抗冻蛋白III(AFPIII)用于冷冻保存各种动物物种的生殖细胞。然而,其冷冻保护的确切机制在分子水平上是未知的。在这项研究中,我们调查了运动性,顶体完整性,和线粒体膜电位(MMP),以及蛋白质组学变化,冷冻保存后的食蟹猴精子。精子运动,顶体完整性,低温保存后MMP较低(p<0.001),但在AFP处理的精子样品(CryoAFP)和未处理的精子样品(Cryo-AFP)之间观察到精子运动和MMP的显着差异(p<0.01)。共有141种和32种差异表达蛋白,分别,与新鲜精子相比,在不含0.1μg/mlAFPIII的情况下冷冻保存的食蟹猴精子中鉴定。这些蛋白质主要参与活性氧(ROS)的线粒体产生,谷胱甘肽(GSH)合成,和细胞凋亡。在精子冷冻培养基中添加AFPIII导致精子中细胞分子功能和/或生物过程的显着稳定,如冷冻和解冻后蛋白质组变化的程度所示。根据差异表达蛋白的蛋白质组学变化,我们假设了一种新的冷冻保护分子机制,即AFPIII可能通过调节线粒体中ROS的产生来减少细胞色素c的释放,从而减少精子凋亡。AFPIII与精子蛋白作用以保护细胞免受冷冻损伤的分子机制有待进一步研究。
Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p < 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo-AFP) (p < 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 μg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.