金黄色葡萄球菌对体外保存猪精子活力及蛋白磷酸化修饰的影响  

Effects of Staphylococcus aureus on Motility and Protein Phosphorylation Modification of Boar Sperm during Storage in vitro

作　　者：李玉华[1] 付杰丽 李培飞 杨强震 王立蕊 谢蔚宜 李新红[1] 

机构地区：[1]上海交通大学农业与生物学院上海市兽医生物技术重点实验室,上海200240

出　　处：《畜牧兽医学报》2017年第9期1665-1673,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：上海市科技兴农攻关项目(沪农科基字(2014)第2-5号)

摘　　要：旨在从精子代谢及蛋白磷酸化修饰水平探讨金黄色葡萄球菌(Staphylococcus aureus)对猪精液17℃保存过程中精子活力的影响,从而为病原菌影响猪精子质量的分子机制研究提供理论基础。本研究采用计算机辅助精子活力分析仪(CASA)及试剂盒测定精子运动性能,3-磷酸甘油醛脱氢酶(GAPDH)活性、环化腺苷一磷酸(cAMP)水平及ATP总量变化,利用Western blot及细胞免疫荧光技术分别检测精子蛋白磷酸化水平变化及磷酸化修饰蛋白亚细胞定位。CASA结果显示,猪精液体外17℃保存过程中,10~3 CFU·mL^(-1)的金黄色葡萄球菌显著降低精子运动性能(P<0.05),培养3d后,随着葡萄球菌增加,猪精子运动性能降低趋势更显著(P<0.01);与精子活力参数相类似,培养3d后,处理组细胞内GAPDH活性及ATP总量显著低于对照组(P<0.05);Western blot结果表明,处理组精子蛋白磷酸化水平与对照组相比存在显著性差异(P<0.05),磷酸化修饰水平变化与cAMP变化具有一致性,预示着金黄色葡萄球菌对精子蛋白磷酸化修饰变化的影响可能遵循sAC-cAMP-PKA信号通路。综上表明,金黄色葡萄球菌抑制糖代谢GAPDH活性,从而降低ATP水平,同时可能遵循sAC-cAMP-PKA信号通路抑制头部核后区、赤道段及鞭毛区与精子活力相关蛋白的磷酸化水平,从而抑制猪精子活力。This study aimed to investigate the effects of S. aureus on boar sperm motility during storage at 17℃ from the perspectives of sperm metabolism and protein phosphorylation modifica- tion levels, and provide a theoretical basis for the molecular mechanisms underlying the study of pathogens on sperm quality. Sperm motility parameters were determined using computer assisted sperm analysis （CASA） and the varying of intracellular glyceraldehyde-3-phosphate dehydrogen- ase （GAPDH） activity, cyclic adenosine monophosphate （cAMP） content and totally ATP level were measured by reagent kits. Protein phosphorylation levels and the localization of phosphoryl- ated targets were analyzed by Western blot and immunofluorescence technique, respectively. The results of CASA showed that 103 CFU· mL-1 S. aureus-treated could significantly inhibit sperm motility during boar semen storage at 17℃ （P〈0.05）. With the increase of S. aureus, sperm motility parameters were significantly decreased after 3 days of inoculation （P〈0.01）. Similarly,GAPDH activity and ATP level in the treatment groups were also markedly decreased compared to that in the control groups after 3 days of inoculation （P〈0.05）. The results of Western blot showed that there were significant differences in sperm protein phosphorylation levels between the treatment groups and the control groups （P〈0.05）. The changing trends of phosphorylation levels were consistent with that of intracellular cAMP level, which suggested that S. aureus af- fecting protein phosphorylation levels might following the sAC-cAMP-PKA signaling pathway. Taken together, S. aureus reduced intracellular ATP content through lowering glycolytic GAPDH activity, meanwhile, S. aureus inhibited the phosphorylation levels of motility-related proteins located in the posterior nucleus, equatorial piece and flagellar regions of boar sperm fol- lowing the sAC-cAMP-PKA signaling pathway. Both the reduction of ATP and the inhibition of protein phosphorylation levels led to

关 键 词：金黄色葡萄球菌 猪精子 活力 糖酵解 蛋白磷酸化 

分 类 号：S828.2[农业科学—畜牧学]