Microinjection Techniques and [Ca2+]/ Monitoring in Mouse Eggs
Microinjection procedures were as previously described. Glass micropipettes were filled by suction from a microdrop containing 0.5 mM fura-2 dextran (fura-2D, dex-tran 10 kDa; Molecular Probes, Eugene, OR), SF/testis extracts (0.5 or 5 mg/ml protein concentration, respectively). IP3 (500 |xM; Molecular Probes), U73122 (30 |xM), CaCl2 (2.0 mM), or 10 ^M adenophostin A, a potent IP3R agonist (kindly provided by Dr. K. Tanzawa, Sankyo Co., Tokyo, Japan). Solutions were expelled into the egg’s cytoplasm by pneumatic pressure (PLI-100 picoinjector; Medical System Corp., Great Neck, NY).
The injection volume was approximately 5-10 pl and resulted in a final intracellular concentration of the injected compounds of approximately 1.5%-3% of the concentration in the injection pipette.
Fura-2D fluorescence was monitored as previously described. [Ca2+]i concentrations, Rmin, and Rmax were calculated according to the methods of Grynkiewickz et al. and Poenie and as previously reported; Rmin and Rmax values were 0.12 and 1.3, respectively. [Ca2+]i monitoring of mouse eggs was initiated 30-45 min after injection of fura-2D, which was approximately 15 h post-hCG, and ceased before eggs had reached 22 h post-hCG.