Fig. 6.

Phosphorylation-dependent sensitization of IP₃R1 during oocyte maturation. (A–D) Inhibition of cdk1 activity was achieved by the addition of roscovitine (Ros). GV oocytes were allowed to undergo GVBD and 50 µM Ros was added 2 hr after the start of maturation. Lysates from GV, GVBD and Ros-treated GVBD oocytes were probed with MPM-2 (A), phospho-S421 (B) and phospho-T779 (C) antibodies (upper panel), and re-probed with IP₃R1 antibody (lower panel), as described above. A representative result of 2–3 independent experiments with similar results is shown. (D) IP₃-induced Ca²⁺ release using cIP₃ was measured in control (solid line) and Ros-treaded (dashed line) oocytes. A 0.001 sec UV pulse was applied to oocytes (open arrow), number of oocytes responding to cIP₃ is shown in parentheses. The bar graphs show comparison of the duration and amplitude of cIP₃-caused Ca²⁺ release. (E–G) RO3306, another cdk1 inhibitor was used to block MPF activity. (E) IP₃R1 MPM-2 (upper panel) and IP₃R1 reactivity (lower panel) in GV, GVBD and RO3306-treated GVBD oocytes. (F) [Ca²⁺]_ER was examined in control (n=9) and RO3306-treated GVBD oocytes (n=10) by the addition of 2 µM ionomycin in Ca²⁺ free medium. (G) IP₃-induced Ca²⁺ release using cIP₃ was measured in control (solid line) and Ros-treaded (dashed line) GVBD oocytes and induced by the application of a 0.001 sec UV pulse (open arrow). The number of oocytes responding to cIP₃ is shown in parentheses. The bar graph shows the comparisons of the duration of the cIP₃-induced Ca²⁺ release.