Strongylocentrotus drobachiensis oocytes maintain a microtubule

MOLECULAR REPRODUCTION AND DEVELOPMENT 74:76–87 (2007)

Strongylocentrotus drobachiensis OocytesMaintain a Microtubule Organizing CenterThroughout Oogenesis: Implications for theEstablishment of Egg Polarity in Sea UrchinsANA L. EGANA, JUDITH A. BOYLE, AND SUSAN G. ERNST*

Department of Biology, Tufts University, Medford, Massachusetts

ABSTRACT Although it has been known forover a century that sea urchin eggs are polarized cells,very little is known about the mechanism responsiblefor establishing and maintaining polarity. Our previousstudies of microtubule organization during sea urchinoogenesis described a cortical microtubule-organizingcenter (MTOC) present during germinal vesicle (GV)migration in large oocytes. This MTOC was localizedwithin the future animal pole of the mature egg. In thisstudy we have used electron microscopy and immu-nocytochemistry to characterize the structure of thisMTOC and have established that this organelle appearsprior to GV migration. We show that the cortical MTOCcontains all the components of a centrosome, includinga pair of centrioles. Although a centrosome proper wasnot found in small oocytes, the centriole pair in thesecells was always found in association with a striatedrootlet, a structural remnant of the flagellar ap-paratus present in precursor germinal cells (PGCs).The centrioles/striated rootlet complex was asymme-trically localized to the side of the oocyte closest tothe gonadal wall. These data are consistent with thepreviously proposed hypothesis that in echinodermsthe polarity of the PGCs in the germinal epitheliuminfluences the final polarity of the mature egg. Mol.Reprod. Dev. 74: 76–87, 2007.� 2006 Wiley-Liss, Inc.

Key Words: sea urchin; oogenesis; centrosome;centrioles; gamma-tubulin; pericentrin; centrin; polar-ity; striated rootlet

INTRODUCTION

Early embryonic development relies on asymmetri-cally localized information found in the mature egg toestablish initial embryonic axes and to specify cell fates.In echinoderms the question of how polarity is estab-lished in the egg has been explored in holothuroids(Frick et al., 1996). However, echinoderm embryogen-esis has been most extensively studied using the seaurchin embryo as a model system. The integration of ourknowledge of how sea urchin development occurs andthe results from studies about cell polarity establish-ment in the sea urchin egg will provide us with a

comprehensive understanding of the mechanismsinvolved in echinoderm embryonic development.

The unequal developmental potential of the seaurchin egg is well known. When unfertilized eggs aredissected equatorially and the fragments fertilized, theanimal half develops into a ciliated ball of cells, while thevegetal half develops into a small sea urchin larva(reviewed in Horstadius, 1973). These classic experi-ments suggested a difference in developmental informa-tion between the animal and vegetal halves of themature egg. After the asymmetric fourth cleavage, themicromeres arise in the most vegetal part of the cleavingembryo. Several recombination and transplantationexperiments (Horstadius, 1973) have shown that themicromeres act as the inductive center of the sea urchinembryo and are directly responsible for the inductionof the endomesoderm (Ransick and Davidson, 1995;Davidson et al., 2002). It has been proposed that micro-meres are specified by the acquisition of vegetally storedmaternal factors requiring that, as in other systems,sea urchin early embryogenesis is dependent on eggpolarity (reviewed in Davidson, 1989; Davidson et al.,2002).

The localization of maternal mRNAs and proteins inthe maturing oocyte of a number of organisms has beenshown to be a result of the activity of the cytoskele-ton (i.e., Theurkauf, 1994; St. Johnston, 1995). In themature sea urchin egg several asymmetrically localizedfactors have been identified to date. For example, bep 1and bep 4 mRNAs, members of a multigene family thatencode cell-surface proteins, are localized in only onehalf of the egg (Di Carlo et al., 1990, 1994, 1996;

� 2006 WILEY-LISS, INC.

Grant sponsor: National Academy of Sciences; Grant sponsor: BettyVernon and Charles Taylor Vernon Fund for Biological Research;Grant sponsor: Tufts University Faculty Research Award.

