Figure 1. TPEN-induced Zn²⁺ deficiency inhibits fertilization-initiated Ca²⁺ oscillations in a dose-dependent manner.

(A) (Left panel) Representative normalized Zn²⁺ recordings of MII eggs loaded with FluoZin-3AM following the addition of increasing concentrations of TPEN (0 μM, DMSO, black trace; 2.5 μM, sky blue; 5 μM, blue; 10 μM, navy). TPEN was directly added to the monitoring media. (Right panel) Representative fluorescent images of MII eggs loaded FluoZin-3AM supplemented with 0, 2.5, and 10 μM of TPEN. Scale bar: 10 μm. (B-D). (B) Representative Ca²⁺ oscillations following ICSI after the addition of 0, 5, 10, or 50 μM TPEN (arrowheads). Insets show representative traces for eggs that resumed Ca²⁺ oscillations after TPEN. (C) As above, but following the addition of 100 μM EDTA, 100 or 500 μM DTPA (time of addition denoted by arrowheads). (D) Ca²⁺ oscillations following ICSI after the addition of 50, 100, and 500 μM TPA (horizontal bars of increasing thickness). (E) Representative bright field images of ICSI fertilized eggs 2.5, 4, and 7 h after sperm injection. Arrows and arrowheads denote the second polar body and PN formation, respectively. Scale bar: 10 μm.