Sotolongo Barbara, Lino Elisabete, Ward W Steven
Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA.
Biol Reprod. 2003 Dec;69(6):2029-35. doi: 10.1095/biolreprod.103.020594. Epub 2003 Aug 20.
Mammalian sperm chromatin is bound by protamines into highly condensed toroids with approximately 50 kilobases (kb) of DNA. It is also organized into loop domains of about the same size that are attached at their bases to the proteinaceous nuclear matrix. In this work, we test our model that each sperm DNA-loop domain is condensed into a single protamine toroid. Our model predicts that the protamine toroids are linked by chromatin that is more sensitive to nucleases than the DNA within the toroids. To test this model, we treated hamster sperm nuclei with DNase I and found that the sperm chromatin was digested into fragments with an average size of about 50 kb, by pulse-field gel electrophoresis (PFGE). Surprisingly, we also found that spermatozoa treated with 0.25% Triton X-100 (TX) and 20 mM MgCl2 overnight resulted in the same type of degradation, suggesting that sperm nuclei have a mechanism for digesting their own DNA at the bases of the loop domains. We extracted the nuclei with 2 M NaCl and 10 mM dithiothreitol (DTT) to make nuclear halos. Nuclear matrices prepared from DNase I-treated spermatozoa had no DNA attached, suggesting that DNase I digested the DNA at the bases of the loop domains. TX-treated spermatozoa still had their entire DNA associated with the nuclear matrix, even though the DNA was digested into 50-kb fragments as revealed by PFGE. The data support our donut-loop model for sperm chromatin structure and suggest a functional role for this type of organization in that sperm can digest its own DNA at the sites of attachment to the nuclear matrix.
哺乳动物精子染色质被鱼精蛋白结合成高度浓缩的超螺旋结构，其中包含约50千碱基（kb）的DNA。它还被组织成大小相近的环状结构域，这些结构域的基部附着在蛋白质性的核基质上。在这项研究中，我们测试了我们的模型，即每个精子DNA环状结构域被浓缩成单个鱼精蛋白超螺旋结构。我们的模型预测，鱼精蛋白超螺旋结构由对核酸酶比超螺旋结构内的DNA更敏感的染色质连接。为了验证这个模型，我们用DNase I处理仓鼠精子细胞核，通过脉冲场凝胶电泳（PFGE）发现精子染色质被消化成平均大小约为50 kb的片段。令人惊讶的是，我们还发现，用0.25% Triton X-100（TX）和20 mM MgCl2过夜处理精子也会导致相同类型的降解，这表明精子细胞核有一种机制可以在环状结构域的基部消化自身的DNA。我们用2 M NaCl和10 mM二硫苏糖醇（DTT）提取细胞核以制备核晕。用DNase I处理过的精子制备的核基质没有附着DNA，这表明DNase I在环状结构域的基部消化了DNA。尽管通过PFGE显示DNA被消化成了50 kb的片段，但TX处理过的精子仍然有其完整的DNA与核基质相关联。这些数据支持了我们关于精子染色质结构的甜甜圈 - 环状模型，并表明这种组织形式具有功能性作用，即精子可以在其与核基质附着的位点消化自身的DNA。
