Shaman Jeffrey A, Yamauchi Yasuhiro, Ward W Steven
Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA.
J Cell Biochem. 2007 Oct 15;102(3):680-8. doi: 10.1002/jcb.21321.
The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.
哺乳动物精子核为研究核结构形成与DNA复制起始之间的关系提供了一个极佳的模型。我们之前证明，哺乳动物精子核含有一种核基质，它以类似于体细胞的方式将DNA组织成环状结构域。在本研究中，我们测试了精子核中对于雄原核形成和DNA合成起始所必需的最小成分。我们用高盐和二硫苏糖醇提取小鼠精子核以去除鱼精蛋白，从而形成核晕。然后用限制性内切酶处理这些核晕以释放不直接与核基质相关的DNA，或者用DNA酶I处理以消化所有DNA。将处理后的精子核注入卵母细胞，并监测父本原核形成和DNA合成情况。我们发现经限制性酶切的精子核晕能够形成父本原核并起始DNA合成。然而，当将分离的小鼠精子DNA或与核基质重构的精子DNA注入卵母细胞时，未观察到父本原核形成或DNA合成。这些数据表明，小鼠父本原核DNA合成需要精子DNA原位的核基质附着组织。
