精子中H-Y抗原的蛋白和mRNA表达背离

目的:进一步验证成熟精子中是否存在H-Y抗原的mRNA,并探讨精子mRNA的保留机制。方法:采集健康男性志愿者的精液,通过上游法纯化精子,使用多克隆抗H-Y抗原的IgY抗体和FITC标记的兔抗鸡IgG对精子进行免疫组化和流式细胞仪检测,观察成熟Y精子膜表面是否带有H-Y抗原。提取成熟精子总RNA,用RT-PCR的方法观察精子mRNA中是否含有H-Y抗原的mRNA。结果:免疫组化实验显示可在许多精子的头部与尾部的交界处非常清晰地看到H-Y抗原的荧光;流式细胞仪检测显示49.86%精子为H-Y抗原阳性。RT-PCR结果显示,未扩增到H-Y抗原的mRNA特异性条带。结论:成熟精子表面存在有H-Y抗原,但未检测到H-Y抗原的mRNA。

Missing of the H-Y Antigen mRNA in Human Ejaculate Spermatozoa

Objective: To investigated whether H-Y antigen mRNA is present in spermatozoal RNA.Methods: Human ejaculates were obtained from healthy volunteers.The ejaculate was purified by swim-up technique to get rid of somatic contaminations.Total RNA of purified spermatozoa was extracted by Trizol RNA isolation reagent.In order to determine whether it had effect on H-Y antigen on the membrane of Y-sperm,the IgY polyclonal antibody against H-Y antigen we had obtained was tested by immunohistochemistry and flow cytometry.We also examined whether mRNA from ejaculate spermatozoa contained H-Y antigen mRNA by RT-PCR analysis.Results: By immunohistochemistry,it is clearly shown many spermatozoa were stained by fluorescence-conjugated second antibody.Through flow cytometric analysis,the positive H-Y antigen percentage of human sperm reached at 49.86%.And RT-PCR analysis reported that there was no H-Y antigen-specific band found in the tests.Conclusion: These results approved the presence of H-Y antigen on the membrane of Y-sperm,but mRNA of H-Y antigen is not detectable in mRNAs in the ejaculate spermatozoa.