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First published online 26 May 2004
doi: 10.1242/dev.01181

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Sperm-triggered [Ca²⁺] oscillations and Ca²⁺ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production

¹ Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK
² Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (45K): [in a new window]   Fig. 11. A model depicting the functional interactions between ER and mitochondria in the mouse egg. After Ca2+ is released from the ER into the cytosol via Ins(1,4,5)P3 receptors it can be taken up by the mitochondria. In the mitochondrial matrix, Ca2+ will stimulate the Kreb’s cycle as well as the electron transport chain and the F0/F1 synthase that phosphorylates ADP. ATP is then transported out of the mitochondria via the activity of the adenine nucleotide translocase (ANT) and the cytosolic ATP will be consumed by the SERCA pumps to restore the resting [Ca2+]c and refill the ER.

View larger version (32K): [in a new window]   Fig. 1. Synchronous oscillations of NADH and FAD++ autofluorescence at fertilisation. (A) Ca2+ fluxes and oxidative phosphorylation in the mitochondria. The supply of substrate (such as pyruvate) to the Kreb’s cycle promotes the reduction of NAD+ to NADH and of FAD++ to FADH2. NADH is then oxidised by complex I in the respiratory chain, whereas FADH2 is oxidised at complex II. The electrons are then transferred to complexes III and IV to reduce O2 to H2O. In the process, protons are translocated across the inner mitochondrial membrane, generating a potential gradient of approximately –150 mV (). ATP synthesis takes place at complex V or F0/F1 synthase, the inward flux of protons through the synthase provides the energy necessary to phosphorylate ADP. ATP is then transported out of the mitochondria by the adenine nucleotide translocase (ANT). Ca2+ enters the mitochondria through the Ca2+ uniporter, while it is extruded via the action of a Na+/Ca2+ exchanger. The actions of CN- and FCCP are also indicated. (B) Variations in NADH (blue trace) and FAD++ (green trace) observed at fertilisation of a mouse egg (n=6). Time 0 corresponds to the time of insemination. F indicates the time of fertilisation. (C) Changes in NADH (blue trace) and FAD++ (green trace) autofluorescence measured inside the ROI shown in (i), upon perfusion of 2 mM CN– (provoking full reduction of NADH and FAD++) and of 1 µM FCCP (provoking full oxidation of NADH and FAD++). (i-iv) Images of FAD++ autofluorescence of a mouse egg at times indicated on the graph.

View larger version (28K): [in a new window]   Fig. 3. Oscillations in FAD++ autofluorescence are stimulated by fertilisation induced Ca2+ transients. (A) Variations in [Ca2+]c (measured with indo 1 AM, blue trace) and FAD++ autofluorescence (green trace) observed at fertilisation of a mouse egg (n=20). Time 0 corresponds to the time of insemination. (B) Variations in [Ca2+]i (measured with rhod 2 AM, red trace) and FAD++ autofluorescence (green trace) in a mature mouse egg injected with caged Ins(1,4,5)P3 (n=10). A UV flash (red arrowhead) releases Ins(1,4,5)P3 in the egg and triggers a Ca2+ transient accompanied by a transient decrease in FAD++ autofluorescence. i and ii show the same egg as in Fig. 2D-F with the FAD++ signal (i) and Rhod2 AM (ii) signal that have markedly different distributions. This suggests that, under our conditions, rhod2 AM does not partition into mitochondria of the mouse egg. The ROI (white circle in i) used to obtain the measurements is drawn. (C) Oscillations of FAD++ autofluorescence are stopped by the addition of 5 µM BAPTA AM to the chamber (the presence of BAPTA AM is indicated by a bar under the graph, n=8)

View larger version (80K): [in a new window]   Fig. 2. Imaging of mitochondrial autofluorescence. (A-C) Simultaneous imaging of FAD++ autofluorescence (green, A) and mitochondria using the potentiometric dye TMRM (red, B). Merging the image of mitochondria and of autofluorescence (C) clearly shows that the FAD++ autofluorescence colocalises with mitochondria. (D-F) Simultaneous imaging of FAD++ (D, green) and NADH (E, blue). Merging the NADH and FAD++ images (F) shows that a significant part of the NADH signal is cytosolic. MS, meiotic spindle.

