外源性激素對日本鰻肝臟卵黃前質表現及卵巢發育調控之研究

Abstract

本論文的研究目的在探討人工誘導日本鰻性腺發育過程中，性類固醇 (estradiol-17β 與 methyltestosterone) 與鮭魚腦下垂體研磨液 (SPH) 分別對於肝臟的卵黃前質生合成與卵巢卵濾胞的影響。雖然人工催熟日本鰻目前已經可以獲得受精卵並且孵化出仔稚魚苗，而魚苗的活存天數可維持數週甚至達250天以上，經變態成為玻璃鰻，但由於受精卵卵質不佳 (受精率與孵化率低)，魚苗活存率也不高，在人工催熟下日本鰻的生殖生理仍有非常多的問題，特別在於卵細胞在卵黃蓄積這個階段，荷爾蒙處理對於卵濾胞的生長及卵細胞成熟影響。以RT-PCR與RACE等分子生物技術，選殖並定序完整之日本鰻卵黃前質核酸序列，並藉北方墨點轉漬法 (Northern blot assays) 檢視性類固醇與鮭魚腦下垂體研磨液對於誘發肝臟卵黃前質的表現。探討以荷爾蒙注射方式所進行的日本鰻人工催熟方法，在短期與長期持續給予下，是否可有效地促進卵黃前質生成以及更進一步地促使卵巢中的卵濾胞完成卵黃蓄積。另一方面，由蛋白質序列分析蛋白質組成，探究日本鰻卵黃前質的生合成在適應其特殊降海生殖行為下所扮演之角色，以及此一基因在演化的過程中與其它硬骨魚類的親源性關係。實驗得到一完整之cDNA基因序列，確定為日本鰻卵黃前質基因 (Japanese eel Vtg cDNA, Genbank Accession Number：AY775788)。基因全長為5395bp，包含15bp 的5端未轉譯區、5229bp的開放讀碼區、135bp 的3端未轉譯區與16bp的多腺嘌呤尾；其所轉譯蛋白質分子約為1743個胺基酸，包含完整的N端lipovitellin-I region、中段的polyserine domain region以及C端的lipovitellin-II。與其他硬骨魚類卵黃前質基因序列比對後相似度約34%-61%。以北方墨點法偵側肝臟卵黃前質基因表現，單獨以雌二醇誘導雖然刺激其合成，但無法有助於促進卵巢中的卵細胞完成卵黃蓄積。另一方面，以鮭魚腦下垂體研磨液併用性類固醇激素誘導卵濾胞發育，檢視對日本鰻卵巢發育之影響。為能有效檢視性類固醇及鮭魚腦下垂體研磨液是否會對於卵濾胞經由細胞凋亡而造成退化的結果，利用原位偵測方法「TUNEL」技術，偵測細胞凋亡作用發生於濾泡層之位置，並進一步觀察在發育過程中，鮭魚腦下垂體研磨液、性類固醇、卵胞數目之減少以及由細胞死亡作用所造成退化作用四者之間的關聯。其結果顯示，在人工催熟卵巢發育早期卵黃蓄積階段 (previtellogenic與early vitellogenic stages)，卵濾胞的確受到不同外源性激素的刺激而誘發由細胞凋亡所引發之退化現象，其發生位置位於鞘細胞 (theca cell)。雖然SPH為卵濾胞發育過程中的必要條件，但是長期催熟結果則是表現在卵胞數目減少、卵徑頻度分佈呈現不同步化。但SPH在配合甲基睪固酮 (MT) 並用的作用下，卵巢卵濾胞發育沒有發現細胞凋亡所引發之退化現象，並可促進卵濾胞生長並維持其存活，並促使卵胞群發育同步化。 本研究希望能夠改善人工誘導性腺成熟對日本鰻性腺發育影響，使用SPH雖然能夠促進卵濾胞的發育，但還是發現卵胞數有減少之趨勢。如果在雄性素參與協同作用下，一方面促進卵濾胞對SPH的感受性，使得卵濾胞進行卵黃蓄積並且同步化生長發育。同時也因為雄性素對卵濾胞的直接作用，促使絕大部分的卵濾胞得以維持存活狀態，減少因為人工給予藥物催熟而使得卵濾胞退化現象發生。

In higher vertebrates, the reproduction of teleost is controlled by the hypothalamus-pituitary-gonadal axis. This study examines the effects of exogenous gonadotropic hormone (salmon pituitary homogenate, SPH) and sex steroids (estradiol-17β and methyltestosterone) on the dynamics of the Japanese eel (Anguilla japonica) ovary during the process of vitellogenesis. This study attempted to obtain the cDNA encoding for Vtg from female Japanese eels, Anguilla japonica. Rapid amplification of cDNA ends (RACE) and polymerase chain reaction (PCR) were used to amplify Vtg cDNA prepared from liver extracts. Obtained PCR products were subcloned and sequenced. The overall sequence of eel Vtg cDNA isolated in this study contained 5395 bp nucleotides. This Vtg sequence encodes 1743 amino acids of the precursor molecule, and is entirely composed of the characteristic N-terminal lipovitellin-I region, an internal polyserine domain region, and a c-terminal lipovitellin-II region. The deduced amino acid sequence from these clones shares 34–61% identity with other teleost Vtgs. Northern blot assays of Vtg gene expression following hormonal treatment demonstrated that this Vtg is synthesized in the liver under stimulation by estradiol injection. However, Vtg synthesis may not be enhanced by salmon pituitary homogenate (SPH) induction for the developing ovarian follicles. Notably, the effect of methyltestosterone, following SPH injection, may be more appropriate for the uptake of Vtg by ovarian follicle maturation during the artificial maturation of Japanese female eels. On the other hand, in situ TUNEL demonstrated that apoptosis-mediated atresia of follicular cells, in the previtellogenic and early vitellogenic stages, display sensitivity for SPH and sex steroids, respectively, during short-term induction; long-term inductions also indicate that the development and growth of ovarian follicles (i.e., total number and size-frequency distribution) responded differently to the sexual hormone. SPH treatment was effective and indispensable for artificially hastening maturation; however, multiple injections of E2 or MT alone failed to promote ovarian development in these cultivated fish. Both the E2 and SPH plus E2 treatment at the time of vitellogenesis led to the failure of ovarian development (i.e., a significant decrease of total numbers) in the present study. Interestingly, the synergistic effect of SPH combined with MT seems to directly affect the gonadal development of female Japanese eels in the present study. This study demonstrated that androgen is not atretogenic and actually enhances the growth and survival of primary follicles, and has a potential effect on the process of vitellogenesis in SPH treatment.