日本鰻青春發育階段卵細胞粒線體之研究

Abstract

日本鰻 (Anguilla japonica) 在淡水養殖環境中無法完成生殖成熟，前人的研究中發現，以海水馴化後，藉由外源性的性腺激素可誘導鰻魚成熟並產卵受精，而孵出的柳葉鰻，可以養成變態至玻璃鰻，甚至到達成鰻，但是由於受精卵的卵質不佳，魚苗的存活率極低，目前還無法商業化養殖鰻苗。魚類青春發育的開啟受到基因遺傳、身體的能量代謝以及環境因子影響的控制，鰻魚的生殖成熟被認為可能與身體的能量代謝比較有關。鰻魚的生殖成熟會伴隨著銀化的發生，無論是銀化現象的發生或是生殖成熟皆需要很大的能量需求，粒線體就扮演很重要的角色。因此，本研究觀察鰻魚在青春發育過程中，隨著卵細胞發育階段的進行粒線體的分佈狀況及數量的變化，藉此瞭解鰻魚青春發育及生殖成熟的機制。 本研究所採用的淡水養殖日本鰻藉由胸鰭和體表的顏色及外部型態的不同將鰻魚分為Y1、Y2、S1及S2四個銀化的階段。淡水養殖鰻，雄鰻體重為462 ± 79公克，雌鰻體重為580 ± 106公克。主要以Y2階段為主，而且未發現有S1和S2的銀鰻出現，經過人工誘導4~8週後，才有S1和S2的銀鰻出現，雌鰻與雄鰻的銀化階段並無差異。此外，在研究中發現有一部分的鰻魚具有Y2和S1兩個階段的形態特徵，胸鰭顏色變黑但外緣並未變色，腹部體色變為灰黑色。而且鰻魚經過海水的馴化，這種銀化階段的鰻魚會增加。 利用粒線體的專一性螢光染MitoTracker標定鰻魚去除濾胞之卵細胞 (defollicular oocyte) 中的粒腺體，隨著卵細胞發育階段的進行，粒線體會呈現不同的分佈形態。在前卵黃生成期 (previtellogenic stage) 剛開始時，粒線體的分佈會集中形成單獨一個或兩個粒線體雲 (mitochondrial cloud, MC) 。但進入卵黃生成期 (vitellogenic stage)，粒線體的數量明顯增加形成多數較小的聚集，並分散於整個卵細胞中。另一方面，以腦下垂體研磨液 (salmon pituitary homogenate, SPH) 和甲基睪固酮 (methyltestosterone, MT) 人工誘導成鰻，卵細胞粒線體的數量亦有增加的趨勢。由結果顯示，日本鰻在青春發育過程中，卵細胞粒線體分佈隨著卵細胞發育的進行呈現明顯的變化，粒線體的數量也有明顯增加，經過人工誘導成熟的成鰻粒線體也有明顯增加。因此，粒線體在日本鰻青春發育過程中很可能扮演重要的角色，詳細的功能還有待進一步研究探討。

Japanese eel, Anguilla japonica, cannot complete its reproductive maturation in freshwater. After seawater acclimation and exogenous gonadotropins induction, the gonad of Japanese eel can mature and produce gametes. Although artificial induction can stimulate eel spermiation, spawning and success in production of glass eels, even to mature eels, the success rate of producing glass eel and survival rate of larvae are still very low. The onset of fish puberty is controlled by genetic, metabolism and environment factors. The reproductive development of eel is considered to be correlated with metabolism. The puberty onset of eel is accompanied by a silvering process. Not only reproductive maturation but the silvering are consuming processes, thus mitochondria may play an important role. So, in this study, we will examine the mitochondrial distribution and content during puberty and oogenesis, and study the reproductive development mechanism. The cultured Japanese eels with body weight (male: 462 ± 79 g ; female: 580 ± 106 g). Depending on the color of pectoral fin, body color and external morphology, it can be distinguished into four silvering stages (Y1、Y2、S1 and S2). In fresh water, the silvering stage is mainly on Y2 stage. After artificial induction, S1 and S2 stage were shown. And there were no difference between male and female eels. Furthermore, parts of the eels possessed the features of Y2 and S1, pectoral fins became darkness, the edge of pectoral fins was colorless and ventral skin became gray and black. Moreover, after seawater acclimation the eels of particular slivering stage were increased. In this study, the mitochondria of defolliclular oocyte were labeled with mitochondrial-specific fluorescent dye MitoTracker. There were various mitochondrial distribution patterns at different oocyte developmental stage. In the initial stage of previtellogenic oocyte, mitochondria assemble as a mitochondrial cloud (MC). There were one or two MC aggregate(s) in the oocyte during this stage. In the vitellogenic oocyte, the number of mitochondria increased significantly, dispersed in whole ooplasm and mitochondria formed small aggregates. Furthermore, the number of mitochondria increased in ovary after artificial induction with salmon pituitary homogenate and methyl testosterone. These results indicated that mitochondrial distribution patterns vary at different stages and mitochondrial population increases significantly during puberty in Japanese eel. Artificial induction also significantly increased mitochondrial population. Therefore, mitochondria may play an important role during puberty in Japanese eel, and their function during oocyte maturation needs further investigation.