Cortical localization of a calcium release channel in sea urchin eggs

We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs. Immunolocalization of the calcium release channel reveals a cortical reticulum or honeycomb staining network that surrounds cortical granules and is associated with the plasma membrane. The network consists of some immunoreactive electron-dense material coating small vesicles and elongate cisternae of the endoplasmic reticulum. The fluorescent reticular staining pattern is lost when egg cortices are treated with agents known to affect sarcoplasmic reticulum calcium release and induce cortical granule exocytosis (ryanodine, calcium, A-23187, and caffeine). An approximately 380-kD protein of sea urchin egg cortices is identified by immunoblot analysis with the ryanodine receptor antibody. These results demonstrate: (a) the presence of a ryanodine-sensitive calcium release channel that is located within the sea urchin egg cortex; (b) an altered calcium release channel staining pattern as a result of treatments that initiate the cortical granule reaction; and (c) a spatial and functional dichotomy of the ER which may be important in serving different roles in the mobilization of calcium at fertilization.     

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.     

The ultrastructure of the sea urchin egg cortex isolated before and after fertilization

The three-dimensional organization of cortices isolated from unfertilized and fertilized Strongylocentrotus purpuratus eggs has been examined by several techniques of light and electron microscopy. It has been found that when moderate shear forces are used, the isolated unfertilized egg cortex, in addition to cortical granules, contains acidic vesicles and an elaborate network of rough endoplasmic reticulum. This network provides a physical link between the cell surface and several kinds of cytoplasmic organelles (mitochondria, yolk granules, acidic vesicles) which are retained as part of the isolated cortex when gentle shear forces are applied. Furthermore a good visualization of actin in the cortex is provided: it is present as short filaments and mostly within the stubby microvilli of the egg. Finally, it has been noted that plaques exist on the inside face of the plasma membrane ready to assemble into typical clathrin coats that prefigure the burst of coated vesicle endocytosis that takes place after fertilization. The cortex isolated soon after fertilization is shown to contain coated pits and a scaffolding of filaments (mostly actin) in which many acidic vesicles are embedded.     

Characterization of sea urchin egg endoplasmic reticulum in cortical preparations

The cortical endoplasmic reticulum (ER) of sea urchin eggs was localized on isolated egg cortices by staining with aqueous suspensions of the dicarbocyanine "DiI." Immunofluorescence localization of a calsequestrin-like protein was essentially identical; this is consistent with a role for the ER in calcium regulation. The ER often encircles cortical granules, making it well-suited for initiating fusion and propagating the calcium wave. Thiazole orange and Hoechst dye 33258 at pH 2 stain ribosomes bound to the ER, providing evidence that the cortical ER is rough ER. High chloride concentrations were found to disrupt ER continuity.     

Subcellular localization of sea urchin egg spectrin: evidence for assembly of the membrane-skeleton on unique classes of vesicles in eggs and embryos

A recent study from our laboratory on the sea urchin egg suggested that spectrin was not solely restricted to the plasma membrane, but instead had a more widespread distribution on the surface of a variety of membranous inclusions. (E. M. Bonder et al., 1989, Dev. Biol. 134, 327-341). In this report we extend our initial findings and provide experimental and ultrastructural evidence for the presence of spectrin on three distinct classes of cytoplasmic vesicles. Immunoblot analysis of membrane fractions prepared from egg homogenates establishes that spectrin coisolates with vesicle-enriched fractions, while indirect immunofluorescence microscopy on cryosections of centrifugally stratified eggs demonstrates that spectrin specifically associates with cortical granules, acidic vesicles, and yolk platelets in vivo. Immunogold ultrastructural localization of spectrin on cortices isolated from eggs and early embryos details the striking distribution of spectrin on the cytoplasmic surface of the plasma membrane and the membranes of cortical granules, acidic vesicles, and yolk platelets, while quantitative studies show that relatively equivalent amounts of spectrin are present on the different membrane surfaces both before and after fertilization. These data, in combination with the localization of numerous spectrin crosslinks between actin filaments in surface microvilli, suggest that spectrin plays a pivotal role in structuring the cortical membrane-cytoskeletal complex of the egg and the embryo.     

The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.     

Synaptotagmin I is involved in the regulation of cortical granule exocytosis in the sea urchin

Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

Modulation of calcium sensitivity by a specific cortical protein during sea urchin egg cortical vesicle exocytosis

A study of the Ca2+ sensitivity of cortical vesicle (CV) discharge has been accomplished using isolated sea urchin egg cortices. Cortices isolated in a medium ionically similar to normal egg cytoplasm discharge 50% of their CVs at 1.6 microM Ca2+ (=[Ca2+]50). Alternatively, cortices isolated in a medium containing 500 mM chaotropic anions (Cl-, Br-, I-, or NO-3) discharge their CVs at 16 microM [Ca2+]50. Incubation with the 500 mM KCl extract of cortices restores high Ca2+ sensitivity and the mode of CV discharge characteristic of cortices before extraction. Fractionation of egg homogenates by differential centrifugation reveals that about 20% of the total restoring activity is associated with the cortex. In eggs of Hemicentrotus pulcherrimus, the factor responsible for this restorative function is a heat and protease labile protein with a molecular weight of 100,000. Similar activity is seen also in the eggs and sperm of other species of sea urchin.     

