Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura Cáceres, Cáceres, Spain.
PLoS One. 2012;7(1):e30688. doi: 10.1371/journal.pone.0030688. Epub 2012 Jan 26.
Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described.
凋亡已被认为是冷冻保存过程中精子死亡的原因之一，也是人类不育的原因之一，但目前尚无关于其在冷藏保存过程中精子死亡中的作用的数据；自噬至今尚未在成熟精子中描述。我们研究了凋亡和自噬在冷却保存种马精子中的作用。从七头种马中采集样本；一半的精液通过单层离心处理，另一半未经处理扩展，并在 5°C 下储存五天。在储存期间，评估精子活力（CASA，每日）和膜完整性（流式细胞术，每日）。使用流式细胞术在第 1、3 和 5 天评估凋亡（活性半胱天冬酶 3、膜通透性增加、磷脂酰丝氨酸易位和线粒体膜电位）。此外，在储存期开始和结束时通过 Western 印迹法研究 LC3B 的加工。在储存过程中精子质量下降在很大程度上归因于凋亡；单层离心选择非凋亡精子，但选择和未选择精子的精子活力没有差异。射出的精子中有很高比例的精子表现出活性半胱天冬酶 3，并且在储存期间，凋亡精子的比例增加，但通过 SYBR-14/PI 检测未观察到活精子的百分比发生变化。与原生精子相比，单层离心后精子中 LC3B 的处理方式不同。在加工后的精子中存在更多的 LC3B-II，而未加工的样本中则不存在；此外，在未加工的精子中，在冷却储存五天后 LC3B-II 增加。这些结果表明，凋亡在冷藏保存过程中精子死亡中起主要作用，自噬在精子存活中起作用，代表了以前未描述的精子中一种新的生存机制。
