| Project/Area Number |
08456141
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied animal science
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
MIYAMOTO Hajime Kyoto University, Graduate School of Agriculture Professor, 農学研究科, 教授 (00026618)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIMOTO Miki Kyoto University, Graduate School of Agriculture Assistant Professor, 農学研究科, 助手 (20243074)
MANABE Noboru Kyoto University, Graduate School of Agriculture Associate Professor, 農学研究科, 助教授 (80243070)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1996: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | Ovary / Follicle / Granulosa cell / Apoptosis / Cell death receptor / Porcine / Monoclonal antibody / Porgram cell death / カスパーゼ様プロテアーゼ / 受容体分子 / モノクロナール抗体 / 卵丘細胞 / 卵胞閉鎖 / 家畜 |
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| Research Abstract |
Porcine ovary samples were prepared for histochemical and ultrastructural observations. In situ analysis of DNA fragmentation was performed on histological sections of follicles using TUNEL method.
1. Healthy follicles : No apoptotic cells were observed among granulosa or cumulus cells, internal or external theca cells, or oocytes.
2. Early stage of atresia : Apoptosis demonstrated histochemically by TUNEL staining was seen in scattered granulosa cells located on the inner surface of the follicular wall, but not in cumulus cells, internal or external theca cells, or oocytes. Typical apoptotic features, i.e. condensed nuclei, were seen in the scattered granulosa cells, but cumulus cells with normal ultrastructure were also seen in the same follicles.
3. Late to final stages of atresia : Granulosa cells scattered on the inner surface of the follicular wall began to undergo apoptosis, but no TUNEL-positive cells were detected among the cumulus cells.
An IgM monoclonal antibody (PFG-1) capable of inducing granulosa cell apoptosis was produced against granulosa cells prepared from healthy antral follicles of pig ovaries. Two-dimensional Western blotting analysis revealed that PFG-l specifically recognized a cell-membrane protein (55 kD, pI 5.9 ; PFG-l antigen). PFG-l immunohistochemically reacted with granulosa cells of healthy follicles but not of atretic follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing PFG-1, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. Specific inhibitors of ICE-like cysteine proteases could stop the apoptotic process induced by PFG-1. Histochemical and biochemical properties of the PFG-l antigen are different from those of Fas or TNF-receptor, and PFG-l antigen is a novel cell death receptor. ICE-like proteases and Ca^<2+>-dependent endonuclease are involved in intracellular signal transmission of this novel receptor.
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