rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization

Rearrangements of sea urchin egg cytoplasmicmembrane domains at fertilization

Philippe Collas1)a, Teresa Baronab, Dominic L. Pocciab

a Institute of Medical Biochemistry, University of Oslo, Oslo/Norwayb Department of Biology, Amherst College, Amherst, MA/USA

Received July 27, 1999Received in revised version October 5, 1999Accepted October 11, 1999

Fertilization ± endoplasmic reticulum ± egg ± lamin B ±lamin B receptor

Fertilization in the sea urchin is accompanied by rapidreorganization of the egg endoplasmic reticulum (ER). ER-derived vesicles contribute to one of three classes of mem-branes used in assembling the male pronuclear envelope invitro. We provide here biochemical evidence for the rear-rangement of sea urchin egg cytoplasmic membrane domainsat fertilization up to the first mitosis, with respect to twonuclear envelope markers, lamin B and lamin B receptor(LBR), using purified vesicles prepared from homogenatesfractionated by floatation on sucrose gradients. In unfertilizedeggs, immunoprecipitation data indicate that most of lamin Band LBR are localized in the same vesicles but do not interact.By 3 min post-fertilization, both proteins are more widelydistributed across the gradients and by 12 min most of lamin Band LBR are localized in vesicles of different densities. Thispartitioning is maintained throughout S phase. At mitosis,most lamin B and LBR remain in distinct vesicles, while asmall proportion of lamin B and LBR, likely derived from thedisassembled nuclear envelope, associate in a minor subset ofvesicles. The results illustrate a dynamic reorganization of eggcytoplasmic membranes at fertilization, and the establishmentof distinct membrane domains enriched in specific nuclearenvelope markers during the first cell cycle of sea urchindevelopment. Additionally, we demonstrate that male pro-nuclear membrane assembly occurs only when both cytosoland membranes originate from fertilized but not unfertilizedeggs, suggesting that fertilization-induced membrane rear-rangements contribute to the ability of the egg to assemble themale pronuclear envelope.

Abbreviations. ER Endoplasmic reticulum. ± LBR Lamin B receptor. ± MWBMembrane wash buffer. ± NE Nuclear envelope.

Introduction

Eukaryotic cell physiology is characterized by a complexpattern of membrane traffic between organelles. One impor-tant example of membrane trafficking involves disassembly ofthe nuclear envelope (NE) at prophase of mitosis and itsreassembly at telophase. Two models of mitotic nuclearmembrane disassembly and reformation have emerged fromstudies of nuclear envelope dynamics. One model suggests thatnuclear membranes reversibly fragment by vesiculation,producing NE-derived vesicles separate from the endoplasmicreticulum (ER) (reviewed in Marshall and Wilson, 1997). Thesecond model proposes that nuclear membranes vanish bydiffusion of their integral proteins through a continuousendoplasmic reticulum (Ellenberg et al., 1997).

An NE disassembly/reassembly phenomenon analogous tothat taking place at mitosis occurs following fertilization whenthe sperm NE is replaced to form the male pronuclearenvelope (Poccia and Collas, 1996). Evidence from the seaurchin suggests that the bulk of the male pronuclear envelopeis derived in vivo from the egg ER (Longo and Anderson,1968; Longo, 1973). Extensive rearrangements of the ER occursoon after fertilization, before or concomitant to appearanceof the male pronucleus in sea urchins (Terasaki and Jaffe, 1991)and starfish (Terasaki et al., 1996), but it is unclear if suchrearrangements are related to the formation of the malepronuclear envelope.

