Extracellular Ca(2+) Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

BACKGROUND: The necessity of extracellular Ca(2+) for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca(2+) entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca(2+) chelator BAPTA. Here we sought to resolve this controversy. METHODOLOGY/PRINCIPAL FINDING: Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca(2+) is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca(2+) chelator, or with BAPTA and saturating CaCl(2). Together these results indicate that fertilization does not occur in high concentrations of Ca(2+) chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca(2+). CONCLUSIONS/SIGNIFICANCE: Taken together, these data are consistent with requirement of extracellular Ca(2+) for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca(2+) ions to foster fertilization in changing extracellular milieu.