Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.
Theriogenology. 2011 Nov;76(8):1450-64. doi: 10.1016/j.theriogenology.2011.05.039.
The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5 nmol/μg protein in fresh samples to 6.7 ± 0.8 nmol/μg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone.
本工作的主要目的是深入了解冻融过程改变猪精子核蛋白结构的机制。为此，通过 Western blot 和免疫细胞化学分析，研究了猪精子头部结构和组蛋白-DNA 结构域位置在冻融过程中的变化。随后，通过 Western blot 分析，研究了冻融是否诱导了鱼精蛋白 1 和组蛋白 H1 的酪氨酸磷酸化水平的变化，这些样本之前已经进行了免疫沉淀。通过分析冻融对精子头部中二硫键总水平的影响，确定了冻融对游离半胱氨酸自由基水平的影响。冻融诱导了组蛋白-DNA 结构的显著变化，表现为出现一条约 35kDa 带的与冻融相关的组蛋白 H1-DNA 聚集体，以及组蛋白 H1 阳性标记从精子头部的尾部区域扩散到更靠前的区域。冻融对鱼精蛋白 1 或组蛋白 H1 的酪氨酸磷酸化水平没有任何显著影响。然而，解冻后的样品显示出游离半胱氨酸自由基含量的显著增加（从新鲜样品中的 3.1±0.5 nmol/μg 蛋白增加到 6.7±0.8 nmol/μg 蛋白）。总之，我们的结果表明，冻融通过与 DNA 中二硫键断裂相关的机制，导致猪精子头部核蛋白结构发生显著改变。这些机制似乎是非特异性的，以类似的方式影响鱼精蛋白-DNA 结合和组蛋白-DNA 结合。此外，结果表明猪精子核结构是不均匀的，存在着一种分区模式，每个区的总 DNA 密度和存在的精确核蛋白结构的紧密程度不同。