Male pronuclear envelope assembly has been studied insome detail in a cell-free system derived from sea urchingametes (Cameron and Poccia, 1994). Following fertilization,the sperm NE is disassembled except in the acrosomal andcentriolar fossa regions where remnants are retained, later tobe incorporated into the new NE. These remnants, originallydescribed in vivo by Longo and Anderson (Longo andAnderson, 1968) are also called lipophilic structures (Collasand Poccia, 1995b). These structures have been shown to berequired to initiate binding of at least three distinct popula-tions of membrane vesicles of the egg cytoplasm to the surfaceof sperm chromatin in vitro (Collas and Poccia, 1996). Most ofthe new NE is derived in vitro from the membrane population

EJCB10 European Journal of Cell Biology 79, 10 ± 16 (2000, January) ´ � Urban & Fischer Verlag ´ Jenahttp://www.urbanfischer.de/journals/ejcb

0171-9335/00/79/01-10 $12.00/0

1) Dr. Philippe Collas, Institute of Medical Biochemistry, Faculty ofMedicine, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo/Norway, e-mail: [email protected], Fax: � 472285 1497.

that contains an ER marker enzyme (Collas and Poccia, 1996).At 10 min post-fertilization, this population (designatedMV2b) consists of at least two functionally distinct membranevesicle subsets, one associated with lamin B and another withlamin B receptor (LBR). Initially, LBR-containing vesiclesbind to the nuclei and fuse with other vesicles to form nuclearmembranes from which lamin B is absent (Collas et al., 1996).Soluble lamin B is transported into the nuclei at a later stage ofNE assembly, and lamina formation, together with additionalmembrane fusion events, results in swelling of the malepronucleus (Collas et al., 1996).

Formation of the male pronuclear envelope does not takeplace in unfertilized eggs (Cothren and Poccia, 1993) or infertilized eggs whose alkalinization response characteristic ofegg activation is blocked (Carron and Longo, 1980). It is notknown whether ER rearrangement following normal activa-tion may be responsible for failure of pronuclear envelopeassembly in unfertilized or incompletely activated eggs.

In this paper, we report the separation of membrane vesiclesof unfertilized eggs and fertilized eggs at various phases of thefirst cell cycle resulting from gentle homogenization andfloatation. We find rearrangements of egg membrane domainscontaining lamin B and LBR markers occur after fertilizationlending biochemical support to the confocal observations ofTerasaki and Jaffe (Terasaki and Jaffe, 1991). In addition, weasked whether the deficiency in pronuclear envelope forma-tion in unfertilized eggs is due to incompetent membranerearrangements or cytosolic deficiencies, or both. Our dataindicate that pronuclear membrane assembly occurs only whenboth cytosol and membranes originate from fertilized eggs.

Materials and methods

Buffers, reagents and antibodiesEgg lysis buffer, nuclear preparation buffer and membrane wash buffer(MWB) were as described previously (Collas and Poccia, 1998). Thelipophilic dyes 3,3'-dihexyloxacarbocyanine iodide (DiOC6, DiO) andoctadecyl indocarbocyanine (DiIC18, DiI) were from Sigma (St. Louis,MO/USA) and Molecular Probes (Eugene, OR/USA), respectively.The chicken polyclonal antibody against sea urchin lamin B ofStrongylocentrotus purpuratus (a gift from Jon Holy, University ofMinnesota, Duluth; Holy et al., 1995) recognizes a 65/68 kDa B-typelamin in Lytechinus pictus gametes and male pronuclei assembled invitro (Collas et al., 1995; 1996). Affinity-purified rabbit polyclonalantibodies against human LBR were a gift from J.-C. Courvalin (CNRS,Institut J. Monod, Paris, France) and were used as described previously(Collas et al., 1996; Buendia and Courvalin, 1997). The anti-LBRantibody recognizes a 56-kDa integral protein of the inner nuclearmembrane in sea urchin NEs (Collas et al., 1996). HRP-, FITC- andTRITC-conjugated secondary antibodies were from Sigma.

Nuclei, egg extracts and membrane vesiclesSperm nuclei of L. pictus were permeabilized with 0.1% Triton X-100 asdescribed previously (Collas and Poccia, 1995b). Demembranatednuclei were washed and resuspended to �108 nuclei/ml. Nuclei werediluted 25-fold and added to egg extracts to a final ratio of �1 spermnucleus per egg equivalent. Embryonic nuclei were purified fromgastrula-stage L. pictus embryos as described previously (Collas, 1998).

