Sea urchin sperm nuclear enlargement and shape transformations are differentially regulated in vitro

Abstract

To elucidate the mechanisms regulating morphogenesis of the sperm nucleus into a male pronucleus after fertilization, we have used an in vitro 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-intensified 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 supported sperm nuclear decondensation to a greater extent than fertilized extract. This was not due to egg activation nor different preparation methods, but was due to the fertilizing sperm nucleus binding or inactivating diffusible decondensation factors within 10 min. Sperm nuclei needed constant exposure to egg cytoplasm for greater than 30 min for decondensation to proceed. Although sperm nuclear decondensation was temperature sensitive, a thermostable (100°C) decondensing factor was present in egg cytoplasm in limited quantities. Sperm nuclear decondensation was ATP-dependent, pH-sensitive, and required kinase activity, including tyrosine kinase activity. Shape transformations in fertilized extract were insensitive to 6-dimethylaminopurine indicating the area and shape alterations are regulated differentially. In addition, alterations of shape, but not area, were sensitive to serine protease inhibitors, suggesting proteases change the sperm nuclear matrix, thereby altering shape. Sperm nuclear decondensation factors and their targets were highly conserved between sea urchin species, demonstrating a common decondensing mechanism is utilized, which may be applicable to other species. J. Exp. Zool. 277:401–416, 1997. © 1997 Wiley-Liss, Inc.