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First published online 9 August 2005
doi: 10.1242/jcs.02506

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Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs

Institute for Cell and Molecular Biosciences, The Medical School, Framlington Place, University of Newcastle, Newcastle, NE2 4HH, UK

View larger version (18K): [in a new window]   Fig. 1. Pharmacological inhibition of CamKII, but not PKC, blocked exit from meiosis. (A) To induce completion of meiosis, eggs were incubated in Sr2+ medium supplemented with doses of KN-93 (CamKII inhibitor), KN-92 (the inactive analogue of KN-93) or BIM1 (PKC inhibitor). Only KN-93 prevented exit from meiosis. (B) Eggs were induced to resume meiosis by PLC cRNA microinjection, in a range of concentrations of KN-93-supplemented media. The CamKII inhibitor blocked meiotic progression over a similar dose range as that observed with Sr2+ medium. (C) KN-93 inhibited Ca2+ spiking. Intracellular Ca2+ changes were recorded in eggs incubated in Sr2+ media supplemented with 10 µM KN-93. All eggs that failed to activate (n=35) also failed to exhibit Ca2+ spiking; however, all those eggs that extruded a second polar body and formed pronuclei (n=10) had one or more Ca2+ spikes. Percentage activation rates were assessed at 8 hours by the formation of pronuclei. The number of eggs used is indicated in parenthesis.

View larger version (15K): [in a new window]   Fig. 2. Constitutively active CamKII and PKC constructs. (A,B) Schematic of wild-type CamKII (A) and PKC (B). Constitutively active (CA) mutants of CamKII and PKC were fused C-terminally to GFP. (C) CA-CamKII::GFP; deletion of residues 291-478. (D) CA-E25PKC::GFP; substitution of alanine for glutamic acid at position 25 and CA-22-28PKC::GFP; deletion of residues 22-28.

View larger version (59K): [in a new window]   Fig. 3. Constitutively active CamKII, but not PKC, induces completion of meiosis. (A) CA-CamKII, but not CA-E25PKC or CA-22-28PKC, was effective at inducing exit from meiotic arrest. The amount of cRNA injected is plotted against the percentage activation rate, as assessed at 8 hours by the formation of pronuclei. The number of eggs used is indicated in parenthesis. (B) The time course of meiotic progression with CA-CamKII is similar to that observed with sperm, with second polar body extrusion (PB2; indicated by arrow) at about 1.5 hours and pronucleus formation (PN; arrowhead) at about 5.5 hours. Scale bar: 20 µm.

View larger version (14K): [in a new window]   Fig. 4. The PKC constructs retain biological activity. Eggs were incubated in Sr2+-containing medium 2 hours after microinjection of CA-E25PKC cRNA (n=20). Eggs that had not been injected with the PKC construct showed a typical spiking pattern, which led to a completion of meiosis (n=20). However, following Sr2+ addition, eggs expressing CA-E25PKC showed an immediate and long-lasting elevation in the fura2 signal, consistent with the reported role of PKCs in SOCE (Halet et al., 2004). We conclude that the PKC constructs used here retain biological activity.

View larger version (71K): [in a new window]   Fig. 5. CA-CamKII could overcome the inhibitory effects of KN-93 on eggs. (A) Experimental design. Eggs were treated with a parthenogenetic stimulus (Sr2+/PLC) but were kept arrested at MetII by 10 µM KN-93 for a period of 2 hours. CA-CamKII cRNA was then microinjected into eggs to determine if it could overcome the inhibitory effects of KN-93. (B) Following microinjections of 3 pg CA-CamKII cRNA all eggs activated (30/30 Sr2+; 30/30 PLC); inset shows decondensed chromatin stained with Hoechst. The vast majority of control eggs not microinjected with CA-CamKII cRNA remained arrested at MetII (rates of resumption of meiosis: 4/26 Sr2+; 0/25 PLC): inset (right) shows the condensed chromatin of a MetII spindle stained with Hoechst. Scale bar: 20 µm.

View larger version (19K): [in a new window]   Fig. 6. CamKII acts downstream of the fertilization Ca2+ signal. (A) Microinjection of CA-CamKII induces meiotic progression in the absence of Ca2+ spiking. Intracellular Ca2+ was recorded in eggs induced to complete meiosis by microinjection of cRNA to CA-CamKII (black, n=12) or PLC (grey, n=8). Second polar body (PB2; arrow) and pronucleus (PN; arrowhead) formation are at the times indicated. (B) Eggs were treated with a parthenogenetic stimulus of Sr2+ medium but were kept arrested at MetII by KN-93 for a period of 2 hours. CA-CamKII cRNA was then microinjected into eggs. After CA-CamKII injection, eggs exited meiosis but Ca2+ spiking was not recovered (n=15). (C) CA-CamKII-induced meiotic progression was not blocked by the Ca2+ chelator BAPTA. BAPTA-loaded eggs were either incubated in Sr2+-containing medium; or microinjected with CA-CamKII cRNA; or were incubated in Sr2+-containing medium followed 2 hours later by CA-CamKII cRNA microinjection. As expected, BAPTA blocked Sr2+-induced egg activation but had no inhibitory effect on meiotic progression induced by CA-CamKII, suggesting that the CA-CamKII works downstream of the sperm Ca2+ signal. Furthermore, in eggs where BAPTA blocked Sr2+-induced meiotic progression, microinjection of CamKII cRNA overcame this block. The number of eggs used is indicated in parenthesis.

View larger version (26K): [in a new window]   Fig. 7. CA-CamKII promotes the destruction of both cyclin B1 and securin. Eggs were microinjected with cRNA to cyclin B1::YFP or securin::CFP and then with cRNA to CA-CamKII. (A) APC/C activity increased following introduction of CA-CamKII as judged by the degradation of cyclin B1 (n=6). Cyclin B1 levels dropped to a minimum at the time of second polar body extrusion and levels remain low until pronuclei formation. Representative brightfield and cyclin B1::YFP images are shown at various times after injection as indicated. (B) A similar degradation profile for securin::CFP is also observed following introduction of CA-CamKII cRNA (n=10). (A and B) CA-CamKII cRNA was microinjected at t=0; second polar body extrusion (PB2; arrow); pronuclei formation (PN; arrowhead). Scale bar: 20 µm.

View larger version (33K): [in a new window]   Fig. 8. CamKII-induced meiotic progression is blocked by a spindle checkpoint. (A-C) Both nocodazole and Mad2 could inhibit Sr2+-induced exit from MetII arrest. (A,B) 100 ng/nl nocodazole completely inhibited parthenogenetic activation by Sr2+ medium as judged by Hoechst staining but 10 ng/ml nocodazole had little effect. (C) Over expression of Mad2::YFP similarly blocked cell-cycle progression. (D) A spindle checkpoint induced by either nocodazole (100 ng/ml) or Mad2 blocked CA-CamKII-induced resumption of meiosis. The number of eggs per treatment group is indicated in parenthesis. In B and C the arrow indicates the MetII spindle and the arrowhead, decondensed chromatin in a pronucleus. Scale bar: 20 µm.

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