Abstract
一、中文摘要 (Chinese Abstract)雌性甲蟲蟑螂羽化交配後咽喉側腺細 胞會週期性的增大細胞體積，然後進行自體吞噬而萎縮的過程(Cheng and Chiang 1995)。在這過程中，高基氏體是許多種酵素與膜蛋白的加工工廠 、合成溶小體酵素與膜蛋白並在自體吞噬初期扮演隔離胞器的角色，( Yang and Chiang 1997)。本實驗應用BODIPY-ceramide螢光染色法及分析 高基氏體輸出端特有的焦磷酸硫氨酵素(Thiamine pyrophosphatase, 簡 稱TPPase) 的活性來顯示高基氏體在咽喉側腺細胞同步自體吞噬中的量的 變化。我們的結果顯示在雌性甲蟲蟑螂羽化交配後5~7天，即咽喉側腺細 胞同步自體吞噬之初期，腺體總TPPase活性最高。第8天TPPase活性快速 地下降，顯示TPPase被分解掉的速度比新生成的速度快。BODIPY- ceramide 染色實驗也可看出在第4~6天高基氏體的顆粒逐漸增多且增大， 第8天高基氏體顆粒數逐漸減少。顯示高基氏體在咽喉側腺細胞進行同步 自體吞噬之初期會增生與膨大，然後被消化掉。以Neutral red染酸性胞 器，觀察在咽喉側腺細胞同步自體吞噬過程中胞器酸化的情形，發現羽化 交配後第六天開始出現大量的大顆粒的酸性融合體(亦即giant autophagolysosome)，顯示此時有明顯的胞器融合與酸化現象。以上說明 了第6天的溶小體活性大增的來源為先一波增生的高基氏體。並說明了先 有高基氏體的增生後有溶小體活性的增加，並且高基氏體與溶小體會參與 自體吞噬的過程，然後逐漸變成大顆粒的消化性自體吞噬體。顯示高基氏 體與胞器酸化現象可作為自體吞噬過程的階段性指標。到第12天時，此時 大部份細胞具有少量且巨大的酸性融合體，但是細胞大部份已變小，顯示 自體吞噬已進行到後期。因此，消化作用與細胞萎縮的必要時段主要發生 於第6~12天之間。利用離體培養測試可能的自體吞噬誘發因子，我們發現 雖然羽化交配後第六天自體吞噬可於離體培養的狀況下持續進行，但是羽 化交配後第4天的咽喉側腺並不會自發性地進行自體吞噬。而且蛻皮激素 及allatostatin-I都不會誘發離體培養的羽化交配後第4天的咽喉側腺進 行同步自體吞噬。切斷未交配的雌性甲蟲蟑螂的一側腦與咽喉側腺的神經 連結nervi corporis allati I(簡稱NCA1) 之後，咽喉側腺仍可增生發育 ，然後進行同步自體吞噬與細胞萎縮。因此，NCA1神經的連結、交配行為 與雄性蟑螂的精包不是誘發咽喉側腺增生與同步自體吞噬的必要因素。然 而，未被切斷神經的一側咽喉側腺並無發育現象。因此，咽喉側腺的發育 受NCA1神經的抑制。另外，切除羽化交配後第四天的卵巢並不能去除誘發 咽喉側腺同步自體吞噬的因子。因此，誘發自體吞噬之因子亦非來自成熟 的卵。 二、英文摘要 (English Abstract)Cheng and Chiang (1995) showed that in Diploptera punctata, volumes of corpora allata (CA) cells fluctuated simultaneously, growth, autophagy and atrophy during ovarian cycle. Although there are four different pathways of autophagy for organelles targeted to destruction, all of them lead to formate Golgi apparatus, lysosomes and giant autophagolysosomes.The aim of this study is to establish assay system for studying possible factors regulating autophagy in CA cells of D.punctata. For this purpose, changes in the functional activity of Golgi apparatus and the number of cells containing giant autophagolysosomes were examined as potential markers of autophagic processes during the ovarian cycle in mated females D.punctata. Two possible inducers of autophagy in CA cells, namely allatostatin I and 20-OH-ecdysone were examined in vitro, and roles of ovary and nerve connection in regulating autophagy were examined in vivo using the assays developed. The functional activity of Golgi apparatus were examined by two methods. In the first methods, changes in the distribution and amount of Golgi complexes were monitored by a modified BODIPY-ceramide staining( Pagano and Martin, 1988). In the second method, changes in CA TPPase activity were estimated, according to Heinonen and Lathi (1981). The distribution and amount of lysosomes and autophagolysosomes was examined by using Neutral red as a staining marker. (Sousa et al. 1996). We found that in mated females, TPPase activity increases on day 4, reaches its maximum between day 5 and day 7, then decreases gradually, during the ovarian cycle. In virgin females, TPPase activity remained at low level. In mated females, the number of Golgi spots increased from day 4, reached their maximum size on day 6, whereas in virgin females it seemed to be on low level. The number of small lysosome spots, stained with neutral red, increased on day 5, and large amount of giant autophagolysosomes occurred on day 6 of ovarian cycle in mated females. In virgin females the large amount of autophagolysosomes did not occur. Occurrence of giant autophagolysosomes was used as a marker of autophagy in further studies. Both allatostatin I and 20-hydroxyecdysone did not induce the autophagic processes in vitro just like control glands from 4-5 days old mated females. However, when the glands taken from 6-day old mated female, autophagy occurred and continued for at least 2 days in vitro. Thus it seems that, autophagy of CA cells was induced between day 5 and day 6 of ovarian cycle. If 4 day old mated females were ovariectomized, autophagy occurred in CA cells on day 6 of ovarian cycle. Thus ovariectomy did not prevent autophagy in CA cells.In order to study the effect of the brain on induction of autophagy of CA, CA of freshly emerged virgin females were denervated, and then tested the autophagic processes. After denervation, the numbers of giant autophagolysosomes and Golgi apparatus increased and reached its maximum on day 7, and then decreased, just like in CAs from normal mated females. In contrary, the cells of the intact CA from the same D. punctata did not grow up, and large amount of autophagolysosomes did not occur. It suggests that nerves transmit inhibitor to suppress cell growth. Some regulatory aspects of autophagy in corpus allatum and possible roles of Golgi apparatus was discussed.The findings mentioned above are generally in agreement with a model for giant autophagolysosome formation, proposed by Cheng and Chiang (1995). Staining of giant autophagolysosomes with neutral red is suggested as a potential tool for an assay for factors regulating autophagy in CA cells.