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.
Highlights
Fertilization represents the union of two distinct cell types: the sperm and the egg
We found that only 12 ± 4% of eggs that were inseminated in 1 mM BAPTA exhibited embryonic development (N = 190 eggs in 5 experimental trials), and that no normal development occurred in eggs inseminated in 3 mM or 5 mM BAPTA (N = 71–179 eggs in 3–5 experimental trials) (Fig 2A)
We found that embryos inseminated in 3 mM BAPTA and 5 minutes later transferred to a Ca2+-containing Modified Ringer (MR)/3 solution, developed doi:10.1371/journal.pone.0170405.g004
Summary
Fertilization represents the union of two distinct cell types: the sperm and the egg. Fertilization and embryonic development reportedly progress normally in the absence of extracellular Ca2+ [9]. It is a widely held belief in the field of reproductive physiology that Ca2+ is unessential for fertilization in X. laevis [10,11,12,13]. 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.
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