Unfertilized Sea Urchin Eggs Research Articles

The Effect of Synthetic Polyamine BPA-C8 on the Fertilization Process of Intact and Denuded Sea Urchin Eggs.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

Possible role of translation in unfertilized sea urchin eggs.

Unfertilized eggs of animals contain maternal messenger RNAs (mRNAs) and proteins, which are required for the maintenance of metabolism and regulation of development during the initial stages of embryogenesis. Unfertilized eggs are transcriptionally and translationally quiescent. After fertilization, activated translation of maternal mRNAs is one of the major forces that direct the early stages of embryogenesis before activation of the zygotic genome. However, a low rate and level of protein synthesis have been detected in unfertilized sea urchin eggs indicating that translation is not completely inhibited. Analysis of translatomes of unfertilized eggs and early embryos detected three sets of maternal mRNAs translated either before or after fertilization, or both before and after fertilization. Proteins encoded by maternal mRNAs, which are translated in unfertilized eggs, perform many different functions required for homeostasis, fertilization, egg activation, and early development. This suggests that translation in unfertilized sea urchin eggs may be required to renew the pool of proteins involved in these processes. Thus, translation may be necessary to maintain the fertility and developmental potential of sea urchin eggs during the long-term storage of eggs in ovaries until spawning begins.

Regulation of the Actin Cytoskeleton-Linked Ca2+ Signaling by Intracellular pH in Fertilized Eggs of Sea Urchin.

In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg’s receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process.

EML proteins in microtubule regulation and human disease.

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy.

Different routes lead to apoptosis in unfertilized sea urchin eggs

Results obtained in various species, from mammals to invertebrates, show that arrest in the cell cycle of mature oocytes is due to a high ERK activity. Apoptosis is stimulated in these oocytes if fertilization does not occur. Our previous data suggest that apoptosis of unfertilized sea urchin eggs is the consequence of an aberrant short attempt of development that occurs if ERK is inactivated. They contradict those obtained in starfish, another echinoderm, where inactivation of ERK delays apoptosis of aging mature oocytes that are nevertheless arrested at G1 of the cell cycle as in the sea urchin. This suggests that the cell death pathway that can be activated in unfertilized eggs is not the same in sea urchin and in starfish. In the present study, we find that protein synthesis is necessary for the survival of unfertilized sea urchin eggs, contrary to starfish. We also compare the effects induced by Emetine, an inhibitor of protein synthesis, with those triggered by Staurosporine, a non specific inhibitor of protein kinase that is widely used to induce apoptosis in many types of cells. Our results indicate that the unfertilized sea urchin egg contain different mechanisms capable of leading to apoptosis and that rely or not on changes in ERK activity, acidity of intracellular organelles or intracellular Ca and pH. We discuss the validity of some methods to investigate cell death such as measurements of caspase activation with the fluorescent caspase indicator FITC-VAD-fmk or acidification of intracellular organelles, methods that may lead to erroneous conclusions at least in the sea urchin model.

Autophagy is used as a survival program in unfertilized sea urchin eggs that are destined to die by apoptosis after inactivation of MAPK1/3 (ERK2/1)

A high MAPK1/3 (also known as ERK2/1, respectively) activity, preventing spontaneous activation, is essential to maintain cell cycle arrest of mature oocytes of mammals, frogs or invertebrates such as starfish. Mature oocytes would undergo a “suicide”-like cell death if not fertilized. We previously have reported that downregulation of MAPK1/3 in unfertilized sea urchin eggs induces a calcium-dependent entry into mitosis. We show here that this event is followed by a series of pseudo-mitotic cell cycles associated with transient Cai increases, preceding CASP3/caspase-3 activation and apoptosis. However, cell death was delayed after inhibition of the Cai transients or of cyclin-dependent kinases (CDK), with roscovitine. In these conditions, eggs enter an autophagy program as suggested by detection of processed LC3B by western blot, immunofluorescence and immunogold staining, visualization of autophagy vesicles by electron microscopy, and an increase in acidic vesicular organelles (AVOs). We found that bafilomycin A1 or an association of leupeptin and pepstatin, which are widely used to study autophagy, may act upon calcium signaling or cell cycle events, respectively, and not only on autophagy events. Finally, inhibition of PtdIns 3-kinase with wortmannin or LY294002 powerfully stimulated cell death of unfertilized eggs, which suggests that this activity does not negatively regulate autophagy as is often reported, but rather stimulates survival in unfertilized eggs. We suggest that apoptosis of unfertilized eggs is the consequence of an aberrant short attempt of development that occurs if MAPK1/3 is inactivated, but these eggs can use autophagy as a survival program when the cell cycle is blocked.

Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs

Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization.

The expression and distribution of Wnt and Wnt receptor mRNAs during early sea urchin development

The protein β-catenin plays a critically important role in establishing axial polarity during early animal development. In many organisms, β-catenin is degraded preferentially on one side of the cleavage stage embryo. On the opposite side of the embryo, β-catenin is stabilized and accumulates in the nucleus, where it functions in concert with members of the LEF/TCF family to activate the transcription of diverse target genes. Genes that are activated by β-catenin play an essential role in the specification of endomesoderm and in the establishment of key signaling centers in the early embryo. In several organisms, the asymmetric distribution of maternal components of the canonical Wnt pathway has been shown to be responsible for the polarized stabilization of β-catenin. In this study, we identified all Wnt and Wnt receptor mRNAs that are present in unfertilized sea urchin eggs and early embryos and analyzed their distributions along the primary (AV) axis. Our findings indicate that the asymmetric distribution of a maternal Wnt or Wnt receptor mRNA is unlikely to be a primary determinant of the polarized stabilization of β-catenin along the AV axis. This contrasts sharply with findings in other organisms and points to remarkable evolutionary flexibility in the molecular mechanisms that underlie this otherwise very highly conserved patterning process.

Calyculin-A Induces Cleavage in a Random Plane in Unfertilized Sea Urchin Eggs

Calyculin-A (CLA), a protein phosphatase inhibitor, has been known to induce cleavage resembling normal furrowing in unfertilized sea urchin eggs. In CLA-treated eggs, actin filaments and myosin assemble to form a contractile ring-like structure in the egg cortex; however, this occurs in the absence of a mitotic spindle or asters. Here, we investigated the relationship between the plane of CLA-induced cleavage and the intrinsic animal-vegetal polar axis in sea urchin eggs. The animal-vegetal axis was established using black ink to visualize the jelly canal located at the animal pole in the jelly coat surrounding the egg. We measured the acute angle between the jelly canal axis and the cleavage plane for both fertilized eggs and CLA-treated unfertilized eggs. Although the acute angle lay within 10 degrees for most of the fertilized eggs, it varied widely for CLA-treated unfertilized eggs. Measurements of the diameter of blastomeres revealed that cleavage of fertilized eggs took place in the mid-plane of the egg, but that CLA-induced divisions were unequal. These results suggest that neither the orientation nor the location of the CLA-induced cleavage furrow is related to the animal-vegetal polar axis of the egg, even though the furrowing mechanism itself is not dissimilar to that in fertilized eggs.

Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca2+-dependent pathway but without completion of S phase

Unfertilized sea urchin eggs that are arrested at G1 phase after completion of meiosis contain a highly phosphorylated mitogen-activated protein (MAP) kinase (MAPK), the ERK-like protein (ERK-LP). Several data including our previous results show that ERK-LP is inactivated after fertilization, which agrees with results obtained in other species including Xenopus, starfish and mammals. The question is to elucidate the function of a high MAPK activity in sea urchin eggs. We report here that dephosphorylation of ERK-LP with very low concentrations of two MEK inhibitors, PD98059 or U0126, triggers entry into mitosis. Under these conditions, recurrent oscillations of the phosphorylation of ERK-LP and of a tyrosine residue in Cdc2 occur, and the intracellular Ca2+ level (Ca2+ i) progressively and slowly increases. Nuclear envelope breakdown and all mitotic events initiated after dephosphorylation of ERK-LP are inhibited when changes in Ca2+ i are prevented; however, they are independent of the intracellular pH. These results suggest that inactivation of a MEK-ERK pathway, normally induced after fertilization of sea urchin eggs, triggers entry into mitosis by altering Ca2+ i but cannot trigger full DNA replication. We discuss the hypothesis that neither inactivation nor activation of a MEK-ERK pathway is required for S phase completion in sea urchin egg.

