精子表面透明带蛋白受体的分析与功能研究

Analysis and functional study of zona pellucida receptors on sperm surface

目的通过对精子细胞膜表面SLEX/透明带结合蛋白进行定量分析,确定精子透明带结合能力和受精能力。方法选取2017年1月至2018年12月我院生殖中心76例精液标本(23例正常生育者,53例不育患者),采用半透明带试验(hemizona assay,HZA)分析不育组是否存在透明带结合障碍;采用络合异硫氰酸荧光素的豌豆凝集素(FITG-PSA)荧光标记法检测精子自发顶体反应率;采用流式细胞仪检测精子C1orf56,ZPBP1,SPACA1的免疫反应性以及与荧光标记的SLEX-牛血清蛋白的结合能力;Western Blot检测精子膜蛋白中C1orf56,ZPBP1,SPACA1蛋白的表达水平。结果不育组53例中32例患者HZI<30,60.4%的比例存在透明带结合障碍;不育组透明带结合障碍患者的精子自发顶体反应率(25.05±6.45)%明显高于对照组(5.12±2.12)%,差异有统计学意义(P<0.001);在不育组中,透明带结合障碍患者的精子自发顶体反应率(25.05±6.45)%明显高于无透明带结合障碍组(15.32±4.76)%,差异有统计学意义(P<0.001);在精子膜表面SLEX结合蛋白中,不育组透明带结合障碍患者的精子细胞中C1orf56,ZPBP1,SPACA1以及与荧光标记的SLEX-牛血清蛋白的结合比例均降低,与对照组比较差异有统计学意义(P<0.05);在精子膜蛋白中,不育组透明带结合障碍患者的精子细胞中C1orf56,ZPBP1,SPACA1蛋白表达水平均降低,与对照组比较差异有统计学意义(P<0.05)。结论受精率低与精子-透明带结合障碍相关,精子透明带结合障碍可能诱导较高水平的自发顶体反应,同时透明带结合异常的精子其细胞膜表面的分子表达受到干扰。

Objective To determine the binding ability of sperm zona pellucida and fertilization ability by using a quantitative analysis of SLEX/Zona pellucida binding protein on sperm cell membrane surface.Methods Seventy-six semen samples(23 normal births and 53 infertile patients)were collected from the reproductive center of our hospital.The hemizona assay(HZA)was used to analyze whether there was zona pellucida binding disorder in the sterile group.The sperm acrosome was detected by fluorescein isothiocyanate labeling of pea lectin(FITG-PSA).The immunoreactivity of spermatozoa C1orf56,ZPBP1,SPACA1 and the binding ability of fluorescently labeled SLEX-bovine serum albumin were detected by flow cytometry.The expression levels of C1orf56,ZPBP1 and SPACA1 in sperm membrane protein were detected by Western blot.Results Of the 53 infertile patients,HZI of 23 patients less than 30,and 60.4%of them had zona pellucida-binding disorder.Spermatozoa spontaneously with zona pellucida acrosome reaction in the infertile patients was(25.05±6.45)%which was higher than control group(5.12±2.12%;P<0.001).In the infertility group,patients with sperm zona pellucida binding acrosome disorders had(25.05±6.45)%of spontaneous reaction rate which was significantly higher than those without zona binding disorder group(15.32±4.76%,P<0.001).In the SLEX sperm surface membrane bound proteins,the percentages of C1orf56,ZPBP1,SPACA1 and fluorescently labeled SLEX-bovine serum albumin in the sperm cells of patients with dysfunction were decreased(P<0.05).In the infertile group,the expression levels of C1orf56,ZPBP1 and SPACA1 in spermatozoa of patients with zona pellucida-binding disorders were significantly lower than those in the control group(P<0.05).Conclusion Low fertilization rate was associated with sperm-zona pellucida binding disorder.Sperm zona pellucida binding disorder may induce a higher level of spontaneous acrosome reaction,while spermatozoon with abnormal zona pellucida binding inhibits interferes the molecular expression of the cell membrane surface.