title: Hybrid nucleoprotein particles containing a subset of male and female histone variants form during male pronucleus formation in sea urchins.
abstract: To determine the changes in chromatin organization during male pronucleus remodeling, we have compared the composition of nucleoprotein particles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated from sea urchin gametes and zygotes containing partially decondensed (15 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) male pronucleus and digested with nucleases. The NP-ps generated were analyzed in agarose gels, and their histone composition was determined. Sperm core histones (SpH) and cleavage stage (CS) variants were identified by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, which migrated in agarose gels between DNA fragments of 1.78-1.26 Kb. Sperm chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type of NP-ps was obtained by digestion of unfertilized egg nuclei, either with 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 containing the female pronucleus and a partially remodeled male pronucleus were digested with ENase, a single NP-ps was generated, which migrated between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested with 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 from 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 (f) 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 CS histone variants. On the basis of the histone compositions and on electrophoretic 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 indicate that at an intermediate stage of male pronucleus remodeling the chromatin is formed by NP-ps containing a subset of both SpH and of CS histone variants, whereas at final stages of male pronucleus decondensation chromatin organization is similar to that of the female pronucleus.
authors.affiliation: Department of Molecular Biology, Universidad de Concepción, Chile. mimschen@buho.dpi.udec.cl
authors.forename: M
authors.initials: M
authors.lastname: Imschenetzky
authors.forename: M I
authors.initials: MI
authors.lastname: Oliver
authors.forename: S
authors.initials: S
authors.lastname: Gutiérrez
authors.forename: V
authors.initials: V
authors.lastname: Morín
authors.forename: C
authors.initials: C
authors.lastname: Garrido
authors.forename: A
authors.initials: A
authors.lastname: Bustos
authors.forename: M
authors.initials: M
authors.lastname: Puchi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry*; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases / Q000494:pharmacology; D005260:Female; D014644:Genetic Variation; D006657:Histones / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D008297:Male; D008836:Micrococcal Nuclease / Q000494:pharmacology; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry; D012617:Sea Urchins; D013094:Spermatozoa / Q000737:chemistry*; D015053:Zygote / Q000737:chemistry*
chemical_list: D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D004247:DNA; D004720:Endonucleases; D008836:Micrococcal Nuclease