Egg activation: upstream of the fertilization calcium signal.

Interaction of sperm and egg at fertilization induces well-coordinated molecular events including specific recognition between species, adhesion and fusion, that lead to the formation of a zygote, a totipotent cell that develops into a new individual. A calcium signal, common to a great number of species, from marine invertebrates to mammals, is essential to activate the metabolism of the unfertilized oocyte. However, how fertilization triggers this calcium signal and initiates development of the early embryo is far from understood. The signaling pathways activated in eggs may be similar to those described in somatic cells, since changes in intracellular free calcium and in mitosis activating protein (MAP) kinase activity occur in both systems after activation. Several hypotheses are currently proposed, implying a spermatic ligand binding to a specific receptor expressed at the egg surface, or where the fused sperm either allows the transit of external calcium into the egg or injects one (or several) activating factor(s). It is still not known which of these ideas is true. We concentrate in this review on the possible signaling pathways involving IP3 (inositol trisphosphate), since its production is involved in most species to generate the fertilization calcium wave.

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.

Detection of phospholipase Cgamma in sea urchin eggs.

Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cgamma (PLCgamma), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCgamma revealed the presence of this protein in cortical regions. It was also observed that PLCgamma displayed calcium-sensitive activity. The present results suggest that PLCgamma may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.

Effect of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, on the first mitotic divisions of the fertilized sea urchin egg.

We have reported earlier that the polyphosphoinositide messenger system may control mitosis in sea urchin eggs. Besides phospholipase C activation and its second messengers, phosphatidylinositol (PI) 3-kinase has been proposed to affect a wide variety of cellular processes in other cellular systems. Therefore, we have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. Our data presented here suggest that PI 3-kinase is present in sea urchin eggs. We found that wortmannin, an inhibitor of PI 3-kinase, led to arrest of the cell cycle. Chromosome condensation, nuclear envelope breakdown, microtubular aster polymerization, protein and DNA synthesis were not affected when fertilization was performed in the presence of the drug. However, maturation-promoting factor (MPF) activation was inhibited and centrosome duplication was perturbed preventing the formation of a bipolar mitotic spindle in wortmannin treated eggs. We discuss how PI 3-kinase might be involved in the cascade of events leading to the first mitotic divisions of the fertilized sea urchin egg.

Evidence for MAP kinase activation during mitotic division.

MAP kinases have been implicated in the control of a broad spectrum of cellular events in many types of cells. In somatic cells, MAP kinase activation seems to be triggered after exit from a quiescent state (in G0 or G2) only and then inactivated by entry into a proliferative state. In oocytes of various species, a one-time activation of MAP kinase that is apparently not repeated during the succeeding mitotic cycles occurs after meiotic activation. However, several reports suggest that a myelin basic protein (MBP) kinase activity, unrelated to that of maturation promoting factor, can sometimes be detected during mitotic divisions in various types of cells and oocytes. We have reinvestigated this problem in order to determine the origin and the role of MBP kinase that is stimulated at time of mitosis in the fertilized eggs of the sea urchin Paracentrotus lividus. We used anti-ERK1 antibodies or substrates specific for different MAP kinases, and performed in-gel phosphorylation experiments. Our results suggest that an ERK1-like protein was responsible for part of the MBP kinase activity that is stimulated during the first mitotic divisions. Furthermore, we observed that wortmannin, an inhibitor of PI 3-kinase that arrests the fertilized sea urchin eggs at the prometaphase stage, inhibited the inactivation of MAP kinase normally observed when the eggs divide, suggesting a role for PI 3-kinase in the deactivation process of MAP kinase. We also discuss how the activities of MPF and MAP kinase may be interconnected to regulate the first mitotic divisions of the early sea urchin embryo.

[Mini-review: oocyte activation mechanisms. Different models, comparison: marine invertebrate and mammal]. / Mini-revue: mécanismes d'activation ovocytaire. Différents modèles, complémentarité: invertébré marin et mammifère.

In most deuterostome eggs, a transient increase in the intracellular free calcium concentration (Cai) occurs after fertilization and is essential for egg activation. How one spermatozoo triggers this calcium signal remains an enigma. In the light of our own results, we suggest a model, leading to the calcium signal and common to invertebrate and mammalian systems, where phosphorylation on tyrosine and phospholipase C gamma (PLC gamma) are interconnected pathways stimulated by a multimolecular complex involving integrins.

