| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
|
|---|
| 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.
|
