金雀异黄酮对冻融猪精子蛋白酪氨酸磷酸化及获能相关基因表达的影响  

Effects of Genistein on Protein Tyrosine Phosphorylation and Capacitation-related Gene Expression of Frozen-thawed Boar Spermatozoa

作　　者：梁莹莹 谢运法 胡传活[1] 陈志英 邓祝新 冯显钤 许春荣 刘德玉 唐胤晟 李铭 LIANG Ying-ying;XIE Yun-fa;HU Chuan-huo;CHEN Zhi-ying;DENG Zhu-xin;FENG Xian-qian;XU Chun-rong;LIU De-yu;TANG Yin-sheng;LI Ming(College of Animal Science and Technology,Guangxi University,Guangxi Nanning 530004,China;Guangxi Work Station of Livestock&Poultry Breed Improvement,Guangxi Nanning 530001,China;Guangxi Institute of Animal Sciences,Guangxi Nanning 530001,China)

机构地区：[1]广西大学动物科技学院,广西南宁530004 [2]广西畜禽品种改良站,广西南宁530001 [3]广西畜牧研究所,广西南宁530001

出　　处：《西南农业学报》2021年第8期1815-1822,共8页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31660700)。

摘　　要：【目的】探究金雀异黄酮对冻融猪精子蛋白酪氨酸磷酸化及获能相关基因的影响,为丰富猪精子冷冻保存技术的理论基础及后续开展蛋白酪氨酸激酶抑制剂对冻融猪精子获能影响研究提供参考依据。【方法】采集猪精液后,设精液冷冻对照组(CK组)、100.0μmol/L金雀异黄酮冷冻处理组(G组)和鲜精组(F组),使用Western blotting、SDS-PAGE、免疫荧光、ELISA和实时荧光定量PCR等检测各组精子的蛋白酪氨酸磷酸化水平、精子不同部位蛋白酪氨酸磷酸化位点变化情况、蛋白激酶A(PKA)和蛋白激酶G(PKG)活性及黏附蛋白家族基因(AQN1、AQN3、AWN、PSP-Ⅰ和PSP-Ⅱ)和精子运动抑制因子(SPMI)基因mRNA的表达水平,分析金雀异黄酮对冻融猪精子的保护机制。【结果】与CK组相比,G组的32和69 kD蛋白酪氨酸磷酸化水平、顶体+顶体后区磷酸化位点、PKA活性显著降低(P<0.05,下同);AWN基因相对表达量极显著升高(P<0.01,下同),PSP-Ⅰ基因相对表达量显著降低,PSP-Ⅱ和SPMI基因相对表达量极显著降低。F组中未检测到32和69 kD蛋白酪氨酸磷酸化,而37和58 kD蛋白酪氨酸磷酸化水平和PKA活性显著降低;AQN1、AQN3和AWN基因相对表达量极显著升高,PSP-Ⅱ基因相对表达量显著降低,SPMI基因相对表达量极显著降低。【结论】金雀异黄酮既能降低冻融猪精子顶体+顶体后区蛋白酪氨酸磷酸化水平,且PKA活性降低可能与p32和p69蛋白表达量降低有关,又可有效提高冻融猪精子中AWN基因及有效降低PSP-Ⅰ、PSP-Ⅱ和SPMI基因的表达量,在一定程度上提高冻融猪精子质量。【Objective】The purpose of the experiment was to investigate the effects of genistein on protein tyrosine phosphorylation and capacitation-related genes expression of frozen-thawed boar sperm,and to lay a theoretical foundation for enriching the cryopreservation technology of boar sperm and to provide a reference on the effects of protein tyrosine kinase inhibitors on the capacitation of frozen-thawed boar sperm.【Method】The sperm was divided into frozen control group(CK group),100.0μmol/L genistein treatment frozen group(G group)and fresh group(F group).The protein tyrosine phosphorylation levels/sites,changes of protein tyrosine phosphorylation sites at different parts of the sperm,the protein kinase A(PKA)and protein kinase G(PKG)activity and adhesion protein family gene(AQN1,AQN3,AWN,PSP-Ⅰ and PSP-Ⅱ)and sperm motor inhibitory factor(SPMI)gene mRNA expression level of frozen-thawed boar sperm was determined by western blotting,SDS-PAGE,immunofluorescent staining,ELISA and qPCR.【Result】The tyrosine phosphorylation levels of 32 and 69 kD proteins,the phosphorylation sites in the acrosome+posterior acrosome zone region and PKA activity in the treatment group were significantly decreased compared with the control group(P<0.05,the same as below).The mRNA expression level of AWN was extremely significantly increased(P<0.01,the same as below),and the expression level of PSP-Ⅰ,PSP-Ⅱ and SPMI were significantly decreased in the treatment group compared with the control group.In the F group,the tyrosine phosphorylation level of 32 and 69 kD proteins were not detected.The tyrosine phosphorylation level of 37 and 58 kD proteins and PKA protein activity were significantly decreased compared with the control group.The mRNA expression level of AQN1,AQN3 and AWN genes were extremely significantly increased,and the expression level of PSP-Ⅱ were significantly decreased and SPMI genes were extremely significantly decreased compared with the F group.【Conclusion】Genistein can reduce the level of protein t

关 键 词：金雀异黄酮 猪精液 冷冻保存 获能相关基因 

分 类 号：S828[农业科学—畜牧学] S814.8[农业科学—畜牧兽医]