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

Mouse sperm-egg fusion was examined using twophoton and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca²⁺-sensitive dyes from egg into sperm)and any change in egg cytoplasmic Ca²⁺. When indo-1 dextran was used to obtain ratiometric two-photon images,there was no detectable local increase 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 cytoplasm Ca²⁺ transient, which suggested that there was a high permeability pathway for Ca²⁺ between egg and sperm. To exclude this pathway from providing trigger Ca²⁺ for the egg transient, we reduced bathing (Ca²⁺] to approx. 18 μM and 13nM (with EGTA). In these conditions the first egg Ca²⁺ transient 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 able 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 during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca²⁺ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.