| 摘要: | 哺乳動物的受精過程起於精子與卵母細胞的接觸，過程包括一系列在配子內所發生的變化，並在 精卵細胞核的融合後終止。精子的生成起始於睾丸內，經由運輸送至附睾中繼續發展，最後在排 出雄性生殖器官外後於雌性生殖道中進行獲能反應及頂體反應，方可成為成熟且具受精能力的精 子。而精子內所含的分子例如醣蛋白，在精子發展成熟過程中密集且大量的合成, 長期以來被認 為在哺乳動物的配子發展及進行受精過程中扮演非常重要的角色。許多研究指出，哺乳類的精子 醣蛋白在精子生成的過程中出現了合成、分解和分佈上之變化。總之，醣化物在精子發展的過程 及精子最終獲得受精能力有著密不可分的關係,因在對精卵結合和受精過程中也扮演了相當重要 的角色。然而我們對哺乳動物精子的生化組成了解仍相當有限。因此，鑒定精子內容物及其相關 分子對了解精子發展、受精及生殖是必要的。 一直以來，生殖學家利用與醣類有專一辨識性的凝集素來研究哺乳類精子醣化內容物。我們的研 究團隊利用花生凝集素(peanut agglutinin/ PNA) 鑑定了小鼠內N- 乙醯半乳糖胺化 （N-acetylgalactosaminylated）蛋白和可能與這些醣蛋白有相互作用的分子。利用PNA 親和層 析法分離出數個小鼠N-乙醯半乳糖胺化蛋白質，再經由 LC/MS/MS 質譜儀技術完成蛋白質鑑定。 在小鼠精子中，我們發現內含一個特殊的蛋白質，methylated in normal thymocytes protein (MENT)，其分子量為38kDa。利用西方墨點法、免疫沉澱法、組織螢光染色法，確認在不同發展 時期 (取於睾丸中、於附睾中、獲能反應後、頂體反應後) 的精子的確有MENT 蛋白的存在。目 前生物學家對MENT 蛋白了解是非常有限的，可參考之研究文獻僅有數篇之少。而目前僅知的MENT 生物功能是與調控腫瘤和加速細胞增生相關，因此探討MENT 在生殖中所扮演的角色及其重要性 是必須且前瞻的。 如上述的結果及發現，我們提出來一系列的試驗，探討MENT 在精子的形成、發展、成熟過程中， 扮演的生物功能角色。此外，我們亦將探討MENT 是否在受精過程中也參與了調控的機制。由於 小鼠精子可能具有與人類同源同功能的蛋白質，故小鼠精子MENT 的研究可讓我們對於人類精子 生物合成有進一步的瞭解。如果實驗證明精子內的MENT 的確具有與生殖相關的生物功能，將對 於檢測精子的生殖能力有正面的助益，而所得到的資訊可以用於男性不孕症的臨床病理診斷。最 後，期望藉此探討精子MENT 對精子發展之影響的研究成果，提供非荷爾蒙避孕藥發展的新方向 及新希望。
Mammalian fertilization is a series of coordinated events involving multiple steps of mutual recognitions between haploid male and female gametes. The synthesis sperm starts in the testes and are later transit to epididymis for maturation. Ejaculated sperm are not fully functional and have to undergo capacitation and acrosome reaction to become fertile. Glycoconjugates are long thought to be essential in mammalian reproduction since the sperm glycoconjugates are extensively synthesized and modified during sperm maturation and transport. Therefore, we know that glycosylated components are important in sperm development and their acquisition of fertilizing ability. Nevertheless, relatively little is known about the exact biochemical composition of mammalian sperm. Therefore, it is a great interest to us to characterize sperm components and to define their reproductive roles and functioning in fertilization. Glycoconjugates are long thought to be essential in mammalian fertilization andtThe most convenient method for detecting sperm glycosylated components is the non-enzymatic lectin assay, in which the lectins can be used to recognize specific carbohydrate moieties. Recently, our research team has characterized new glycoconjugates in mouse sperm using lectin peanut agglutinin (PNA), which specifically recognizes N-acetylgalactosaminylated molecules using LC/MS/MS proteomic approach. Of analyzed samples, band with molecular weight 38 kDa was identified to be methylated in normal thymocytes protein (MENT). The protein is encoded by gene MENT and is a tumor modifier and accelerates lymphomagenesis. In our preliminary study, we showed, using western blotting， immuoprecipitation, and immunofluorescent staining, confirmed the presence of MENT protein in mouse sperm from developmental stages (testicular, epididymal, capacitated, and acrosome reacted sperm). This newly identified molecule has not yet been characterized with reproductive functions. Our finding of MENT protein present in sperm raises the question whether this molecule plays a role in sperm generation, development and maturation, during which is a window of time requiring rapid and tighly regulated cell division. Data obtained from this study will allow us to have a better understanding the molecular basis and changes associated with reproduction. In this proposed study, we will conduct a series of experiments to delineate the exact reproductive function of MENT protein. Identification and characterization of sperm components, such as MENT, may give us insights into the constituents of human sperm since homologous components are likely to be present. We believe that sperm component MENT may be used as indicative parameter(s) in assessing the maturation and quality (fertilizing ability) of sperm for patients under assisted reproduction therapy. Finally, it is tempting to envision the possibility that MENT involved in sperm generation and development may be used as new targets for developing non-hormone based contraceptive control. |
