Inhibition of oocyte maturation by ectopic XeWee1 expression. (A) Quantification of ectopically expressed XeWee1. Immature stage VI oocytes were left uninjected or injected with either 0.04 or 0.15 ng of XeWee1 mRNA and cultured for 10 hr; the levels of ectopically expressed XeWee1 were compared with that of endogenous XeWee1 in mature oocytes (MO; 4 hr after GVBD) by Western blotting. (B) Kinetics of GVBD. Thirty immature oocytes uninjected (Cont.) or injected with XeWee1 mRNA (+Wee1) as above were treated with progesterone 10 hr after the injection, cultured, and scored for the percentage GVBD. In parentheses are shown the amounts of ectopically expressed XeWee1 relative to that of endogenous XeWee1, which were determined from the data in A.

Induction of nucleus reformation and DNA replication by ectopic XeWee1 expression. (A) Cytology. Oocytes were prepared as in Fig. 3 and cytologically examined at the indicated times after GVBD. Bar, 10 μm. (B) Analysis of DNA synthesis. Immature oocytes were injected with [α-³²P]dCTP, treated with progesterone, and 1.5 hr later, injected with either buffer or XeWee1 mRNA as described in Fig. 3. DNA was extracted at the indicated times (after progesterone treatment) and analyzed by agarose gel electrophoresis. Only the nuclear DNA, but not the mitochondrial DNA (which migrates more slowly; Furuno et al. 1994), is shown.

Direct involvement of Myt1 in prophase I arrest and the effect of its overexpression on the initiation of maturation. (A) Induction of maturation by ectopic expression of A14/F15 Cdc2. Thirty immature stage VI oocytes were injected with mRNA (10 ng/oocyte) encoding either wild-type Cdc2 or a Thr-14/Tyr-15-nonphosphorylatable A14/F15 Cdc2 mutant, and then scored for the percentage GVBD. (B) Induction of maturation by injection of anti-Myt1 antibody. Thirty immature oocytes were left uninjected or injected with 3 ng of Myt1 mRNA (+Myt1); 10 hr later, they were injected with either anti-Myt1 antibody (α-Myt1) or preimmune antibody (IgG) (each at 200 ng/oocyte) and then scored for the percentage GVBD. The kinetics of Cdc2 Tyr-15 dephosphorylation after injection of α-Myt1 alone (see inset) was determined by Western blotting with anti-phospho Cdc2 (Y15) antibody. (C) Effects of overexpression of Myt1 on the initiation of maturation. Thirty immature oocytes were injected with buffer (Cont.), 0.1 or 1.5 ng of Myt1 mRNA (+Myt1), or 0.15 ng of Wee1 mRNA (+Wee1); 10 hr later, they were treated with progesterone and scored for the percentage GVBD. The levels of ectopically expressed Myt1 (10 hr after the mRNA injection) are shown in the inset and its relative amounts to that of endogenous Myt1 are shown in parentheses. (For Wee1, cf. Fig. 2).

Model for interphase/S phase omission between the two meiotic divisions. (A) Model for meiosis in Xenopus oocytes. Both the Mos/MAPK pathway and the absence of Wee1 are involved in the omission of interphase/S phase between meiosis I (MI) and meiosis II (MII). The Mos/MAPK pathway may function to rapidly reactivate Cdc2 after MI, thereby suppressing interphase/S phase, whereas the absence of Wee1 in MI directly omits interphase/S phase. Either pathway would act to suppress inhibitory Tyr-15 phosphorylation of Cdc2 during the MI/MII transition, thereby ensuring direct entry into MII. See text for details. (B) Model for meiosis in general. Absence of Wee1 is assumed to be the primary mechanism of interphase/S-phase omission between MI and MII. The S-phase omission may in turn activate the DNA replication checkpoint pathway, which, however, would be cancelled immediately also by the absence of Wee1. Large animal oocytes such as Xenopus oocytes may not activate such a replication checkpoint pathway due to constraints specific to them. In the other cases, however, the pathway may be actually activated, and its cancellation by the absence of Wee1 may be important for entry into MII. Particularly in cases where the differentiated meiotic cells no longer have the ability to replicate DNA, cancellation of the replication checkpoint by the absence of Wee1 may be essential for normal entry into MII. (See text for details.)

Expression of XeWee1 during oogenesis and oocyte maturation. (A) Western blot analysis of XeWee1 during oocyte maturation. Immature stage VI oocytes were treated with progesterone (PG), collected at the indicated times, and subjected to either Western blot analysis with anti-XeWee1 antibody (Wee1) or histone H1 kinase assays of Cdc2 (H1). The time of GVBD and periods of meiosis I (MI) and meiosis II (MII) are indicated. (B) Western blot analysis of XeWee1 during oogenesis. Twenty micrograms of proteins each from stage I to VI oocytes and mature meiosis II oocytes (MO) was subjected to Western blot analysis of XeWee1, Cdc25C, or Cdc2. In MO, Cdc25C and Cdc2 showed prominent mobility shifts due to phosphorylation. (C) RT–PCR analysis of XeWee1 transcripts during oogenesis. Total RNA from four oocytes at each stage was subjected to RT–PCR analysis using XeWee1, Cdc25C, or Cdc2 oligonucleotides as primers. (D) Stability of (ectopic) XeWee1 in prophase I arrest. Immature stage VI oocytes injected with XeWee1 mRNA (0.7 ng/oocyte) were cultured, treated with cycloheximide (CHX) at 12 hr, and collected at the indicated times (after the mRNA injection) for Western blot analysis of XeWee1. At 10 hr, (ectopic) XeWee1 protein was expressed in three- to fourfold excess over endogenous XeWee1 in mature oocytes.

Effects of ectopic XeWee1 expression on entry into meiosis II. (A) Quantification of ectopically expressed XeWee1. Immature stage VI oocytes were injected with 0.35 ng of XeWee1 mRNA 1.5 hr after progesterone treatment, and then collected at the indicated times (after the progesterone treatment) for Western blot analysis. As a standard, endogenous XeWee1 in mature oocytes (MO; 4 hr after GVBD) was used. (B) Kinetics of GVBD and activation phenotypes. Thirty oocytes were treated with progesterone, injected at the indicated time (or at 1.5 hr; Inj.) with either buffer (Cont.) or 0.35 ng of XeWee1 mRNA (+Wee1) as described above, and then scored for the percentages GVBD and activation phenotypes (or cortical changes in pigmentation; see below). (C) Typical external morphology. The oocytes described above were photographed at 4 hr after GVBD. Note that XeWee1 mRNA-injected oocytes show distorted pigmentation and a cleavage furrow-like streak on the animal half, while control oocytes show a regular white spot (indicative of GVBD). (D) Kinetics of Cdc2 activity. Cdc2 kinase activities in the oocytes prepared as in B were measured by histone H1 kinase assays and are shown in an arbitrary unit. (E) Kinetics of Cdc2 Tyr-15 phosphorylation. The oocytes prepared as in B were analyzed by Western blotting using anti-phospho Cdc2 (Y15) antibody.