FIGURE 1:

[Ca²⁺]_e and Ca²⁺ influx are required to fill [Ca²⁺]_ER in oocytes. The underlying Ca²⁺ influx mechanism(s) are inactivated during maturation and are sensitive to 2-APB and TG at the GV stage. (A) The contribution of extracellular Ca²⁺ to [Ca²⁺]_ER content was estimated in GVBD-stage oocytes after culturing GV oocytes for 4 h in media supplemented with 1.7 mM CaCl₂ or without supplementation, nominal Ca²⁺-free medium. Release of [Ca²⁺]_ER was induced by addition of 10 μM TG. All [Ca²⁺]_i responses are shown in the graphs, and the bold trace in each graph represents the mean response; bar graphs to the right of each Ca²⁺ panel denote mean ± SEM of [Ca²⁺]_ER content estimated as area under the curve. (B) Spontaneous Ca²⁺ influx was measured in GV oocytes and MII eggs. Oocytes and eggs were placed in Ca²⁺-free conditions, after which 2 and 5 mM CaCl₂ were successively added. Given that only GV oocytes showed Ca²⁺ influx, they were pretreated with 50 μM 2-APB for 5 min before addition of CaCl₂ to prevent influx. (C) Ca²⁺ influx was promoted by addition of CaCl₂ into GV oocytes with and without prior treatment with TG. GV oocytes were placed in nominal Ca²⁺-free media or exposed to 10 μM TG for 30 min in nominal Ca²⁺-free medium to deplete [Ca²⁺]_ER, after which 5 mM CaCl₂ was added. Representative traces are shown, and bold trace represents mean response.