Biology of Reproduction Highlights …

Sea urchin and human sperm contain receptors for neurotransmitters and psychoactive drugs, including cannabinoid receptors (CNRs). Anandamide, arachidonoylethanolamide (AEA), is a lipid-signal molecule that is an endogenous agonist for CNRs. AEA is enyzmatically released from membrane phospholipids when neurons are stimulated. Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses in mammalian brain. Analogous processes regulate sperm functions during fertilization in sea urchins. AEA and (-)delta9tetrahydrocannabinol [(-)delta9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (-)delta9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.

A MAPK pathway is involved in the control of mitosis after fertilization of the sea urchin egg

Activation and role of mitogen-activated protein (MAP) kinase (MAPK) during mitosis are still matters of controversy in early embryos. We report here that an ERK-like protein is present and highly phosphorylated in unfertilized sea urchin eggs. This MAPK becomes dephosphorylated after fertilization and a small pool of it is transiently reactivated during mitosis. The phosphorylated ERK-like protein is localized to the nuclear region and then to the mitotic poles and the mitotic spindle. Treatment of eggs after fertilization with two different MEK inhibitors, PD 98059 and U0126, at low concentrations capable to selectively induce dephosphorylation of this ERK-like protein, or expression of a dominant-negative MEK1/2, perturbed mitotic progression. Our results suggest that an ERK-like cascade is part of a control mechanism that regulates mitotic spindle formation and the attachment of chromosomes to the spindle during the first mitosis of the sea urchin embryo.

Integrins on eggs: the βC subunit is essential for formation of the cortical actin cytoskeleton in sea urchin eggs

Eggs of several metazoans have been demonstrated to express integrins; however, their function is unclear. Previous studies have shown that the βC integrin subunit is expressed on unfertilized sea urchin eggs and proteolytically removed at fertilization. Here we report that the βC subunit is reexpressed on the egg surface immediately after fertilization. Using morpholino antisense oligonucleotides to block translation, we show that without βC expression, eggs undergo cleavage resulting in loosely adherent cells that fail to develop beyond a blastula. Without βC containing integrins, the cortical actin network of the egg does not form, yet contractile rings appear. Coinjection of RNA encoding the βC or chicken β1 subunit, but lacking the morpholino target sequence, rescues the cortical actin network and normal embryos result. Coinjection of RNA encoding the βC subunit lacking the cytoplasmic domain fails to rescue. These studies demonstrate that the cortical actin cytoskeleton is anchored by βC integrins and contractile ring actin is not. We suggest that one important function of egg integrins is to organize the actin cortex.

Calyculin A causes the activation of histone H 1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation-promoting factor

Calyculin A is known to inhibit the type-1 and type-2A phosphatases. We previously reported that calyculin A induces contractile ring formation in unfertilized sea urchin eggs, an increase in histone H 1 kinase activity, and chromosome condensation in the calyculin A-treated unfertilized eggs, and the changes induced by calyculin A are not affected by emetine, an inhibitor of protein synthesis. These observations suggest that the mechanism by which histone H 1 kinases are activated by calyculin A is different from that of maturation-promoting factor (MPF), which is activated by a molecular modification of existed cdc2 and newly synthesized cyclin B. We report here that no cyclin B was detected by immunoblotting of unfertilized calyculin A-treated eggs. In addition, no DNA synthesis was induced by calyculin A. As well, butyrolactone I (an inhibitor of cdc2 and cdk2 kinase) had no effect on the increase in histone H 1 kinase activity nor the chromosome condensation, both of which were induced by calyculin A. Thus, we conclude that calyculin A induces histone H 1 phosphorylation in an MPF-independent manner through inhibition of type-1 phosphatase, and that the chromosome condenses as a result of histone H 1 phosphorylation.

Molecular cloning and characterization of ryanodine receptor from unfertilized sea urchin eggs.

