Abnormal Sea Urchin Fertilization Envelope Assembly in Low Sodium Seawater

9 Pages·1991·3.6 MB·English

by  S D Cheng

Reference: Uinl Bull 180: 346-354. (June, 1991)
Abnormal Sea Urchin Fertilization Envelope Assembly
in Low Sodium Seawater
SOU-DE CHENG1, PATRICIA S. GLAS2, AND JEFFREY D. GREEN*
Department ofAnatomy, Louisiana State UniversityMedical Center, New Orleans, Louisiana 70112
Abstract. The structuralization ofthe sea urchin fertil- with the change from egg to embryo. The irreversible
ization envelope (FE), a model for extracellular macro- transformation ofthe relatively thin, soft vitelline layer
molecular assembly, was found to require sodium ions, (VL = glycocalyx), investing the unfertilized egg, into a
the predominant cation ofseawater. Eggs from Strongy- hardened, insoluble fertilization envelope (FE), elevated
locentrotuspurpuratitsactivated in seawaterswith sodium from the egg surface, is a critical step for the protection
chloride substitutes (choline or Tris chloride) elevated ofthe developing embryo. Furthermore, this process has
incomplete FEs. In addition, the conversion of the mi- been thesubjectofnumerousinvestigationsasanexample
crovillar casts ofthe FE from blunt (I-form) to angular ofregulated extracellular matrix assembly. Several recent
(T-form)did not occur. The permeability ofthe abnormal reviews (Kay and Shapiro. 1985; Shapiro et al., 1989;
FEs wasalso compromised, asapproximately eight times Somers and Shapiro, 1989) contain the details of such
more protein than normal was released into the ambient investigations: therefore, the process will be summarized
seawater. There were also significant increases in the es- only briefly here.
cape oftwo cortical granule (CG) enzymes, 0-1,3-gluca- Transglutaminase has an important role in the earliest
nase and ovoperoxidase. Furthermore. FEs elevated in stages ofVL modification (Battaglia and Shapiro, 1988).
choline chloride (ChCl) seawaterappeared to bedeficient It is located on the egg surface and catalyzes the incor-
in the incorporation ofovoperoxidase, an enzyme that is poration ofprimary amines into the nascent FE during
normally bound to the FE and that cross-links structural thefirst4 minofeggactivation. Thisprocessisapparently
proteins in the nascent FE. The morphology ofFEs ele- related tothe I-T transition ofthe microvillarprojections
vated in potassium chloride-substituted seawater was ofthe VL (in 5. purpuratiis). because the transition does
similar to those in normal sodium seawater. Thus, it ap- not occur in the presence oftransglutaminase inhibitors.
pears that sodium, or at least a similar ion, is necessary Duringthe nextfew minutes, ovoperoxidasesecreted from
fortheproper functioningofovoperoxidaseand structural thecortical granules(CG)catalyzesthe insertion ofstruc-
proteins in the elevation and normal assembly ofthe sea tural proteins from the CGs into the VL by cross-linking
urchin FE. tyrosyl residues between polypeptides (Foerder and Sha-
piro, 1977; Hall, 1978). Besides this catalytic reaction,
Introduction ovoperoxidase itselfis incorporated into the nascent FE
CG
Sea urchin fertilization hasbeen intensivelystudied for via a specific interaction with another protein, pro-
the dramatic intra- and extracellular events concomitant teoliaisin (Weidman et al., 1985). These enzyme activities
result in a hardened, insoluble, fully formed FE within
Received 29August 1990:accepted 24January 1991. the first 10 min following egg activation.
' Genetics Division. Children's Hospital, 300 Longwood Avenue. Several recent reports have been concerned with ionic
Boston, MA02115. requirements for FE formation. Carroll and Endress
2 DepartmentofZoologyand Physiology. LouisianaStateUniversity. (1982) demonstrated that formation of mature FEs re-
B(cahtAooblnbirneRevociuhaglteoi.roindLse:A);S7F0WE80((3fa.errttifiilciizaaltisoenaewnavteelr)o;peC);GFP(co(rfetritciallizgartainounlep)r;odCuchtC)l; tqhueirseesawtahteern.orBmyalom9itmtiMng t[hCeas2e+]ioannsdth4e8ymcoMuld[Mpgro2+d]ucien
VL an "intermediate envelope" that was much thinner than
(vitelline layer).
* Towhom correspondence shouldbesent. the normal FEanddid notincorporatestructuralproteins.
346
FERTILIZATION ENVELOPE ASSEMBLY 347
nDoefticoinelnycyintoefrCfelre,stwhietmhotshteanbournmdaalntI-aTnitornanisnfosremaawtaitoenr,, 0s.u1spAeInsTiroinssH(Cv1/v()KwleebraenomfatdVe/ aain.d1i9n79c)u.baTteend pienrc5.e6ntmeAg/g
but also increases the permeability ofthe FE (Lynn ct ai, 3M,3-diaminobenzidine (DAB;Sigma)in0.45 A/NaCl-0.1
1988: Green et ai. 1990). Furthermore, the normal ex- Tris HC1 in an ice bath for 10 min. The experimental
ternal Na+ concentration (419 mA/) is not only essential eggs(low Na+) were handled identically except that NaCl
forpreventing polyspermy and hardeningthe FE, but also was replaced with ChCl. Activated eggs were then fixed
TEM
forthe normalembryonicdevelopmentoftheseaurchins and prepared for as described below.
Arbacia pnnctiilata and Strongylocentrotus purpwatiis
(Schuel ct ai. 1982). Transmission electron microscopy
To better understand the mechanism ofFE elevation
Eggs were fixed with 2% glutaraldehyde in the appro-
awne icmopnocretnatrnatteedarloynetvheentefofefctemobfrNyoan+icdefdiecvieelnocypmoenntthe priate seawaters for 1 h at room temperature and washed
formation ofthe FE. In the present study, the elevation with0.1 A/sodium cacodylate(pH 7.4). Postfixation with
and morphology ofFEs from normal and low sodium (2 1% osmium tetroxide (OsO4) was performed for 30 min.
mA/) seawaters were investigated. Envelopes were ob- Eggs were washed with double distilled water and dehy-
smeircvreodscwoiptyh.pThoatsael, ssoclaunbnliengs,ecarnetdedtrparnostmeiisnsiaonndetlheecteronn- wdraasterdepilnacaesEdcMewnidtihngprcoopnycleennteraotxiiodnes aonfdetehgagnsolw.erEethiannfioll-
zymaticactivitiesofovoperoxidase(FoerderandShapiro. tTrhaetedblwoictkhs werebecdu-r8e1d2a(tEl5e8ct-r6on0CMifcorros3cdoapyys.ScSieecntcieos)n.s
W1e9s77s;elHaeltla,i.1917988)7a)nidn^t-hle,f3e-rgtliulcizaantaisoen(pSrcohduuecltest(aFiP.)1w9e7r2e; werecutwithglass knives on a Reichert-JungUltracut E;
compared. Portions ofthis investigation have been pre- mountedon coppergrids;stainedwith leadcitrate(Reyn-
sented in a preliminary form (Cheng et ai. 1989). olds, 1963)and uranyl acetate;andobservedwitha Philips
301 transmission electron microscope at 60 kV.
Materials and Methods
Scanning electron microscopy
Handling ofgametes
Sea urchin (5. piirpuratits) eggswerecollected andacid Fixations were accomplished at 20C by mixing equal
dejelliedasdescribedpreviously(Greenetai. 1990). Eggs volumesofeggswith 4% glutaraldehydein seawater(with
were divided into four equal portions, the three low Na+ 10 mMTAPS pH 8.3) before activation and at 1, 3, 10,
groups: KC1-, Tris-, andChCl-substituted seawaters(SW) 30, and 60 min posMtactivation. Eggs were fixed for 1 h
and the control: normal Na+-SW group. They were and washed in 0.1 sodium cacodylateM(pH 7.4). Post-
washed and incubated in the appropriate SW for 15-40 fixation took place in 1% OsO4 in 0.1 sodium caco-
min. AllseawaterformulationswerebasedonCavanaugh dylate (pH 7.4) for 30 min. They were then washed with
(1956). For the low Na+-SWs, equimolar concentrations double distilled water and dehydrated in an ascending
ofKG, Tris HC1, orChCl(Sigma) replacedNaCl, yielding seriesofethanol. Absoluteethanolwasreplacedgradually
acalculated residual [Na+ ofapproximately 2 mA/. Nor- by acetone. Eggs were transferred to porous containers
]
mal and low Na+ SWs were buffered with 10 mA/ TAPS (Bio-Rad) and processed for CO: critical point drying
(Sigma) and adjusted to pH 8.3 with NaOH or KOH, (Samdri-790, Tousimis Research Corp.). The eggs were
respectively. Toavoidcontamination from sperm proteins then attached to aluminum mountscoated with colloidal
+
and secretions, eggs were activated by adding the Ca' silverliquid(TedPella, Inc.)forsputtercoatingwithgold:
ionophoreA23187 (Chambers etai, 1974; Steinhardtand palladium (60:40; Electron Microscopy Sciences) for 3
Epel, 1974) to a final concentration of 38 ^Af in 1% di- min inaHummerVI (Technics). Eggswereobserved with
methyl sulfoxide (DMSO). Experiments were performed a JEOL JSM-35CF scanning electron microscope at 25
at 20C. kV and a condenser lens setting of 3. The photos were
taken with Polaroid type 55 (4 X 5 in.) positive/negative
Light microscopy
instant sheet film.
Eggs activated in normal and low Na+-SWs were ob-
served continuously on a 1 X 3 inch microscope slide Totalprotein assay
under a coverslip supported by a ring ofpetroleumjelly.
Photographs were taken with phase contrast optics on lonophore-activated eggs were allowed to settle forap-
Kodak Technical Pan Film 2415. proximately 10 min and the supernatant (secreted FP)
wascollected and centrifuged by hand to remove the few
Ovoperoxidase localization remaining eggs. Ionophore activation resulted in at least
Activated eggs ofthe control group (normal Na4-SW) 95%elevated FEs. Forproteindeterminationtheproteins
were rinsed at 75 minpostactivation with 0.45 A/ NaCl- in 1.0mlofFP(5%eggsuspension, v/v)wereprecipitated
348 S.-D. CHENG ET AL
by adding 1 10 n\ ice-cold 50% trichloroacetic acid (TCA) Results
and centrifuged for 20 min at 8800 X g(Eppendorfcen-
trifuge 5413) at 10C. Tubes were drained by inversion Observations with light microscopy
and the protein pellets air dried. Bovine serum albumin The elevation ofFEs in normal and low Na+-SWs are
(BSA; Sigma) was the standard. All the precipitates were compared in Figure 1. Figure 1A-Ddepictseggsat 1 min
assayed according to Lowry el til. (1951). As a control, postactivation. Although difficult to quantify from the
protein determinationswereperformed with BSA in each photomicrographs in the normal Na+-SW control group
ofthe seawaters to ascertain their influences, ifany, on (Fig. 1A, E), the FE appeared relatively thin at the end of
the Lowry procedure. the first minute and thickened with increasing time.
However, it appeared thicker (more refractive) than the
0-1,3-glucanase assay low Na+ groups, especially Tris and ChCl (Fig. 1C, D).
Glucanase activity was measured according to Green Atlatertimepoints, theFEofthecontrolgroupremained
and Summers 1980). The FP was collected as described more refractive than those ofthe low Na+ groups. These
(
above. FP (0.2 ml; 5% egg suspension, v/v). normal Na+- differences were more striking at 3 min postactivation
or ChCl-substituted SW (0.2 ml), and laminarin (0.2 ml when the FE ofChCl eggs began to shrink and some col-
ofa 2.5 mg/ml SW solution) were incubated for 1 h at lapsed, while those ofthe control remained spherical. By
37C. Then0.2 mlofthismixture,0.4 mlenzymesolution 30 min postactivation, the FEs remained robust in the
(0.4 ml glucose oxidase and 3 mg horseradish peroxidase normal and K+-substituted SWs (Fig. IE, F), while those
in 50 ml of25 mM phosphate bufTer, pH 6) and 0.4 ml of the Tris- and choline-substituted SWs had collapsed
o-dianisidine(40 mgin 50 mldoubledistilled water)were back nearer to the egg surface (Fig. 1G, H).
incubated for 10 min at 37 C. The reaction was stopped An interesting attribute ofthe activated eggs is that of
bytheaddition withrapid vortexingof0.8 ml4A'sulfuric their increased stickiness in the Tris and ChCl groups. In
acid. Spectrophotometric readingswere taken at 530 nm. the first 3 min there was no apparent difference among
Controls lacking the substrate laminarin were used to the 4 groups. At approximately 4 min postactivation,
check for the presence ofglucose (the product ofthe glu- however, the eggs of the Tris and ChCl groups formed
canase-laminarin reaction) in the FP. Glucose was gen- extensive clumps.
erated onlyin the presence ofboth the FPand laminarin.
A glucose solution was the standard. Control glucose de- Ultrastructural changes
terminations were performed in normal Na+-SW and Unactivated eggs incubated in normal Na+- or ChCl-
ChCl-substituted-SW to ascertain the effects, if any, of substituted-SWs and observed by SEM displayed similar
ChCl substitution on the glucose oxidase-peroxidase re- surface morphology(Fig. 2A,E).At 1 min postactivation,
action. All the chemicals for this assay were purchased the FEs in both SWswereelevated with rounded (I-form)
from Sigma. microvillar projections (Fig. 2B, F). In contrast to FEs in
SW
ChCl-substituted (Fig. 2G), the typical I-T ("Igloo-
Ovoperoxidase assay Tent") transformation of S. purpuratus FEs in normal
Ovoperoxidase assayswereperformed in 1 ml contain- Na+-SWwascompleted by 3 min (Fig. 2C)and resembled
i1n0gm18MmAT/AgPuaSiaactoplH(S8i.g0maa)n,d0.230mCA/(DHei2Ots:e(tSiag/.m,a)1,98a4n)d. dtihdosenootfluantedrertgiometphoeinttrsa(nFsifgo.rm2aD)t.ioHnoweevveenr,byCh3C0lmFiEns
The reaction was started by adding enzyme (in the FP), (Fig. 2H).
and the increase in absorbency at 436 nm was recorded Asjudged with TEM (Fig. 3), FEs that elevated in nor-
spectrophotometrically with astripchart recorder. All re- mal Na'-SW resulted in thewell-defined, angularT-form
ported values are initial rates, because the reaction slows projections (Fig. 3A) characteristic ofthis species. How-
after 15-30s. A unitofOvoperoxidasewasdefined asthat ever, those in K'-substituted-SW appeared to be inter-
which is required to oxidize 1 ^mole ofguaiacol per min mediate in form (Fig. 3B),compared tothosein Tris-and
in a 1-ml assay volume (Deits et ai, 1984). ChCl-substituted SWs, which were similar to each other
FP including the Ovoperoxidase was collected from a in retaining rounded projections (Fig. 3C, D).
5% (v/v) suspension ofactivated eggs. Ten minutes after The above observations ofthe "soft" FEs (incomplete
activation, eggs were settled by low speed centrifugation formation) suggested that their permeability, as well as
and the supernatant (FP) was removed for the assay. their morphology, might be altered. Therefore, several
measurements ofpermeability were undertaken.
Statistics
Statisticalanalysesfortotalprotein,ft-1,3-glucanaseand Totalprotein secretion
Ovoperoxidase assayswere performed usingthe Student's Soluble secreted protein that leaked through the FEs
I test. ofeggsactivated in normal orlow Na+ SWswas measured
FERTILIZATION ENVELOPE ASSEMBLY 349
Figure 1. Phase microscopyofStrongylocentrotuspiirpiiranixeggsactivated in normal (A. E)and Na*
depleted SWs (B-D and F-H). A. E: Normal Na+-SW. B, F: KCl-substituted-SW. C, G: Tns-substituted-
SW. D, H: ChCl-substituted-SW. A-D: 1 min postactivation. E-H: 30 min.These micrographsweretaken
focusing on the fertilization envelopes, fe = fertilization envelope; PVS = perivitelline space. Scale bar
= 50^m.
(Fig. 4). FPswerecollected at 10 min postactivation from activity was found in the FP from the ChCl eggs. This
eggs pooled from several females. The FPs ofthe normal difference was significant (P < 0.05).
and K+-substituted SW eggs had 22.7 1.8 Mg and 22.2 Control incubationsofglucosewereassayed in normal
5.7 Mg(Mean S.E.M.) ofprotein/ml FP, respectively. and ChCl-substituted SWs, and no significant differences
They were not significantly different. However, the FPs (95% confidence level)were observed. Therefore, it is un-
ofTris-andChCl-substitutedSWscontained 162.1 25.9 likely that the ChCl interfered with the glucose determi-
Mg and 168.7 3.4 ^g- respectively. This 7- to 8-fold in- nation.
crease over normal and KC1 was highly significant (P
< 0.0001). Ovoperoxidase secretion
Control assays demonstrated no significant difference Ovoperoxidase released from ChCl-SW eggs (3.72
(n9o5rm%aclonafniddelncoewlNevael+)SiWnsT.CAT-hperreecfiopriet,abtlheeBvSarAiobuestwSeWesn 0.78 ^moles guaiacol oxidized/min/ml ofFP) had sig-
nificantly higher activity (see Fig. 6) than that released
hsuapdernnoataadnvterpsreoteefifnecftsroomnutnhaectLiovawtreydeasgsgasy.inIDnMadSdOi~tiwoan,s ofxriodmasneoracmtailvitSieWs eigngsKC(01.-8S2W (01..2019jum0o.l3e0/m/iLnj/mmoll)es./mPienr/-
measuhresedeaanldsofGournedentoetcoanit,ri1b9u9t0e).littletothetotal ( 1.4 imnlt)earmneddiTarties-bSeWtw(e1e.6n8con0t.r3o0l^amnodleCsh/Cmiln/gmrolu)psF.PsThweerree
were significant differences between normal and ChCl (P
ft-1,3-glucanase secretion < 0.001), and Tris (P < 0.05). However, Ovoperoxidase
Glucanase activity was measured (in normal and ChCl KreCl1ea(sPe>w0a.s3n5o)t.significantly different between normal and
SWs) by the amount ofglucose hydrolyzed from the /8-
1,3-glucan polysaccharide laminarin by egg-derived-glu- Ovoperoxidase localization
canase in the FP (Fig. 5). Aliquots ofFP ofexperimental
and control groups were taken at 10 min postactivation. Because Ovoperoxidase is incorporated into the FE
The glucose measurements from ChCl and normal SW (Somers et ai, 1989) and more enzyme activity was ob-
were 1.62 0.19 and 0.93 0.19 ^moles glucose per ml served in the FP ofthe low Na+ treatments (see above),
ofFP, respectively. Approximately 75% more glucanase it is possible that the higher activity was not only due to
350 S.-D. CHENG ET AL.
Figure2. SEMofStrongylocentrotuspurpwatuseggsurfaces.A.VLofegg(unactivatedlinnormalNa*-
SW. B-D. FEs ofnormal Na+-SW eggs at 1,3, and 60 min postactivation. E. VL ofegg (unactivated) in
ChCl-substituted-SW.F-H.FEsofChCl-suhstituted-SWeggsat 1,3,and30min.Themicrovillarprojections
oftheChCl-suhstituted-SW eggsdid not undergothe I-T transformation. Scale bar = 1 ^m.
FE permeability, but that the ovoperoxidase was not in- sembled and intercalated under the influence ofseveral
corporated efficiently into the structure ofthe FE. There- enzymes. A requisite for proper structuralization is the
fore, DAB localization ofovoperoxidase was performed. presence ofseveral ions in the seawater, e.g.. Ca2+, Mg:4
Comparing the TEM micrographs (Fig. 7) ofFEs after (Carroll and Endress, 1982), Cl~ (Lynn etal. 1988;Green
DAB incubation, the normal SW FE (Fig. 7C) is con- et al.. 1990), and Na+ (Schuel etal. 1982). In the present
spicuously darker than that of the ChCl FE (Fig. 7D). study, we focused on the effects ofNa+-depletion on the
Although both normal and ChCl FEsstained more inten- elevation and structuralization ofthe FE.
sivelythan thecontrols(Fig. 7A, B), the intensity ofstain- There issome information on thesea urchin eggduring
ingwas higher in the normal FEs. Presumably, there was fertilization in Na+-depleted seawater. It was found that
more ovoperoxidase incorporated intothe FEsin normal the fast block to polyspermy decayed concurrently with
Na+-SW than in ChCl-substituted SW. the retardation of the depolarization of the egg plasma
membrane, aNa+-dependent process(Jaffe, 1980; Schuel
Discussion and Schuel, 1981). Additional investigations have shown
that Na+ accounts for the release ofacid from the egg,
Sea urchin eggs are excellent material for many bio- resulting in an increased intracellular pH. This increase
DNA
logical studies because they can be harvested in large isnecessary forincreased protein synthesis, synthe-
numbers, cultured in a well-defined medium (artificial sis, and cell division (Nishioka and Cross, 1978). In re-
seawater), andtheydevelopsynchronously. Theyarewell lation totheassemblyofthe FE, Schuel etal. (1982)dem-
suited for the study of extracellular self-assembly (Kay onstrated that low Na+ (19 mM), ChCl-substituted sea-
and Shapiro, 1985; Somers and Shapiro, 1989; Shapiro waterresulted in FEsthatcollapsed and failedto undergo
el al., 1989). The complexity ofthe transition ofthe vi- normal structuralization. including the I to T transfor-
telline layer (VL) glycoprotein to the FE tempts one to mation ofthe microvillarcasts. The inhibition ofthenor-
CG
trytodissect the myriadofsequential processes involved. mal hardeningprocesswasattributed to the failureof
The VL is not merely an inert cell coat, but it serves as a structural proteins to impregnate or insert into the VL.
template orscaffolding upon which otherproteins are as- However,thisimpairment ofthehardeningprocessisdis-
FERTILIZATION ENVELOPE ASSEMBLY 351
2.00-1
1.5O-
1.OO-
O.5O-
o.oo
MBL ChCl
sea water treatment
Mean +/- S.EM
Figure 5. 0-1,3-glucanase secretion. Glucanase activity released
through FEs was assayed as described in Materials and Methods. The
Mean + S.E.M. are shown for four trials. The * denotes a statistically
significantdifference from thecontrol, normal SW.
Inthe present study we loweredthe Na+ concentration
to approximately 2 m.Mand observed an earliercollapse
oftheFE, 3 minasopposed to 30 min. Itisnotsurprising
that our FEs collapsed earlier than those ofSchuel et al.
1982). because our Na+ concentration was ten-fold less.
(
Additional evidenceofthe failureofthesodium-deficient
15Fmiigunrepo3s.tacTtiEvaMtioofnSitnrotnhgeylfooclelnotwriontgusSWpisi:rpAu.ranioursmFaEls.NEag+g.sBw.erK.eC1f.ixCe.d FEs to harden is that initially they expanded more than
Tris. D. ChCl. Scalebar = 0.5 urn. the normal FEs. This greater distension may also be re-
latedtothefailureofproteinstoinsertintotheFE,thereby
raising the hydrostatic pressure in the perivitelline space
tinct from thephenomenon of"cross-linking," in thatthe (Schuel et al., 1974; Green and Summers, 1980). However,
latter isassayed by the disruption ofFEs in urea. In their within a few minutes, the FE began to shrink, suggesting
experiments, cross-linking was not affected. Furthermore, that the FE was permeable to the secreted proteins, and
K+ and Li+ substituted for Na+ in normal structuraliza- this allowed for a decrease in the hydrostatic pressure
tion, while ChCl and Tris did not.
5-1
200-
150-
3-
<U 1OO
O
Q
Dl 5O-
MBL KCI Tris ChCl MBL KCI Tris ChCl
sea water treatment
sea water treatment
Mean +/- S.EM. Mean +/- S.E.M.
Figure4. Protein releasethrough FEs. Proteinconcentrationswere Figure6. Ovoperoxidase secretion. Ovoperoxidaseactivityreleased
determinedbytheLowryassay.EachmeasurementrepresentstheMean throughFEswasmeasuredasdescribedin MaterialsandMethods. Mean
S.E.M.ofthreetrials.The*denotesastatisticallysignificantdifference S.E.M. are shown (n = 3-7) and * denotes statistically significant
from thecontrol, normal SW. differences from thecontrol, normal SW.
352 S.-D. CHENG AT AL.
^;
L.- . . . - - >*,LV>--;
* <yv* '
!
;-
7C
Figure7. Ultrastructural localization ofovoperoxidase. Thepointed T-form microvillarprojectionson
normal Na*-FE (15 min afteractivation) and the rounded I-form microvillar projections on ChCl-FE are
demonstrated(Aand B, respectively).AfterDABlocalizationofovoperoxidasetheFEs(C,normal Na+;D,
ChCl)stainedmoreintenselythanthoseofthecontrols(AandB)inbothnormalNa*andChCl-substituted-
SW. Furthermore, the stainingofthe normal Na* wasdarkerthan thatoftheChCl. Thissuggeststhat less
ovoperoxidase was incorporated into the FE ofeggs activated in ChCl-suhstituted SW. D is a montage
showingthe FE and cortexofthesameegg. Scalebar = 0.5//m.
FERTILIZATION ENVELOPE ASSEMBLY 353
within theperivitellinespace. Thisconclusion isstrength- byan EdwardG. SchliederEducational FoundationGrant
enedbythefactthatweobservedagreater(approx. eight- toJ.D.G.
fold) increase in total proteins secreted into the ambient
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(Somers and Shapiro, 1989). the increase in soluble ovo- tJ.raCneslgllBuitoalm.in1a0s7:e2i4n4e7a-r2ly45s4t.ages offertilization envelope assembly.
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into the nascent FE. The substitution ofK+ for Na+ did sembly and isolation ofan envelope intermediate from Strongy/o-
not significantly interfere with hardening (structuraliza- Cavacennaturgoht,usGp.urMp.u,raedt.us19e5m6b.ryosPp..D6e7v.-6B9ioiln.F94o:rm2u5l2a-2e5a8n.dMethodsof
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the result ofChCl or Tris addition, rather then exclusion concavity at fertilization in the sea urchin egg. DOT Growth Differ
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Another interesting observation of these experiments 355.
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