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p90Rsk is not involved in cytostatic factor arrest in mouse oocytes.
Dumont J
,
Umbhauer M
,
Rassinier P
,
Hanauer A
,
Verlhac MH
.
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Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos-MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos-/- strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos-/- oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes.
Figure 1. Constitutively active Rsk1 or 2 does not induce CSF arrest in early mouse embryos, but does so in early Xenopus embryos. (AâC) Phase-contrast image of mouse embryos injected at the two-cell stage into one blastomere and observed 24 h later. (A) Cleavage arrest induced after Mos-RNA injection (black arrowheads). No arrest is observed after the injection of RNA-encoding Rsk2wt or Rsk2ca (B and C, respectively). Bar, 50 μm. (DâF) Phase-contrast image of two-cell Xenopus embryos injected into one blastomere with RNA-encoding Mos (D), Rsk2wt (E), and Rsk2ca (F). Embryos were observed 3 h after injection. The black arrowheads point toward blastomeres, which undergo cell cycle arrest after injection. Bar, 100 μm. (G) Percentage of cleavage arrest (%bloc) induced in one blastomere of a late two-cell mouse embryo after an injection of RNA-encoding Mos, Rsk1ca, Rsk2wt, and Rsk2ca. The number in parentheses corresponds to the number of embryos that were analyzed. Two-cell embryos were injected between 48 and 50 h after a human chorionic gonadotropin injection, which corresponds to â¼4 h before the two- to four-cell division. Error bars are the SD from three experiments. (H) Immunoblotting of 30 two-cell mouse embryos that were either noninjected (NI) or injected with Rsk2wt or Rsk2ca RNA into one blastomere and collected 24 h after injection. Left panel is revealed with an anti-Rsk2 antibody. Right panel is revealed with an anti-Flag antibody. The anti-Rsk2 recognizes endogenous Rsk2, but not Rsk2ca.
Figure 2. Constitutively active Rsk1 does not rescue the CSF arrest of oocytes from the mosâ/â strain. (A) Time-lapse microscopy of maturing oocytes from the Mos-deficient strain, either noninjected (white stars) or injected with RNA-encoding Rsk1ca. Time points correspond to time after meiosis resumption. Black dots show extrusion of the second polar body. Gray dots correspond to the second polar body moving out of focus. Bar, 50 μm. (B) Immunoblotting of 30 oocytes from the experiment shown in A, either noninjected (NI) or injected with RNA-encoding Rsk1ca. Rsk1ca is overexpressed about five times (top) after injection of the corresponding RNA into oocytes from the mosâ/â strain. The top panel is revealed with an anti-Rsk1 antibody. The bottom panel is revealed with an anti-ERK antibody and serves as a loading control.
Figure 3. Constitutively active Rsk1 or 2 does not rescue the CSF arrest of oocytes from the mosâ/â strain. (AâC) Phase-contrast images of oocytes from the Mos-deficient strain, either noninjected (A), injected with RNA-encoding Mos (B), or injected with Rsk2ca (C). Arrowheads point toward the polar bodies (two in A and C, and one in B). Bar, 50 μm. (D) Overexpression of Mos, but not of Rsk1ca or Rsk2ca, rescues the MII arrest in oocytes from mosâ/â mice, as evidenced by spontaneous second polar body extrusion (%PB2). The number in parentheses corresponds to the number of embryos analyzed. Error bars are the SD from three experiments. (E) Immunoblotting of 50 oocytes either noninjected (NI) or injected with RNA-encoding Rsk1ca or Rsk2ca. Rsk1ca and Rsk2ca are overexpressed (bottom) and active (top) after injection of the corresponding RNA into oocytes from the wild-type or mosâ/â strain. The top panel is revealed with an anti-phosphoRsk antibody. The bottom panel is revealed with an anti-Flag antibody.
Figure 4. Oocytes from the Rsk (1, 2, 3)–deficient strain arrest in MII like control oocytes. (A–C) Immunofluorescent images show the microtubules in green and the chromosomes in red of control wild-type oocytes (A) and oocytes from the Rsk (1, 2, 3)–deficient strain (B and C). Oocytes were observed 19 h after GVBD (∼7 h in CSF arrest). (C) An MII spindle of an oocyte from the Rsk (1, 2, 3)–deficient strain is observed under a 63× objective to show the sister chromatids. Bar, 10 μm. (D) Immunoblotting of protein extracts from the mitotic embryonic fibroblasts of control mice (lane 1) or of Rsk (1, 2, 3)–deficient mice (lane 3), and of protein extracts from the liver of control mice (lane 2) or of Rsk (1, 2, 3)–deficient mice (lane 4). Extracts were probed using antibodies specific for Rsk1–3 and ERK1 and 2. (E) Table showing the percentage of oocytes undergoing meiosis resumption (GVBD) and MII arrest in control mice versus that in Rsk (1, 2, and 3)–deficient mice. (F) Scheme of the pathway leading to CSF arrest in the mouse oocyte, which does not go through p90Rsk to maintain a high maturation-promoting factor activity.
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