Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

Abstract

BackgroundThe 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 findingUsing 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/SignificanceTaken 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.

Zinc protection of fertilized eggs is an ancient feature of sexual reproduction in animals

“…laevis fertilization. The jelly is known to bind to and buffer other metals [ 38 ] and may trap zinc near the egg. We assayed for the appearance of cleavage furrows in jelly-free X .…”

Ca2+-Induced Mitochondrial ROS Regulate the Early Embryonic Cell Cycle

“…To determine if Ca 2+ influx was necessary for ROS production, we transferred oocytes to calcium-free OR2 medium containing the Ca 2+ chelator EGTA (100 μM) for 5 min prior to activation. Extracellular Ca 2+ has been shown to be required for fertilization in Xenopus ( Wozniak et al., 2017 ). In the presence of calcium-free OR2 medium containing EGTA, the Ca 2+ wave was completely inhibited in laser-activated oocytes ( Movie S5 ), and ROS production was also inhibited 42% and 42.2% at 40 and 60 min after prick activation (n = 32–36; p < 0.0001, compared to control, two-way ANOVA) ( Figures 2 C and 2D).…”

Ca2+-induced mitochondrial ROS regulate the early embryonic cell cycle

“…To determine if Ca 2+ influx was necessary for ROS production, we incubated oocytes in the presence of the Ca 2+ chelator EGTA (100 µM) prior to activation. Extracellular Ca 2+ has been shown to be required for fertilization in Xenopus (Wozniak et al, 2017). In the presence of EGTA, the Ca 2+ wave was completely inhibited in laser-activated oocytes (Movie S5), and ROS production was also inhibited 42% and 42.2% at 40 min and 60 min after prick activation (n = 32-36, p < 0.0001, compared to control, two-way ANOVA) (Figures 2C and 2D).…”