SEA-URCHIN SPERM NUCLEAR ENLARGEMENT AND SHAPE TRANSFORMATIONS ARE DIFFERENTIALLY REGULATED IN-VITRO

To elucidate the mechanisms regulating morphogenesis of the sperm nucl eus into a male pronucleus after fertilization, we have used an in vit ro cell-free system derived from sea urchin eggs. Demembranated sperm nuclei were added to extracts prepared from either unfertilized eggs ( unfertilized extract) or fertilized eggs (fertilized extract), and the area and shape of the sperm nuclei were measured using video-intensif ied fluorescence microscopy coupled with image analysis. Sperm nuclei did not decondense in buffer used to prepare the extracts, indicating decondensation factors are of maternal origin. Unfertilized extract su pported sperm nuclear decondensation to a greater extent than fertiliz ed extract. This was not due to egg activation nor different preparati on methods, but was due to the fertilizing sperm nucleus binding or in activating diffusible decondensation factors within 10 min. Sperm nucl ei needed constant exposure to egg cytoplasm for greater than 30, min for decondensation to proceed. Although sperm nuclear decondensation w as temperature sensitive, a thermostable (100 degrees C) decondensing factor was present in egg cytoplasm in limited quantities. Sperm nucle ar decondensation was ATP-dependent, pH-sensitive, and required kinase activity, including tyrosine kinase activity. Shape transformations i n fertilized extract were insensitive to 6-dimethylaminopurine indicat ing the area and shape alterations are regulated differentially. In ad dition, alterations of shape, but not area, were sensitive to serine p rotease inhibitors, suggesting proteases change the sperm nuclear matr ix, thereby altering shape. Sperm nuclear decondensation factors and t heir targets were highly conserved between sea urchin species, demonst rating a common decondensing mechanism is utilized, which may be appli cable to other species. (C) 1997 Wiley-Liss, Inc.