Unfertilized eggs of sea urchins (Hemicentrotus pulcherrimus) demonstrated cyclic ADP-ribose (cADPR)-induced Ca(2+) release and caffeine-induced Ca(2+) release, both of which were considered to be mediated through the ryanodine receptor (RyR). We cloned cDNAs for sea urchin egg RyR (suRyR), which encode a 597-kDa protein of 5,317 amino acids. suRyR shares common structural features with known RyRs: the well-conserved COOH-terminal domain, which forms a functional Ca(2+) channel, and a large hydrophilic NH2-terminal domain. suRyR shows amino acid sequence identity (43-45%) similar to the three mammalian RyR isoforms. Phylogenetic analysis indicates that suRyR branched from three isoforms of vertebrates before they diverged, suggesting that suRyR may be the only RyR isoform in the sea urchin. Four in-frame insertions were found in suRyR cDNAs, one of which was novel and unique, in that it had a cluster of serine residues. The transcripts with and without these insertions were found in the egg RNA. These results suggest that suRyR may be expressed as a functional Ca(2+)-induced Ca(2+) release channel, which might also be involved in cADPR-induced Ca(2+) release.

Calyculin‐A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggs

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex “actin cup” since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a 32P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. Cell Motil. Cytoskeleton 48:245–261, 2001. © 2001 Wiley-Liss, Inc.

EIF4E Association with 4E-BP Decreases Rapidly Following Fertilization in Sea Urchin

The eukaryotic translation initiation factor (eIF) 4F facilitates the recruitment of ribosomes to the mRNA 5′ end. The 4E-BPs are small proteins with hypophosphorylated forms that interact with the cap binding protein eIF4E, preventing its interaction with eIF4G, thereby preventing ribosome interaction with mRNA. In sea urchin, fertilization triggers a rapid rise in protein synthesis. Here, we demonstrate that a 4E-BP homologue exists and is associated with eIF4E in unfertilized eggs. We also show that 4E-BP/eIF4E association diminishes a few minutes following fertilization. This decrease is correlated with a decrease in the total amount of 4E-BP in combination with an increase in the phosphorylation of the protein. We propose that 4E-BP acts as a repressor of protein synthesis in unfertilized sea urchin eggs and that 4E-BP/eIF4E dissociation plays an important role in the rise in protein synthesis that occurs shortly following fertilization.

Calyculin-A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggs.

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a (32)P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis.

Cortical Granule Exocytosis and Fertilization Coat Elevation is Reduced in Aging Sea Urchin Eggs

Abstract Cortical granules are specialized Golgi-derived membrane-bound secretory granules that are located beneath the plasma membrane in unfertilized sea urchin eggs. Upon fertilization cortical granules discharge in a reaction induced by calcium and release their contents between the plasma membrane and a thin vitelline layer that lines the plasma membrane. Microvilli at the plasma membrane elongate incorporting cortical granule membranes during elongation. The vitelline layer elevates and becomes the egg's fertilization coat that hardens and serves as physical block to polyspermy. While we do not understand the precise mechanisms that participate in cortical granule discharge it is believed that actin plays a role in this process. Because actin and calcium metabolism is affected in aging cells we investigated if cortical granule secretion is affected in aging sea urchin eggs. Lytechinus pictus eggs were obtained by intracoelomic injection of 0.5M KCI to release the eggs into sea water at 23°C.

The Relationship between Calcium, MAP Kinase, and DNA Synthesis in the Sea Urchin Egg at Fertilization

Fertilization releases the brake on the cell cycle and the egg completes meiosis and enters into S phase of the mitotic cell cycle. The MAP kinase pathway has been implicated in this process, but the precise role of MAP kinase in meiosis and the first mitotic cell cycle remains unknown and may differ according to species. Unlike the eggs of most animals, sea urchin eggs have completed meiosis prior to fertilization and are arrested at the pronuclear stage. Using both phosphorylation-state-specific antibodies and a MAP kinase activity assay, we observe that MAP kinase is phosphorylated and active in unfertilized sea urchin eggs and then dephosphorylated and inactivated by 15 min postinsemination. Further, Ca2+ was both sufficient and necessary for this MAP kinase inactivation. Treatment of eggs with the Ca2+ ionophore A23187 caused MAP kinase inactivation and triggered DNA synthesis. When the rise in intracellular Ca2+ was inhibited by injection of a chelator, BAPTA or EGTA, the activity of MAP kinase remained high. Finally, inhibition of the MAP kinase signaling pathway by the specific MEK inhibitor PD98059 triggered DNA synthesis in unfertilized eggs. Thus, whenever MAP kinase activity is retained, DNA synthesis is inhibited while inactivation of MAP kinase correlates with initiation of DNA synthesis.