载脂蛋白A1抗体通过抑制Ca2+载体A23187诱导的Ca2+内流降低精子活力

doi:10.3969/j.issn.1673-8640.2024.04.007

摘要/Abstract

摘要：

目的分析载脂蛋白A1（apo A1）抗体对Ca²⁺载体A23187诱导的精子Ca²⁺内流的影响，探讨apo A1抗体与精子活力的关系。方法收集健康男性精液样本38份。采用免疫荧光法观察apo A1在人精子中的定位和apo A1抗体对精子顶体反应的影响。采用免疫印迹法检测精子获能相关蛋白酪氨酸磷酸化情况。分别采用正常兔IgG（正常兔IgG组）和40 μg·mL^-1 apo A1抗体（apo A1抗体组）处理样本，以正常精液样本作为空白对照组，比较各组精子总活力和前向运动精子百分率差异。分别采用正常兔IgG和10、20、40 μg·mL^-1 apo A1抗体处理已经10 μmol·L^-1 A23187诱导30 min的Fluo-4AM负载的精子样本，以未作处理的Fluo-4AM负载的精子样本作为空白对照，采用流式细胞术检测精子内Ca²⁺水平。结果 apo A1蛋白定位于人精子头部。apo A1抗体组精子总活力和前向运动精子百分率均显著低于空白对照组和正常兔IgG组（P<0.001），空白对照组与正常兔IgG组之间2项指标差异无统计学意义（P>0.05）。流式细胞术结果显示，A23187诱导后，精子内Ca²⁺荧光强度升高；采用apo A1抗体处理后，Ca²⁺浓度显著降低（P<0.01），且呈剂量依赖性。40 μg·mL^-1 apo A1抗体+A23187组顶体反应率显著低于A23187组和正常兔IgG+A23187组（P<0.001），且apo A1抗体对精子自发顶体反应无影响。免疫印迹法结果显示，apo A1抗体处理前后精子的蛋白酪氨酸磷酸化无显著变化。结论 apo A1抗体可以通过降低Ca²⁺内流使精子活力下降，并抑制Ca²⁺载体A23187诱导的顶体反应。

关键词: 载脂蛋白A1, 精子, 细胞内钙离子, 精子活力, 顶体反应, 酪氨酸磷酸化

Abstract:

Objective To analyze the influence of apolipoprotein A1（apo A1） antibodies on ionophore Ca²⁺ A23187-induced Ca²⁺ influx，and to investigate the relation of apo A1 antibodies with sperm motility. Methods Totally，38 healthy male semen samples were collected. Immunofluorescence was used to observe the localization of apo A1 in human spermatozoa and the effect of apo A1 antibody on sperm acrosome reaction. Western blot was used to determine the tyrosine phosphorylation of proteins related to sperm capacitation. Samples were treated with normal rabbit IgG（normal rabbit IgG group） and 40 μg·mL^-1 apo A1 antibody（apo A1 antibody group）. Normal semen samples were used as blank control group，and the difference of total sperm motility and the percentage of progressively motile sperm were compared. Fluo-4AM-loaded semen samples induced by 10 μmol·L^-1 A23187 for 30 min were treated with normal rabbit IgG and 10，20 and 40 μg·mL^-1 apo A1 antibodies，respectively. Fluo-4AM-loaded sperm in blank control group were incubated，and Ca²⁺ levels in sperm were determined by flow cytometry. Results The apo A1 protein is localized in the head of human sperm. The total sperm motility and the percentage of progressively motile sperm in apo A1 antibody group were lower than those in blank control group and normal rabbit IgG group（P<0.001）. There was no statistical significance between blank control group and normal rabbit IgG group（P>0.05）. Flow cytometry showed that the fluorescence intensity of intracellular Ca²⁺ increased after induction with A23187 and decreased after treatment with apo A1 antibody in a dose-dependent manner（P<0.01）. The acrosome reaction rate of 40 μg·mL^-1 apo A1 antibody+A23187 group was lower than those of A23187 group and normal rabbit IgG+A23187 group（P<0.001），and apo A1 antibody had no effect on the spontaneous acrosome reaction of sperm. The results of western blot showed that the protein tyrosine phosphorylation of sperm before and after apo A1 antibody treatment did not change significantly. Conclusions The apo A1 could decrease sperm motility by decreasing Ca²⁺ influx and inhibit the acrosome reaction induced by ionophore Ca²⁺ A23187.

Key words: Apolipoprotein A1, Spermatozoa, Intracellular calcium ion, Sperm motility, Acrosome reaction, Tyrosine phosphorylation

中图分类号:

R446.1

林静, 池秀平, 梁臻龙, 向代军, 王成彬. 载脂蛋白A1抗体通过抑制Ca²⁺载体A23187诱导的Ca²⁺内流降低精子活力[J]. 检验医学, 2024, 39(4): 351-357.

LIN Jing, CHI Xiuping, LIANG Zhenlong, XIANG Daijun, WANG Chengbin. Apolipoprotein A1 antibodies reduce sperm motility by inhibiting ionophore Ca²⁺ A23187-induced Ca²⁺ influx[J]. Laboratory Medicine, 2024, 39(4): 351-357.

图/表 6

图1 apo A1在人精子中的免疫荧光定位注：（a）FITC标记apo A1抗体处理后的免疫荧光颜色（绿色）；（b）DAPI染色液染色的免疫荧光颜色（蓝色）；（c）绿色荧光（FITC）与蓝色荧光（DAPI）重合图；（d）精子的相位对比图。

表1 apo A1抗体孵育2 h后精子总活力和前向运动精子百分率 %

组别	精子总活力	前向运动精子百分率
空白对照组	66.11±12.27^*	56.80±10.65^*
正常兔IgG组	67.68±13.07^*	57.91±12.72^*
apo A1抗体组	31.31±11.38	21.49±11.33

图2 各组精子内Ca2+水平比较和流式图注：（a）各组精子内Ca2+水平比较；与A23187组比较，*P<0.01；（b）空白对照的流式图；（c）A23187单独诱导的流式图；（d）经A23187诱导后与40 μg·mL-1 apo A1抗体孵育的流式图。

图3 获能过程中apo A1抗体对酪氨酸磷酸化的影响注：条带1为未获能精子，蛋白酪氨酸磷酸化无变化；条带2为获能精子，蛋白酪氨酸磷酸化增强（相对分子质量为55 000~125 000）；条带3为加入apo A1抗体孵育后的获能精子，蛋白酪氨酸磷酸化增强（相对分子质量为55 000~125 000）；条带4为加入正常兔IgG孵育后的获能精子，蛋白酪氨酸磷酸化增强（相对分子质量为55 000~125 000）。

图4 apo A1抗体对A23187诱导的顶体反应的影响注：与40 μg·mL-1 apo A1抗体+A23187组比较，*P<0.001。

图5 A23187诱发的精子顶体反应荧光染色图（×100）注：（a）经A23187单独诱导的精子顶体反应；（b）经A23187诱导后加入40 μg·mL-1 apo A1抗体孵育的精子顶体反应；A为顶体完整精子，B和C均为发生顶体反应的精子。

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载脂蛋白A1抗体通过抑制Ca²⁺载体A23187诱导的Ca²⁺内流降低精子活力

Apolipoprotein A1 antibodies reduce sperm motility by inhibiting ionophore Ca²⁺ A23187-induced Ca²⁺ influx

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