Date Published: January 23, 2017
Publisher: Public Library of Science
Author(s): Katherine L. Wozniak, Brianna L. Mayfield, Alexis M. Duray, Maiwase Tembo, David O. Beleny, Marc A. Napolitano, Monica L. Sauer, Bennett W. Wisner, Anne E. Carlson, Michael Klymkowsky.
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.
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+.
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.
Fertilization represents the union of two distinct cell types: the sperm and the egg. Although a substantial amount of work on various species has already been done, many of the signaling pathways and molecular events required for fertilization are unknown. Ca2+ is an example of an incompletely understood signaling molecule in fertilization and early embryonic development. Fertilization evokes increased intracellular Ca2+ in the zygote, and this Ca2+ is essential for egg activation and the earliest events of embryonic development in all sexually reproducing species studied thus far [1, 2]. Moreover, intracellular Ca2+ regulates various cellular events in both gametes [3–7]. By contrast, the importance of extracellular Ca2+ during fertilization is controversial. For example, entry of extracellular Ca2+ into sperm signals processes required for fertilization such as the acrosome reaction , and extracellular Ca2+ is required for robust motility in X. laevis sperm . However, fertilization and embryonic development reportedly progress normally in the absence of extracellular Ca2+ . Moreover, it is a widely held belief in the field of reproductive physiology that Ca2+ is unessential for fertilization in X. laevis [10–13].