Ana L. Egana’s present address is New England Biolabs, 240 CountyRoad, Ipswich, Massachusetts.

Judith A. Boyle’s present address is Department of Biochemistry andMolecular Biology, Colorado State University, Fort Collins, Colorado.

*Correspondence to: Susan G. Ernst, Department of Biology, TuftsUniversity, Medford, MA 02155. E-mail: [email protected]

Received 8 January 2006; Accepted 27 February 2006Published online 23 August 2006 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/mrd.20511

Romancino et al., 1992). Another mRNA that is asym-metrically localized in the mature sea urchin egg isSpCOUP-TF, which encodes a homologue of the verte-brate nuclear receptors COUP-TFs (Chan et al., 1992;Vlahou et al., 1996). The fibrillar collagen COLL1mRNA also localizes asymmetrically in the mature seaurchin egg (Gambino et al., 1997). Although no studieshave been published on the role of the cytoskeleton in thelocalization of SpCOUP-TF mRNA, it has been demon-strated that microtubules play a key role in facilitatingthe proper asymmetrical distribution of bep1, bep4 andCOLL1 mRNAs in the mature sea urchin egg (Roman-cino et al., 1998, 2000).

Sea urchin oocytes contain complex networks ofmicrofilaments, microtubules, and intermediate fila-ments (Boyle and Ernst, 1989; Bonder and Fishkind,1995). In a previous study we described a microtubule-organizing center (MTOC) present at the time of GVmigration in sea urchin oocytes (Boyle and Ernst, 1989).The MTOC found in sea urchin oocytes is located in theanimal cortex. Thus, the microtubular composition ofthe cortical area where the MTOC is located during GVmigration is different from that of any other area of thesea urchin oocyte cortex (Boyle and Ernst, 1989). Thesefindings beg the question of whether the assembly of thecortical MTOC occurs prior to or after the migration ofthe GV. If the MTOC is assembled after the movement ofthe GV towards the cortex, this would indicate that themechanisms establishing egg polarity in sea urchins actduring the late stages of oogenesis. However, if theMTOC is assembled prior to GV migration, then theorigin of cell polarity is to be found in the early stages ofoogenesis.

In the context of when the MTOC is formed, and howthis relates to the establishment of egg polarity duringoogenesis, it is also important to understand the natureand dynamics of MTOC assemblage. Unlike vertebratesystems in which the centrosome gets disassembledduring oogenesis (i.e., Gard et al., 1995), some inverte-brate systems appear to maintain or resurrect thecentrosome during meiosis. Conklin’s classic work withCrepidula fornicata oocytes showed that Crepidulaoocytes partially disassemble their centrosomes duringoogenesis resulting in the apparent loss of their cen-trioles. However, during meiosis I the oocytes are able toassemble maternal asters and eventually direct theformation of centrioles within the structures, knownas protocentrosomes, that drive the formation of thematernal meiotic asters (Conklin, 1902). Some evidencesuggests that the same is true for Spisula oocytes(reviewed in Palazzo et al., 2000). Also, in starfishoocytes the meiotic asters are organized by centriolarcentrosomes (Sluder et al., 1989; Kato et al., 1990;Uetake et al., 2002). Our previous observations that aMTOC was present during GV migration (early pro-phase of meiosis I) did not clarify whether the MTOCpresent at this stage in sea urchin oocytes had themolecular composition of a classical centrosome. How-ever, the ability of the GV-migration stage MTOC toassemble a robust microtubule aster (Boyle and Ernst,

1989) suggested that this MTOC probably was nothomologous to the protocentrosome identified by Con-klin in Crepidula.

The results reported here on the study of the natureand time of formation of the sea urchin MTOC in thecontext of sea urchin oogenesis is an additional step inunderstanding the mechanisms involved in generatingegg polarity and centrosome dynamics during echino-derm oogenesis.

MATERIALS AND METHODS

Animals

Strongylocentrotus drobachiensis were obtained fromMarine Biological Laboratory (MBL), Woods Hole, MA.They were kept at 88C in sea water aquariums and fedseaweed three times per week.