Membrane-containing cytoplasmic extracts and membrane-freecytosolic extracts were prepared from unfertilized or fertilized eggsas described previously (Collas and Poccia, 1998). Briefly, dejelliedunfertilized or fertilized eggs were homogenized through a 22-gaugeneedle in lysis buffer and the lysate was cleared at 10000g for 10 min.The supernatant, referred to as cytoplasmic extract or S10, consists of

cytosol and cytoplasmic membrane vesicles. The S10 was either usedfresh or frozen in liquid nitrogen and stored at ÿ 80 8C. Alternatively,the S10 was fractionated at 150000g for 3 h in a Beckman SW41 rotor at4 8C over a 2 M sucrose cushion made in MWB. The supernatant,referred to as cytosolic extract or S150, was free of cytoplasmicmembrane vesicles, and was either used fresh or frozen. Sedimentedmembrane vesicles were processed as described below. Mitoticcytosolic extracts were prepared from L. pictus embryos at 100 minpost-fertilization at 15 8C as described (Collas, 1998). In order to be ableto detect solubilized pronuclear lamin B in the cytosol, the mitoticextracts were immunodepleted of soluble lamin B prior to nuclearenvelope disassembly reactions as described earlier (Collas et al.,1996).

Membrane vesicles sedimented onto a 2 M sucrose cushion at150000g from unfertilized or fertilized egg cytoplasmic extracts werecollected, resuspended in MWB, washed twice in MWB (Collas andPoccia, 1996) and resuspended in 1 ml of 2 M sucrose made in MWB.Vesicles were fractionated by floatation to density equilibrium into a2.0 ± 0.2 M linear sucrose gradient at 150000g in a Beckman SW28 rotorat 4 8C for 24 h. Twenty-three fractions of 0.5 ml were recovered bydripping from the bottom of the tube. Protein concentrations andcontents of each fraction were determined as described in Results.

Membrane vesicle binding and fusion assaysDemembranated nuclei were decondensed for 50 min at room tem-perature in S10 or S150 extracts from unfertilized or fertilized eggs, asindicated, containing an ATP-generating system (Cameron and Poccia,1994). Membrane vesicles from either unfertilized or fertilized eggswere also added to S150 extracts. GTP (100 mM) was added to thereactions to promote vesicle fusion (Collas and Poccia, 1995b). Tovisualize vesicle binding to chromatin or fusion, the extracts wereunderlaid with 1 M sucrose, nuclei were sedimented for 15 min at 500g,resuspended in fresh S150, labeled with 0.1 mg/ml DiO and examined byfluorescence microscopy. Vesicle binding was characterized by unevenmembrane labeling on the chromatin surface, whereas fusion wasmanifested by thinner and even membrane labeling, reflecting smooth-ing of the nuclear membranes (see Results). Fusion was verified by asubsequent nuclear swelling assay as described previously (Collas andPoccia, 1995a). Only nuclei with fused membranes were capable ofswelling. Fluorescence microscopy and photography were carried outas described (Collas and Poccia, 1995b).

Nuclear envelope disassemblyEmbryonic nuclei were incubated in mitotic cytosol (5� 105 nuclei per500 ml cytosol) at room temperature. Nuclear disassembly was initiatedby the addition of an ATP-generating system (Collas, 1998). After 1 h,the chromatin was sedimented at 1000g for 10 min and disassemblednuclear envelope-derived vesicles collected by centrifugation at100000g for 1 h after diluting the supernatant with an equal volumeof MWB. Sedimented membranes were washed twice in MWB andeither dissolved in SDS sample buffer or resuspended in 500 ml ofimmunoprecipitation (IP) buffer for immunoprecipitation experiments(see below).

Immunological proceduresImmunoblotting analysis was performed as described (Collas et al.,1996). Briefly, proteins were resolved by SDS-PAGE in 10% poly-acrylamide gels, transferred to nitrocellulose and detected with anti-lamin B (1 :1000 dilution) or anti-LBR antibodies (1 :500 dilution),followed by HRP-conjugated secondary antibodies. Blots were scannedwet, and signals were quantified from duplicate blots using the UVPGel-Blot Pro software (UVP, Cambridge, UK).

