ISOLATION OF HISTONES AND RELATED CHROMATIN STRUCTURES FROM SPERMATOZOA NUCLEI OF A DASYURID MARSUPIAL, SMINTHOPSIS-CRASSICAUDATA

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, ha ve two distinct nuclear regions: uniformly electron-dense chromatin (C 1) in the interior and fissured chromatin (C2) at the periphery. To in vestigate whether the differences in morphology are due to incorporati on of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis ( PAGE) and fractionated by reverse-phase high-pressure liquid chromatog raphy (HPLC). The main protein component was protamine I, but a comple te histone complement (H1, H2A, H2B, H3, and H4) was also detected. Im munocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chro matin. The fissures in the chromatin of this region disappeared follow ing incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiest er charges remain negative in nucleohistone DNA, whereas all DNA charg es are neutralised in highly compact nucleoprotamines. Treatment of sp ermatozoa with micrococcal nuclease showed that the C1 chromatin was r esistant to digestion, whereas the C2 region was cleaved into 30- to 3 8-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this st udy demonstrates that spermatozoa nuclei of this marsupial species con tain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with t he rest of the nucleus. (C) 1997 Wiley-Liss, Inc.