In vitro development of the sea urchin male pronucleus

Abstract

We have developed a cell-free extract from fertilized or unfertilized sea urchin eggs which promotes formation of male pronuclei from exogenously added permeabilized sperm nuclei. Using a buffer to simulate egg cytoplasmic conditions, three states of nuclear condensation can be distinguished: condensed (conical), partially decondensed (conical or ovoid), and decondensed (spherical). The in vitro system meets several in vivo criteria established by microinjection experiments. Decondensation is promoted at elevated pH and in activated egg cytoplasm, but does not require Ca2+. Pronuclear development is supported to > 100 male nuclei per egg-equivalents as in vivo. Pronuclear development requires addition of an ATP-generating system and is blocked by two kinase inhibitors (6-DMAP and staurosporine) at the same concentrations effective in vivo. Decondensed nuclei form by 40 min of incubation and acquire a putative nuclear envelope shown by exclusion of 150 kDa FITC-dextran by 1-2 hr. The rates of decondensation and nuclear envelope formation are accelerated by addition of GTP. Protease inhibition experiments suggest a role for nonhistone protein degradation in pronuclear progression. This system should prove useful for investigating mechanisms of the postmeiotic sea urchin male chromatin remodeling which follows fertilization, previously accessible only in vivo.