Mechanisms of ROS-induced mitochondria-dependent apoptosis underlying liquid storage of goat spermatozoa

Liquid storage of spermatozoa is important for artificial insemination and herd genetic breeding. However, the extending time of storage inducing rapid decline of spermatozoa quality limits the development of this technology. The molecular mechanisms underlying liquid storage of spermatozoa remain largely unexplored. In this study, the effects of liquid storage on functional quality of spermatozoa were assessed in goat (Capra hircus). The time-dependent decline of spermatozoa motility showed a strong correlation with the significant increase of apoptosis. Moreover, apoptosis-related ultrastructural changes were observed, especially the defect in mitochondria. Meanwhile, significant decrease of mitochondrial membrane potential and expression changes of mitochondrial apoptosis-related proteins indicated mitochondrial dysfunction and activation of mitochondrial apoptotic pathway. Notably, the abnormal high level of reactive oxygen species (ROS) caused by liquid storage resulted in the oxidative damage to mitochondria and accelerated mitochondria-dependent apoptosis, which was demonstrated by addition of ROS scavenger N-acetylcysteine. Furthermore, critical differentially expressed proteins involved in mitochondria-dependent apoptosis and antioxidant defense were identified and profiled by quantitative proteomic analysis, facilitating the understanding of molecular regulation of ROS-induced mitochondria-dependent apoptosis. These outcomes provide insights into the mechanisms underlying liquid storage of goat spermatozoa and enhance the progress of semen storage technology.

精子液态保存技术对于人工授精及群体遗传育种具有重要意义。然而，随着保存时间延长，精子品质快速下降，限制了该技术的推广应用。目前，精子液态保存背后的分子机制仍未得到充分阐明。本研究以山羊（Capra hircus）为研究对象，评估了液态保存对精子功能品质的影响。研究发现，精子活力随保存时间逐渐下降，这与细胞凋亡水平显著升高呈强相关性。此外，观察到与细胞凋亡相关的超微结构变化，尤以线粒体结构异常最为显著。与此同时，线粒体膜电位显著降低，以及线粒体凋亡相关蛋白的表达变化，均提示线粒体功能障碍与线粒体凋亡通路被激活。值得注意的是，液态保存过程中产生的过量活性氧（ROS）会造成线粒体氧化损伤，进而加速线粒体依赖性细胞凋亡，这一结论通过添加ROS清除剂N-乙酰半胱氨酸得到了验证。本研究通过定量蛋白质组学分析，筛选并鉴定出参与线粒体依赖性凋亡与抗氧化防御的关键差异表达蛋白，有助于进一步理解ROS诱导的线粒体依赖性凋亡的分子调控机制。本研究结果为阐明山羊精子液态保存的分子机制提供了新的见解，同时也将推动精液保存技术的发展进程。