For immunoprecipitation experiments, sucrose was dialyzed out ofeach vesicle fraction overnight at 4 8C against MWB, and fractions werediluted to 1 ml with IP buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl,1 mM PMSF and 10 mg/ml each of aprotinin, leupeptin, pepstatin A)without or with 1% Triton X-100, as indicated. LBR was immunopre-cipitated from intact vesicles or Triton X-100-solubilized vesicles withanti-LBR antibodies (1 :50 dilution) at room temperature for 2.5 h,

11Egg membrane rearrangements at fertilizationEJCB

followed by incubation with protein A/G-agarose for 1 h and centri-fugation at 4000g for 10 min. Immune complexes were washed threetimes in IP buffer and proteins were eluted in boiling 2� SDS samplebuffer.

Results

Uniform distribution of egg cytoplasmicvesicles following floatation in sucrosegradientsWe have previously fractionated fertilized sea urchin eggcytoplasmic membrane vesicles by sedimentation to densityequilibrium into populations of different densities, whichharbored specific biochemical markers and displayed distinctchromatin binding and fusion properties (Collas and Poccia,1996). In the present study, to avoid possible carry over effectsof vesicles of low density into fractions of higher density,vesicles were fractionated by floatation to density equilibriuminto linear sucrose gradients. To determine whether distinctfractions were obtained, as by sedimentation (Collas andPoccia, 1996), the distribution of vesicles from unfertilizedeggs, eggs at 12 min post-fertilization or mitotic embryos(100 min post-fertilization) was determined. Twenty-threefractions were collected, labeled with the lipophilic dye DiI,vesicles were washed, and fluorescence intensity was deter-mined by fluorometry. Fig. 1a shows that, regardless of theorigin of the vesicles, little variation in fluorescence intensitywas observed across the gradients (fractions 1 ± 20), indicatingthat vesicles were distributed uniformly in each gradient. Thisobservation was supported by the profile of protein concen-tration measured in each fraction (Fig. 1b). Thus, in contrast toour previous sedimentation results, vesicle fractionation byfloatation led to a uniform distribution of vesicles rather thanclearly distinct populations.

Segregation of lamin B and LBR into differingmembrane domains at fertilizationExamination of fertilized sea urchin eggs in vivo aftermicroinjection of a fluorescent lipophilic dye (DiI) previouslysuggested extensive reorganization of the egg ER at fertiliza-tion (Terasaki and Jaffe, 1991). To provide biochemicalevidence for such ER reorganization, we determined thedistribution of lamin B and LBR before and after fertilization.Both proteins were previously identified in ER-enriched eggvesicles (Collas and Poccia, 1996) and are markers of thenuclear lamina and of the inner nuclear membrane, respec-tively. To this end, vesicles from unfertilized eggs or fertilizedeggs homogenized at 3 min, 12 min (G1 phase) or 60 min (Sphase) post-fertilization were fractionated by floatation asabove. Fractions were collected, proteins immunoblotted usinganti-lamin B or anti-LBR antibodies and relative proportionsof each marker determined by densitometric analysis of theblots. In unfertilized eggs, most of lamin B and LBR weredetected in vesicles of similar densities (Fig. 2a). As early as3 min post-fertilization, both proteins were more widelydistributed across the gradients (Fig. 2b) whereas by 12 min,most of lamin B and LBR were found in vesicles of differentdensities (Fig. 2c). These results indicate a dramatic redistri-bution of lamin B and LBR within minutes of fertilization,reflecting a rapid reorganization of cytoplasmic membranedomains. Nevertheless, partitioning of lamin B and LBRestablished in G1 was maintained throughout S phase

(Fig. 2d), suggesting that no or little membrane rearrangementoccurs during this stage of the first embryonic cell cycle.