View larger version (25K): [in a new window]   Fig. 4. Inhibition of sperm-triggered [Ca2+] oscillations by mitochondrial inhibitors. Variations of [Ca2+]i observed at fertilisation (measured with fura red AM in A and B or with fura 2 AM in C). 1 µM FCCP (A), 2 mM CN– (B) or 60 µM oligomycin (C) are added to the chamber as indicated by the bar under the graph.

View larger version (16K): [in a new window]   Fig. 8. Mitochondrial inhibitors cause an increase in resting [Ca2+]c by depleting ATP levels in mature eggs. (A) Simultaneous measurement of [Ca2+]i (using fura 2 AM) and of m [using Rhod 123 (compare with Materials and methods)] in mature eggs. Where indicated, oligomycin (60 µM) and FCCP (1 µM) are added to the bath. (B) Photon emission of a single egg injected with luciferase. Where indicated, oligomycin (60 µM) and FCCP (1 µM) are added to the bath. The background luminescence count was of six photons per 10 seconds in this experiment. (C) Schematic representation of a mitochondria and the effect of oligomycin on the proton fluxes generated at the F0/F1 synthase.

View larger version (27K): [in a new window]   Fig. 5. Effect of energetic substrates starvation on sperm-triggered [Ca2+] oscillations and cellular [ATP]. (A,B) Mature eggs are incubated in M2 medium without lactate, pyruvate, glucose and amino acids for 2 hours and then fertilised. Where indicated, 2 mM pyruvate is added to the dish. The lack of energetic substrates disrupts the sperm-triggered [Ca2+] oscillations with either [Ca2+]i remaining high after a Ca2+ transient (A, n=12) or with [Ca2+]i remaining low (B, n=11). (C) Changes in [ATP]i upon withdrawal of energetic substrates (n=7). An egg was placed in M2 medium without energetic substrates for 30 minutes before the start of the recording, then, where indicated, 2 mM pyruvate or 1 µM FCCP was added.

View larger version (30K): [in a new window]   Fig. 6. Fertilisation does not cause a decrease in ATP levels in single eggs. (A,B) Measurements of bulk cytosolic [ATP] in fertilised eggs (A, n=9; B, n=16). Luminescence recording of an egg injected with firefly luciferase. Sperm (S) was added when indicated by the arrow and, in B, FCCP was added when indicated by the second arrow. The time of fertilisation is 15-30 minutes after sperm addition. Fertilisation was confirmed by the presence of the second polar body 90 minutes after sperm addition.

View larger version (23K): [in a new window]   Fig. 7. Mitochondrial inhibitors cause an increase in resting [Ca2+]i in mature eggs. (A,B) Increase in [Ca2+]i induced by addition to an unfertilised egg of FCCP (1 µM) in normal H-KSOM medium (A) or in H-KSOM without Ca2+ and supplemented with 2 mM EGTA (B). (C,D) Increase in [Ca2+]i induced by addition to an unfertilised egg of CN– (2 mM) in normal H-KSOM medium (C) or in H-KSOM without Ca2+ and supplemented with 2 mM EGTA (D). (E,F) Variations of [Ca2+]i induced by sequential addition to an unfertilised egg of 20 µM thapsigargin and FCCP (1 µM) in normal H-KSOM (E) or in H-KSOM without Ca2+ (F).

View larger version (28K): [in a new window]   Fig. 9. Mitochondrial potential can be maintained by the reversal of the ATP synthase. (A-C) Simultaneous measurement of [Ca2+]c (using fura 2 AM) and of m (using Rhod123) in mature eggs. Where indicated, oligomycin (60 µM), FCCP (1 µM), malonate (20 mM) and rotenone (5 µM) are added to the bath. (D,E) Schematic representations of a mitochondria and the effect of mitochondrial inhibitors on the proton fluxes generated at the respiratory chain (D, CN–, rotenone, malonate) or at the F0/F1 synthase (E, oligomycin).

View larger version (113K): [in a new window]   Fig. 10. Confocal imaging reveals close apposition of mitochondria and ER in a mature egg. (A) General view of a mature egg stained for mitochondria (green) and ER (red) showing mitochondrial aggregates and an ER network dispersed in the whole cytoplasm except in the region of the meiotic spindle (MS) marking the animal pole of the egg. (B,C) Higher magnification view of a mitochondrial aggregate and its relationship with the ER network. It can be clearly seen that strands of ER are intermingled in the aggregate of mitochondria (two of them are indicated by white arrowheads).

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