The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse

Mouse sperm-egg fusion was examined using two-photon and confocal microscop y. A delay of several minutes occurred between the first observable event o f fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sper m) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increas e in egg cytoplasmic Ca2+ near the site of sperm fusion, However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cy toplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from provid ing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx . 18 mu M and 13 nM (with EGTA). In these conditions the first egg Ca2+ tra nsient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were a ble to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the 'pore(s)' that form durin g sperm-egg fusion must be at least 8 nm in diameter. These data are compat ible with the idea that a diffusible sperm protein could trigger the observ ed changes in intracellular Ca2+ in the egg, but do not exclude the possibi lity that other second messengers are generated during sperm-egg fusion.