To unambiguously determine whether lamin B and LBRsegregated into different vesicle populations at fertilization,LBR was immunoprecipitated from vesicle fractions display-ing at least partially overlapping lamin B and LBR distributionboth before and at 12 min after fertilization (fractions 12 ± 17;Fig. 2a, c). Immunoprecipitations were carried out with orwithout 1% Triton X-100 and immune precipitates wereimmunoblotted using anti-LBR or anti-lamin B antibodies.Immunoblotting of immune precipitates with anti-LBR anti-bodies revealed that LBR was successfully immunoprecipita-ted (Fig. 3a ± c, upper panels). Furthermore, Fig. 3a (lowerpanel) shows that, without detergent, lamin B co-precipitatedwith LBR in fractions 12 to 17 from unfertilized eggs,indicating that both proteins co-localized in the same vesicles.Following solubilization of these vesicles with 1% Triton X-100however, lamin B did not co-precipitate with LBR (Fig. 3b,lower panel), indicating that, although they resided in the samevesicles, lamin B and LBR did not interact. In vesicles fromfertilized eggs (Fig. 3c, fractions 12 ± 17), lamin B did not co-precipitate with LBR in the absence of detergent, arguing thatthese proteins were localized into distinct vesicles. Thus, in

12 P. Collas, T. Barona, D. L. Poccia EJCB

Fig. 1. Uniform distribution of sea urchin egg cytoplasmic membranevesicles across gradients following floatation to density equilibrium. a.Intensity of the lipophilic dye DiI across gradients. Membrane vesiclesprepared from unfertilized eggs (.), eggs at 12 min post-fertilization(&) or mitotic eggs at 100 min post-fertilization (~) were fractionatedby floatation to density equilibrium in sucrose gradients. Twenty-threefractions were collected and labeled with 10 mg/ml DiI. Vesicles werewashed and fluorescence intensity was measured by fluorometry. P,residual pellet. b. Mitotic membrane vesicles were fractionated byfloatation as above, protein concentration of each fraction (0.5 ml) wasdetermined (.) and fraction density measured by weight (*).

unfertilized eggs, most of lamin B and LBR are localized in thesame vesicles but are not physically associated. Subsequentmembrane rearrangements at fertilization result in segregationof these components into distinct membrane domains.

Lamin B and LBR interact in a minor subset ofvesicles at mitosisFractionation of vesicles prepared from embryos at firstmitosis (100 min post-fertilization at 15 8C) showed that littleegg membrane rearrangement had occurred compared to Sphase. Immunoblotting and densitometric analyses revealedthat, as in S phase, the majority of lamin B and LBR re-mained localized in vesicles of different densities (Fig. 4a).Nevertheless, immunoprecipitation of LBR in the presenceof 1% Triton X-100 from fractions exhibiting partiallyoverlapping lamin B and LBR (fractions 12 ± 17; Fig. 4a,graph) did co-precipitate lamin B in a subset of thesevesicles (Fig. 4b, fraction 14). This indicates that, while laminB and LBR mostly remain segregated into distinct vesicles atmitosis, a small proportion of each protein physically interactsin a minor subset of vesicles. It is also noteworthy that,although lamin B and LBR were found in vesicles of similardensities at the bottom of the sucrose gradient (Fig. 4a,fractions 1 ± 5), they did not associate in these vesicles (datanot shown).

To determine the origin of mitotic vesicles containing bothLBR and lamin B vesicles, purified L. pictus gastrula nucleiwere disassembled in a mitotic cytosolic extract previouslyimmunodepleted of endogenous lamin B (Collas, 1998). Afterremoving the chromatin, NE-derived vesicles were sediment-ed by ultracentrifugation, dissolved in IP buffer containing1% Triton X-100, and LBR was immunoprecipitated. Asexpected, both LBR and lamin B were detected in thesevesicles (Fig. 5a). Furthermore, immunoprecipitation ofLBR (Fig. 5b, top panel) co-precipitated lamin B (Fig. 5b,bottom panel). Thus, at least a proportion of vesiclesderived from disassembled NEs at mitosis contains physical-ly associated lamin B and LBR (see also Meier and Georga-tos, 1994). Similar results were obtained from analysis ofvesicles derived from male pronuclear envelopes disassem-bled in a mitotic extract (data not shown). Consequently, it islikely that the mitotic vesicle fraction harboring both lamin Band LBR (fraction 14; Fig. 4b) is enriched in NE-derivedvesicles.


