Immunofluorescence techniques for determining the numbers of inner and outer blastomeres in mouse morulae

Methods are described for distinguishing inner and outer blastomeres of compact 8- to 32-cell mouse morulae. The first involves the selective labelling by immunofluorescent reagents of the exposed surface of the compact morula, disaggregation of the morula into single blastomeres and separation of these blastomeres into partially labelled (presumptive outer) and unlabeled (presumptive inner) populations. The second involves labelling blastomeres after disaggregation and is based on the recent observation that the sera on an isolated outer blastomere which originally contributed to the exposed surface of the intact morula labels more intensely than the remaining (non-exposed) surface of the blastomere. Analysis of the labelling patterns obtained from individual disaggregated morulae indicated that inner blastomeres were absent from compact 8-cell morulae, but increased in number throughout the next cleavage division until at the 16-cell stage the mean number of these blastomeres varied from 4.6 to 6.6 (with a range of 1--8) depending on the technique used. At later stages, the numbers of inner blastomeres can probably be accounted for by division of the inner cells at the 16-cell stage.