[Morphological and biochemical characterization of the developmental stages of fertilized eggs inSphaerechinus granularis Lam : II. DNA content, DNA polymerase activity and DNase activity].

The investigations were performed with the eggs of the sea urchin speciesSphaerechinus granularis Lam. They were kept at 22° C under continuous aeration for up to 45 hours with stirring to compensate for sedimentation. 1. The change in DNA content, 2. the change in DNA dependent DNA polymerase activity, and 3. the change in DNase activity with time have been evaluated. 1. DNA Content of Embryos. The DNA content of the embryo development was determined by two different methods. Before and immediately after fertilization DNA content has been found to be 1.7±0.5·10-10 g per egg. This amount is about 100 times higher than in diploid nuclei. Three periods with different rates of DNA synthesis may be distinguished: a) the first one, lasting from fertilization to about the time of the volume maximum just before the onset of gastrulation with an average rate of synthesis of 1.2·10-10g DNA per minute per embryo; b) a second one, lasting from then on to the gastrula stage with a lower average rate of synthesis of about 0.7·10-12 g DNA per minute per embryo; c) a third one, starting from the gastrula stage up to the experimental end point in the pluteus stage. The rate of synthesis in this case is 2.3·10-12 g DNA per minute per embryo. On a relative base the rates of synthesis are 100∶58∶192. The cytoplasmic, extramitochondrial DNA persists through the stage of the first period of the embryogenesis, up to the blastula stage. The amount of extranuclear DNA increases in the first 6 hours of embryo development; then the cytoplasmic DNA disappears. 2. DNA Dependent DNA Polymerase Activity. The DNA polymerase has been isolated from embryos. Its activity has been determined in relation to the activity of the total embryo as well as per embryonic cell. The polymerase activity is much higher at the start of the development than in later stages, reaching a minimum in the blastula stage, the time at which cytoplasmic DNA has been exhausted. In the subsequent period the polymerase activity parallels the rate of DNA synthesis in vivo. The level of the DNA polymerase activity per cell remains constant. 3. DNase Activity. The DNase activity has been determined using the Lanthanum-Nitrate-Method. Three distinct maxima were found: A first maximum is reached immediately upon fertilization. The second one coincides with the onset of mesenchyme formation in the blastula, and the third one coincides with the end of gastrulation. The average specific activity is roughly equivalent to about 10-6 g DNase I per g of embryo. The possibility is discussed that rises in nucleolytic activities may trigger differentiation events in the developing egg. The influence of DNA polymerase activity and DNase activity on in vivo DNA synthesis is discussed.