The nucleocytoplasmic hybrid combination performed by nuclear graft between B. bufo nucleus and B. calamita egg is 95% lethal during gastrulation mostly at the young gastrula stage (75%) and to a lesser extent at the yolk plug stage (20%). The heterologous B. calamita cytoplasm was treated or fractioned by different techniques including centrifugation, nucleic acid extraction, ammonium sulfate treatment and heating.
The treated cytoplasmic fractions were injected in B. bufo fertilized eggs in order to check their influence on morphogenetic capacities of the B. bufo nucleus. Injection of hyaline cytoplasmic fraction obtained from B. calamita centrifuged eggs blocks the B. Bufo fertilized egg gastrulation in similar rates as in nucleocytoplasmic association performed by interspecific nuclear grafting. Gastrulation, however, is not affected by injection of total nucleic acids extracted from this hyaline fraction. The soluble cytoplasmic fraction obtained after 60% ammonium sulfate precipitation blocks the development at the beginning of gastrulation, this property being inactivated by heating for 5 min at 60°C. Supernatant from 70% ammonium sulfate precipitation blocks the development at the yolk plug stage and is inactivated by heating for 5 min at 80°C.
These experiments suggest that two distinct factors in B. calamita eggs are involved in developmental arrest of injected B. bufo fertilized eggs, these two factors respectively acting on the beginning and on the end of gastrulation.