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).