Abstract

The main objective of this study was to investigate the expression of acrosin inhibitor (AI), ubiquitin (Ub), and small ubiquitin-related modifier 1 (SUMO1) proteins during in vitro capacitation of pig sperm. Duroc pig sperm was divided into fresh sperm and capacitation treatment groups. Protein expression was evaluated using computer-assisted sperm analysis (CASA) systems, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and immunofluorescence. The results showed that the expression of AI (30 kDa) incapacitated sperm was significantly lower than that in fresh sperm (P < 0.05), and that the levels of ubiquitinated and SUMO1-ylated proteins in capacitated sperm were significantly higher than those in fresh sperm (P < 0.05). Immunofluorescence results showed that AI, Ub, and SUMO1 were located in the acrosome region of the fresh and capacitated sperm heads. After capacitation, the fluorescence intensity of AI and SUMO1 decreased, while that of Ub increased. The protein band at 30 kDa represented the AI-Ub-SUMO1 complex, indicating that this complex was involved in sperm capacitation. Furthermore, SUMO1 increased the stability of AI at 30 kDa, preventing its complete decomposition, while at 46 kDa, in the absence of SUMO1, AI is bound to ubiquitin, and was completely degraded.

摘要

本研究的主要目的是研究猪精子体外获能过程中顶体蛋白抑制剂 (AI)、泛素 (Ub) 和小泛素相关修饰剂 1 (SUMO1) 蛋白的表达。杜洛克猪精子分为新鲜精子和获能处理组。使用计算机辅助精子分析 (CASA) 系统、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE)、蛋白质印迹和免疫荧光评估蛋白质表达。结果表明，AI（30 kDa）失能精子的表达量显着低于新鲜精子（P <  0.05），且失能精子中泛素化和SUMO1-ylated蛋白水平显着高于新鲜精子。 ( P < 0.05）。免疫荧光结果表明，AI、Ub 和 SUMO1 位于新鲜和获能精子头部的顶体区域。获能后，AI 和 SUMO1 的荧光强度降低，而 Ub 的荧光强度增加。30 kDa 的蛋白质条带代表 AI-Ub-SUMO1 复合物，表明该复合物与精子获能有关。此外，SUMO1 在 30 kDa 时增加了 AI 的稳定性，防止其完全分解，而在 46 kDa 时，在没有 SUMO1 的情况下，AI 与泛素结合，并完全降解。