Insemination or phosphatidic acid induces an outwardly spiraling disk of elevated Ca2+ to produce the Ca2+ wave during Xenopus laevis fertilization.

During Xenopus fertilization, the initial intracellular calcium ((Ca2+)i) release at the sperm-egg binding site (hot spot) has not been described without the use of inhibitors, nor related to underlying ER structure. Without inhibitors, we now report that sperm induce an initial hot spot after sperm addition to Xenopus eggs that was ~25 µm. This area is consistent with the size of ER patches and clusters of IP3 receptors that have enhanced activity. Furthermore, we find a new mechanism for the fertilization (Ca2+)i wave; instead of outward diffusion of inositol 1,4,5-trisphosphate (IP3), we find that the wave was generated by an outward, clockwise rotation of a ~63 µm disk of elevated (Ca2+)i moving very rapidly at ~65 µm/s. We also suggest a new mechanism for the acceleration of the fertilization (Ca2+)i wave as the disk accelerated and was joined by other rotating disks (some rotating counterclockwise) at a time when the speed of the (Ca2+)i wave increases. To examine the role of phosphatidic acid (PA) in the release of (Ca2+)i during Xenopus fertilization, we find that two inhibitors of PA production delayed the appearance of fertilization hot spots by ~9-12 min but did not reduce the size of hot spots and actually accelerated the later (Ca2+)i wave. Surprisingly, global addition of PA to Xenopus eggs induced localized hot spots at a time and size that was similar to those induced after sperm addition. In contrast, sperm induce a rapid (Ca2+)i wave (~4 µm/s) within ~30 s after hot spot appearance, whereas hot spots induced by PA required an ~32 min to induce a very slow (~1 µm/s) (Ca2+)i wave with a lower peak of (Ca2+)i. Thus, PA may not be required for the initial release of (Ca2+)i at the sperm-egg binding site, but mimics sperm by inducing a similarly sized localized (Ca2+)i release. As compared with sperm, PA may induce a weak, slow (Ca2+)i wave by slowly increasing IP3 receptor clustering. Addition of PA to Xenopus oocytes, or Ca2+ ionophore to either Xenopus oocytes or eggs, did not induce hot spots but a global (Ca2+)i wave that rapidly moved at ~12 µm/s.