1995 Fiscal Year Final Research Report Summary
The mechanisms by which sperm activate oocyte in mouse fertilization
| Project/Area Number |
06671648
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Osaka University |
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Principal Investigator |
MASUMOTO Nobuyuki Osaka Univ., Med.School, Assistant Professor, 医学部, 助手 (70243235)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Cortical granule / Exocytosis / Fertilization / Intracellular calcium / small G protein / Rab |
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| Research Abstract |
Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently, we developed a method to evaluate the kinetics of exocytosis in single living cells, using an impermeable fluorescent membrane probe, TMA-DPH.In this study, we evaluated cortical granule (CG) exocytosis in living mouse eggs with TMA-DPH using digital imaging and confocal laser scanning microscopy. Time-related changes of CG exocytosis were estimated as the % increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate and ceased at 45-60 min. While the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next, we determined some intracellular mediators of exocytosis in the egg using this method. An intracellular calcium chelator, BAPTA-AM,and a microfilament inhibitor, cytochalasin B,blocked sperm-induced exocytosis. A GTP-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, micorofilament and GTP-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying of exocytosis in living eggs.
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[Publications] Tahara, M., Tasaka, K., Masumoto, N., Mammoto, A., Ikebuchi, Y., Miyake, A.: "Dynamics of cortical granule exocytosis at fertilization in living mouse eggs" American Journal of Physiology. (in press).
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