Imschenetzky M, Oliver M I, Gutiérrez S, Morín V, Garrido C, Bustos A, Puchi M
Department of Molecular Biology, Universidad de Concepción, Chile.
J Cell Biochem. 1996 Dec 15;63(4):385-94. doi: 10.1002/(sici)1097-4644(19961215)63:4<385::aid-jcb1>3.0.co;2-p.
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.
为了确定雄性原核重塑过程中染色质组织的变化，我们比较了用内源性核酸酶（ENase）和微球菌核酸酶（MNase）消化后产生的核蛋白颗粒（NP-ps）的组成。从海胆配子和受精卵中分离出完整的细胞核，这些受精卵含有部分解聚的（授精后15分钟，p.i.）或完全解聚的（授精后40分钟，p.i.）雄性原核，并用核酸酶进行消化。对产生的NP-ps在琼脂糖凝胶中进行分析，并确定其组蛋白组成。通过用特异性抗体进行Western免疫印迹鉴定精子核心组蛋白（SpH）和卵裂期（CS）变体。用MNase消化精子核后产生单个NP-ps，其在琼脂糖凝胶中迁移到1.78 - 1.26 Kb的DNA片段之间。在ENase激活缓冲液中孵育后，精子染色质未被消化，表明这些细胞核不含ENase。用ENase或MNase消化未受精卵细胞核可获得一种类型的NP-ps；该NP-ps位于琼脂糖凝胶中对应于3.4 - 1.95 Kb DNA片段的区域[Imschenetzky等人（1989年）：《实验细胞研究》182:436 - 444]。当用ENase消化含有雌性原核和部分重塑的雄性原核的受精卵的完整细胞核时，产生单个NP-ps，其在2.5 - 1.9 Kb的DNA片段之间迁移。该颗粒仅包含CS组蛋白变体。或者，当用MNase消化这些细胞核时，产生两个NP-ps；迁移较慢的NP-ps（s）位于琼脂糖凝胶中的位置与ENase消化产生的NP-ps相同，而迁移较快的NP-ps（f）在1.95 - 1.26 Kb的DNA片段之间迁移。发现NP-ps（s）仅包含CS组蛋白变体，而NP-ps（f）由一部分SpH和CS组蛋白变体组成。当用ENase或MNase消化含有完全解聚的雄性原核的受精卵的细胞核时，观察到单一类型的NP-ps，其在琼脂糖凝胶中的迁移位置与NP-ps（s）相同。该颗粒仅包含CS组蛋白变体。基于组蛋白组成和电泳相似性，得出结论：NP-ps（s）起源于雌性原核，而NP-ps（f）由部分重塑的雄性原核产生。因此，我们的结果表明，在雄性原核重塑的中间阶段，染色质由同时包含一部分SpH和CS组蛋白变体的NP-ps形成，而在雄性原核解聚的最终阶段，染色质组织与雌性原核相似。
