FIGURE 3.

Vg1RBP phosphorylation is a late event in meiotic maturation and correlates with release of Vg1 mRNA into the soluble fraction. (A) Full phosphorylation of Vg1RBP is a late event in meiotic maturation. Random groups of 10 cells out of ∼150 stage VI oocytes treated with progesterone were taken at the time of progesterone addition (T0), when the first GVBD was observed (0%), when the indicated proportion of cells has undergone GVBD (10%–100%) and after an overnight incubation with progesterone (O/N). Control oocytes were incubated for the duration of the experiment in the absence of progesterone (VI). Oocytes were then subjected to Western blot analysis using antisera directed against the proteins indicated on the left. Asterisk: ∼40% phosphorylation; arrow: earliest visible phospho-Vg1RBP. (B) Phosphorylation of Vg1RBP coincides with solubilization of Vg1 mRNA in low-salt conditions. Groups of synchronized stage VI oocytes undergoing maturation were harvested 30 min (G + 30′), 3 h (G + 3 h), or 12 h (G + 12 h) after synchronization. Control oocytes were incubated for the duration of the experiment in the absence of progesterone (VI). Oocytes were then either subjected to Western blot analysis using antisera directed against the indicated proteins (top panel) or fractionated into soluble (S) and insoluble (P) fractions in different buffers (see Experimental Procedures for more details). Equivalent proportions of RNA extracted from these fractions was then assayed for the presence of Vg1 and histone H3 mRNAs using nonsaturated RT-PCR. Arrows indicate the diagnostic PCR product. The yield and integrity of extracted RNA was examined by electrophoresis in the presence of ethidium bromide (rRNA).