Johnston S D, López-Fernández C, Arroyo F, Roy R, Holt W V, Gosálvez J
School of Agriculture and Food Sciences, The University of Queensland, Gatton, Qld 4343, Australia; and Corresponding author. Email:
Department of Biology, Autonomous University of Madrid, Cantoblanco, Madrid, 28049, Spain.
Reprod Fertil Dev. 2019 Sep;31(10):1558-1566. doi: 10.1071/RD18512.
To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynamics over 24h of post-thaw incubation. Following validation of a wombat sperm chromatin dispersion test, wombat DNA showed a rate of SDF that was 6-fold higher than for koala spermatozoa (P=0.038). Finally, we examined whether expected differences in chromatin compactness, associated with protamine sequence, had an effect on restriction site accessibility of sperm DNA. Thawed spermatozoa were exposed to Alu I and EcoR1 endonuclease restriction enzymes and the SDF dynamics were observed. Koala spermatozoa exposed to Alu I showed a greater rate of SDF (P=0.01), whereas wombat spermatozoa exposed to EcoR1 showed a greater rate of SDF (P=0.032). We conclude that restriction sites in these species are differentially present or exposed and potentially account for differences in SDF dynamics. Although differences in the arginine composition of protamine may explain relative differences in SDF following cryopreservation, they do not support the hypothesis that increased arginine composition increases DNA stability; rather, increased arginine composition in the wombat may reduce post-thaw chromatin swelling.
为了研究考拉和袋熊精子解冻后DNA稳定性的差异，对鱼精蛋白氨基酸序列进行了比较，发现袋熊的精氨酸残基多三个。使用相同方案冷冻保存的考拉和袋熊精子，在解冻后孵育的24小时内检测精子DNA碎片化（SDF）动态变化。在验证袋熊精子染色质扩散试验后，发现袋熊DNA的SDF速率比考拉精子高6倍（P=0.038）。最后，我们研究了与鱼精蛋白序列相关的染色质紧密程度的预期差异是否对精子DNA的限制性酶切位点可及性有影响。将解冻的精子暴露于Alu I和EcoR1内切酶，观察SDF动态变化。暴露于Alu I的考拉精子显示出更高的SDF速率（P=0.01），而暴露于EcoR1的袋熊精子显示出更高的SDF速率（P=0.032）。我们得出结论，这些物种中的限制性酶切位点存在差异或暴露程度不同，这可能是SDF动态变化差异的原因。虽然鱼精蛋白中精氨酸组成的差异可能解释冷冻保存后SDF的相对差异，但它们并不支持精氨酸组成增加会提高DNA稳定性的假设；相反，袋熊中精氨酸组成的增加可能会减少解冻后染色质肿胀。
