In female pronuclei of starfish eggs, chromatin is loaded with Cdc6 and Mcm2 before fertilization, and then with Cdc45 after fertilization at G1/S-phase transition. (A) Mature eggs, which were arrested at G1 phase after completion of meiosis II, were inseminated. To monitor DNA replication, every 5 min, fertilized eggs were pulse labeled for 5 min with BrdU, extracted, and immunostained with anti-BrdU antibody (green). DNA was stained with DAPI (red). Insets indicate male chromatin, and main figures indicate female chromatin or fused chromatin. (B–D) Every 5 min, isolated eggs were extensively extracted and then immunostained with anti-starfish Cdc6 antibody (B, green), anti-starfish Mcm2 antibody (C, green), and anti-starfish Cdc45 antibody (D, green). (Scale bar, 10 μm.) (E) Cdc45 protein is detectable in immature oocytes and its levels remain constant during meiotic and cleavage cycles.

Cdc45 is loaded onto chromatin and DNA replication is initiated in the absence of Cdk activity when G1-phase eggs are fertilized. (A) In starfish eggs, Cdk1 associates with cyclin A and cyclin B, and Cdk2 associates predominantly with cyclin E. Immature oocytes were treated with 1-MeAde to resume meiosis, and then at 50 min, meta-I oocytes were inseminated to initiate embryonic cycles after completion of meiosis II. Oocyte or egg extracts were prepared at 0 min (immature), 30 min (GVBD), 60 min (interkinesis), 120 min (the first M phase in cleavage cycle), and 160 min (the second M phase) (Fig. S3I). Whole-cell extracts (WCE), and their immunoprecipitates with anti-Cdk2 (Cdk2 IP) or anti-Cdk1 (Cdk1 IP) were immunoblotted with each of cyclins A, B, and E, and Cdk1 and Cdk2. Lower band of Cdk1 corresponds to its active form. (B–E) A single knockdown of Cdk2 (B and C) or a triple knockdown of cyclins A, B, and E (D and E) does not prevent DNA replication induced by fertilization. Immature oocytes were injected with morpholino oligonucleotides against Cdk2 or cyclins A and E, and then treated with 1-MeAde to resume meiosis. At 120–150 min after 1-MeAde addition, mature eggs that had been injected with cyclins A and E morpholino oligonucleotides were further injected with morpholino oligonucleotide against cyclin B. At 180 min, all eggs were inseminated in the presence of BrdU. Successful insemination was confirmed by elevation of fertilization envelope. Fifty (B and C) or 80 (D and E) min later, eggs were recovered to confirm knockdown of Cdk2 (B, MO Cdk2) or cyclins A, B, and E (D, MO CycABE) with immunoblots and to examine BrdU incorporation (C and E). As controls, respective control morpholino oligonucleotides were injected (MO Control). In the triple knockdown (E), significant delay was observed in BrdU incorporation into female pronucleus chromatins. MAPK was used as a loading control for immunoblots (B and D). Knockdown of Cdk2 or cyclins A and E did not affect the meiotic cell-cycle progression. (F) Inhibition of Cdk activity does not affect Cdc45 loading onto chromatin after fertilization. G1 eggs were inseminated in the presence or absence of 10 μM roscovitine, followed by examination of Cdc45 loading at 30 min. Note that BrdU incorporation (E) or Cdc45 loading (F) was observed separately in female and male pronuclei. Such a failure in pronuclear congression is evidence for sufficient knockdown of cyclin B (E) or sufficient inhibition of Cdk (F), because pronuclear congression requires low levels of cyclin B-Cdk1 activity (12).

