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

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

【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对精卵结合和卵裂率的影响。【结果】硫醇酯分析结果表明,TAK-243处理组精子未形成Ub-E1复合物,说明TAK-243抑制了E1对Ub的激活作用。与鲜精组相比,获能组精子的曲线速度(VCL)和平均鞭打频率显著升高(P<0.05)。3个浓度TAK-243组与获能组精子的各项动力学参数差异均不显著(P>0.05)。与获能组相比,3个浓度TAK-243组酪氨酸磷酸化水平均显著降低(P<0.05),且随着TAK-243浓度的增加,精子蛋白的酪氨酸磷酸化水平逐渐降低。后续试验使用7.2 nmol/L TAK-243进行精子中Zn2+水平和顶体膜重构的研究,其结果表明,与获能组相比,7.2 nmol/L TAK-243组Zn2+水平、顶体膜完整性及AQN-1和AWN蛋白的表达水平均显著增加(P<0.05),精子与卵母细胞的黏附率和卵裂率均显著降低(P<0.05)。【结论】7.2 nmol/L TAK-243显著降低了精子获能期间蛋白酪氨酸磷酸化水平、精卵结合率和卵裂率,显著增加了Zn2+含量、顶体膜完整率及顶体表面蛋白AWN和AQN-1的表达,说明泛素-蛋白酶体信号通路参与了精子的体外获能。

【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.