An early increase in cGMP follows fertilization of sea urchin eggs.

It has been proposed that both inositol trisphosphate and ryanodine receptors contribute to the Ca signal generated at fertilization of the sea urchin egg. Pharmacological studies indicate that cyclic adenosine diphosphate-ribose (cADPr) is the endogenous modulator of Ca release by the ryanodine-like receptor in eggs and that cADPR cyclase, the enzyme responsible for cADPR synthesis, can be stimulated by 3',5'-cyclic guanosine monophosphate (cGMP). Also, recent results show that the gaseous transmitter nitric oxide (NO) releases calcium in eggs via a mechanism linked to cGMP and cADPR production. Results reported here show that fertilization induces a rapid and transient increase in the intracellular concentration of cGMP. This increase occurs during the latent period, before the major increase in cytoplasmic free calcium (Cai), consistent with the hypothesis that cGMP production may play a key role in the Ca signal seen at fertilization.

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.

Effects of progesterone on human spermatozoa prepared for in-vitro fertilization.

Progesterone has been tested in vitro with human spermatozoa to verify its physiological effects and its possible therapeutic use in cases of male infertility. Progesterone induced a rapid, dose-dependent influx of calcium in capacitated and non-capacitated spermatozoa with a half-maximally effective dose of 30 nM. The agonist, 19-nortestosterone, was much less potent that progesterone itself. Progesterone-induced calcium influx was not inhibited by a similar concentration (0.1 microgram/ml) of RU 486, a classical progesterone antagonist. The increase in intracytoplasmic calcium levels was unable to induce the acrosome reaction (AR) even after incubation for 5 h, when this was evaluated by double staining, using a monoclonal antibody GB24 raised against the inner acrosome membrane and ethidium homodimer as a vital probe. However, after incubation for 5 h, progesterone was able to enhance the tyrosine phosphorylation of a 95 kD sperm protein, which is phosphorylated progressively during capacitation in well-defined culture media. Incubation of spermatozoa with 1 or 10 micrograms/ml progesterone for 3 or 30 min did not induce major modifications of hyperactivated movement when analysed by computer-assisted semen analysis. Progesterone secreted by cumulus cells may physiologically increase sperm intracytoplasmic free calcium during capacitation. This priming effect may facilitate the acrosome reaction, induced by binding to the zona pellucida, without enhancing spontaneous acrosome reaction prematurely. It therefore seems useful to propose progesterone as a means of accelerating capacitation during in vitro fertilization in cases of male infertility.

Follicle cell regulation of amino acid uptake and polyphosphoinositide metabolism in oocytes of the limpet Patella vulgata.

We investigated the role of follicle cells during meiosis reinitiation in the oocytes of Patella vulgata. Germinal vesicle breakdown, chromosome condensation, and metaphase-1 spindle formation were artificially induced by treating oocytes with 10 mM NH4Cl. By following the accumulation of 32PO4 and [3H]inositol in polyphosphoinositides (PPI) and by measuring chemical amounts of these lipids, we found that the presence of follicle cells caused PPI turnover to remain at a low level in the immature oocyte. On the other hand, the presence of these cells was necessary for maximal activation of the metabolism of these lipids during maturation. We also examined the nutritive role of follicle cells by studying the transport of leucine and found that the presence of these cells was necessary for optimal transport of this amino acid. We conclude that follicle cells can exert either a stimulatory or an inhibitory effect on the oocyte metabolism according to its state of maturity.

Effect of nordidemnin on the cell cycle of sea urchin embryos. Role in synthesis and phosphorylation of proteins and in polyphosphoinositide turnover in mitosis progression.

Nordidemnin (NorD) is a cyclic depsipeptide isolated from a Caribbean tunicate. This drug is thought to affect cell proliferation by acting on protein, RNA, and DNA syntheses. We studied the ability of NorD to arrest sea urchin embryos at the prophase stage. We tested whether NorD could alter the synthesis and phosphorylation of proteins as well as polyphosphoinositide (PPI) metabolism, as the activation of these processes is necessary for progression through the cell cycle. The dose-response effect on protein synthesis and cell cleavage suggests that NorD acts in the same way as emetine, a well-known protein synthesis inhibitor. We observed that treatment of eggs with emetine or NorD, even at concentrations that inhibited DNA and protein synthesis as well as phosphorylation of proteins, led to modifications in the incorporation of 32P into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate without any alteration in the chemical amounts of these lipids. However, fluctuations in the PPI messenger system that occur during the cell cycle were maintained in the presence of either drug. We suggest that proteins and PPI are linked in an intricate network to control mitosis and cellular proliferation.

