title: Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Wozniak
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Brianna L
authors.initials: BL
authors.lastname: Mayfield
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Alexis M
authors.initials: AM
authors.lastname: Duray
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Maiwase
authors.initials: M
authors.lastname: Tembo
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: David O
authors.initials: DO
authors.lastname: Beleny
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Napolitano
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Monica L
authors.initials: ML
authors.lastname: Sauer
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Bennett W
authors.initials: BW
authors.lastname: Wisner
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Anne E
authors.identifier: http://orcid.org/0000-0003-2724-1325
authors.initials: AE
authors.lastname: Carlson
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000737:chemistry; D005260:Female; D005306:Fertilization; D008297:Male; D013084:Sperm-Ovum Interactions; D014982:Xenopus laevis / Q000196:embryology / Q000502:physiology*
chemical_list: D002614:Chelating Agents; D002118:Calcium