Electron Microscopy—Chemical Fixation

Gonads were dissected from S. drobachiensis femalesinto calcium-free sea water (CFSW) (MBL formula) andcut into small fragments. Tissue fragments and looseoocytes were fixed for immunocytochemistry (see below)or electron microscopy. Gonadal tissue and oocytes usedfor electron microscopy were fixed in 1% glutaraldehydeand 1% paraformaldehyde in CFSW pH 6.8 for 1.5 hr at15–188C (Begg et al., 1982). They were washed 3� for15 min in CFSW at 15–188C, postfixed on ice with 0.5%osmium tetroxide in 0.1 M sodium phosphate buffer pH6.0 for 30 min, rinsed several times in ice cold distilledwater, and left overnight at 48C. The tissue was staineden bloc in 1% aqueous uranyl acetate for 1 hr at roomtemperature, washed with distilled water, dehydratedin a graded ethanol series (10%, 20%, 30%, 50%, 70%,95%, and three changes in 100%), and embedded in LRWhite resin (Polysciences, Inc. Warrington, PA). Theembedding procedure was as follows: 2:1 (resin: 100%ethanol) for 2 hr at room temperature with gentlerotation; 100% resin overnight at room temperaturewith gentle rotation; a fresh change in 100% resin thenext day for 1–2 hr at room temperature with gentlerotation before embedding. Tissue pieces wereembedded in gelatin capsules while oocytes and eggswere flat embedded in a single layer between Tefloncoated slides. Both thin (90 nm) and thick (0.25–0.5 mm)sections were cut with a diamond knife on a ReichartOMU2 ultramicrotome, collected on formvar-coatedcopper slot grids, and stained in 1% aqueous uranylacetate followed by Sato’s lead stain (Sato, 1968).Sections were viewed and photographed using a JEOL100B transmission electron microscope (TEM) or thehigh-voltage electron microscope (HVEM) at the Uni-versity of Colorado in Boulder.

Electron Microscopy—HighPressure Fast Freezing

Gonadal tissue fragments and oocytes were collectedmanually by micropipette, mixed with 1-hexadecane, acryoprotectant, and loaded into specimen holders. Theywere then subjected to freezing under high pressure

Molecular Reproduction and Development. DOI 10.1002/mrd

CENTRIOLESASMARKERSOFOOCYTEPOLARITYDURINGSEAURCHINOOGENESIS 77

using a Balzars HPM 010 (Balzars Corporation,Hudson, NH), freeze substituted and embedded asdescribed by McDonald and Morphew (1989). Tissuefragments were embedded in gelatin capsules, andoocytes flat embedded, sectioned, and viewed asdescribed above.

Immunocytochemistry

Eggs and oocytes obtained from gonadal dissectionswere treated with 50 mM DL-dithiothreitol (DTT) inartificial sea water (MBL formula) for 7 min to removethe vitelline layer. They were allowed to settle on slidescoated with 1 mg/ml poly-L-lysine (Sigma, St. Louis, MO).Eggs and oocytes were then fixed at room temperaturefor 10 min in �208C 90% methanol. Material wasrehydrated overnight in PBS (0.13 M NaCl, 2.2 mMKCl, 0.1 M Na2HP04, 1.7 mM KH2P04) at 48C.Rehydrated material was blocked for 1 hr at roomtemperature in 5% bovine serum albumin (BSA) in PBS.Slides were washed in PBS 4� for 15 min and incubatedin 1 M hexylene glycolþ 0.15% Triton X-100 for 35 min.After a brief wash in PBS, slides were incubated with a1:200 dilution of g-tubulin monoclonal antibody (Sigma)or a 1:100 dilution of a pericentrin polyclonal antibody(Covance, Berkeley, CA) or a 1:50 dilution of the centrin20H5 monoclonal antibody (gift from J. Salisbury) for 1hr at room temperature. Slides were washed 3� for 20min at room temperature in PBS, incubated with a 1:200dilution of rhodamine-labeled anti-mouse or anti-rabbitIgG (Jackson, WestGrove, PA,ImmunoResearch Labora-tories, Inc.), and washed overnight at 48C in PBS. Theslides were then incubated with a 1:200 dilution of a-tubulin antibody (Sigma) or a 1:200 dilution of the g-tubulin antibody for 1 hr at room temperature, washedfor 2 hr (6� for 20 min) at room temperature, incubatedwith a 1:200 dilution of FITC-labeled anti-mouse IgG(Sigma), and washed overnight at 48C in PBS. Slideswere mounted using the Slowfade Light Antifade Kit(Invitrogen-Molecular Probes, Carlsbad, CA) and

