人类精子中H-Y抗原mRNA的缺失与精子mRNA功能探讨

发布时间：2018-11-06 13:51

【摘要】： 作为生殖细胞的人类精子,承担着将男性的遗传物质传递给下一代的重要任务。传统观点认为精子唯一的目的就是传递父本基因组,本身是终末分化细胞,已经关闭基因的转录与表达,但最近有越来越多的研究发现成熟精子中存在有大量而复杂的mRNA群体。本项研究旨在通过在精子mRNA中寻找H-Y抗原mRNA,来探讨精子mRNA的保留机制以及精子mRNA可能执行的功能。 本项研究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。为研究成熟精子mRNA是否含有H-Y抗原的mRNA,我们提取精子总RNA,用RT-PCR的方法进行了分析。 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,通过流式细胞仪检测,被荧光素抗体标记的H-Y抗原阳性精子的比例为49.86%。经RT-PCR分析没有从成熟精子mRNA中得到H-Y抗原mRNA特异的条带。 以上结果证实了H-Y抗原存在于Y精子膜表面,但成熟精子mRNA中不含H-Y抗原的mRNA,表明H-Y抗原表达于精子发生的早期阶段,该结果与健康人类精子SAGE表达谱的结果一致。并且H-Y抗原mRNA缺失的现象提示了保留在精子中的mRNA,进入卵母细胞以后会表现至关重要的功能,而非仅仅是精子发生过程中遗留的残存物。
[Abstract]:Human spermatozoa, as germ cells, undertake the important task of transferring male genetic material to the next generation. The traditional view is that the sole purpose of spermatozoa is to transmit the paternal genome, which is a terminal differentiated cell, which has already turned off the transcription and expression of genes. However, more and more studies have recently found that there are a large number of complex mRNA populations in mature sperm. The aim of this study was to investigate the retention mechanism of sperm mRNA and the possible function of sperm mRNA by looking for H-Y antigen mRNA, in sperm mRNA. The semen of healthy male volunteers was collected and purified by upstream method. The sperm was detected by immunohistochemistry and flow cytometry using polyclonal IgY antibody against H-Y antigen and rabbit anti-chicken IgG labeled by FITC. The surface of mature Y sperm membrane was examined for H Y antigen. In order to study whether mature sperm mRNA contains H-Y antigen mRNA, total RNA, extracted from sperm was analyzed by RT-PCR. Immunohistochemistry showed that the fluorescence of H-Y antigen could be seen very clearly at the junction of head and tail of many spermatozoa. The percentage of H-Y antigen-positive sperm labeled by fluorescein antibody was 49.86 by flow cytometry. H-Y antigen mRNA specific bands were not obtained from mature sperm mRNA by RT-PCR analysis. These results confirmed that H-Y antigen existed on the surface of Y sperm membrane, but mRNA, without H-Y antigen in mature sperm mRNA indicated that H-Y antigen was expressed in the early stage of spermatogenesis. The results were consistent with the results of SAGE expression profile of healthy human sperm. Moreover, the absence of H-Y antigen mRNA suggests that mRNA, retained in spermatozoa may perform a vital function when it enters the oocyte, rather than being a remnant of spermatogenesis.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321

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