哺乳類動物受精過程中必要之醣蛋白的鑑定與特性

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题名:	哺乳類動物受精過程中必要之醣蛋白的鑑定與特性 Identification and Characterization of Glycoproteins Essential for Mammalian Fertilization
作者:	楊惠婷
贡献者:	生物科技研究所
关键词:	卵子皮質顆粒胚胎發育 oocyte cortical granules embryogenesis
日期:	2014
上传时间:	2014-09-18 14:13:56 (UTC+8)
摘要:	先前研究證實，LCA (Lens Culinaris Agglutinin)這種凝集素可以辨識小鼠皮質顆粒。我們實驗室曾利用質譜儀分析小鼠卵子中LCA所辨識的醣蛋白，而其中有一醣蛋白為WNT4。本研究假設WNT4在受精時會伴隨著皮質顆粒而釋放及影響到精子與卵子間的受精作用，並觀察及研究WNT4醣蛋白在早期胚胎之表達及功能分析。利用免疫螢光染色法，西方墨點法，及功能分析法證實WNT4在卵子及多種時期早期胚胎之多項生物學上特點。免疫螢光染色結果顯示，WNT4與LCA同在原泡核卵母細胞(germinal vesicle intact)的皮脂顆粒及及受精卵上的卵膜(oolemma)。在二細胞胚胎週期、八細胞胚胎週期，WNT4會一直存在所有胚葉細胞(blastomere)及其細胞膜上。特別的是，在囊胚期(blastocysts) ，WNT4只辨識到滋胚層(trophoblast)。西方墨點法實驗結果發現，在小鼠減數分列第二中期卵子的皮質顆粒中有一主要蛋白質被WNT4抗體所辨識，其分子量約為66kDa，此蛋白質確認源自於皮脂顆粒。在原泡核卵母細胞、受精卵、二細胞胚胎週期、八細胞胚胎週期以及囊胚期都有兩種分子量的蛋白質可被WNT4抗體所辨識，分別約為66 kDa及32 kDa。功能分析實驗中證實，在體外受精(in vitro fertilization)培養中加入WNT4抗體，會抑制精卵結合並抑制其形成二細胞胚胎；在體外胚胎培養(in vitro embryo culture)中，將WNT4抗體加入培養液，證實二細胞胚胎無法發育至囊胚期。因此，WNT4參與受精過程，並且會調控著床前胚胎發育。 Mammalian fertilization is a complex process which takes place in female reproductive track. In mammals, the ovulated oocytes are arrested at metaphase II, which are highly specialized containing an unique structures termed cortical granules (CG). Cortical granules are membrane-bound organelles, located in the cortex of the unfertilized oocytes cortical granule reaction, is thought to function in blocking polyspermy and early embryogenesis. The purpose of this study was to characterize the LCA-binding cortical granule component WNT4 and to determine its distribution in unfertilized oocytes and fertilized oocytes, as well as in preimplantation embryo. Our immunefluorescent data showed that WNT4 was present in the cortex of the germinal vesicle intact (GVI) oocytes and metaphase II (MII) oocytes. Following fertilization, WNT4 remained to be associated with the plasma membrane of zygotes, and each blastomere in 2-cell and 8-cell embryos. Prior to implantation, WNT4 appeared to be only detectable in the trophoblast cells with reduced amount. Moreover, our western blot results showed that WNT4 appeared as two major proteins of 66 and 32 kDa in germinal vesicle intact oocytes. Our functional study results showed that the addition of WNT4-specific antibody significantly reduced the percentage of 2-cell embryo formation during in vitro fertilization, indicating that WNT4 played a role in cleavage of the zygote. In the embryo culture experiments, the addition of WNT4-specific antibody greatly inhibited the development of preimplantation embryo into blastocyst stage, indicating WNT4 not only plays a role in fertilization but also involved in regulating preimplantation embryogenesis. In conclusion, we have identified a mouse cortical granule component as WNT4, which function in regulating zygote cleavage and early embryogenesis.
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