Effect of arachidonic acid on Na+/H+ exchange and neutral amino acid transport in sea urchin eggs.

We have investigated the role of arachidonic acid (AA) in regulation of sea urchin egg metabolism activated after fertilization. We show in this paper that addition of exogenous AA to fertilized eggs triggered a transitory stimulation of three ionic events related to the Na+/H+ exchange, H+ excretion, and increase in Nai and in pHi. AA also induced a complete inhibition of neutral amino acid uptake. We found that alterations in this Na(+)-dependent amino acid uptake induced by AA came from modifications in the intracellular sodium concentration. We discuss how AA or derivates may play a role in regulating intracellular free calcium concentration and pH.

Cell-cycle calcium transients driven by cyclic changes in inositol trisphosphate levels.

Transient changes in intracellular calcium ([Ca2+]i) have been shown to punctuate the cell cycle in various types of cells in culture and in early embryos. The [Ca2+]i transients are correlated with cell-cycle events: pronuclear migration, nuclear envelope breakdown, the metaphase-anaphase transition of mitosis, and cytokinesis. Mitotic events can be induced by injecting calcium and prevented by injecting calcium chelators into the sea urchin embryo. Cell-cycle calcium transients differ from the transients linked to membrane signal transduction pathways: they are generated by an endogenous mechanism, not by plasma membrane receptor complexes, and their trigger is unknown. We report here that the phosphoinositide messenger system oscillates during the early embryonic cell cycle in the sea urchin, leading to cyclic increases in inositol trisphosphate that trigger cell-cycle [Ca2+]i transients and mitosis by calcium release from intracellular stores.

Effect of lithium on ionic balance and polyphosphoinositide metabolism during larval vegetalization of the sea urchin Paracentrotus lividus.

Treatment of Paracentrotus lividus embryos with lithium, which is known to block the polyphosphoinositide (PPI) cycle, induces their development into vegetalized larvae. We have investigated how ionic balance and PPI metabolism were affected in such vegetalized embryos. Exposure to 80 mM lithium for 3 1/2 hr at the morula stage induced an accumulation of lithium into the embryos, concomitant with a decrease in intracellular sodium and potassium contents. Intracellular lithium was totally expelled 6 hr after removal of this ion from the external medium. We found that intracellular levels of all PPI-cycle intermediates were altered after vegetalization: PIP2 and PIP increased, while PI decreased. Finally, vegetalized embryos showed a significant depression of IP3 mass. The degree of change in inositol phosphates increased as the intracellular accumulation of lithium increased. The change in morphology (vegetalization) also correlated well with the increase in lithium accumulation. This is consistent with the hypothesis that lithium alters the PPI cycle.

Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs.

A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of polyphosphoinositide metabolism at artificial maturation of Patella vulgata oocytes.

The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella vulgata. Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mM NH4Cl, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of 32PO4 and [3H]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol 4,5-bisphosphate (PIP2). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol 1,4,5-trisphosphate (IP3) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Patella oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP3, the messenger responsible for the Ca2+ signal at fertilization.

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 Ca(2+)-sensitive pathway. The timing and Ca2+ sensitivity suggest a role in the primary signal transduction events of fertilization.

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.

Cyclic-GMP-dependent refilling of calcium stores in macrophages.

The effects of cyclic GMP on the release of calcium from intracellular stores, induced in murine peritoneal macrophages by either ATP or platelet-activating factor, were studied by microfluorimetry with fura-2. When macrophages were incubated for 10-20 min with 10 microM LY83583, an inhibitor of guanylate cyclase, the rise in intracellular calcium induced by agonist application was strongly depressed. This inhibition of the response to platelet-activating factor could be reversed by the addition of 0.1 mM cyclic 8-bromo-GMP. In the presence of cyclic 8-bromo-GMP, the decay of the calcium transient was speeded. Furthermore, when two calcium transients were evoked within 1 min by stimulating the cells with 10 microM ATP, the second calcium transient was more depressed than the first one in the presence of LY83583. These findings are compatible with the hypothesis that cyclic GMP is necessary for the activation of the calcium pump of the intracellular stores.

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TTPA) 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).