viewed using an Olympus fluorescent microscope. Thespecificity of the antibodies for centrosomal proteins wasconfirmed by immunocytochemical analysis with theantibodies of mitotic and meiotic cells. Reversal in theorder of primary antibody treatment did not affect thestaining pattern exhibited by the antibodies.

RESULTS

The MTOC identified previously by immunofluores-cence microscopy (Boyle and Ernst, 1989) was analyzedultrastructurally by TEM. When individual largeoocytes (140–150 mm in diameter) containing an asym-metrically localized GV were sectioned, the MTOCexhibited the same basic morphology whether theoocytes had been fixed chemically or by high-pressurefast freezing (Fig. 1A,B). The MTOC was found to becomposed of electron-dense granules (Fig. 1C). Electron-dense granules 80–100 nm in diameter called micro-tubule-organizing granules (MTOGs) have also beenfound in the centrosome of sea urchin eggs undergoingthe first cleavage (Endo, 1979). The MTOGs observed byEndo (1979) and those described here are surrounded byamorphous pericentriolar material, which appearsdarker than the surrounding cytoplasm (Fig. 1A,C),and from which microtubules (MTs) emanate (Fig. 1C).The MTs are better preserved in the high-pressure fastfrozen oocytes (Fig. 1C) than in the chemically fixedoocytes (Fig. 1A), as was previously observed in seaurchin embryonic cells (McDonald, 1994).

The MTOC occupies a distinct area of the cytoplasm.The plasma membrane and cytoplasm in this regioncharacteristically bulge out from the oocyte surface. Ashas been shown for other echinoderms (Smiley, 1990)and for mammals (Messinger and Albertini, 1991) largeorganelles such as cortical granules, yolk, and lipiddroplets are excluded from this region (Fig. 1B). Inaddition, a larger electron-dense structure was fre-quently observed within the MTOC region of oocytesfixed using the two techniques mentioned above


Fig. 1. Analysis of the ultrastructure of large premeiotic oocyteMTOCs by electron microscopy. Electron micrographs of thin sectionstaken approximately half-way through the MTOC in large oocytes(140–150 mm in diameter). Low magnification views of the MTOCin chemically fixed (A) and high-pressure fast frozen (B) oocytesdemonstrate that the MTOC is made up of amorphous material (PCM)that surrounds electron-dense granules (or MTOGs). Numerousvesicles lie within the central region of the MTOC and within the largemicrovilli. An electron-dense structure is also present within the

MTOC (A, marked by arrow). B: The exclusion of large organelles fromthe MTOC area is illustrated in this view of a high-pressure fast frozenoocyte. C: In a higher magnification view of the pericentriolar materialof a high-pressure fast frozen oocyte the MTs (marked by small arrow)that emanate from the PCM (marked by long arrow) are seen. TheMTOGs (80–100 nm in diameter—marked by arrowhead) have astarburst appearance due to freezing damage. A: 24,000�; (B) 10,000�;(C) 100,000�.

78 A.L. EGANA ET AL.

(Fig. 1A arrow, and data not shown). Since the largeelectron-dense structure appears to correspond in size toa centriole, immunocytochemistry investigations werecarried out as described below to clarify the presence ofcentrioles in the MTOC.

