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Chesnel F
,
Vignaux F
,
Richard-Parpaillon L
,
Huguet A
,
Kubiak JZ
.
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The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases.
Fig. 1. Molecular characterization of the first two embryonic M-phases in a cycling extract. M II oocytes laid in 110 mM NaCl overnight were dejellied and activated parthenogentically by a calcium ionophore 30 min later. The low-speed cytoplasmic extract was prepared 60 min post-activation, incubated at 21�C and sampled every 5 min for 100 min. (A) Samples (10 μg cytoplasmic proteins) were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-Phospho-tyr15 CDK1 antibody and the membranes were reprobed with anti-CDK1 antibody (top panel). Signals were detected using ECF reagent and quantified using ImageQuant� software. The ratio P-CDK1/total CDK1 was calculated (histogram bars) and compared to the profile of H1 kinase activity (-○-) assayed in parallel in the extract samples (bottom panel). Double-headed arrows show the duration of the first two peaks of H1 kinase activity. (B) Samples were analyzed by 8% (12% for ERK2) SDS-PAGE followed by immunoblotting with anti-cyclin B2, anti-MCM4, anti-Eg3, or anti-ERK2 antibodies. Signals were detected using NBT/BCIP or ECF reagent. Vertical arrows indicate the time of cyclin B degradation, while horizontal double-headed ones show the duration of maximal up-shift of Eg3 and MCM4. For ERK2 Western blots, the membranes were first probed with anti-P-ERK2 and than stripped and reprobed with anti-ERK2 antibody. Signals were detected using ECF reagent and quantified using ImageQuant� software. (C) Quantification of the Western blot of cyclin B2 seen in Fig. 2B using ImageQuant� software. Results are expressed as percentage, with the value measured at t0 arbitrarily set to 100%.
Fig. 2. Molecular characterization of the first two embryonic M-phases in extracts specific for the first and the second embryonic M-phase made from parthenogenetically activated embryos obtained from the same batch of oocytes. M II oocytes laid in 110 mM NaCl overnight were dejellied and activated parthenogenetically by calcium ionophore 30 min later. The low-speed extracts were prepared from half of the embryos 72 min post-activation (left panels), and from the remaining half, 108 min post-activation (right panels), and were incubated in 21�C and sampled every 5 min for 60 min. (A) Samples (10 μg cytoplasmic proteins) were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-phospho-tyr15 CDK1 antibody. The membranes were reprobed with anti CDK1 antibody (top panel). Signals were detected using ECF reagent and quantified using ImageQuantTM software. The ratio P-CDK1/total CDK1 was calculated (histogram bars) and compared to the profile of H1 kinase activity (-○-) assayed in parallel in the extract samples (bottom panel). (B) Samples were analyzed by 8% SDS-PAGE followed by immunoblotting with anti-cyclin B2 and anti-MCM4 antibodies. Signals were detected using NBT/BCIP. Double-headed arrows show the duration of maximal up-shift of MCM4. (C) Samples were analyzed by 12% SDS-PAGE followed by immunoblotting with anti phospho-ERK2 antibody. The membranes were reprobed with anti-ERK2 antibody (top panel). Signals were detected using ECF reagent and quantified using ImageQuant� software. The ratio P-ERK2/total ERK2 was calculated (histogram bars) and compared to the profile of H1 kinase activity (-○-) (bottom panel). Results are representative of three independent experiments.
Fig. 3. Molecular characterization of the first two embryonic M-phases in extracts specific for the first and the second embryonic M-phase made from in vitro fertilized embryos. M II oocytes obtained from egg-laying females were fertilized in vitro and dejellied 30 min later. The low-speed extracts were prepared 75 min post-fertilization for the first M-phase (left panel) and 99 min post-fertilization for the second one (right panel), then incubated at 21�C and sampled every 5 min for 60 min. Analyses of extracts were performed exactly as described in Fig. 2. Results are representative of three independent experiments.
Fig. 6. Modulation of the first embryonic M-phase with protein synthesis inhibitor, cycloheximide (CHX). The low-speed extract was prepared from parthenogenetic embryos 63 (A) or 70 min post-activation (B�D) and incubated at 21�C. CHX was added to the extract at the beginning of incubation (A) and/or at 9 min of incubation (A�samples beginning at 15 min of incubation and B�arrow). (A) In this experiment, the peak of histone H1 kinase appeared at 20 min from the beginning of incubation. The peak of histone H1 kinase in the extract treated with CHX at the beginning of incubation (t0) was delayed for 15 min (35 min starting from the beginning of incubation), and in the extract treated at 9 min of incubation (t9) was delayed for 5 min (25 min starting from the beginning of incubation). (B) In this experiment, the peak of histone H1 kinase in the control extract appeared at 30 min from the beginning of incubation. The peak of histone H1 kinase in CHX-treated extract (t9) was delayed for 5 min (35 min starting from the beginning of incubation). (C) Western blots of cyclin B2, Cdc25, and MCM4. Analyses of extracts were performed as described in Fig. 2. Double-headed arrows show periods of maximal phosphorylation of Cdc25 and MCM4. (D) Samples corresponding to the maximum levels of cyclin B2 (25- and 30-min samples for the control, lanes 1 and 2; and 30- and 35-min samples for the CHX-treated extract, lanes 3 and 4, marked also with squares in anti-cyclin B2). Western blot in panel C was transferred to the same nitrocellulose membrane and revealed for cyclin B1 and B2 (D, top panel). Quantification of the Western blots (D, lower panel). Results are representative of three independent experiments.
Fig. 7. Modulation of the second embryonic M-phase by exogenous cyclin B addition to the extract. The low-speed extract was prepared from parthenogenetic embryos 112 min post-activation and incubated at 21�C. Recombinant GST-cyclin B2 of Xenopus was added to the extract at the beginning of incubation to a final concentration of 15 and 30 nM and Δ90 sea urchin cyclin B to 15 nM final concentration. (A) Histone H1 kinase activity peaked at 30 min in the control extract and in 15 nM GST-cyclin B2-supplemented one. It was advanced by 5 min in 30 nM GST-cyclin B2 and by 15 min in Δ90 cyclin B-treated extract. Note that the slight increase in histone H1 kinase activity was observed only in 15 nM GST-cyclin B2-treated extract. In two others, the advancement of the peak of this activity was not accompanied by the increase of its amplitude (when compared to the control). (B) MCM4 Western blots. Samples in which the up-shift of MCM4 reaches the maximal phosphorylation state are marked with horizontal arrows. Comparable dephosphorylation states of MCM4 are marked with vertical arrows. (C) Cyclin B2 Western blots. Endogenous cyclin B is marked with double arrowheads, exogenous GST-cyclin B2 with single arrowheads. Results are representative of three independent experiments.