A COMPARATIVE STUDY OF THE ISOLATION OF THE CORTEX AND THE ROLE OF THE CALCIUM-INSOLUBLE PROTEIN IN SEVERAL SPECIES OF SEA URCHIN EGG

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.     

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.     

Ryanodine Activates Sea Urchin Eggs

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP₃, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner ⁴⁵Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP₃ stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg⁺⁺, ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.     

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.     

Differentiation of a calsequestrin-containing endoplasmic reticulum during sea urchin oogenesis

We have used light and electron microscopic immunolocalization to study the distribution of a sea urchin calsequestrin-like protein (SCS) during sea urchin oogenesis. SCS was localized exclusively in the lumen of the endoplasmic reticulum (ER) and in the nuclear envelope of oocytes of all maturation stages. Immunoelectron microscopy also revealed that SCS is not present in golgi complexes of oocytes. Double label immunofluorescent staining of frozen sections of ovary with the SCS antiserum and an antibody to the cortical granule protein hyalin indicated a dramatic morphogenesis of the SCS-containing ER (SCS-ER) coincident with oocyte maturation. This differentiation included an apparent increase in the amount and complexity of the cytoplasmic SCS-ER network, the transient appearance of stacks of SCS-ER cisternae in synthetically active vitellogenic oocytes, and the restructuring of the SCS-ER in the cortex. Immunofluorescence of isolated oocyte cortices showed a plasma membrane-associated SCS-ER which was much less dense and regular than that found surrounding the cortical granules in the mature unfertilized egg cortex. Cytoplasmic and cortical microtubule arrays are present in oocytes and may provide the basis for the SCS-ER distributional dynamics. The results of this study underscore the dynamic nature of ER and how it's organization reflects cellular functions. We suggest that the establishment during oogenesis of the dense SCS-ER tubuloreticulum provides the egg with the calcium sequestration and release apparatus that regulates calcium fluxes during egg activation and early development.     

Movements of Echinochrome Granules during the Early Development of Sea Urchin Eggs

ECHINOCHROME pigment granules in unfertilized eggs of the sea urchin Arbacia punctulata undergo randomly-directed saltatory movements^1,2. After fertilization, nearly all these granules migrate to the egg cortex and become embedded. Subsequent pigment granule movements may represent mass cortical changes rather than independent granule movements^2,3. At the fourth cleavage, a quartet of micromeres containing little or no pigment forms at the vegetal pole. By the two or four-cell stage, pigment granules have begun to move out of this region, leaving a “clear area” on each blastomere (Fig. 1 and refs. 4, 5). To investigate possible mechanisms for these movements and their relation to cortical events     

Ultrastructural localization of intracellular calcium stores by a new cytochemical method

We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods.     

Evidence that the Mg-ATPase in the cortical layer of sea urchin egg is dynein

Indirect immunofluorescence staining of cleaving sea urchin eggs with an antiserum against a tryptic fragment of dynein 1 (fragment 1A) from sea urchin sperm flagella suggested the presence of dynein in the cortex as well as in the mitotic apparatus. In the present study, we found that the Mg²⁺-ATPase activity of the isolated cortices from sea urchin eggs, which exhibited similar characteristics to those of flagellar dynein, was inhibited by 60–80% with the anti-fragment 1A serum. Faintly stained bands corresponding to the A-band (dynein 1) and the B-band of the sperm flagella was detected on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis of the isolated cortices. Furthermore, the SDS-gel electrophoresis revealed the presence of a polypeptide band corresponding to dynein 1 in the antigen-antibody complex precipitated from the KCl-extract of the cortices with the antiserum.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

Evidence for an acidic compartment in sea urchin eggs (Paracentrotus lividus): role at fertilization

The characteristics of [14C]methylamine accumulation by isolated cortices were measured in eggs from three species of sea urchins: Paracentrotus lividus, Arbacia lixula and Sphaerechinus granularis. In all cases, the results pointed to the existence of an acidic compartment in the cortical zone. In P. lividus eggs, cortical granules did not participate in proton storage which likely took place in pigment granules. [14C]Methylamine accumulation was dramatically reduced by monovalent cation ionophores (monensin and nigericin) and by NH4Cl, but not by valinomycin. ATP depletion only partially affected the isotope uptake. Simultaneous measurements of intracellular pH and of external titratable acidity during ammonia treatment of eggs, indicate that after fertilization, eggs increased their capacity to concentrate hydrogen ions in an intracellular store. Following insemination, cortices from P. lividus eggs exhibited a 3-fold increase in [14C]methylamine accumulation. It is concluded that the egg cortical area contains acidic organelles sequestering hydrogen ions by means of an electrogenic H+ pump, and that this mechanism, enhanced at fertilization, participates in a local alkalinization. The role of such a mechanism is discussed.