To determine when the MTOC first appears duringoocyte maturation, we looked at younger oocytes for thepresence of MTOCs. Ovarian tissue fragments werethick sectioned (0.25–0.5 mm) for HVEM. Sections cutfrom these fragments contain oocytes that are stillattached to the ovarian wall as well as oocytes of varyingsizes that are no longer attached but remain withinthe tissue during the preparation process. Severalintermediate stage oocytes >115 mm and <140 mm indiameter were observed to have a MTOC. By HVEMthese MTOCs had the same general characteristics andmorphology as of those observed in the larger oocytes140–150 mm in diameter (Fig. 2B). Sections throughthese MTOCs also revealed a large electron-densestructure similar to the one seen in sections of theMTOC present in large premeiotic oocytes (Fig. 2A).

In small oocytes less than 115 mm in diameter and stillattached to the gonadal wall no MTOC, as the onedescribed above, was observed. Instead, microtubuleswere found associated with a basal flagellar apparatuscomposed of a basal bodies/striated rootlet complex(Fig. 3B–D). In all oocytes examined at this stage thecentriole/striated rootlet complex was present onthe side of the oocyte attached to the gonadal wall(Fig. 3A). These results suggest the possibility of thecontinuous presence of a microtubule-organizing orga-nelle throughout oogenesis, thus indicating that echi-noderm oocytes may maintain their cell polarity duringtheir differentiation process.

To begin investigating the proposal that a microtu-bule-organizing organelle is maintained during oogen-esis, we analyzed the localization of several centrosomalproteins during oogenesis. The first protein analyzed was g-tubulin. Gamma-tubulin is a component of the

centrosome of many cell types (Oakley and Oakley,1989; Stearns et al., 1991; Zheng et al., 1991; Liu et al.,1993) and plays an important role in microtubule-nucleation (Joshi et al., 1992; Felix et al., 1994; Stearnsand Kirschner, 1994; Li and Joshi, 1995; Shu and Joshi,1995). When whole-mount immunofluorescence wascarried out on large oocytes using a-tubulin and g-tubulin monoclonal antibodies, it was observed that theg-tubulin antibody recognized one or two structures(Fig. 4B) present within the MTOC (Fig. 4A). This samepattern of expression, with one or two structures beingidentified in the centrosome, also was observed wheneggs undergoing polar body formation were analyzedwith the g-tubulin antibody (data not shown). Controltreatment of oocytes with the secondary antibodyconjugated to rhodamine to the g-tubulin monoclonalantibody in combination with the a-tubulin primary andsecondary antibodies resulted in no rhodamine stainingof any structure within the MTOC (Fig. 4F). Likewise,no MTOC-localized FITC staining was observed whenoocytes were reacted with only the FITC-conjugatedsecondary antibody to the a-tubulin antibody in combi-


Fig. 2. MTOCs present in medium size oocytes (>115mm and<140–150 mm in diameter) are morphologically very similar to the MTOCfound in larger more mature oocytes. A, B: High-voltage electronmicrographs of thick sections through the central region of the MTOCin medium size oocytes>115 mm and<140–150 mm in diameter. B: Thepericentriolar material as well as the MTOGs are visible. A: Anothersection through the same MTOC as in (B), showing a large electron-dense structure. (A) 24,000�; (B) 57,000�.

Fig. 3. Small oocytes still attached to the gonadal wall containcentrioles associated with a striated rootlet. A,B: Chemically fixed and(C, D) high-pressure fast frozen ovarian tissues were thick sectionedand viewed by TEM. B, D: Higher magnification images of A and C,respectively. A: shows the striated rootlet/centriole complex on the sideof the oocyte that is closest to the gonadal wall (gw) and opposite to thegonadal lumen (ac-acinus). The striated rootlet extends through thecytoplasm and is found in close proximity to the germinal vesicle (gv) (A,C). The striation of the rootlet is seen (B, D). Microtubules (MT) arefound associated with the centriole/striated rootlet complex (D). In A, B,C, and D the centrioles are marked by arrowheads and the striatedrootlets are marked by arrows. A: 5,000�; (B) 14,000�; (C) 9,000�; (D)41,000�.


nation with the g-tubulin primary and secondaryantibodies (data not shown). When the double-labelimmunocytochemistry analysis using the a- and g-tubulin antibodies was extended to younger premeioticoocytes, which included oocytes that contain centrallylocated GVs, in all cases where an MTOC was identifiedwith the a-tubulin antibody (Fig. 4C), a g-tubulinpositive structure was observed within that MTOC(Fig. 4D). However, we were unable to determine thelocalization of g-tubulin in small oocytes in which it wasnot possible to clearly visualize the MTOC with thea-tubulin antibody. These results demonstrate thatthe MTOC shown to be present during GV migration(Boyle and Ernst, 1989) is assembled prior to GVmigration.

