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Abstract
Immunohistochemical studies with antiserum against the protamines of the toad, Bufo
japonicus, revealed that the sperm nucleus loses protamines within 5 min after entry
into the egg. Likewise, lysolecithin-permeabilized sperm incubated with the egg extract
lose the protamines within 1 min, accompanied by nuclear decondensation. The activities
that induce both protamine removal and decondensation in sperm nuclei were found in
extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula
embryos or adult tissues. SDS-PAGE analyses revealed that the egg extract removed
not only protamines from the Bufo sperm, but also selectively the sperm-specific basic
proteins from sperm nuclei of Xenopus laevis. The protamine-removing activity (PRA)
was partially purified from egg extracts as negatively charged macromolecules by anion-exchange
chromatography and gel filtration. The PRA was heat-stable (100 degrees C, 10 min)
and sensitive to proteinase K, but not to RNase A and DNase I. Immunoblot analysis
of the supernatant after incubation of Bufo sperm in the fraction with the PRA revealed
that protamines derived from sperm nuclei were associated with a major protein of
the fraction. This protein exhibited mobilities of 140 and 36 kDa on native- and SDS-PAGE,
respectively, with the isoelectric points in the range 4.2 to 4.5 and possessed an
amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We
propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin
by binding to but not by enzymatic degradation of the protamine.