HYBRID NUCLEOPROTEIN PARTICLES CONTAINING A SUBSET OF MALE AND FEMALEHISTONE VARIANTS FORM DURING MALE PRONUCLEUS FORMATION IN SEA-URCHINS

To determine the changes in chromatin organization during male pronucl eus remodeling we have compared the composition of nucleoprotein parti cles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated fro m sea urchin gametes and zygotes containing a partially decondensed (1 5 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) mal e pronucleus and digested with nucleases. The NP-ps generated were ana lyzed in agarose gels, and their histone composition was determined. S perm core histones (SpH) and cleavage stage (CS) variants were identif ied by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, whic h migrated in agarose gels between DNA fragments of 1.78-4.26 Kb. Sper m chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type o f NP-ps was obtained by digestion of unfertilized egg nuclei, either w ith ENase or MNase; the NP-ps was located in the region of the agarose gel corresponding to DNA fragments of 3.4-1.95 Kb [Imschenetzky et al . (1989): Exp Cell Res 182:436-444]. When whole nuclei from zygotes co ntaining the female pronucleus and a partially remodeled male pronucle us were digested with ENase, a single NP-ps was generated, which migra ted between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested wi th MNase, two NP-ps were generated; the slower migrating NP-ps (s) was located in the same position of the agarose gel as those resulting fr om ENase digestion and the faster migrating NP-ps (f) migrated between DNA fragments of 1.95-1.26 Kb. It was found that Np-ps (s) contained only CS histone variants, whereas NP-ps (fl were formed by a subset of SpH and by CS histone variants. When nuclei from zygotes containing a fully decondensed male pronucleus were digested either with ENase or MNase, a single type of NP-ps was observed, which migrated in the same position as NP-ps (s) in agarose gels. This particle contained only C S histone variants. On the basis of the histone compositions and on el ectrophoretic similarities, it was concluded that NP-ps (s) originated from the female pronucleus and that NP-ps (f) were generated from the partially remodeled male pronucleus. Consequently, our results indica te that at an intermediate stage of male pronucleus remodeling the chr omatin is formed by NP-ps containing a subset of both SpH and-of CS hi stone variants, whereas at final stages of male pronucleus decondensat ion chromatin organization is similar to that of the female pronucleus . (C) 1996 Wiley-Liss, Inc.