【摘要】： 作為生殖細胞的人類精子,承擔著將男性的遺傳物質傳遞給下一代的重要任務。傳統(tǒng)觀點認為精子唯一的目的就是傳遞父本基因組,本身是終末分化細胞,已經(jīng)關閉基因的轉錄與表達,但最近有越來越多的研究發(fā)現(xiàn)成熟精子中存在有大量而復雜的mRNA群體。本項研究旨在通過在精子mRNA中尋找H-Y抗原mRNA,來探討精子mRNA的保留機制以及精子mRNA可能執(zhí)行的功能。 本項研究采集健康男性志愿者的精液,通過上游法純化精子,使用多克隆抗H-Y抗原的IgY抗體和FITC標記的兔抗雞IgG對精子進行免疫組化和流式細胞儀檢測,觀察成熟Y精子膜表面是否帶有H-Y抗原。為研究成熟精子mRNA是否含有H-Y抗原的mRNA,我們提取精子總RNA,用RT-PCR的方法進行了分析。 免疫組化實驗顯示可在許多精子的頭部與尾部的交界處非常清晰地看到H-Y抗原的熒光,通過流式細胞儀檢測,被熒光素抗體標記的H-Y抗原陽性精子的比例為49.86%。經(jīng)RT-PCR分析沒有從成熟精子mRNA中得到H-Y抗原mRNA特異的條帶。 以上結果證實了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抗原表達于精子發(fā)生的早期階段,該結果與健康人類精子SAGE表達譜的結果一致。并且H-Y抗原mRNA缺失的現(xiàn)象提示了保留在精子中的mRNA,進入卵母細胞以后會表現(xiàn)至關重要的功能,而非僅僅是精子發(fā)生過程中遺留的殘存物。
[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.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R321

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