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PLoS One
2017 Jan 23;121:e0170405. doi: 10.1371/journal.pone.0170405.
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Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis.
Wozniak KL
,
Mayfield BL
,
Duray AM
,
Tembo M
,
Beleny DO
,
Napolitano MA
,
Sauer ML
,
Wisner BW
,
Carlson AE
.
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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.
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28114360
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Fig 1. X. laevis embryos developed normally in the absence of added Ca2+.(A) Averaged percentage of embryos that developed cleavage furrows from eggs inseminated in MR/3 or DVF/3 (N = 153–160 eggs in 5 experimental trials). (B) Representative images of a developed X. laevis embryo at the 4-cell stage (top), an undivided egg (middle), and an embryo with faint cleavage furrows (bottom); scale bar = 250 μm.
Fig 2. Extracellular Ca2+ is required for early embryonic development in X. laevis.Plots of averaged percentage of embryos that developed from eggs inseminated in DVF/3 with increasing chelator or CaCl2 concentrations. Each plot was fit with a sigmoidal function. (A) BAPTA concentrations ranged from 10 μM—5 mM (N = 71–190 eggs in 3–5 experimental trials). (B) Varying concentrations of added CaCl2 ranging from 10 μM—5 mM, with 1 mM BAPTA (N = 74–102 in 3–5 experimental trials). (C) Various EGTA concentrations ranging from 3 μM—3 mM (N = 80–167 in 4–6 experimental trials).
Fig 3. Extracellular Ca2+ important for the earliest events of embryonic development in X. laevis.Incidence of cleavage furrow development from eggs inseminated in DVF/3 either with 0 or 3 mM BAPTA. After 30 minutes, inseminated eggs were washed twice and moved to a new solution of DVF/3 with 0 or 3 mM BAPTA, as indicated. Embryos were assessed for the appearance of cleavage furrows 60–90 minutes after transfer (90–120 minutes after sperm addition) (N = 75–85 eggs in 4 experimental trials).
Fig 4. Sperm penetrate jelly but not the vitelline envelope of X. laevis eggs inseminated in BAPTA.(A) Inseminated eggs were incubated in 0 or 3 mM BAPTA, and with 0 or 3 mM CaCl2, were stained with Hoechst to visualize the sperm. 20 minutes following insemination, eggs were dejellied and imaged using fluorescence and bright-field microscopy to assess sperm penetration of the vitelline envelope. Representative images document the presence of Hoechst-stained sperm within the vitelline envelope of eggs inseminated in DVF/3 alone (top) or DVF/3 with 3 mM BAPTA and 3 mM CaCl2 (bottom) (N = 33–56 eggs in 4 experimental trials). By contrast, no Hoechst-stained sperm were evident within the vitelline envelope of eggs inseminated in DVF/3 with 3 mM BAPTA (middle). Scale bars represent 25 μm. Red, dashed line on overlay indicates location of envelope. (B) Incidence of cleavage furrow development of eggs inseminated in DVF/3 with 0 or 3 mM BAPTA, washed after five minutes, and transferred to a final solution as indicated (N = 75–87 eggs in 3 experimental trials).
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