QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 August 2006
doi: 10.1242/jcs.03082

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zhang, W. L. Articles by Ciapa, B. Search for Related Content PubMed PubMed Citation Articles by Zhang, W. L. Articles by Ciapa, B. Social Bookmarking                         What's this?

Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca²⁺-dependent pathway but without completion of S phase

Wen Ling Zhang^1,*, Philippe Huitorel^2,*, Anne-Marie Geneviere³, Sandrine Chiri¹ and Brigitte Ciapa^1,

¹ UMR 7622 CNRS, Université Paris 6, 9 Quai St Bernard, Case 24, 75252 Paris cedex 05, France
² UMR 7009 CNRS, Université Paris 6, Station Zoologique, Observatoire Océanologique, 06234 Villefranche-sur-Mer cedex, France
³ UMR 7628 CNRS, Observatoire Océanologique de Banyuls, Université P. et M. Curie / CNRS / INSU, BP 44, 66651, Banyuls-sur-mer cedex, France

View larger version (100K): [in a new window]   Fig. 1. Effect of two MEK inhibitors on egg morphology. (A) Light-microscopy images of changes in the general morphology of eggs from two sea urchin species, P. lividus (top) and L. pictus (bottom), induced after a 3-hour treatment with U0126 or PD98059. Untreated eggs showed a clear nucleus and no apparent fertilization membrane (upper left inset), whereas figures of constrictions and blisters, NEB and a thin fertilization membrane for a few eggs (lower left and right insets) were induced in treated eggs of both species. Bars, 50 µm. (B) Confocal microscopy images of microtubule polymerization and chromatin condensation induced in unfertilized eggs of P. lividus after treatment with 1 µM U0126. Chromatin was labeled with Hoechst 123 (blue) and microtubules were labeled with an anti-tubulin antibody (red). Eggs were observed (a) before treatment with U0126 or (b) 30 minutes, (c) 1 hour, (d) 2 hours, (e) 3 hours or (f) 4 hours afterwards. Bar, 50 µm. Similar results were obtained in the presence of PD98059 (supplementary material Fig. S1, Table S1).

View larger version (90K): [in a new window]   Fig. 2. Effect of U0126 on DNA synthesis. DNA replication was monitored by incubating P. lividus sea urchin eggs with BrdU as described in Materials and Methods. Untreated eggs, analyzed 1 hour after fertilization, show major BrdU incorporation (a). This was not observed when eggs were treated with 20 µM aphidicolin (b). Unfertilized eggs treated with 1 µM U0126 for 3 hours, and then incubated (d) with or (c) without 20 µM aphidicolin showed similar BrdU incorporation. Bar, 50 µm.

View larger version (60K): [in a new window]   Fig. 3. Effect of PD98059 or U0126 on phosphorylated MAPK (P-MAPK) and MPF activity in unfertilized eggs of P. lividus and L. pictus. (A) Comparison of ERK-LP in eggs of two sea urchin species using three different antibodies. (Aa) Samples 2, 3, 5 and 7 were cut in two halves after transfer and before blotting with various antibodies, and then re-aligned before ECL. The anti-ERK1 (ERK1) and the anti-panERK (panERK) antibodies detected in both species a doublet at approximately 44 kDa (small arrows) with an upper band corresponding to the protein detected with the anti-phosphoMAPK42/44 antibody (P-ERK). The anti-panERK antibody also revealed a single protein of approximately 81 kDa (large arrow). (Ab) Detection in P. lividus eggs of changes in the phosphorylation of the 44 kDa ERK-LP using the anti-phosphoMAPK42/44 antibody (left panel). A decrease in this signal was observed during the 1-hour treatment with 5 µM PD98059 but no change (small arrow) was observed after stripping and re-blotting with the anti-panERK antibody (right panel). A protein was detected by both antibodies (especially by the anti-pan ERK antibody) at approximately 81 kDa (large arrow). (B) Effect of 2.5 µM PD98059 on P-MAPK and MPF activity. (Ba) Fluctuations in the phosphorylation of ERK-LP were detected in P. lividus eggs with the anti-phosphoMAPK42/44 antibody (P-MAPK) and that of Cdc2-TyrP using the anti-Cdc2-TyrP antibody (upper panel). H1 kinase activity (lower panel) slowly increased after addition of the inhibitor, with transient decreases at times when Cdc2 was phosphorylated at tyrosine residue 15 (arrows). (Bb) Progressive loss of PD98059 activity after dilution in ASW. Changes after 1 µM PD98059 addition in P-ERK-LP after western blot using the anti-phosphoMAPK42/44 antibody. The inhibitor was added in the egg suspension at t=0. The supernatant, containing the inhibitor, was taken and added to fresh eggs at different times (arrow) that were then analyzed 30 minutes later (star). PD98059 was unable to act on the MAPK pathway after a 60-minute dilution in ASW. (C) Effect of 1 µM U0126. (Ca) L. pictus. U0126 addition induced oscillations in ERK-LP and in Cdc2-TyrP phosphorylation similar to those observed with PD98059 (Fig. 3B). No change was detected with the anti pan-ERK antibody after stripping and reprobing of the western blot performed with the anti-phosphoMAPK42/44 antibody. The same gel was cut around 38 kDa after transfer in order to blot the upper part for P-MAPK and the lower part for Cdc2-TyrP detection. (Cb) P. lividus. The upper panel shows that ERK-LP was dephosphorylated after addition of U0126, but was not phosphorylated again. Oscillations in Cdc2-TyrP similar to those observed in (Ca, upper panel) or after PD98059 (Fig. 3B) corresponded to a gradual increase in H1 kinase activity (lower panel) that showed decreases at times when Cdc2-TyrP level was high (arrows). (Cc) Progressive loss of U0126 activity after dilution in ASW. Changes after 0.5 µM U0126 addition in P-ERK-LP after western blot using the anti-phosphoMAPK42/44 antibody. The inhibitor was added in the egg suspension at t=0. The supernatant containing the inhibitor was taken and added to fresh eggs at different times (arrow) that were analyzed 30 minutes later (*). A complete loss of U0126 activity was observed after a 3-hour dilution in ASW.

