Occurrence of H1 subtypes specific to pronuclei and cleavage-stage cell nuclei of anuran amphibians

Abstract

Male pronuclei were induced in vitro by incubating the lysolecithin-permeabilized sperm nuclei of anuran amphibians Bufo japonicus and Xenopus laevis with the extracts from homologous eggs, and nuclear basic proteins were analyzed by two-dimensional gel electrophoresis. By 5 min after the incubation, the sperm nuclei had completely lost sperm-specific proteins but possessed a whole set of nucleosomal core histones, except that H2A was mostly replaced by a putative H2A.X. The pronuclei did not possess any H1 histone subtypes found in differentiated somatic cell nuclei, but instead possessed components showing electrophoretic mobilities distinct from other H1 histones. This protein, designated as H1X, was regarded as an H1 subtype on the basis of its solubility in acids or salt and its lysine-rich amino acid composition. Peptide mapping using V8 protease further indicated that H1X is not a post-transcriptionally modified form of other somatic type H1 histones. By the time of chromosome condensation, pronuclear histones comprised H1X, H2A.X, H2B, H3, and H4. During embryonic development, H1X persisted in the nuclei up to the blastula stage, but mostly disappeared at the gastrula stage concomitant with the appearance of H1 subtypes of differentiated cells and the replacement of H2A.X by H2A. We conclude that H1X represents a maternally stored histone variant specific to nuclei of cleaving embryos.