School of Agriculture and Food Science, The University of Queensland, Gatton, Queensland 4343, Australia.
Reproduction. 2012 Jun;143(6):787-97. doi: 10.1530/REP-11-0436. Epub 2012 Apr 12.
This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.
本研究旨在探讨在模拟渗透通量的结构和生理效应的实验模型中，是否可以解释冷冻诱导的树袋熊精子损伤。解冻冷冻保存精子后原位缺口翻译的 DNA 标记显示，核松弛染色质的面积与核苷酸标记程度之间存在正相关关系（r=0.573；P<0.001；n=50）。虽然一些精子的染色质增加了正常大小的八倍以上，但并非所有松弛染色质的精子核都显示出核苷酸掺入的证据。与精子 DNA 碎片化（SDF）相关的优先染色通常位于精子头部的顶体周围和周边区域以及精子的基部，在冷冻保存后，这些区域似乎是 DNA 损伤的“热点”。比较各向异性介质和冷冻保存对树袋熊精子完整性的影响的结果表明，暴露于渗透通量引起的损伤实质上模仿了冷冻保存后的结果。质膜完整性、染色质松弛和 SDF 似乎特别容易受到极端低渗环境的影响。虽然线粒体膜电位（MMP）容易受到极端低渗和高渗环境的影响，但当返回等渗条件时，它显示出从高渗应激中恢复的能力。暴露于 64 mOsm/kg 介质的树袋熊精子显示出与冷冻解冻精子相当或更严重的结构和生理损伤程度，支持冷冻损伤主要与低渗效应相关的假设。解冻冷冻保存精子的 SDF 与暴露于 64 mOsm/kg 偏移的精子的 SDF 进行直接比较显示出显著相关性（r=0.878；P<0.05；n=5）；然而，当比较松弛染色质的精子百分比时，没有发现相关性。虽然冷冻诱导的渗透损伤模型似乎可以解释冷冻解冻后考拉 SDF 的变化，但导致松弛染色质的机制需要进一步研究。松弛染色质的精子百分比与 SDF 之间缺乏相关性表明这些病变的时间不同步。我们提出了一种冷冻诱导的渗透损伤综合模型，该模型涉及结构损伤（膜破裂）和氧化应激的结合，首先导致 MMP 减少和染色质松弛，随后最终导致 DNA 碎片化增加。
