NUCLEAR LAMINA AND NUCLEAR MATRIX ORGANIZATION IN SPERM PRONUCLEI ASSEMBLED IN XENOPUS EGG EXTRACT

Nuclear lamina and matrices were prepared from sperm pronuclei assembl ed in Xenopus egg extracts using a fractionation and extraction proced ure, Indirect immunofluorescence revealed that while chromatin was eff iciently removed from nuclei during the extraction procedure, the dist ribution of lamins was unaffected, Consistent with this data, the ammo unt of lamin B-3, determined by immunoblotting, was not affected throu gh the extraction procedure, Nuclear matrices were visualised in DGD s ections by TEM, Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nu cleus (internal nuclear matrix filaments), To improve resolution, nucl ear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM), This techniqu e revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filame nts were readily labelled with monoclonal anti-lamin B-3 antibodies, F ilaments lying within the body of the nuclear matrix were highly branc hed but were not readily labelled with anti-lamin B-3 antibodies, Nucl ear matrices were also prepared from sperm pronuclei assembled in lami n B-3 depleted extracts. Using FEISEM, filaments were also detected in these preparations, However, these filaments were poorly organised an d often appeared to aggregate, To confirm these results nuclear matric es were also observed as whole mounts using TEM. Nuclear matrices prep ared from control nuclei contained a dense array of interconnected fil aments, Many (but not all) of these filaments were labelled with anti- lamin B-3 antibodies, In contrast, nuclear matrices prepared from 'lam in depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B-3 antibodies.