Intracellular Ca2+ during fertilization and artificial activation in mouse oocytes.
OBJECTIVE
To study the mechanism of oocyte activation in mammals.


METHODS
Mouse oocytes arrested at metaphase of the second meiotic division were loaded with Fura 2-AM and then activated with ethanol, calcimycin, electric pulse or fertilization. Intracellular free Ca2+ during activation were measured by Spex AR-CM-MIC cation system. Cortical granule exocytosis after activation was detected under electron microscopy.


RESULTS
Sperm penetration initiated a long lasting Ca2+ oscillation in Ca2+ containing IVF medium in mouse ova. The Ca2+ oscillation lasted for over 3-4 h until the ova developed to pronuclear stage. The Ca2+ oscillated faster as extracellular Ca2+ concentration was increased from normal 1.7 mmol.L-1 to 5.0 mmol.L-1 and ceased to oscillate when extracellular Ca2+ was removed. The ova resumed Ca2+ oscillation after transferred back to IVF (Ca2+ 1.7 mmol.L-1). The ova which exhibited Ca2+ oscillation all extruded second polar body and formed pronuclei. Suppression the Ca2+ oscillation by intracellular injection of egtazic acid (2-10 pL, 200 mumol.L-1) blocked the activation of oocytes. Heparin (100 mumol.L-1) injection failed to prevent the Ca2+ oscillation. In artificial activation, ethanol, calcimycin, and a single electric pulse usually induced a monotonic Ca2+ rise and resulted in the activation only in older oocytes (> 18 h after CG injection). Activation of freshly ovulated oocytes required multiple intracellular Ca2+ increases induced by repetitive electric pulses. Artificial activation elicited the similar cortical granule exocytosis in oocytes as occurring at fertilization. Suppression of the intracellular Ca2+ elevation by introduction of egtazic acid into the oocytes blocked the activation process.


CONCLUSIONS
The increase of intracellular free Ca2+ is the primary signal responsible for the initiation of oocyte activation but there are distinct differences between fertilization and artificial activation both in Ca2+ change patterns and Ca2+ sources. Young oocytes require oscillatory Ca2+ signals for activation.
Deng
M Q
MQ
Jiangsu Academy of Agricultural Sciences, Nanjing, China.
Fan
B Q
BQ
D000818:Animals; D000001:Calcimycin; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004558:Electric Stimulation; D005260:Female; D005306:Fertilization; D008297:Male; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*
D000001:Calcimycin; D002118:Calcium