Inactivation of p90Rsk is necessary and sufficient for loading of Cdc45 onto chromatin and the subsequent initiation of DNA replication in G1-phase-arrested starfish eggs. (A and B) Maintenance of p90Rsk activity prevents loading of Cdc45 onto chromatin and the subsequent DNA replication in fertilized eggs. Mature eggs after completion of meiosis II were uninjected or injected with 0.6 ng of either constitutively active p90Rsk (CA-Rsk-EE) or its control kinase-dead form (CA-Rsk-EE KD). Thereafter, these eggs were inseminated and incubated in the presence of BrdU and then fixed either at 30 min for anti-Cdc45 immunostaining or at 50 min for anti-BrdU immunostaining. Successful fertilization was confirmed by formation of fertilization membrane. (C and D) Inhibition of p90Rsk activity causes loading of Cdc45 onto chromatin and the subsequent DNA replication in the absence of fertilization. Unfertilized mature eggs were injected in the presence of BrdU with 3 ng of either neutralizing anti-p90Rsk antibody or control preimmune IgG, followed by incubation. Eggs were fixed either at 30 min for anti-Cdc45 immunostaining or at 60 min for anti-BrdU immunostaining. (E–G) Constitutively active p90Rsk restores G1-phase arrest in eggs injected with neutralizing anti-p90Rsk antibody. Unfertilized mature eggs were injected with neutralizing anti-p90Rsk antibody along with CA-Rsk-EE or control CA-Rsk-EE KD. After 60 min incubation in the presence of BrdU, eggs were fixed for anti-BrdU immunostaining (F and G). Egg lysates were either immunoblotted with anti-MAPK antibody, or assayed for phosphorylation of GST-S6 (S6K activity) (E). DNA was stained with DAPI. Number of eggs examined for Cdc45 loading or BrdU incorporation are indicated in parentheses (B, D, and G).

Loading of Mcm2 onto chromatin during meiosis is blocked by geminin. (A) At each end of meiosis I and II, Mcm2, but not Cdc45, is loaded onto chromatin. Immature oocytes were treated with 1-MeAde to undergo meiotic maturation. Beginning at 50 min, every 5 min, oocytes were either fixed for immunostaining with anti-Mcm2 (green) or anti-Cdc45 antibodies along with DAPI (red) staining, or extracted for histone H1 kinase assay to monitor meiotic cell cycle progression (Top). Note that Mcm2 was undetectable on chromatin until 55 min (metaphase I) and at 90 min (metaphase II). Arrow indicates the second polar body. (B–D) Nondegradable geminin inhibits loading of Mcm2 onto chromatin during meiosis, and hence prevents DNA replication after fertilization. Immature oocytes were injected with nondegradable form of geminin (geminin^DEL, Gem), wild-type (degradable) geminin (B, control; C, Cont), or control buffer (D, control) and then treated with 1-MeAde to undergo meiotic maturation. At 40 min (metaphase I), 60 min (anaphase I), or 150 min (G1 phase after completion of meiosis II), oocytes were fixed for staining with anti-Mcm2 antibody or with DAPI (B). Number of eggs examined for loading of Mcm2 onto chromatin is indicated in parentheses (C). Alternatively, 150 min after 1-MeAde addition, mature eggs were inseminated in the presence of BrdU, and 40 min later, DNA replication was examined (D, GV). For reference, after meiotic maturation, G1-phase eggs were injected with nondegradable form of geminin and then inseminated (D, PN). Note that BrdU incorporation (red) was undetectable both in female and male (arrowhead) pronuclei (D, GV), though detectable only in female pronucleus (D, PN), showing that only the female, but not the male, pronucleus is licensed for DNA replication before fertilization.

Cdc45 is loaded onto chromatin and DNA replication is initiated in the absence of new synthesis and accumulation of Cdc7 when G1-phase eggs are fertilized. (A) On immunoblots, Cdc7 protein was slightly detectable in immature oocytes. Its levels increased during meiotic maturation and remained elevated in the first cleavage cycle. (B and C) Delayed loading of Cdc45 occurs in Cdc7-knocked-down eggs after fertilization. Immature oocytes (GV) were injected with morpholino oligonucleotide against Cdc7 and then treated with 1-MeAde to resume meiosis. Female pronucleus stage eggs (PN) were either processed to examine Cdc7 protein levels (B) or inseminated in the presence or absence of 10 μM roscovitine to examine Cdc45 loading at 30 and 40 min or BrdU incorporation at 50 min (C). As controls, control morpholino oligonucleotide was injected (MO Cont) or not (−). Note that paternal DNA is discerned as a bright spot within zygotic nucleus in the absence of roscovitine, whereas male (Inset) and female pronuclei are separated in the presence of roscovitine. Asterisks in the Cdc7 blots indicate nonspecific bands, and upper and lower bands of MAPK correspond to active and inactive form, respectively (B).