Fig. 10. Effect of antisense oligonucleotide and anti-pancytokeratin C11 antibody injection on the ultrastructure and molecular composition of germinal granules in four-cell embryos. (A) Electron microscopy of sections of germ plasm islands from four-cell embryos hybridized in situ, as whole mounts, with digoxigenin-labeled Xcat2 RNA probe. Hybridization signal was detected using nanogold-conjugated anti-digoxigenin antibody and silver enhancement (see Kloc et al., 2002a). In control uninjected embryos (1, inset 1') and embryos injected with antisense Xpat ODN (2, inset 2'), the germinal granules (arrows) labeled with black silver grains (representing Xcat2 mRNA) are visible between mitochondria (m) and yolk platelets (Y). In embryos injected with antisense Xlsirts ODNs (3, inset 3') the germinal granules (small arrows) are devoid of label, and Xcat2 mRNA (black silver grains, large arrows) is located outside the germinal granules. In embryos injected with antisense VegT ODN (4), the germinal granules (small arrows) form huge aggregates containing low level of Xcat2 mRNA (small black silver grains, large arrows). In embryos injected with anti-pancytokeratin C11 antibody (5) the germinal granules form huge aggregates (small arrows), similar to aggregates in antisense VegT embryos, but heavily labeled with black silver grains (large arrows). Scale bars: 320 nm in 1-5, and 160 nm in 1'-3'. (B) Student's t-test statistical analysis of the number of silver grains (Xcat2 mRNA) associated with germinal granules. Each bar represents the average (with the standard deviation) number of silver grains counted in 10 independent samples in 0.25 µm² area of germinal granule in each experimental group. Number above the graph represents statistically significant (P<0.05) P value. The graph shows that the decrease in the number of silver grains (Xcat2 mRNA) present in the germinal granules of antisense Xlsirts and antisense VegT embryos is statistically highly significant (P=0.00001 and P=0.001, respectively).