View larger version (71K): [in a new window]   Fig. 4. Role of Ca2+ in mitosis entry after MAPK inactivation in P. lividus eggs. (A) The time course of Ca2+i changes in (a) unfertilized control eggs, (b) fertilized eggs or (c) unfertilized eggs treated with 1 µM U0126. The fluorescence ratio (340 nm to 380 nm) measured using fura-2 dextran indicate relative Ca2+i changes. Each color corresponds to one egg: a, no clear change was recorded in unfertilized control eggs during 3 hours; b, all Ca2+i signals were recorded in eggs showing elevation of the fertilization membrane, whereas absence of Ca2+i signal corresponded to eggs that were not fertilized; c, eggs treated with U0126 showed a slow increase in Ca2+i; calculations of average ratios indicated with an arrow at (1) 62 minutes, (2) 82 minutes and (3) 106 minutes (enlarged in inset) are given in the text and suggest small oscillations of Ca2+i level. (B) Ca-EGTA inhibits entry in mitosis triggered by 1 µM U0126. The injection buffer containing Ca-EGTA and carboxyfluorescein allowed to visualize the injected eggs (left panels). All eggs, including the injected ones, were treated with the MEK inhibitor (UO126) or not treated (Control) and observed 3 hours after treatment. Injected eggs did not show NEB, whereas non-injected eggs entered mitosis and sometimes showed constrictions. (C) Effect of U0126 or PD98059 on egg activation induced by injection of Ins(1,4,5)P3. MEK inhibitors reduced egg activation, as seen by elevation of the fertilization membrane, in eggs injected with less than 10 µM Ins(1,4,5)P3. (D) Effect of the absence of external Ca2+ on U0126-treated eggs. Eggs were treated with 5 µM U0126 (UO126) or not (Control, upper panels) in ASW containing 10 mM Ca2+ (Ca, left panels) or not (0 Ca, right panels) for 3 hours. Entry into mitosis was induced by U0126 even at zero external Ca2+, with deformations of the eggs that were even more severe.

View larger version (31K): [in a new window]   Fig. 5. No effect of PD98059 was seen on pHi and P-MAPK. (A) Change in pHi. Eggs were fertilized (, ) or not (, ) in the presence (, ) or not (, ) of 50 µM PD98059. PD98059 did not modify the increase in pHi observed after fertilization, nor did it change the small acidification observed in unfertilized eggs. (B) Changes in the phosphorylation of the ERK-LP detected by western blot using the anti-phosphoMAPK42/44 antibody after fertilization (F) or treatment with 10 mM ammonium (A) or with 10 µM A23187 (I). MAPK was rapidly dephosphorylated after fertilization or activation with the Ca2+ ionophore, but no change was observed after treatment with ammonium.

View larger version (22K): [in a new window]   Fig. 6. Models of the relation between Ca2+i, MAPK and MPF activity changes during egg activation. (A) Schematic representation of egg activation at fertilization. Activated MAPK and MPF activities, and Ca2+i levels are indicated in green, blue and red, respectively. Changes induced by each of them are given by arrows of corresponding color. A high MAPK activity (green line) is present in unfertilized egg. A t=0, the sperm triggers (large yellow arrow 1) a sharp Ca2+i signal (red line) that desactivates MAPK by a Ca-dependent pathway (red arrow 2) (Carroll et al., 2000). The decreasing MAPK activity brings the Ca2+i level (green arrow 3) higher (*), to an almost undetectable point (Wilding et al., 1996), that precludes the mitotic Ca2+i transients at 60 and 90 minutes, times of first (M1) and second (M2) mitosis, respectively. This change in Ca2+i level (red arrow 4), together with inactivation of MAPK (green arrow 3), induces oscillations of MPF activity (blue line). These MPF oscillations would be shaped in the fertilized eggs by the sperm: (1) The huge Ca2+i fertilization signal would first desactivate the low amount of active MPF already present in the unfertilized egg by a Ca-dependent pathway (red arrow 2') (Tunquist and Maller, 2003); (2) factor(s) of spermatic origin would act at mitosis by undetermined pathway(s), perhaps involving the polyphosphoinotide messenger system (thin yellow arrow 5) (Ciapa et al., 1994) to generate the Ca2+i mitotic transients that would themselves induce the huge MPF mitotic activation (red arrow 6 and 6'); this pathway of spermatic origin could also act directly on MPF oscillations (thin yellow arrow 5). MPF oscillations would induce MAPK oscillations (blue arrow 7). (B) Egg activation after treatment with a MEK inhibitor. Only pathways 3, 4 and 7 are activated, pathways 1, 2, 5 and 6, under sperm control, being absent. The MEK inhibitor induces MAPK inactivation, which triggers a slow rise in Ca2+i (arrow 3), itself responsible for MPF oscillations (arrow 4). Those oscillations would themselves induce MAPK oscillations (arrow 7). This would explain why MPF and MAPK oscillations are wider and of smaller amplitude in this condition. The level of Ca2+i reached in such treated eggs (red line) reaches a high level that cannot be attained in fertilized eggs.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter