Proc. Nat. Acad. Sci. USAVol. 72, No. 11, pp. 4474-4478, November 1975Cell Biology

Relationship between release of surface proteins and metabolicactivation of sea urchin eggs at fertilization

(cell surface/cell transformation/protein synthesis/glycoproteins)

JAMES D. JOHNSON AND DAVID EPELMarine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, Calif. 92093

Communicated by S. J. Singer, September 4, 1975

ABSTRACT Macromolecular components are releasedfrom sea urchin eggs when their metabolism is activated atfertilization or by incubation in ammonia. When the re-leased material is dialyzed, concentrated, and added back topartially activated eggs the rate of protein synthesis is sup-pressed to the level oFthe unactivated egg. The surface pro-teins of the unfertilized eggs can be labeled with 125I by alactoperoxidase procedure. When fertilized or activated withvarious parthenogenetic agents, 15-25% of the total labeledprotein is released; most of the label is associated with a150,000-dalton glycoprotein. The extent of metabolic activa-tion, as assessed by measuring increased protein synthesis, iscorrelated with the amount of surface label released. Severalother proteins are released during activation but are not la-beled by the lactoperoxidase procedure in the intact cell. Wehave not yet identified which of these components is respon-sible for suppressing protein synthesis, nor do we know ifany of the other metabolic changes of fertilization such asK+ conductance and DNA synthesis are also suppressed. Wesuggest that these released components are surface moleculesinvolved in maintaining the low metabolic state occurring atthe end of oogenesis and that removal of these componentsduring fertilization results in the release of the suppressionof the egg.

Fertilization of sea urchin eggs results in a cascade of eventsoccurring in a defined temporal sequence. As part of theensuing metabolic transformation, there occurs a massivereorganization of the plasma membrane resulting from thefusion of approximately 15,000 secretory granules (corticalgranules) with the egg plasma membrane (1). This exocytosisoccurs about 30 sec after fertilization, but is not a prerequi-site for the array of metabolic events that follow, since incu-bating eggs in 1-10 mM NH4Cl initiates a number of themetabolic changes such as K+ conductance of the plasmamembrane and protein and DNA synthesis (2-4), but doesnot initiate the cortical granule exocytosis (2). Also, develop-ment of eggs is activated when fertilized in the presence ofinhibitors of the cortical exocytosis (5, 6).

Using a lactoperoxidase-125I technique for labeling theegg surface, we had previously observed that fertilizationand several different types of parthenogenetic activation re-sulted in the release of a labeled, large molecular weight sur-face protein (7). We here show that release of labeled pro-tein and also nonlabeled proteins occurs when the eggs areactivated with a resultant cortical granule exocytosis or aretreated with agents such as ammonia, nicotine, or procaine,which only activate cytoplasmic events such as protein andDNA synthesis. This finding suggested a relationship be-tween the release of protein and the subsequent metabolicactivation of the egg. This correlation is directly supportedby experiments showing that adding released surface com-ponents to activated eggs suppresses the protein synthetic ca-pacity of the cell back to the level of the unfertilized egg.

Loss of surface protein accompanying a metabolic activa-tion has also been reported for vertebrate somatic cells upon

viral, proteolytic (8), or chemical (9) transformation andsuggests that metabolic repression by loosely held surface(peripheral) proteins is a common feature of cells. Wesuggest that in oocytes the metabolic stasis occurring at theend of oogenesis results from addition of proteins or othermaterials to the oocyte surface and that the removal of thesesurface components at fertilization results in the metabolicderepression of the oocytes.

MATERIALS AND METHODSHandling of Gametes. Ovulation of Strongylocentrotus

purpuratus eggs was induced by intracoelomic injection 0.5M KCl. Egg jelly was removed by exposure to pH 5.0 seawa-ter with swirling for 2 min. The pH was then titrated backto 8.0 with 1.0 M Tris-HCl (pH 8.0) in seawater, and the de-jellied eggs were then washed several times in filtered sea-water. Temperature in all experiments was 16°.

Activation of Eggs with Ionophore. Ionophore A23187was obtained from R. L. Hamill, Eli Lilly Co., Indianapolis,Ind. Eggs were activated as described by Steinhardt andEpel (10).

Labeling of Surface Proteins with 125I. The procedure ofHuber and Morrison (11) was followed, modified as de-scribed below. To 1 ml of a seawater-lactoperoxidase (0.3mg/ml, Calbiochem) solution in a 10-ml beaker was added10 gl of NaI25I (2 mCi/ml in 0.01 M NaOH, New EnglandNuclear). Sodium sulfite (10 ,uM) was added to reduce any 12to I-. After thorough mixing, 1 ml of a 10% suspension ofdejellied eggs was added. Ten microliters of a 0.06% solutionof H202 in filtered seawater was added at zero time, andfurther 10-il additions were made at 2-min intervals until 8min had elapsed. The eggs were kept in suspension by swirl-ing during the entire procedure. After a total time of 10min, the eggs were poured into 40 ml of filtered seawater ina conical centrifuge tube and washed four times by gentlehand centrifugation and resuspension in filtered seawater.Eggs iodinated by this procedure exhibited normal develop-ment upon fertilization. Approximately 5% of the total 125Iadded was bound. The radioactivity of the 125I-labeled ma-terial was determined by placing a portion of the protein so-lution or egg suspension in 10 ml of Aquasol (New EnglandNuclear) and counting in a Beckman LS-230 Scintillationcounter.Removal of the Vitelline Layer. The vitelline layer was

removed by the dithiothreitol method of Epel et al. (12) orby a new procedure developed by Carroll et al.* In the lat-ter technique a partially purified protease fraction from thecortical granules of S. purpuratus is used to detach the vitel-line layer from the plasma membrane; a subsequent incuba-

* E. J. Carroll, J. D. Johnson, E. W. Byrd, and D. Epel, in prepara-tion.

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tion of the eggs in the 10 mM dithiothreitol in seawater atpH 9.1 completely removes the vitelline layer. This proce-dure is more reliable than the original dithiothreitol method(12), which variably removed 20-40% of the 125I from eggslabeled as above. It is more specific than proteolytic proce-dures utilizing trypsin or Pronase, which alter many surfaceproteins; the cortical granule proteases carry out a very lim-ited proteolysis of the vitelline layer*.Measurement of Protein Synthesis. The extent of meta-

bolic activation of a 0.2% suspension of eggs or embryos wasdetermined by measuring the rate of incorporation of L-[3H]valine into trichloroacetic acid-insoluble protein after a5-min pulse, as described by Epel et al. (3). Radioactivity inboth the trichloroacetic acid-soluble and insoluble fractionswas measured (3), and total dpm in each fraction was calcu-lated by standard procedures. Incorporation into protein isexpressed as percent incorporation, using the ratio (dpm inacid-insoluble)/(dpm in acid-soluble and acid-insoluble).Radioactivity in the trichloroacetic acid-soluble fraction wasdetermined as above; radioactivity of the protein pellet wasmeasured by liquid scintillation counting after solution inNCS and counting in a toluene-based cocktail. Counting ef-ficiency was determined by addition of internal standards.

Preparation of Released Proteins for Bioassay or GelElectrophoresis. A 10% suspension of eggs was incubatedfor 15 min in 10 mM NH4Cl, pH 8.0, the eggs were re-moved by hand centrifugation, and the supernatant fluidwas then centrifuged at 48,000 X g for 30 min. For bioas-says, the resulting supernatant was dialyzed against severalchanges of filtered seawater at 4°. For electrophoresis, sam-ples were dialyzed against distilled water, frozen, and lyo-philized. The resulting residue was weighed and solubilizedin sodium dodecyl sulfate buffer (14) to a concentration ofapproximately 1-2 mg/ml.

Polyacrylamide Gel Electrophoresis with Sodium Do-decyl Sulfate. Electrophoresis on polyacrylamide slab gelswas as described by Studier (13) with 7.5% acrylamide in therunning gel and 4% acrylamide in the stacking gel. All gelswere run at a constant 45 V. Gels were stained for proteinwith Coomasie blue according to Laemmli (14) and for car-bohydrate with the periodic-acid Schiff stain as described bySegrest and Jackson (15). For molecular weight determina-tion and for quantitating the radioactivity of 125I-labeledprotein, 7.5% sodium dodecyl sulfate disc gels were run bythe procedure of Laemmli (14). Standards for molecularweight included Escherichia coli f3-galactosidase (130,000),rabbit muscle glycogen phosphorylase (94,000), bovineserum albumin (68,000), rabbit muscle aldolase (40,000),and bovine pancreas carboxypeptidase A (34,000). For de-termining radioactivity, gels were sliced into 0.20-cm slices,placed in scintillation vials with 0.5 ml of NCS tissue solubil-izer, and heated for 4 hr at 55°. After 12 hr at room temper-ature, 2,5-diphenyloxazole (PPO)-1,4-bis[2(5-phenyloxaz-dyl)]benzene(POPOP)-toluene scintillation fluid was addedand radioactivity determined in a LS-230 Beckman liquidscintillation system.

RESULTS

The iodination procedure as described had no adverse ef-fects on development through the pluteus stage. Incubationof labeled eggs in 1 mg/ml of Pronase for 1 hr removed88-92% of the label, indicating the bulk of iodination was atthe cell surface and available to these enzymes. Similar re-sults were earlier obtained with eggs from another species

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FIG. 1. Time course of release of 1251-labeled protein from eggsurface after treatment with ionophore A23187 or ammonia. lodin-ated eggs were treated with either 5 gM A23187, or 10 mM NH4Cl,pH 8.0. Two-milliliter samples, taken at the indicated times, werehand-centrifuged; the supernatant fluid was decanted and recen-trifuged at 48,000 X g for 30 min and then dialyzed against severalchanges of distilled water for 24 hr. Radioactivity of aliquots of thedialyzed supernatnat was determined in Aquasol as described inMaterials and Methods. Ionophore-treated eggs 0; ammonia-treated eggs X; controls 0.

(Arbacia punctulata), and here surface labeling was furtherconfirmed by autoradiography (16).When eggs were fertilized with sperm or activated with

ionophore A23187, approximately 25% of the 125I label wasreleased into the seawater in a nondialyzable form (Fig. 1).These released counts were covalently bound to protein, asindicated by precipitation in 10% trichloroacetic acid andmigration as a single Coomassie blue staining band on sodi-um dodecylsulfate-polyacrylamide gel electrophoresis.

This release could result from the membrane vesiculationaccompanying exocytosis of the cortical granules or be relat-ed to some aspect of the metabolic activation accompanyingfertilization. These two alternatives were distinguished byincubating eggs in 10 mM NH4Cl, pH 8.0, which activatesmetabolism but does not initiate cortical exocytosis. Asshown in Fig. 1, incubation in ammonia also releases 125Ilabel at a rate slower than that accompanying ionophore ac-tivation or fertilization. This suggests that the release is relat-ed to activation. Metabolic activation in ammonia is slowerthan that induced by ionophore or normal fertilization (3).

Correlation of protein release and metabolic activationIf the release of protein is related to metabolic activation,then there should be a correlation between the activation ofmetabolic processes and the release of protein by agentsother than ammonia. Carroll (personal communication)found that 1 mM nicotine would activate chromosome con-densation, but not the cortical reactions. Vacquier and Bran-diff (6) have reported similar observations for procaine. Wecompared release of protein and metabolic activation bythese agents, assessing activation by measuring percent in-corporation of amino acid into protein. As shown (Fig. 2),there is strong correlation between increased protein synthe-sis and release of iodinated surface protein. This correlationalso applies to incubation in ammonia at pH 6.0, which nei-ther activates protein synthesis nor results in release of sur-face label.

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D PROTEIN SYNTHESIS

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FIG. 2. Correlation between protein synthesis and release of1251-labeled surface protein. Amino-acid incorporation, measuredduring a 5-mmn pulse beginning at 30 min after treatment, is ex-pressed as percent amino acid incorporation (trichloroacetic acid-insoluble)/trichloroacetic acid-soluble and trichloroacetic acid-in-soluble). Release of surface protein is expressed as percent of totallabeled protein released after 30 min of treatment. Eggs were fer-tilized with a 0.01% suspension of sperm. Concentration of ammo-nia and procaine (PROC.) was 10 mM, nicotine (NICO.) was 1 mM(all at pH 8).

Characterization of proteins released duringincubation of eggs in ammoniaThe major component released during incubation of eggs inammonia is a 150,000-dalton glycoprotein, as judged by pos-itive staining with the Coomassie blue and periodic acid-Schiff stain. This same molecular weight was observed overa 5-10% range of polyacrylamide gel concentrations. A de-tectable amount of this protein is released after only 3 minof incubation in ammonia; other lower molecular weightproteins become apparent after 20 min of incubation in am-monia (Fig. 3), and two of these (A and B) stain positivelywith the periodic acid-Schiff stain (Fig. 3). However, onlythe 150,000-dalton protein is labeled with 125I (Fig. 3); ei-ther this protein is the only one containing exposed tyrosineresidues or is the only protein released during the ammoniatreatment that is sufficiently exposed on the egg surface tobe iodinated.Site of the 1251-labeled protein released by ammoniaThe following experiments indicate that the iodinatable pro-tein released by ammonia is most likely on the plasma mem-brane itself and is not a component of the vitelline layer.This latter layer contains major surface proteins of the un-fertilized egg and elevates at fertilization as the template ofthe fertilization membrane. The vitelline layer of iodinatedeggs was removed with either the dithiothreitol (12) or corti-cal granule protease* procedure (see Materials and Meth-ods). Eggs denuded by either of these procedures were notmetabolically activated, as judged by measurement of rateof protein synthesis. The eggs were then activated by incu-bation in ammonia; labeled surface protein was released thatwas identical in amount and in molecular weight to that re-leased by normal eggs.

Results of experiments in which eggs were incubated intrypsin or Pronase suggest that only a small portion of the150,000-dalton protein is susceptible to proteolysis by exog-enous proteases. Eggs were treated in 1 mg/ml of trypsin for1 hr and incubated in ammonia. The proteins released into

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FIG. 3. Sodium dodecyl sulfate gel electrophoresis pattern of125I-labeled proteins and Coomassie blue, stained proteins thatwere released by eggs during a 20-min incubation in 10 mMNH4CI, pH 8.0. Lower figure, cpm contained in 0.2-cm slices;upper insert, Coomassie blue stained gel from same extract. Themajor band coincident with the 1251 label and bands A and B allstained positively with the periodic acid-Schiff stain.

the seawater were collected and electrophoresed on sodiumdodecyl sulfate gels as described. A major protein of ap-proximately 150,000 daltons was released from these eggsafter incubation in ammonia. Eggs treated with 1 mg/ml oftrypsin or Pronase for 30 min were not metabolically acti-vated, as judged by measurement of protein synthesis.Biological activity of nondialyzable componentsreleased upon activationThe above results indicate that release of labeled surfaceprotein is closely correlated to metabolic activation. The fol-lowing experiment asks if the activation is reversible, i.e.,can the nondialyzable material released by eggs upon acti-vation be added back and suppress the metabolism of al-ready activated eggs? Three samples of eggs (0.2% suspen-sion) were activated by incubation in 10 mM NH4Cl, pH8.0. At 30 min the percent incorporation of [3H]valine intoprotein was measured. The samples were then washed twicein filtered seawater and resuspended in ammonia solution,filtered seawater, or the solution of nondialyzable materialfrom activated eggs (10-30 gg/ml of protein). This repre-sents a 50-fold concentration of material (see Materials andMethods). The rate of protein synthesis of each group wasthen measured at 60 and 90 min. In five experiments, therate of protein synthesis of eggs incubated in the nondialyz-able material was significantly suppressed when comparedto the eggs resuspended in seawater only or resuspended in10 mM NH4C1 in filtered seawater (Fig. 4). Ammonia (10mM, pH 8.0) reversed the suppressive effect. When ammo-nia-activated eggs were incubated with 50,ug/ml of bovineserum albumin or ovalbumin, no suppression of protein syn-thesis was observed. Heating the solution of nondialyzablematerial in a boiling-water bath for 15 min did not inacti-vate the activity. If protein is responsible for the suppressoractivity, then the heat stability of the molecule would beconsistent with the characteristics of glycoproteins (17). Wehave not yet directly established, however, that the iodinata-ble glycoprotein is rebound to the surface of already activat-ed eggs.

DISCUSSIONFertilization normally initiates a programmed sequence ofchanges, which can be temporally divided into two distinctphases (18). The initial or "early" changes occur in the first

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FIG. 4. The suppression of protein synthesis by nondialyzablecomponents released by eggs. A 0.2% suspension of eggs was incu-bated in 10 mM ammonia for 30 min, at which time a 2-ml sampleof eggs was pulsed with [3H]valine for 5 min. Eggs were thenwashed twice in filtered seawater, and separate groups were incu-bated in ammonia (X), filtered seawater (0), or components re-

leased by eggs (0). Controls remained in filtered seawater through-out (A). Samples (2 ml) from each group were pulsed for 5 minwith [3H]valine beginning at 60 and 90 min. Error bars are stan-dard deviation of five experiments.

60 sec after sperm-egg interaction and appear to involve therelease of Ca+2 from intracellular stores (10) and the resul-tant exocytosis of cortical granules (19). The "late" changes,which begin at 5 min after insemination, normally followthe early changes as a part of the programmed sequence offertilization. The early changes, however, are not a prere-

quisite for the late changes, since they can be bypassed byincubation of eggs in agents such as ammonia (2-4), nicotine(this paper), and procaine (6). Also, these late changes act in-dependently of each other, as opposed to the early changeswhich are all dependent upon one another (3). On thesegrounds one could argue that the early and late changes are

regulated by different factors.The results of this paper suggest that one of these regulat-

ing factors is a protein (or proteins) released by cells upon

activation and which reversibly controls at least one of thelate events, a post-transcriptional increase in protein synthe-sis. These proteins appear to act in a suppressive fashion, andtheir loss from the cell surface promotes an increased syn-

thetic rate. Kinetic data of this paper and a similar study byShapiro (20) indicate that during activation by fertilizationor by ionophore a maximal release of labeled protein occurs

coincident with or shortly after the cortical reaction. Tryp-sin-like proteases of the cortical granules do not appear to beinvolved in the release, since inhibitors such as N-a-p-tosyl-L-lysine chloromethyl ketone (10 mM concentration), L-1-tosylamide-2-phenylethyl chloromethyl ketone (1 mM con-

centration), and phenylmethyl sulfonylfluoride (5 mM con-

centration) do not prevent the release of the labeled surfaceprotein and do not affect activation of protein synthesis. Par-tial metabolic activators such as ammonia, nicotine, and pro-

caine result in a slower release of this protein, but in the ab-sence of cortical granule breakdown. No apparent structuralanalogy exists among these three agents except that all are

amines (primary, secondary, or tertiary amines). These acti-vating agents may perturb the plasma membrane, releasingthe proteins.

