Brigitte Ciapa

A rapid change in phosphorylation on tyrosine accompanies fertilization of sea urchin eggs

Alterations in protein phosphorylation, particularly phosphorylation on tyrosine, frequently accompany cell change and are important agents in the cascades initiated by extracellular signals. This paper examines whether the activation of the sea urchin egg at fertilization involves an early and rapid phosphotyrosine response. Using an anti‐phosphotyrosine antibody and a rapid sampling technique, we find a very early increase in the phosphorylation on tyrosine of two proteins of approximately 91 kDa and 138 kDa. A similar phosphorylation occurs after activation of the eggs by the calcium ionophore, ionomycin, suggesting the stimulation of a Ca2+‐sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization.

Mechanisms regulating intracellular pH in sea urchin eggs

Intracellular pH (pHi) of sea urchin eggs (Paracentrotus lividus) was determined using DMO (dimethyloxazolidinedione) and a rapid filtration technique (P. Payan, J.P. Girard, R. Christen and C. Sardet (1981). Exp. Cell Res. 134, 339-344). Transfer of unfertilized or fertilized eggs from normal sea water into Na+-free artificial sea water leads to a progressive acidification and fall of intracellular Na+ content. A step rise in external Na+ to 10 meq causes a rapid but transient Na+ entry coupled to an excretion of H+, giving rise to a pHi increase. It is shown that the plasma membrane of unfertilized eggs contains a permanent and reversible Na+/H+ exchanger which contributes to the regulation of pHi. This exchange occurs with a 1:1 stoichiometry and is independent of metabolic energy. Proton excretion and sodium entry follow saturable kinetics with respect to external Na+ and are completely inhibited by amiloride. At fertilization, pHi increases from 7.38 to 7.64 and is maintained at this level by two separate mechanisms: (1) a Na+/H+ exchange with the same characteristics as in unfertilized eggs; (2) a H+-excreting system that is dependent on external Na+, amiloride sensitive, and requiring metabolic energy. The relationship between the permanent Na+/H+ exchange involved in pHi regulation and the transient Na+/H+ exchange occurring at fertilization is discussed.

Characterisation and role of integrins during gametic interaction and egg activation.

It has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By performing immunofluorescence labelling and Western blots with antibodies directed against some of the alpha and beta subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, alpha 2 and alpha 5 were also present on in vitro preparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that alpha 2 and alpha 5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich in three cytoskeletal proteins known to be linked to the beta chain of integrins: talin, vinculin and alpha-actinin. Moreover, we found that talin and alpha-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.

Characterization of valine transport in sea urchin eggs

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na+-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na+-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na+-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in Vmax with no change in Km. These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na+-dependent valine uptake and the ionic events triggered by fertilization is discussed.

The calcium content of cortical granules and the loss of calcium from sea urchin eggs at fertilization

In many species, fertilization triggers a wave of cortical granule exocytosis in the egg that is the consequence of an increase in intracellular free calcium concentration. We have measured the total calcium content of cortical granules from two species of sea urchins by quantitative X-ray microanalysis and spectrometric measurements. Our results show that cortical granules: (1) contain a high concentration of total calcium (around 30 and 95 mM for Paracentrotus lividus and Arbacia lixula, respectively), (2) represent a major cortical storage site of calcium in the egg (5 and 11% of total egg calcium for P. lividus and A. lixula, respectively), and (3) exchange part of their accumulated calcium by an ATP dependent mechanism. In addition we have confirmed that at fertilization, sea urchin eggs lose a sizeable amount of their calcium (7% for P. lividus and 15% for A. lixula). The kinetics and magnitude of the loss suggest that some of this calcium could be provided by cortical granules during exocytosis.

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs☆

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.

Effect of Cytochalasin B on the Development of Membrane Transports in Sea Urchin Eggs after Fertilization

Investigations were made on the role of the cytoskeleton in the onset of ionic events following fertilization of sea urchin eggs. Events which depend upon phosphoinositide metabolism, such as the cortical reantion and acid release are affected by cytochalasin B (CB) after fertilization but not after activation of eggs with the ionophore A23187. These findings suggest that the sequence of events following sperm‐egg attachment depends on the cytoskeleton. CB also inhibits the Na+ pump and alanine uptake when added before insemination and during the following 30 min. These results argue for a role of the egg cortex cytoskeleton in activation of the Na+ pump by fertilization. We propose that the inhibitory effect of CB on the development of amino‐acid uptake after fertilization may result from an increase in the Na+ content of the egg resulting from Na+ pump suppression rather than from direct blockage of the carrier.

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

SummaryFreeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive 86Rb uptake and amiloride-sensitive 24Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na+/H+ and Na+/K+ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na+/H+ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na+/H+ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na+ pump, Na+/H+ exchange) without eliciting corresponding regulatory mechanisms (Na+ stat, pH stat).

Role of integrins and polyphosphoinositide metabolism during fertilization in sea urchin egg and hamster oocyte

Summary In almost all species studied to date, a transient increase in the intracellular free calcium concentration (Cai) occurs after fertilization and is essential for egg activation. How the sperm triggers this calcium signal remains, however, to be determined. In this brief review, we compare the mechanisms that are common to mammalian and invertebrate systems. In the light of our own data, we discuss how integrins that have recently been proposed to mediate sperm-egg binding and fusion in mammals might trigger egg activation through cytoskeletal structures. We suggest a model, leading to the calcium signal and common to all species, where phosphorylation on tyrosine and phospholipase Cγ (PLCγ) are interconnected pathways stimulated by a multimolecular complex involving integrins.