Figure 5

Induction of S phase between the two meiotic M phases by increasing the Wee1 levels in meiotic extracts. (A and B) Meiotic extracts added with demembranated sperm and BSA (0.75 mg/ml) (open circles) or His–Wee1 (12.0 nM) (solid squares) were examined for histone H1 kinase activity (A) and immunoblotted with cyclin B2, Wee1 and phosphotyrosine antibodies (B) at 15 min intervals from the start of incubation. The cell-cycle phase determined by sperm chromatin morphology is indicated in (D). M, condensed chromosomes; T, telophase-like chromatin mass; S, nuclei. (C) Demembranated sperm were incubated in meiotic extracts that had been added with Cy3-dUTP and His-Wee1 (a and b) or BSA (c), and stained with Hoechst 33342 (upper panels) at 75 (a) and 180 min (b and c) after incubation. The distribution of dUTP is visualized by the Cy3 signal (lower panels). Scale bar indicates 10 m. Note that the replicated chromosomes formed in the His–Wee1-supplemented extract are thicker than the unreplicated ones formed in the BSA-supplemented control extract. (D) Demembranated sperm (2 10⁵ sperm/ml extract) were mixed with meiotic and egg extracts to which His–Wee1 (12.0 nM) or BSA (0.75 g/ml) had been added. Following 180 min incubation in the presence of [-³²P]CTP, sperm DNA was separated on an agarose gel and processed for autoradiography.