Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra (Barcelona), Spain.
Andrology. 2017 Nov;5(6):1153-1164. doi: 10.1111/andr.12410. Epub 2017 Sep 21.
Cryopreservation is the most suitable method to preserve boar spermatozoa over long-term storage. However, freeze-thawing protocols inflict extensive damage to sperm cells, reducing their viability and compromising their fertilizing ability. In addition, high individual variability is known to exist between boar ejaculates, which may be classified as of good (GFE) or poor (PFE) freezability. While conventional spermiogram parameters fail to predict sperm cryotolerance in fresh spermatozoa, high levels of certain proteins, also known as freezability markers, have been found to be related to the sperm resilience to withstand freeze-thawing procedures. In this context, the hypothesis of this study was that aquaporins AQP3, AQP7, and AQP11 could be linked to boar sperm cryotolerance. Twenty-nine ejaculates were evaluated and subsequently classified as GFE or PFE based upon their sperm viability and motility at post-thawing. Fourteen ejaculates resulted to be GFE, whereas the other fifteen were found to be PFE. Relative abundances of AQP3, AQP7, and AQP11 and their localization patterns were evaluated in all fresh and frozen-thawed ejaculates through immunoblotting and immunocytochemistry. Prior to cryopreservation, relative amounts of AQP3 and AQP7 were found to be significantly (p < 0.05) higher in GFE than in PFE. In contrast, no significant differences (p > 0.05) between freezability groups were found for AQP11, despite GFE tending to present higher levels of this protein. The localization of AQP7, but not that of AQP3 or AQP11, was observed to be affected by cryopreservation procedures. In conclusion, these results suggest that AQP3 and AQP7 are related to boar sperm cryotolerance and may be used as freezability markers.
冷冻保存是长期储存猪精子最适宜的方法。然而，冻融过程会对精子细胞造成广泛的损伤，降低其活力，损害其受精能力。此外，已知猪精液个体间存在很大的变异性，可分为良好（GFE）或不良（PFE）的冷冻可育性。虽然传统的精子参数不能预测新鲜精子的抗冻性，但高水平的某些蛋白质，也称为冷冻可育性标记物，已被发现与精子对冻融过程的抵抗力有关。在这种情况下，本研究的假设是水通道蛋白 AQP3、AQP7 和 AQP11 可能与猪精子的抗冻性有关。评估了 29 个精液样本，并根据解冻后的精子活力和运动性将其分为 GFE 或 PFE。14 个精液样本被归类为 GFE，而另外 15 个被归类为 PFE。通过免疫印迹和免疫细胞化学评估了所有新鲜和冷冻解冻的精液中 AQP3、AQP7 和 AQP11 的相对丰度及其定位模式。在冷冻保存之前，发现 GFE 中的 AQP3 和 AQP7 的相对含量明显（p<0.05）高于 PFE。相比之下，AQP11 在冷冻可育性组之间没有发现显著差异（p>0.05），尽管 GFE 倾向于表现出更高水平的这种蛋白质。AQP7 的定位，但不是 AQP3 或 AQP11 的定位，被观察到受到冷冻保存过程的影响。总之，这些结果表明 AQP3 和 AQP7 与猪精子的抗冻性有关，可用作冷冻可育性标记物。