Fig. 2. Redistribution of lamin B andlamin B receptor (LBR) in sea urchin eggcytoplasmic membranes at fertilization.Membrane vesicles were isolated fromhomogenates of (a) unfertilized eggs, and(b ± d) pronuclear stage embryos at (b)3 min, (c) 12 min (G1 phase) and (d)60 min (S phase) post-fertilization. Vesicleswere floated to density equilibrium insucrose gradients. Fractions were collectedas in Fig. 1 and immunoblotted using anti-lamin B (.) and anti-LBR (*) antibodies.Relative proportions of each protein weredetermined by densitometric analysis ofduplicate blots. Fractions with the greatestamount of lamin B or LBR were given anarbitrary value of 100. P, residual pellet.Bars underline vesicle fractions used forimmunoprecipitation experiments shownin Fig. 3.

Fig. 3. Lamin B and LBR segregate into distinct membrane domainsat fertilization. Vesicles were isolated from (a, b) unfertilized eggs or (c)eggs at 12 min post-fertilization, and fractionated by floatation insucrose gradients as in Fig. 2. Vesicles of fractions 12 ± 17 wereimmunoprecipitated using affinity-purified rabbit anti-LBR antibodiesin (a, c) IP buffer without Triton X-100 (ÿTX) or (b) in IP buffercontaining 1% Triton X-100 (�TX). Immune complexes wereimmunoblotted using either anti-LBR or anti-lamin B antibodies.HC, IgG heavy chain.

Vesicles or cytosol from unfertilized eggs donot support male pronuclear envelopeassemblyTo address the biological significance of the redistribution ofegg cytoplasmic membrane vesicles at fertilization, we exam-ined the ability of vesicles derived from unfertilized orfertilized eggs to assemble a male pronuclear envelope.Demembranated sperm nuclei were incubated in membrane-free S150 extracts from fertilized or unfertilized eggs that weresupplemented with vesicles of either origin. An ATP-generat-ing system and 100 mM GTP were added to promote chromatindecondensation, vesicle binding to chromatin and fusion.Vesicle binding to chromatin and fusion were allowed toproceed for 50 min, nuclei were sedimented through a sucrosecushion and chromatin-bound or fused membranes werelabeled with DiO. The results are shown in Fig. 6a and Table I.In fertilized egg cytosol, vesicles from both fertilized orunfertilized eggs were capable of binding to chromatin, butonly vesicles from fertilized eggs were capable of fusing in thepresence of GTP. This was shown by the more even distribu-tion of the lipophilic dye around the chromatin periphery,reflecting smoothing of the nuclear membranes as a result offusion (Fig. 6a, top left panel), as well as subsequent nuclear

swelling, a characteristic of nuclei containing sealed mem-branes (Collas et al., 1996) (data not shown). The inability ofunfertilized egg cytosol or membranes to support NE assemblywas verified by allowing vesicle binding and fusion in S10


Fig. 4. Lamin B and LBR are localized mostly in distinct vesicles atmitosis, but also interact in a minor subset of vesicles. a. Vesiclesprepared from mitotic eggs were fractionated by floatation in a sucrosegradient and the distribution of lamin B and LBR across the gradientwas examined by immunoblotting as in Fig. 2 (upper panels). Relativeproportions of each protein were determined by densitometry (graph).P, residual pellet. b. Vesicles of fractions 12 ± 17 were immunoprecipi-tated using anti-LBR antibodies in the presence of 1% Triton X-100and immune complexes immunoblotted using anti-lamin B antibodies.Lamin B and LBR associate primarily in fraction 14 (note that lamin Band LBR were not found to be associated in fractions 1 ± 5; data notshown). HC, IgG heavy chain.

Fig. 5. Lamin B and LBR co-localize and associate in NE-derivedmitotic vesicles. a. Immunoblotting analysis of lamin B and LBR in NE-derived vesicles. Purified L. pictus gastrula nuclei were disassembled inmembrane-free mitotic cytosolic extract, the chromatin was removed,NE-derived vesicles were sedimented and their proteins immunoblot-ted using anti-LBR (a-LBR) and anti-lamin B (a-LB) antibodies. b.NE-derived vesicles were solubilized in detergent-containing IP bufferand LBR was immunoprecipitated. Immune complexes and controlprecipitates (Pre-imm.) were analyzed by immunoblotting using anti-LBR (top panel) or anti-lamin B (bottom panel) antibodies. HC, IgGheavy chain.

Fig. 6. Vesicles or cytosol prepared from unfertilized eggs do notsupport nuclear vesicle fusion and NE assembly. a. Membrane vesiclesand cytosolic extracts were prepared from fertilized eggs at 12 minpost-fertilization (Fert.) or from unfertilized eggs (Unfert.). Demem-branated sperm nuclei were incubated for 50 min in either extractcontaining vesicles of either origin as indicated, an ATP-generatingsystem and GTP. Vesicle binding to chromatin and fusion were allowedto proceed, nuclei were purified through a sucrose cushion andmembranes labeled with 0.1 mg/ml of the lipophilic dye DiO. b. Tocontrol for binding and fusion activities, sperm nuclei were incubated inS10 extracts from fertilized (b'') or unfertilized (b'''') eggs. Membranebinding and fusion were assessed as in (a). Note the lack of fusion(nuclear membrane smoothing) with either extract or vesicles fromunfertilized eggs in (a) and (b). Bars, 5 mm.

membrane-containing extracts from fertilized or unfertilizedeggs. As anticipated from the previous results, only thefertilized S10 extract was capable of promoting nuclear vesiclefusion and NE formation (Fig. 6b). Because neither egg cytosolnor membranes of unfertilized eggs support fusion under theconditions tested, these results suggest that soluble andmembrane-associated components essential for male pronu-clear envelope formation are missing or inactive in unfertilizedeggs.

Discussion

Unfertilized sea urchin eggs are arrested in a G0 state aftercompletion of both meiotic divisions. Upon fertilization, theyenter the first embryonic cell cycle with a 30 min G1 phase andan S phase of approximately 50 min, after which they entermitosis, with a brief or no G2 phase (Hinegardner et al., 1964).Soon after fertilization, the egg ER undergoes a globalreorganization resulting in an apparently more fragmentedstructure, which then is restored during G1 (Terasaki and Jaffe,1991). Likewise, in starfish, fluorescence recovery afterphotobleaching data using DiI or a green fluorescent proteintargeted to the ER lumen provided evidence for a transientloss of continuity of the ER at fertilization (Terasaki et al.,1996). The significance of this apparent membrane rearrange-ment is unknown.

We had previously shown that the bulk of the sea urchin eggcytoplasmic membranes remaining in the supernatant aftergentle egg homogenization of G1-phase fertilized eggs (frac-tion MV2b) contained a marker enzyme for ER (a-glucosi-dase) (Collas and Poccia, 1996). MV2b also contains twomarker proteins associated with the inner nuclear membrane,lamin B and LBR (Collas and Poccia, 1996). We find that inunfertilized egg homogenates, these markers are contained inthe same vesicles but do not physically interact. Withinminutes of fertilization, lamin B and LBR are associatedwith vesicles of a range of densities and by 12 min they are inclearly separate vesicles of different densities. This distributionremains unchanged during S phase but at mitosis, a minorfraction of membrane vesicles contains both lamin B and LBRphysically associated, which most likely results from NEbreakdown. Our biochemical results are consistent with thosederived from previous confocal microscopy observations in seaurchins (Terasaki and Jaffe, 1991) and starfish (Terasaki et al.,1996), and suggest that a major qualitative rearrangement of

egg ER occurs in response to fertilization. Since sea urchineggs are fertilized after completion of both meiotic divisions,no data were obtained in the present study on the distributionof NE markers in the oocyte during meiosis. Nevertheless,recent results from Gajewski and Krohne (Gajewski andKrohne, 1999) suggest that reorganization of ER membranesalso takes place during resumption of meiosis in Xenopus eggs,as illustrated by alterations in the distribution of p58/LBR andlamin B in total cytoplasmic egg membranes fractionated bysucrose step gradient centrifugation.