The g-tubulin staining area that was observed usingthe g-tubulin monoclonal antibody did not appear tocoincide completely with the entire area of the MTOC.Centrosomal g-tubulin is distributed in two pools: acentriolar pool (Dibbayawan et al., 1995; Fuller et al.,1995; Komarova et al., 1997) and a pericentriolar pool. Ithas been proposed that the stable g-tubulin pool isassociated with the centrioles, while the other poolexchanges rapidly with the g-tubulin cytoplasmic pool(Khodjakov and Rieder, 1999). The less stable pool of g-tubulin is the pericentriolar pool. The pericentriolar poolof g-tubulin forms a lattice, known as the centromatrix(Schnackenberg et al., 1998; Palazzo et al., 2000), withpericentrin, another centrosomal protein proposed to bea structural component of the pericentriolar material


Fig. 4. Gamma-tubulin is localized to the MTOC in large (140–150mm in diameter) and medium (between 115 and 140mm in diameter)oocytes. Double-label immunocytochemistry was carried out on largeoocytes using a-tubulin (green) and g-tubulin (red) antibodies. The toppanels (A, B) show a representative large oocyte while the mid-levelpanels (C, D) correspond to a representative medium size oocyte. Thea-tubulin staining (A, C) shows the cortical MTOCs. The g-tubulin

antibody recognizes a structure within the MTOCs in the large (B) andmedium (D) size oocytes (marked by arrows). Control oocytes treatedwith only the a-tubulin primary antibody and both the g-tubulin and a-tubulin secondary antibodies (E, F) show the MTOC being recognizedby the a-tubulin primary and secondary antibodies (green) and not thesecondary antibody to g-tubulin (red). A–F: 1,575�.


(Doxsey et al., 1994; Dictenberg et al., 1998). To testwhether the g-tubulin monoclonal antibody used wasrecognizing the g-tubulin present within the centriolesor the g-tubulin that interacts with pericentrin, double-label experiments were carried out in maturing oocytesusing the g-tubulin antibody and a pericentrin anti-body. The g-tubulin antibody recognized a smaller area(Fig. 5B) than the area recognized by the pericentrinantibody, which includes the entire MTOC (Fig. 5A).These results indicate that the g-tubulin monoclonalantibody used in these studies recognizes the centriolarg-tubulin and not the pericentriolar g-tubulin, which isconsistent with previous reports that some g-tubulinantibodies recognize only the centriolar g-tubulin, andnot the pericentriolar g-tubulin (Dibbayawan et al.,1995).

To better investigate the continuity of the MTOCduring oocyte differentiation, the distribution of peri-centrin during oogenesis was analyzed. We reasonedthat since pericentrin localizes to a larger area of thecentrosome than g-tubulin, the centrosome in smalleroocytes may be more easily visualized using the peri-centrin antibody than using the g-tubulin antibody.Oocytes of varying sizes were double labeled with a-

tubulin and pericentrin antibodies. Pericentrin localizesto the MTOC in large and medium size oocytes (Fig. 6A–D). In small oocytes, even when MTOC was visualizedwith difficulty using the a-tubulin antibody, pericentrinwas seen to localize to the MTOC (Fig. 6E,F). These dataprovide the first evidence indicating that in sea urchinsthe MTOC is assembled very early during oogenesis andit is maintained throughout the rest of the maturationprocess. Control treatment of oocytes with only therhodamine-conjugated secondary antibody to pericen-trin in combination with the a-tubulin primary andsecondary antibodies resulted in no rhodamine-specificcentrosomal staining (Fig. 6G,H).

