Figure 4. Zn²⁺ depletion alters Ca²⁺ homeostasis and increases Ca²⁺ store content independent of IP3R1 mass.

(A, B) Representative Ca²⁺ traces of MII eggs after the addition of Tg and Io in the presence or absence of TPEN. Blue trace recordings represent TPEN-treated eggs whereas gray traces represent control, untreated eggs. (A) Io was added to fresh MII eggs once Ca²⁺ returned to baseline after treatment with Tg. Comparisons of mean peak amplitudes after Tg and Io are shown in the bar graphs in the right panel (P < 0.001). (B) MII eggs were aged by 2h. incubation supplemented or not with TPEN followed by Io addition and Ca²⁺ monitoring (P < 0.001). (C) Western blot showing the intensities of IP₃R1 bands in MII eggs freshly collected, aged by 4h. incubation without TPEN, and with TPEN. Thirty eggs per lane in all cases. This experiment was repeated three times. (D, E) (Left panels) Representative traces of Ca²⁺ values in eggs loaded with the Ca^2+-sensitive dye Rhod-2 AM and the ER Ca²⁺reporter, D1ER (1 μg/μl mRNA). TPEN was added into the media followed 10 min later by (D) 10 μM Tg and (E) 50 μM Ach. (Right panel) Bars represent the difference of FRET value between at the time of Tg/Ach addition and at 3 and 5 min later of the addition (P < 0.05). Experiments were repeated two different times for each treatment. Black and green traces represent cytosolic Ca²⁺ and Ca²⁺-ER, respectively. Blue and black arrowheads indicate the time of addition of TPEN and Tg/Ach, respectively. (F) Basal Zn²⁺ level comparison in WT (open bar) and Trpv3^−/−/Trpm7^−/− (dKO, orange bar) MII eggs. Each plot represents the Fluozin3 measurement at 5 min after starting monitoring. (G) (Left panel) Representative Ca²⁺ traces of WT (black trace) and dKO (orange trace) MII eggs after adding Tg. Insets represent the magnified traces at the peak of Ca²⁺ spike from different sets of eggs. (Middle panel) Individual traces of WT and dKO eggs after Tg addition. Dashed circles represent the flection point in dKO traces. (Right panel) Comparisons of mean peak amplitudes after Tg and the time between Tg addition and the Ca²⁺ peak are shown in the bar graphs in the right panel (P < 0.001).