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

First published online 23 November 2004
doi: 10.1242/jcs.01567

This Article Summary Full Text Full Text (PDF) 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Chang, H.-Y. Articles by Jones, K. T. Search for Related Content PubMed PubMed Citation Articles by Chang, H.-Y. Articles by Jones, K. T. Social Bookmarking                         What's this?

Degradation of APC^cdc20 and APC^cdh1 substrates during the second meiotic division in mouse eggs

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

View larger version (55K): [in a new window]   Fig. 1. Cdc20 and cdh1 protein in mouse eggs. Western blots of MII eggs. Anti-cdh1 (lane 1, antibody ab3242; lane 2, AR38) and anti-cdc20 (lanes 3 and 4, sc8358). In lane 4, germinal-vesicle (GV) stage oocytes were microinjected with cdc20::GFP cRNA and allowed to mature to MII. The upper band in lane 4 is at the predicted molecular mass of cdc20::GFP. The number of eggs used for each lane is as stated; and the migration of protein standards (kDa) are marked.

View larger version (80K): [in a new window]   Fig. 2. Egg progression through meiosis II. By inseminating eggs with low numbers of sperm we were able to monitor accurately both second polar body formation (pb2), which occurred within 3 hours of insemination, and pronucleus (pn) formation, at about 4-6 hours. Sperm were added at time 00:00 (hours:minutes). Sample images at 20-minute intervals are shown but images were captured every 15 seconds. Scale bar: 20 µm.

View larger version (31K): [in a new window]   Fig. 3. Degradation of securin and cyclin B1 is initiated simultaneously. Eggs were microinjected with the Ca2+ reporter fura2-dextran and cRNA to either securin::GFP (A,B) or cyclin B1::GFP (C). Sperm were added at the beginning of recording and following gamete fusion a series of Ca2+ spikes were observed that were responsible for initiating exit from MII arrest. Degradation of cyclin B1 and securin occurred at the same time: approximately 10-20 minutes after the initiation of Ca2+ spiking. (A) Representative securin::GFP images captured from an inseminated egg every 2 hours at the times indicated (in hours); time is relative to the addition of sperm, and in this egg polar body extrusion occurred at 2.3 hours and pronucleus formation at 6.5 hours. Scale bar: 20 µm. (B,C) Sperm induced a series of Ca2+ spikes that lasted for several hours (black trace), simultaneous imaging of eggs for GFP showed that degradation of both GFP constructs were complete by second polar body formation. Levels increased again as the pronuclei formed in the 1-cell embryo. A single representative recording from 16 eggs is shown for both constructs. GFP levels are represented as the mean egg average fluorescence (in arbitrary units) from a region-of-interest defined on the Metafluor program.

View larger version (9K): [in a new window]   Fig. 4. CDK1 activity declines rapidly with polar body extrusion during meiosis II. Small groups of eggs were assayed, at the times indicated, for CDK1 activity by the ability of CDK1 to phosphorylate histone H1 in an in vitro assay. Eggs sampled at 1 hour had extruded their second polar body and eggs sampled at 8 hours had pronuclei. The shaded bars indicate that the polar body (pb2) and pronucleus (pn) formation must have occurred during this time. CDK1 activity declined rapidly in activating eggs, such that the largest decrease occurred in eggs that had extruded their pb2. Values are means ± standard deviation (n=5, separate experiments) in H1 kinase activity normalised with respect to non-activated eggs.

View larger version (36K): [in a new window]   Fig. 5. Delay in securin degradation by removal of its D box. Mouse eggs expressing securindm were inseminated and egg activation monitored. The destruction of securin was significantly delayed by removal of its D box. (A) Representative securindm::GFP images captured from an inseminated egg every 2 hours at the times indicated (in hours). In this egg, polar body extrusion occurred at 2.8 hours and pronucleus formation at 5.6 hours. Scale bar: 20 µm. (B) The securindm::GFP profile in a fertilized egg. Ca2+ spiking, initiated at 1.5 hours, induced second polar body extrusion at 2.3 hours but during this time securindm was stable. At the time of polar body extrusion degradation of securindm began, and this continued until pronucleus formation at 7 hours.

View larger version (16K): [in a new window]   Fig. 6. CDH1 during egg activation. Western blots of cdh1 were consistent with the reported bandshift in cdh1 following dephosphorylation. Mouse oocytes at MI and MII are compared with activated eggs at the pronculeate stage (PN). An actin loading control is as indicated. The number of eggs used for each lane is indicated above.

View larger version (29K): [in a new window]   Fig. 7. Meiosis II degradation of cdc20 is KEN-box dependent. (A) Wild-type cdc20 coupled to GFP was expressed in MII eggs that were subsequently inseminated. Following sperm-triggered Ca2+ spiking cdc20 degradation was observed around the time of second polar body extrusion and continued until pronuclei formed in the 1-cell embryo. (B) Degradation of cdc20 was observed to be dependent on the presence of a KEN box, since its removal rendered cdc20 stable. Thus despite fertilization of these eggs, as observed by a series of Ca2+ spikes, cdc20km:: GFP levels continued to rise throughout the completion of meiosis II. Recordings are representative of 8 eggs for both constructs. (C) Endogenous cdc20 was observed to be degraded at the two time points sampled: 2 hours (+2hr) and 6 hours (+6hr) after addition of Sr2+-containing medium to induce parthenogenetic activation. Polar bodies were extruded after about 1 hour, and pronuclei were visible at around 5 hours. (i) western blot; (ii) Coomassie Blue stained membrane; (iii) eggs at time 0 hours and 6 hours probed for cdc20 and actin as a loading control. All lanes were loaded with 50 eggs.

View larger version (9K): [in a new window]   Fig. 8. Model of APCcdc20 and APCcdh1 activation during meiosis II. Unfertilized MII eggs, which have high CDK1 levels, have low APCcdc20 and APCcdh1 activity. A sperm-derived Ca2+ signal initiates activation of APCcdc20 at gamete fusion, which is responsible for cyclin B1/securin degradation and an associated loss in CDK1 activity. Degradation of these APCcdc20 substrates permits second polar body formation and at this time APCcdh1 activity appears. APCcdh1 is responsible for degradation of KEN-box substrates such as cdc20. APCcdh1 activity drops when pronuclei form and the zygote enters S phase of the first embryonic cell cycle.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter