泛素激活酶抑制剂对猪精子获能状态、膜重构及体外受精的影响

【目的】评估泛素激活酶(E1)对猪精子获能、膜重构和体外受精的影响。【方法】选取5头长白猪采集精液，以硫醇酯法评估泛素激活酶(ubiquitin activating enzyme, E1)抑制剂(TAK-243)对精子中泛素(ubiquitin, Ub)与E1结合的影响；将精子分为鲜精组(Fresh)、DMSO组(DMSO)、获能组(Capacitated)及2.4、4.8和7.2 nmol/L TAK-243组，鲜精组用2 mL PBS重悬精子沉淀；获能组用2 mL获能液重悬精子沉淀；DMSO组及2.4、4.8和7.2 nmol/L TAK-243组分别用2 mL含0.07%DMSO及2.4、4.8和7.2 nmol/L TAK-243的获能液重悬精子沉淀，使用微生物动(静)态图像检测系统检测精子的动力学参数；以Western blotting法检测精子的酪氨酸磷酸化水平；Zn²⁺荧光探针检测精子的Zn²⁺含量；花生凝集素染色检测精子的顶体膜重构；免疫荧光法检测顶体表面精子黏附蛋白(AQN-1、AWN)的表达；体外受精法检测TAK-243...

Effects of Ubiquitin Activating Enzyme Inhibitor on Capacitation Status,Membrane Remodelling and in vitro Fertilization of Pig Sperm

【Objective】 The main purpose of this study was to evaluate the effects of ubiquitin activating enzyme(E1) inhibitor (TAK-243) on pig sperm capacitation, membrane remodelling and fertilization.【Method】 Semen was collected from 5 Landrace pigs, the effect of TAK-243 on the binding of ubiquitin (Ub) and E1 in sperm was evaluated by thiol ester method.Sperm were divided into fresh group(Fresh), DMSO group(DMSO), capacitated group (Capacitated)and 2.4, 4.8 and 7.2 nmol/L TAK-243 groups.The fresh group was resuspended in 2 mL PBS for sperm precipitation.Sperm precipitation was resuspended with 2 mL capacitation solution in capacitation group.In DMSO group and 2.4, 4.8 and 7.2 nmol/L TAK-243 groups, sperm precipitation was resuspended with 2 mL capacitated solution containing 0.07% DMSO and 2.4, 4.8 and 7.2 nmol/L TAK-243, respectively.The kinetic parameters of sperm were detected by microbial dynamic (static) image detection system.The tyrosine phosphorylation of sperm was detected by Western blotting.Zn²⁺ fluorescence probe was used to detect the content of Zn²⁺ in sperm.Acrosomal membrane remodeling was detected by peanut lectin staining.The expression of acrosomal surface sperm adhesion protein AQN-1 and AWN was detected by immunofluorescence assay.The effect of TAK-243 on sperm-egg binding and cleavage rate was detected by in vitro fertilization.【Result】 Thiol ester analysis showed that sperm in TAK-243 treatment group did not form Ub-E1 complex, which indicated that TAK-243 inhibited the activation of Ub by E1.Compared with fresh sperm group, the curve velocity (VCL) and mean whipping frequency of sperm in capacitated group were significantly increased (P<0.05).There were no significant differences in the kinetic parameters between the three concentrations of TAK-243 and capacitated groups (P>0.05).Compared with the capacitated group, the tyrosine phosphorylation level of the three concentrations of TAK-243 group was significantly decreased (P<0.05), and the tyrosine phosphorylation level of sperm protein gradually decreased with the increase of TAK-243 concentration.Therefore, In subsequent experiments, 7.2 nmol/L TAK-243 was used to investigate the Zn²⁺ level and acrosomal membrane remodeling in sperm.The results showed that the Zn²⁺ level, acrosomal membrane integrity, AQN-1 and AWN protein expression levels in 7.2 nmol/L TAK-243 group were significantly increased compared with those in the capacity group (P<0.05), and the adhesion rate and cleavage rate of sperm and oocytes were significantly decreased (P<0.05).【Conclusion】 7.2 nmol/L TAK-243 significantly decreased the protein tyrosine phosphorylation level, sperm-egg binding rate and cleavage rate during sperm capacitation, and significantly increased the content of Zn²⁺, acrosomal membrane integrity rate and acrosomal surface proteins AWN and AQN-1 expression, indicating that ubiquitin-proteasome signaling pathway was involved in sperm capacitation in vitro.