RESUMEN
Fertilization of an egg by multiple sperm presents one of the earliest and most prevalent obstacles to successful reproduction. Eggs employ multiple mechanisms to prevent sperm entry into the nascent zygote. The fast block to polyspermy uses a depolarization to inhibit sperm entry. For some external fertilizers, fertilization and the fast block require actin polymerization. Here we explored whether the fast block to polyspermy in the external fertilizer, Xenopus laevis, requires actin polymerization. Inseminating in the presence of inhibitor cytochalasin B, here we demonstrate that actin polymerization is not required for the fast block to polyspermy in X. laevis.
RESUMEN
One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago.
Asunto(s)
Fertilización , Oocitos/metabolismo , Zinc/metabolismo , Ambystoma mexicanum , Animales , Femenino , Hidrozoos , Masculino , Strongylocentrotus purpuratus , Xenopus laevis , Pez CebraRESUMEN
BACKGROUND: The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+. CONCLUSIONS/SIGNIFICANCE: Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.