The localization pattern of the centrosomal proteincentrin was also analyzed. Centrin was originallyidentified as one of the predominant components ofstriated rootlets (Salisbury et al., 1984). Subsequently,it was shown that centrin is also present in centrosomesin a number of systems (Baron and Salisbury, 1992;Salisbury, 1995). Centrosomal centrin is present pri-marily in the centrioles, and thus centrin has been usedas a molecular marker for centrioles, although somecentrin has been reported to be present in the pericen-triolar material (Baron et al., 1992; Paoletti et al., 1996).As described above, early S. drobachiensis oocytescontain a basal flagellar apparatus composed of basalbodies and a striated rootlet (Fig. 3). If centrin is presentin sea urchin striated rootlets, then centrin could beused as a marker of the MTOC in early oocytes. Double-label experiments were performed on varying sizeS. drobachiensis oocytes using a-tubulin and centrinantibodies. Large and medium premeiotic oocytescontain centrin in the cortical centrosome (Fig. 7A,B;data not shown). In small oocytes, even in those in whicha MTOC is not visible by a-tubulin staining (Fig. 7C),centrin localizes to a distinct structure (Fig. 7D). It isnecessary to point out that unlike in large and mediumsize oocytes, in which the centrin positive structure wasobserved in a single focal plane, the centrin positivestructure seen in small oocytes was visible consistentlyin several focal planes. Using epifluorescence it was notpossible to determine if centrin localized to the striatedrootlet in young oocytes, although the presence ofcentrin in a large area of the MTOC in small oocytesseems to suggest this possibility. The presence of centrinin the MTOC throughout oogenesis provides additionalevidence in support of the hypothesis that the MTOC isassembled very early during oogenesis and maintainedduring premeiotic oogenesis. Furthermore, it suggeststhatS. drobachiensis oocytes establish cell polarity veryearly and that this polarity is maintained during thedifferentiation of the oocytes into mature eggs.

DISCUSSION

Sea Urchin Oocytes Contain a MicrotubuleOrganizing Structure Throughout Oogenesis

The studies presented here indicate that sea urchinoocytes maintain a microtubule-organizing structurethroughout their differentiation process. Our ultra-


Fig. 5. Gamma-tubulin recognizes the centrioles present in theoocyte MTOC. (A, B) Two images of the same MTOC double labeled byimmunocytochemistry with pericentrin (A-red) and g-tubulin (B-green) antibodies. Pericentrin is localized to the entire MTOC surface(A) while g-tubulin is localized to a smaller area within the MTOC,which most probably corresponds to the centrioles (B). A, B: 7,700�.


structural observations show that young oocytes thatare still attached to the gonadal wall organize theirmicrotubules from a basal flagellar apparatus thatcontains centrioles (basal bodies) and an associatedstriated rootlet. This basal flagellar apparatus mostlikely corresponds to the structural remnant of theflagellar apparatus present in the flagellated precursorgerminal cells (PGCs) (Houk and Hinegardner, 1980). Inlater stages of oogenesis microtubules are organizedfrom a cortical centrosome. In most animal cells the

centrosome acts as the main MTOC and it consists of apair of centrioles surrounded by pericentriolar material(PCM) that contains the microtubule nucleating activity(reviewed in Balczon, 1996; Palazzo et al., 2000). Otherstructures found in centrosomes include: a structurethat physically links both centrioles (Bornens, 1992);basal feet that extend from the centrioles into the PCM(Baron and Salisbury, 1992); and aggregates that areknown as MTOGs or ‘‘electron-dense granules’’ (Endo,1979). Ultrastructural observations reported here de-


Fig. 6. Pericentrin localizes to the MTOC in S. drobachiensisoocytes. Double-label immunocytochemistry on S. drobachiensisoocytes of varying sizes using the pericentrin and a-tubulin antibodies.A, C, E: Large, medium, and small oocyte, respectively, reacted withthe a-tubulin antibody (green) showing the cortical MTOC. B, D, F:Same oocytes as in A, C, and E, respectively, reacted with the

pericentrin antibody (red) showing the localization of pericentrin tothe MTOC. Control oocytes treated with only the a-tubulin primaryantibody and both the pericentrin and a-tubulin secondary antibodies(G, H) show the MTOC being recognized by the a-tubulin primary andsecondary antibodies (green) and not the secondary antibody topericentrin (red). A–F: 600�.


