Injection of SF Stimulates IP3 Production in Xenopus Oocytes
To determine whether SF injection stimulates IP3 production, we measured the intracellular concentration of IP3 ([IP3]i) in single Xenopus oocytes injected with SF using a biological detector cell combined with capillary electrophoresis. The Xenopus oocyte was chosen because of its large size, thus allowing simultaneous Ca2+ monitoring and cytoplasmic sampling for [IP3]i quantification. First, we determined whether injection of SF was able to induce Ca2+ release in Xenopus oocytes. As shown in Figure 3, approximately 20 sec after SF injection, [Ca2+]i was increased. This Ca2+ increase occurred initially at the SF injection site, at the peripheral region of the oocyte, and then spread across the ooplasm over 15-20 min, and this time course was similar to that reported for normally fertilized Xenopus eggs.
In contrast, injection of SF buffer alone did not result in the generation of a [Ca2+]i rise (data not shown). Five to 8 min after SF injection, a sample of ooplasm was removed from each oocyte (n = 6) and electro-phoresed onto the detector cell for [IP3]i measurement. The SF induced a significant rise in [IP3]i of Xenopus oocytes from 40 nM to 1.3 ± 0.5 ^M (range, 0.5-2.5 ^M). Addition of SF directly onto the detector cell did not elicit a [Ca2+]i rise, suggesting that the active component of SF is not IP3.
FIG. 3. Confocal Ca2 + images of a Xenopus oocyte after injection of SF. The number presented below each image denotes the time (in seconds) from injection of SF. The image at Time 0 shows the oocyte’s basal level of Ca2 +. Approximately 20 sec after SF injection, [Ca2+]j began to increase concentrically around the SF injection pipette, and values returned to baseline approximately 23 min later (1450 sec).