U73122 Inhibits SF-Induced [Ca2+]-: Oscillations U73122, a membrane-permeable inhibitor of events leading to PLC activation and/or of PLC itself, was used in this study to evaluate whether SF-induced [Ca2+]i oscillations are mediated by activation of the PI pathway. Incubation of mouse eggs in 20 ^M U73122 for 15 min completely suppressed the ability of SF to initiate [Ca2+]i oscillations (n = 0/7; Fig. 1B). Incubation of eggs with lower concentrations of the inhibitor was not as effective, and SF-induced Ca2+ responses were blocked in only 1/5 (10 ^M) and 7/14 (15 ^M) eggs (Fig. 1E), although in the few eggs in which the responses were inhibited the inhibition was complete.
Incubation with higher U73122 concentrations (25 ^M), although effective in blocking Ca2+ responses, resulted in cell death within a couple of hours after exposure, and this higher concentration was not used in our studies. Injection of SF induced normal [Ca2+] oscillations in untreated eggs (n = 8/8; Fig. 1A) or into eggs incubated for 15 min in 20 ^M U73343 (n = 4/4), the inactive analogue of U73122 (Fig. 1C; P < 0.05). In spite of successful inhibition of SF-induced Ca2+ responses by U73122, several detrimental effects have been reported in cells exposed to this inhibitor, including a rise in basal [Ca2+]i levels and emptying of IP3-sensitive internal Ca2+ stores. Thus, to determine if the inhibition of the Ca2+ responses in our studies was due to emptying of the Ca2+ stores, eggs incubated with 20 ^M U73122 were injected with 500 ^M IP3 (concentration in the pipette). This concentration of IP3 was chosen because it closely reproduced in frequency and duration the oscillations evoked by injection of 0.5 mg/ml SF. Injection of IP3, which acts directly on the IP3R in the ER, induced [Ca2+]i oscillations in all eggs (Fig. 1D; n = 4/4). These results suggest that the inhibitory effect of U73122 on SF-induced [Ca2+]i oscillations is most likely due to its ability to prevent activation of the PI pathway and subsequent IP3 production. FIG. 1. Incubation of mouse eggs in U73122 inhibits SF-induced [Ca2+] oscillations. A) Control eggs show normal [Ca2+]j oscillations upon SF injection. B) Incubation of eggs in U73122 (20 ц,М) inhibits SF-induced [Ca2+] oscillations. C) Incubation in U73343 (20 ц,М) fails to inhibit [Ca2+] oscillations. D) U73122 (20 ц,М) did not inhibit [Ca2+] oscillations induced by injection of IP3 (500 ц,М). SF concentration was 0.5 mg/ml in the injection pipette throughout these studies, resulting in intracellular SF amounts representing 1-2.5 sperm equivalents. Arrows indicate times of SF or IP3 injection, respectively. The pipette was removed immediately after the injection in all cases. E) Percentage inhibition of [Ca2+] oscillations when eggs are incubated in various concentrations of U73122. Error bars represent SEM. Different superscript letters denote significant differences (P < 0.05; chi-square test).