The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China.
Reproduction. 2014 Feb 3;147(3):321-30. doi: 10.1530/REP-13-0313. Print 2014 Mar.
Cryodamage is a major problem in semen cryopreservation, causing changes in the levels of proteins that influence the function and motility of spermatozoa. In this study, protein samples prepared from fresh and frozen-thawed boar spermatozoa were compared using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled to 2D LC-MS/MS analysis. A total of 41 differentially expressed proteins were identified and quantified, including 35 proteins that were present at higher levels and six proteins that were present at lower levels in frozen-thawed spermatozoa by at least a mean of 1.79-fold (P<0.05). On classifying into ten distinct categories using bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant to sperm premature capacitation, adhesions, energy supply, and sperm-oocyte binding and fusion. The expression of four of these proteins, SOD1, TPI1, ODF2, and AKAP3, was verified by western blot analysis. We propose that alterations in these identified proteins affect the quality of cryopreserved semen and ultimately lower its fertilizing capacity. This is the first study to compare protein levels in fresh and frozen-thawed spermatozoa using the iTRAQ technology. Our preliminary results provide an overview of the molecular mechanisms of cryodamage in frozen-thawed spermatozoa and theoretical guidance to improve the cryopreservation of boar semen.
冷冻损伤是精液冷冻保存中的一个主要问题，会导致影响精子功能和活力的蛋白质水平发生变化。在这项研究中，使用等重同位素标签相对和绝对定量（iTRAQ）标记技术与 2D LC-MS/MS 分析相结合，比较了新鲜和冷冻解冻猪精子的蛋白质样品。共鉴定和定量了 41 个差异表达的蛋白质，其中 35 个蛋白质在冷冻解冻精子中至少高出 1.79 倍（P<0.05），而 6 个蛋白质则低于新鲜精子。通过生物信息学分析将这 41 个差异表达的蛋白质分为十个不同的类别，发现其中大多数与精子过早获能、黏附、能量供应以及精子-卵母细胞结合和融合密切相关。通过 Western blot 分析验证了其中 4 种蛋白质（SOD1、TPI1、ODF2 和 AKAP3）的表达。我们提出，这些鉴定出的蛋白质的变化会影响冷冻保存精液的质量，最终降低其受精能力。这是首次使用 iTRAQ 技术比较新鲜和冷冻解冻精子中的蛋白质水平。我们的初步结果提供了冷冻解冻精子中冷冻损伤的分子机制的概述，并为提高猪精液的冷冻保存提供了理论指导。
