de Mateo S, Ramos L, van der Vlag J, de Boer P, Oliva R
Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Barcelona, Spain.
Int J Androl. 2011 Jun;34(3):256-67. doi: 10.1111/j.1365-2605.2010.01080.x.
A two-step gradient density centrifugation system has been set up to isolate two contrasting sperm populations of normozoospermic and oligoasthenoteratozoospermic (OAT) men. High- and low-density fractions were characterized by total and free thiol fluorescence as determined by monobromobimane-flow cytometry and by protamine/DNA ratios after protamine extraction and polyacrylamide acid-urea gel electrophoresis. Further chromatin characterization was performed through immunofluorescence (IF) with specific antibodies to nucleosomes, histone subtypes (H3.1/H3.2 and TH2B), histone modifications (KM-2 and H4K8ac) and precursor protamine 2. The native sperm samples from normozoospermic and OAT patients showed a biphasic distribution of total thiol levels, which changed in the sperm fractions obtained using the density isolation protocol presented here. Moreover, significant differences were detected in the protamine content in the different fractions of OAT and fertile donor samples. In addition, in the high-density fractions from OAT and normozoospermics, higher IF levels for H4K8ac and TH2B were seen. These results would be consistent with the intended beneficial effect on chromatin maturity of the density selection techniques currently being used in assisted fertilization procedures. However, most nucleosome and related proteins/modifications differ between OAT and normozoospermic men, even after gradient centrifugation, providing evidence for incomplete nuclear maturity in OAT patients.
已建立一种两步梯度密度离心系统，以分离正常精子症男性和少弱畸精子症（OAT）男性的两种对比鲜明的精子群体。通过单溴代马来酰亚胺流式细胞术测定的总硫醇荧光以及鱼精蛋白提取和聚丙烯酰胺酸尿素凝胶电泳后的鱼精蛋白/DNA比率，对高密度和低密度部分进行了表征。通过使用针对核小体、组蛋白亚型（H3.1/H3.2和TH2B）、组蛋白修饰（KM-2和H4K8ac）和前体鱼精蛋白2的特异性抗体进行免疫荧光（IF），进一步对染色质进行了表征。正常精子症患者和OAT患者的天然精子样本显示总硫醇水平呈双相分布，在使用本文介绍的密度分离方案获得的精子部分中发生了变化。此外，在OAT和可育供体样本的不同部分中检测到鱼精蛋白含量存在显著差异。此外，在OAT和正常精子症患者的高密度部分中，观察到H4K8ac和TH2B的IF水平较高。这些结果与目前辅助受精程序中使用的密度选择技术对染色质成熟度的预期有益效果一致。然而，即使经过梯度离心，OAT男性和正常精子症男性之间的大多数核小体及相关蛋白质/修饰仍存在差异，这为OAT患者核成熟不完全提供了证据。
