in vitro

Two explants of shell were removed from each of several fertile eggs of domestic fowl at different times during incubation. The chorioallantoic membrane (CAM) was removed from one of the explants (SHELL ONLY) and was left in situ on the other (SHELL+CAM). Explants were cultured for 24, 48 or 96 h at 37 degrees C and 5% CO2 in air in individual Petri dishes containing Dulbecco's modified Eagle's medium, bovine serum albumin, penicillin and streptomycin. Both SHELL+CAM and SHELL ONLY explants released calcium into the culture medium, but the former released considerably more calcium than the latter. More calcium was released by SHELL+CAM explants taken from older eggs than from younger ones, but the age of the donor eggs did not affect release of calcium by SHELL ONLY explants. In addition, release of calcium by SHELL+CAM explants exceeded that shown by SHELL ONLY explants for multiple 24 h intervals. However, the capacity for sustained release of calcium by SHELL+CAM explants declined with age and maturity of the CAM. Manipulations that lead to the death of the CAM abolish the capacity for SHELL+CAM explants to release more calcium than SHELL ONLY explants. Differential release of calcium by SHELL+CAM explants was not attributable to calcium present in the CAM at the onset of culture or to non-specific degradation of the shell by intracellular constituents released as a result of the death of the CAM. Taken in concert, these results indicate that the CAM mobilizes calcium from the eggshell during in vitro culture.