Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30598
標題: 免疫組織化學分析東方果實蠅卵黃形成作用
Immunohistochemical analysis of vitellogenesis in the oriental fruit fly, Bactrocera dorsalis (Hendel)
作者: 呂曉玲
Lu, Hsiao ling
關鍵字: vitellogenesis;卵黃形成作用;vitellogenin;vitellin;endocytosis;Bactrocera dorsalis;卵黃原蛋白;卵黃蛋白;內食作用;東方果實蠅
出版社: 昆蟲學系
摘要: 
本研究主要以免疫組織化學(immunohistochemistry)技術分析東方果實蠅(Bactrocera dorsalis(Hendel))的卵黃形成作用(vitellogenesis)。以經50-70% 硫酸銨鹽析沉澱及利用SDS-PAGE分離的東方果實蠅卵內之卵黃蛋白(vitellins, Vns)為抗原製備抗體,並以此抗體偵測東方果實蠅卵黃原蛋白(vitellogenins, Vgs)之合成、分泌、吸收及其在組織內之分佈。由西方墨點法(Western blotting)偵測第0至第8日齡血液得知,羽化後第4日齡的雌成蟲血液內始可檢測到卵黃原蛋白,並持續至第8日齡。以免疫螢光(immuno- fluorescence)標定腹部組織切片,發現第4與第5日齡雌成蟲的血液、脂肪體以及發育中的卵細胞皆顯示有螢光標定,且在卵黃形成時期的卵囊(follicle)有螢光標定存在。進一步以免疫金(immunogold)標定並以電子顯微鏡觀察卵黃形成時期之卵囊,發現在卵黃形成早期(early-vitellogenesis),卵囊細胞(follicle cells)本身會製造卵黃原蛋白,並自細胞端部分泌於卵囊細胞間隙以及卵囊細胞與卵細胞間之空腔。之後卵囊細胞會停止製造卵黃原蛋白,並且細胞間會有通道(patency)產生,此通道可供脂肪體所合成之卵黃原蛋白由血液進入卵囊。在卵細胞膜上類似被膜小窩(coated pit)之內陷以及卵細胞內類似被膜小泡(coated vesicle)之構造皆有免疫金標定,由此推測卵黃原蛋白到達卵細胞表面時,是以內食作用吸收至卵細胞內,另外卵細胞內之內食泡(endosome)、轉變卵黃體(transitional yolk body)與成熟卵黃體(matured yolk body)皆有免疫金標定,推測卵黃原蛋白由細胞膜內食形成類似被膜小泡後,先聚集為內食泡再形成轉變卵黃體,最後形成成熟卵黃體,在卵內沉積。

The process of vitellogenesis of the oriental fruit fly, Bactrocera dorsalis (Hendel), was investigated by immunohistochemical methods. The purified vitellins (Vns) were collected by salting out the egg homogenate with 50-70% ammonium sulfate and further separated in SDS-PAGE, and used as antigens to produce polyclone antibody. The antibody, which was confirmed to specifically against vitellogenins (Vgs), was used for analyzing the synthesis, secretion and storage of Vgs and distribution of Vgs in the tissues in the adult females of B. dorsalis. Western bolting examination indicated that Vgs were first detected in the 4-day-old female hemolymph, and continuously existed in after observations. Immunofluorescence localization of Vgs and Vns in the abdomenal sections of the 4- and 5-day-old adult females demonstrated that Vgs distributed in the hemolymph, fat body and vitellogenic follicles. Immunogold electron microscopy analysis of follicle sections revealed that follicle cells synthesized and secreted Vgs during early-vitellogenesis and the Vg production stopped in the cells before patency formation between follicle cells. Follicle cells secreted Vgs from the apical sides of the cells into inter-cellular space of follicle cells and the space between follicle cells and oocyte. The Vgs in hemolymph, which produced by the fat body, reached the oocyte through an enlarged patency. The Vgs were taken up by oocytes via endocytosis, which involved formation of coated pit-like invagination, coated vesicles, endosomes, transitional yolk bodies and matured yolk bodies, sequentially, and finally were deposited in the yolk of eggs.
URI: http://hdl.handle.net/11455/30598
Appears in Collections:昆蟲學系

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