It is not yet known whether protein synthesis is regulatedby one or several of the nondialyzable components, nor is itclear whether any of these components regulate the otherlate events, such as DNA synthesis and chromosome conden-

sation. Correlative arguments support the concept that alllate events are regulated by some similar mechanism; agentsreleasing iodinatable protein or various treatments perturb-ing the surface (21) activate all the late events. Assumingsurface proteins are the regulators, it is most provocativethat the metabolism of the egg can literally be "titrated" byammonia; low concentrations will increase protein synthesis,but higher concentrations are needed to induce chromosomecondensation (3). In light of the present results, this couldmean that increasing concentrations of ammonia release in-creasing amounts of some general regulatory molecule, i.e.,that the regulation is quantitative and depends on theamount of cell surface component that is released; or thiscould mean that increasing concentrations of ammonia re-lease different species of regulatory molecules, i.e., thatthere are qualitatively different cell surface regulators forcell processes such as protein or DNA synthesis.

Concepts of membrane-mediated regulation of cell activi-ty, as through hormone receptors, are widely accepted (22).Recent work has suggested that the promotion of cell activi-ty, as occurs during various types of transformation, is relat-ed to the loss of a large molecular weight glycoprotein (8, 9).Also, glycopeptides derived from the surface of HeLa cellswill repress the protein synthesis of these cells (23).We see two major possibilities as to how loss of the surface

components of the egg might be translated into activation ofthe metabolic processes of the cell. One of these is that thissurface molecule is regulating some enzyme or enzyme sys-tem. In analogy to models of hormone action (22), this en-zyme could be a nucleotide cyclase and the effect mediatedthrough cyclic nucleotides to protein-modifying enzymes(e.g., protein kinase). Although cAMP and cGMP do notchange after fertilization (24-26), the possibility of a tran-sient increase at the time of the late changes (5 min after in-semination) has not been eliminated. Alternatively, regula-tion could be directly on a protein kinase. This enzyme ispresent in the cortex of sea urchin eggs (27).A second possibility is that the surface components regu-

late cell structure and that their loss during fertilization oractivation results in cell recompartmentation and increasedavailability of enzymes to their substrates. Glucose-6-phos-phate dehydrogenase (28) and aldolase (29) are present inparticulate fractions in unfertilized eggs and are translocat-ed to the soluble fraction in the fertilized egg. Also, studieson enzyme activation suggest that substrates and enzymesare unavailable to each other until after fertilization (30, 31).Such recompartmentation may occur in two dimensions, asby changes in membrane fluidity, or in three dimensions, asthrough changes in the structure of the cytoplasm. A recom-partmentation mechanism is appealing since it would ex-plain the generalized promotion of many processes as are in-duced during partial activation by ammonia or during fertil-ization by sperm.

We thank Ms. Bonnie Dunbar for her assistance with developingthe iodination procedure. We also thank Dr. S. J. Singer for helpfuldiscussions, Drs. Daniel Mazia, Richard Steinhardt, Victor Vac-quier, and Bennett Shapiro for sharing their unpublished resultswith us, and Franklin Collins and Dr. Miles Paul for criticism ofthis manuscript. Ms. Elizabeth Baker provided excellent technicalassistance. Initial stages of this work were performed in the Fertil-ization and Gamete Physiology Program at the Marine BiologicalLaboratory, Woods Hole (NIH Grant 3-TO1-HD0026-1251). Thiswork was supported by a grant from the National Science Founda-tion.

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Dev. Biol. 40,245-255.4. Mazia, D. & Ruby, A. (1974) Exp. Cell Res. 85,167-172.5. Longo, F. J. & Anderson, E. (1970) J. Cell Biol. 47, 646-665.6. Vacquier, V. & Brandiff, D. (1975) Dev. Biol., in press.7. Johnson, J. D., Dunbar, B. S. & Epel, D. (1974) Biol. Bull. 147,

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