Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract

Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure, Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected, Consistent with this data, the ammount of lamin B-3, determined by immunoblotting, was not affected through the extraction procedure, Nuclear matrices were visualised in DGD sections by TEM, Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments), To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM), This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B-3 antibodies, Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with anti-lamin B-3 antibodies, Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B-3 depleted extracts. Using FEISEM, filaments were also detected in these preparations, However, these filaments were poorly organised and often appeared to aggregate, To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments, Many (but not all) of these filaments were labelled with anti-lamin B-3 antibodies, In contrast, nuclear matrices prepared from 'lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B-3 antibodies.