The segregation of two marker proteins of the NE uponqualitative rearrangement of the sea urchin egg ER inresponse to fertilization may have functional implications.The contribution of the MV2b fraction to the NE is accom-plished in vitro in two separate steps. In the first, MV2bvesicles containing LBR (which targets them to the chromatin)assemble and fuse. In the second, addition of a lamina fromboth the soluble pool and the MV2b-associated store of laminB occurs (Collas et al., 1996). This implies that the two markerproteins exist in separate vesicles at least prior to envelopeformation in G1 which would facilitate their independenttargeting. Such sorting of ER domains by protein type mayalso be characteristic of other important membrane-associatedproteins of the NE and possibly for other membrane traffic aswell. If this sorting were extensive, it would perhaps accountfor the morphological observations of Terasaki and Jaffe(Terasaki and Jaffe, 1991).

Our second set of experiments was prompted by theobservation that unfertilized or unactivated eggs appear tobe deficient in assembling the male pronuclear envelope(Carron and Longo, 1980; Cothren and Poccia, 1993). Onepossible explanation for this deficiency would be an inability ofunsorted membranes to bind to the chromatin or fuse. Asecond possibility is that alkalinization or other cytosolicconsequences of egg activation were unrealized. We thereforetested the ability of sorted membranes from G1 fertilized eggsto form NEs in unfertilized egg cytosol but at the higher pH ofthe activated fertilized eggs. This was unsuccessful, indicatingthat unsorted membranes cannot be the sole deficiency inunfertilized eggs. In addition, cytosol from fertilized eggs didnot bring about fusion of unsorted membranes from unfer-tilized eggs. These results indicate that both ªactivatedºcytosol and sorted membranes are needed to form the malepronuclear envelope, and that pH is not the factor missing inunfertilized egg cytoplasm.

Another intriguing observation of Terasaki and Jaffe(Terasaki and Jaffe, 1991) was that during male pronuclearmigration, a concentration of cytoplasmic membranes sur-rounds the male pronucleus and eventually surrounds thezygote nucleus. This membrane concentration does not seemto emanate from the centrosome. It was not ascertainedwhether this membrane cloud was a reflection of a concentra-tion of membrane vesicles or a distortion of the otherwisefairly evenly distributed endoplasmic reticular network. Ineither case, however, the observation points to a spatialrelationship between cytoplasmic membranes and the malepronucleus. It will be of interest to determine which of the twocomponents orchestrates this remarkable association.

Acknowledgements. The authors are thankful to Dr. J.-C. Courvalin(CNRS, Institut Jacques Monod, Paris, France) for the generous gift ofanti-LBR antibodies, Dr. J. Holy (University of Minnesota, Duluth) forthe gift of anti-lamin B antibodies, and Christina Wiedl (AmherstCollege) for assistance in the laboratory. This work was supported by


Tab. I. Proportions of binding to chromatin and fusion ofcytoplasmic vesicles from unfertilized or fertilized eggs in unfer-tilized or fertilized egg cytosol.

% Binding % FusionSource of cytosol Source of vesicles (� SD)a (� SD)a

Fert. S10 ± 89� 10.4 84� 11.5Unfert. S10 ± 88� 15.6 0� 0Fert. S150 Fert. 87� 7.6 82� 6.9Fert. S150 Unfert. 85� 3.5 0� 0Unfert. S150 Fert. 76� 5.2 0� 0Unfert. S150 Unfert. 87� 10.5 0� 0

a Vesicle binding to sperm chromatin and fusion were assessed asdescribed in the text. Data are from 3 ± 4 separate experiments includingover 100 nuclei per treatment.

NIH grant GM55970 and an Amherst College Faculty Research Awardto D. L. Poccia, and grants from the Norwegian Cancer Society and theNorwegian Research Council to P. Collas.

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rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization

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