IN-VITRO DEVELOPMENT OF THE SEA-URCHIN MALE PRONUCLEUS

We have developed a cell-free extract from fertilized or unfertilized sea urchin eggs which promotes formation of male pronuclei from exogen ously added permeabilized sperm nuclei. Using a buffer to simulate egg cytoplasmic conditions, three states of nuclear condensation can be d istinguished: condensed (conical), partially decondensed (conical or o void), and decondensed (spherical). The in vitro system meets several in vivo criteria established by microinjection experiments. Decondensa tion is promoted at elevated pH and in activated egg cytoplasm, but do es not require Ca2+. Pronuclear development is supported to > 100 male nuclei per egg-equivalents as in vivo. Pronuclear development require s addition of an ATP-generating system and is blocked by two kinase in hibitors (6-DMAP and staurosporine) at the same concentrations effecti ve in vivo. Decondensed nuclei form by 40 min of incubation and acquir e a putative nuclear envelope shown by exclusion of 150 kDa FITC-dextr an by 1-2 hr. The rates of decondensation and nuclear envelope formati on are accelerated by addition of GTP. Protease inhibition experiments suggest a role for nonhistone protein degradation in pronuclear progr ession. This system should prove useful for investigating mechanisms o f the postmeiotic sea urchin male chromatin remodeling which follows f ertilization, previously accessible only in vivo. (C) 1994 Academic Pr ess, Inc.