Localized accumulation of cytosolic calcium near the fused sperm is associated with the calcium- and voltage-dependent block of sperm entry in the sea urchin egg

AbstractIntracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of cytosolic [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelop and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation/repolarisation produced by an upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to pHi, voltage and [Ca2+]i fluctuations. The upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelop and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent, [Ca2+]i-inactivated CatSper dynamics alone. The model predicts that these two modules interfere with each other and produce unreasonable dynamics when present at similar proportions, which suggests that one may predominate over the other in vivo. To assess these alternatives, several quantitative predictions were derived from each module and confronted to experimental observations. We show that the [Ca2+]I dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper module but not by one including the pH-independent CaV and BK module. We conclude in favour of the module containing CatSper.

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The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.

The Journal of Cell Biology ◽

10.1083/jcb.103.6.2333 ◽

1986 ◽

Vol 103 (6) ◽

pp. 2333-2342 ◽

Cited By ~ 163

Author(s):

K Swann ◽

M Whitaker

Keyword(s):

Sea Urchin ◽

Calcium Ions ◽

Calcium Concentration ◽

Calcium Release ◽

Inositol Trisphosphate ◽

Lytechinus Pictus ◽

Sea Urchin Egg ◽

Sperm Entry ◽

Fertilization Membrane

Sea urchin egg activation at fertilization is progressive, beginning at the point of sperm entry and moving across the egg with a velocity of 5 microns/s. This activation wave (Kacser, H., 1955, J. Exp. Biol., 32:451-467) has been suggested to be the result of a progressive release of calcium from a store within the egg cytoplasm (Jaffe, L. F., 1983, Dev. Biol., 99:265-276). The progressive release of calcium may be due to the production of inositol trisphosphate (InsP3), a second messenger. We show here that a wave of calcium release crosses the Lytechinus pictus egg; the peak of the wave travels with a velocity of 5 microns/s; microinjection of InsP3 causes the release of calcium within the egg; calcium release (as judged by fertilization envelope elevation) is abolished by prior injection of the calcium chelator EGTA; neomycin, an inhibitor of InsP3 production, does not prevent the release of calcium in response to InsP3 but does abolish the wave of calcium release; the egg cytoplasm rapidly buffers microinjected calcium; the calcium concentration required to cause fertilization membrane elevation when microinjected is very similar to that required to stimulate the production of InsP3 in vitro; and the progressive fertilization membrane elevation seen after microinjection of calcium buffers appears to be due to diffusion of the buffer across the egg cytoplasm rather than to the induction of the activation wave. We conclude that InsP3 diffuses through the egg cytoplasm much more readily than calcium ions and that calcium-stimulated production of InsP3 and InsP3-induced calcium release from an internal store can account for the progressive release of calcium at fertilization.

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Fusion of membranes during fertilization. Increases of the sea urchin egg's membrane capacitance and membrane conductance at the site of contact with the sperm.

The Journal of General Physiology ◽

10.1085/jgp.99.2.137 ◽

1992 ◽

Vol 99 (2) ◽

pp. 137-175 ◽

Cited By ~ 72

Author(s):

D H McCulloh ◽

E L Chambers

Keyword(s):

Sea Urchin ◽

Plasma Membranes ◽

Membrane Conductance ◽

Membrane Potentials ◽

Membrane Capacitance ◽

Cortical Granules ◽

Abrupt Increase ◽

Sea Urchin Egg ◽

Sperm Entry ◽

Whole Egg

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.

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Assembly of unfertilized sea urchin egg tubulin at physiological temperatures.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)32654-1 ◽

1983 ◽

Vol 258 (7) ◽

pp. 4518-4525 ◽

Cited By ~ 2

Author(s):

K A Suprenant ◽

L I Rebhun

Keyword(s):

Sea Urchin ◽

Sea Urchin Egg

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A Ribonucleoprotein Which Catalyzes Thiol-Disulfide Exchange in the Sea Urchin Egg

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)96114-4 ◽

1967 ◽

Vol 242 (7) ◽

pp. 1458-1461

Author(s):

Hikoichi Sakai

Keyword(s):

Sea Urchin ◽

Disulfide Exchange ◽

Sea Urchin Egg

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Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs

Biochemical Journal ◽

10.1042/bj3460743 ◽

2000 ◽

Vol 346 (3) ◽

pp. 743-749 ◽

Cited By ~ 32

Author(s):

Keith T. JONES ◽

Miho MATSUDA ◽

John PARRINGTON ◽

Matilda KATAN ◽

Karl SWANN

Keyword(s):

Phospholipase C ◽

Sea Urchin ◽

Tissue Extracts ◽

Sea Urchin Egg ◽

Boar Sperm ◽

Specific Activities ◽

Mammalian Eggs ◽

Mouse Eggs

A soluble phospholipase C (PLC) from boar sperm generates InsP3 and hence causes Ca2+ release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca2+ oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca2+ oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P2, the ability of sperm extracts to release Ca2+ was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca2+-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCβ1, -γ1, -γ2, -∆1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca2+ in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca2+ oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P2 in eggs.

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