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

doi: 10.1242/10.1242/dev.00340

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 Marangos, P. Articles by Carroll, J. Search for Related Content PubMed PubMed Citation Articles by Marangos, P. Articles by Carroll, J.

Ca²⁺ oscillations at fertilization in mammals are regulated by the formation of pronuclei

Department of Physiology, UCL, Gower Street, London WC1E 6BT, UK

View larger version (135K): [in a new window]   Fig. 1. The correlation between Ca2+ transients, Cdk1-cyclin B, MPF and MAP-kinase activities and pronucleus (Pn) formation. (A) Fertilization stimulates a series of Ca2+ transients that persist for about 4 hours, stopping close to the time of pronucleus formation. The schematics show the state of the eggs during the time course of the Ca2+ transients (A, inset). During the timecourse of the Ca2+ oscillations, Cdk1-cyclin B activity was determined by measuring H1-kinase activity and MAP-kinase activity by measuring phosphorylation of myelin basic protein (MBP). Kinase activities were recorded in unfertilized oocytes arrested at MII, in fertilized eggs that had extruded the second polar body (Pb2) within 2 hours of insemination and after Pn formation 4-6 hours after insemination. Data are from two experiments each with two replicates. Data are normalized to 100% activity in unfertilized eggs. The time that the Ca2+ oscillations stopped relative to the time of Pn formation is shown in Bi (n=20) and Bii (n=18). The zero time point is defined as the point at which the pronuclei were visible under bright-field observation (Bi) or by accumulation of FITC-NLS-BSA (Bii). (C) Fluorescent images of FITC-NLS-BSA (left column) and bright-field images (right column) illustrating the assessment of Pn formation. The sperm fusion site, or fertilization cone, can be seen in the first bright-field image (arrow). The first evidence of Pn formation is evident in the FITC-NLS-BSA image (arrows, Cii). The first evidence of pronuclei in the bright field optics is some 20 minutes later (arrow, Civ).

View larger version (17K): [in a new window]   Fig. 2. Inhibition of MAP-kinase activity does not inhibit Ca2+ oscillations. Treatment with UO 126 inhibited MAP-kinase activity in MII eggs and maintained low levels of MAP kinase up until Pn formation when it would normally decline. Kinase assays as for Fig. 1. Ca2+ oscillations in UO 126-treated eggs (n=38) were similar to controls (B). The cessation of oscillations correlated tightly with Pn formation (C) (compare with Fig. 1C, see Table 1).

View larger version (18K): [in a new window]   Fig. 3. Injection of excess cyclin-GFP leads to long-lasting Ca2+ oscillations. Cyclin-GFP fusion protein was microinjected into eggs prior to monitoring Ca2+ at fertilization. Cyclin-injected eggs produced long-lasting Ca2+ oscillations at fertilization (A). The schematic diagrams show the state of the eggs under bright-field observation. Cyclin-injected eggs showed no sign of second polar bodies or pronuclei (A). The duration of Ca2+ signalling in cyclin-injected eggs (n=15) is significantly longer than in controls (n=18) (B). Data show the mean±s.d.

View larger version (13K): [in a new window]   Fig. 4. Low levels of Cdk1-cyclin B activity do not explain persistent Ca2+ oscillations after extrusion of the second polar body. To inhibit cyclin synthesis after polar body extrusion, cycloheximide (CHX) was added to eggs 90 minutes after fertilization. In the presence of CHX, Ca2+ transients were generated as in controls (Ai). The time that the Ca2+ oscillations stop relative to Pn formation is shown in Aii (n=17). Note that the distribution is similar to that shown for controls in Fig. 1Bii (see also Table 1). (B) Cdk1-cyclin B activity was measured in groups of 50 unfertilized eggs and in eggs 3 hours after fertilization that had extruded a second polar body and 6 hours after fertilization when they had formed pronuclei. Data are from two experiments, each with two replicates. No significant difference in Cdk1-cyclin B activity is seen before and after Pn formation.

View larger version (62K): [in a new window]   Fig. 5. Inhibition of Pn formation leads to persistent Ca2+ oscillations after inactivation of Cdk1-cyclin B and MAP kinase. Microinjection of WGA inhibits Pn formation in mouse oocytes (A). Hoechst (i and ii) and Bright field images (iii and iv) of WGA-injected (i and iii) and uninjected (ii and iv) eggs are shown 6 hours after fertilization. Note the lack of any discernable pronuclei in WGA-injected eggs. Cdkl-cyclin B (Bi) and MAP kinase (Bii) activity declines at fertilization with similar kinetics in WGA-injected (grey columns) and control eggs (black columns). Data are from two experiments, each with two replicates. Fertilization of WGA-injected eggs leads to persistent Ca2+ oscillations (n=16) (Ci) that last significantly longer than controls (n=13) (Cii,D). Data show the mean±s.d.

View larger version (12K): [in a new window]   Fig. 6. Inhibition of importin ß-mediated nuclear transport inhibits pronucleus formation and prolongs Ca2+ oscillations. Oocytes were injected with dominant-negative importin ß45-462 and fertilized to record the effects of inhibition of nuclear transport on Ca2+ oscillations at fertilization. Importin ß45-462-injected eggs continued oscillating for nearly 12 hours, whereas controls stopped after 4 hours (P<0.01).

View larger version (28K): [in a new window]   Fig. 7. At the first mitosis the nuclear membrane becomes permeable to high molecular weight molecules before the first Ca2+ transient is generated. FITC-dextran was injected into one of the pronuclei to monitor the permeability of the nuclear membrane in relation to the Ca2+ transient at NEBD. Ca2+ was monitored simultaneously using fura red. Nuclear FITC-dextran is shown in the top row and fura red images are shown in the bottom row. Note that the pronuclei can be seen in the fura red images because of fluorescence bleed-through from the FITC emission to the fura red emission collected using a 600 long-pass filter. Fluorescence traces of the FITC-dextran and fura red ratio are shown. The time scale in the images corresponds to that in the traces. Note the Ca2+ transient takes place after the nuclear fluorescence has started to decrease (n=24). See text for details.

View larger version (22K): [in a new window]   Fig. 8. Mitotic Ca2+ transients in one-cell embryos do not continue beyond the reformation of the nuclei in the two-cell embryo. Mitotic one-cell embryos were co-injected with fura 2-dextran and FITC-NLS-BSA to simultaneously record Ca2+ and reformation of the nuclei. A representative sample is shown (n=13). Note that Ca2+ transients are detected prior to the reformation of the nuclei but not after.

View larger version (19K): [in a new window]   Fig. 9. Model depicting the nuclear localization and release of sperm-derived Ca2+-releasing activity. At fertilization, the sperm introduces a Ca2+-releasing activity. This activity, which may be a PLC or an activator or substrate of PLC (see text), is depicted by black dots or black shading. After fertilization, the Ca2+-releasing activity is proposed to localize to the pronuclei (dark stippling). The nuclear localization inhibits the ability to generate Ins(1,4,5)P3 and so the Ca2+ oscillations stop. Other factors also appear to be at play to desensitize Ins(1,4,5)P3-induced Ca2+ release in pronucleate embryos, as depicted by the grey shading of the cytoplasm (see text for more details). The pronuclei migrate to the centre of the embryo and NEBD takes place, marking the start of the first mitotic division. NEBD leads to the factor responsible for Ca2+-releasing activity to disperse in the cytoplasm, where it has the capacity to generate Ca2+ transients. The oscillations stop again at the two-cell stage when the nuclei form. This model of nuclear compartmentalization of Ca2+-releasing activity, including PLCs, may be important for regulating mitotic Ca2+ transients in a variety of cells (see text).