Involvement of Rho family G protein in the cell signaling for sperm incorporation during fertilization of mouse eggs: inhibition by Clostridium difficile toxin B.

Sperm-egg interaction was investigated in mouse eggs freed from the zona pellucida and injected with Clostridium difficile toxin B, the inhibitor of Rho family small G proteins. Toxin B reduced in a dose-dependent manner the percentage of eggs associated with sperm fusion on the surface or sperm nucleus decondensation in the ooplasm, examined by injection of a DNA-staining dye into the egg and transfer of the dye to the fused sperm head after recording intracellular Ca(2+) responses for 100 min postinsemination. The mean number of decondensed sperm nuclei per egg was remarkably decreased by approximately 1 microg/ml toxin B in the ooplasm. This was because spermatozoa were arrested at the fusion state without developing to sperm incorporation and tended to lose cytoplasmic continuity to the egg. The fusion-arrested spermatozoa caused transient small Ca(2+) oscillations in most of eggs, while an injected spermatozoon produced repetitive large Ca(2+) spikes unaffected by toxin B. A decrease in the rate of fused spermatozoa and decondensed sperm nuclei was also caused by 20-40 microM cytochalasin D, the inhibitor of actin polymerization. Immunostaining of Rho proteins showed that Rac1 and RhoB are present in the cortical ooplasm, but Cdc42 is absent. Actin filaments in the cortex appeared to be reduced in toxin B-injected eggs. This study suggests that Rho protein(s) regulating actin-based cytoskeletal reorganization is involved in the process leading to sperm incorporation.