Formation of the sea urchin male pronucleus in cell-free extracts

Abstract

At fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear transformation observed in vitro is the disassembly of the sperm nuclear lamina as a result of lamin B phosphorylation mediated by egg protein kinase C. The conical sperm nucleus then decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP- dependent fusion of vesicles with one another. Three cytoplasmic vesicle fractions with distinct properties are required for the formation of the male pronuclear envelope. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the NE. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus, formation of the male pronuclear envelope involves a highly ordered series of reactions. (C) 2000 Wiley-Liss, Inc.