Lipovitellin Ⅱ為鯉魚卵膜組成之鑑定與其前質－卵黃生成前質素基因之選殖

Abstract

卵膜是水生卵生動物很重要的構造，它防護卵細胞、受精卵及胚胎不遭受水生環境的各種物理化學性損害，因此它的存在有著極重要的生理意義。對於卵膜的組成，尚未完全被研究清楚，本實驗室先前對於鯉魚卵膜的研究已發現許多組成分子，包括ZP2、ZP3、cystaitin、cathepsin S like substance、fibroin like substance、FEO-1，並對其性狀加以探討。然而卵膜上仍有許多未被發現的組成分子，本篇論文的主要目的即在鑑識鯉魚卵膜萃取液中分子量為 25kDa 及 30kDa 之組成分子。 本論文首先從鯉魚的卵膜萃取液中，純化出 p25 及 p30，藉由胺基端的胺基酸定序，發現胺基端序列和鯉魚的卵黃生成前質素的羧基端極為類似，藉由進一步的基因還殖與比對其他物種相似的胺基酸區域，我們確定p25 為卵黃生成前質素的lipovitellin II 部分。經由使用免疫組織化學的方法觀察 p25 在鯉魚卵細胞的分佈位置，發現在表層反應前的卵膜上即有 p25 的存在，而在表層反應後， p25在卵膜的量明顯地增加。至於 p25 是如何釋放到卵膜上，其機制還需更進一步的探討。 此外我們也將卵黃生成前質素的全長基因還殖出來，目前已完成 3500bp 的定序，此基因將可作為日後研究除了 lipovitellin Ⅱ 外，是否還有其他部分的卵黃生成前質素組成卵膜蛋白之用。

In aquatic oviparous animals, the chorion is a critical structure. It protects the fertilized egg and early embryo from chemical and physical damage. The knowledge about the components of piscine chorion is still limited. In our preliminary study on the components of carp chorion, some components have been identified, including ZP2、ZP3、cystatin、cathepsin S-like substance、fibroin-like substance and FEO-l. However, there are still a lot of unidentified chorion proteins. In this study, the two components, p25 and p30, from carp chorion are found. A series of studies are performed to characterize them. The N-terminal amino acid sequence of p25 is very similar to the partial sequence of fathead minnow vitellogenin. By 3'-RACE, the p25 cDNA was cloned and analyzed. The deduced amino acid sequence of p25 cDNA is also homologous to lipovitellin II that is located at the C-terminal of the vitellogenin from various vertebrates. The result of immunocytochemical staining reveals that the p25 is present in the carp chorion before ovulation. Moreover, the signal intensity of p25 in the chorion of cortical reacted eggs is higher than that of oocytes and ovulated eggs. The mechanism of translocation of p25 to the chorion remains to be elucidated. A cDNA of carp vitellogenin of 3.5kb is cloned. The cDNA may provide further information regarding whether other parts of vitellogenin may or may not be a component of carp chorion.