monstrate that the MTOC present in medium and largeoocytes (>115 mm in diameter) of S. drobachiensis iscomposed of amorphous material (PCM) surroundingMTOGs. The PCM has been shown to be primarily madeout of pericentrin and g-tubulin embedded in a filamen-tous structure known as the centromatrix (Schnacken-berg et al., 1998; reviewed in Palazzo et al., 2000). In ourstudy, pericentrin is present and localizes to the MTOCin small, medium, and large oocytes, in which the GV ismigrating towards the animal pole. In addition topericentrin, the MTOC contains g-tubulin and centrinin medium and large oocytes. These stages correspond tothose in which by electron microscopy the presence of acentrosome was observed. In small oocytes we wereunable to establish the presence of a MTOC by a-tubulinstaining, but centrin localized to a large structure.

The observation that in all stages examined oocytescontain a microtubule-organizing structure suggeststhe continuous presence of a polarized microtubulenetwork in differentiating sea urchin oocytes. This is inmarked contrast to what has been reported for othernonechinoderm deuterostome systems. For example,during Xenopus oogenesis, early oocytes contain acentrosome, which later gets degraded (Gard et al.,1995). After the disassembly of their centrosome,Xenopus oocytes do not seem to have a microtubuleorganizing structure until the stage at which the GVbegins its migration towards the animal pole. At thispoint a MTOC, that does not exhibit the morphologicalcharacteristics of a centrosome, appears at the vegetalside of the GV (Gard, 1991). Also, recent studies oncentrosoma dynamics in ascidian oocytes have demon-

strated that after GV breakdown and meiotic spindleformation in the center of the oocyte, the spindlemigrates in a random direction towards the cortex(Prodon et al. 2006).

The organization of the MTOC prior to GV migrationis consistent with the idea that the MTOC plays anactive role in the movement of the GV. In a number ofsystems microtubules and/or the actin cytoskeleton areresponsible for positioning the nucleus and maintainingits localization (reviewed in Reinsch and Gonczy, 1998).It has been proposed that nuclear movement alongmicrotubules towards MTOCs is driven by dynein, aminus-end directed motor (Rouviere et al., 1994;Schatten, 1994; Allan, 1996; Reinsch and Karsenti,1997). In sea urchins the MTOC is cortical suggestingthat the minus ends of the microtubules are located inthe cortex, while the plus ends extend inside the cell.Microtubules that emanate from the MTOC have beenobserved to be long enough to cover the GV (A.L. Eganaand S.G. Ernst, unpublished results). Thus dyeninmotors associated with those MTs could contact thesurface of the GV. Recent studies on starfish oocyteshave shown that the cortical centrosome present inoocytes anchors the GV to the cell surface of the pre-sumptive animal pole (Miyazaki et al., 2000), providingthe first evidence that in starfish there is an associationbetween the cortical centrosome and the GV. In sea-cucumber oocytes not only does the cortical MTOCanchor the GV as in starfish, but in addition, treatmentwith nocodazole results in the inhibtion of GV migration(Miyazak et al., 2005). We propose that the same is truein sea urchins. If the location of the MTOC is driven by apreexisting polarity in the oocyte and the MTOC isinvolved in GV migration, then the dynein-drivenmovement of the GV towards the cortical MTOC wouldensure the correct location of the meiotic apparatusrelative to the preexisting polarity in the maturingoocyte (Fig. 8).

The Sea Urchin Oocyte CentrosomesContains Centrioles

The studies on the localization of centrosomal proteinspresented here also suggest the continuous presence ofcentrioles during sea urchin oogenesis. The ultrastruc-tural analysis demonstrated the presence of basal bodiesin early oocytes and the identification of a large electron-dense structure suggested the presence of centrioles inthe centrosome found in later stages of oogenesis. Theproposal that centrioles are present in the centrosome inlater stages of oogenesis is supported by our analysisof the distribution of g-tubulin and centrin. Althoughmost antibodies raised against g-tubulin identify thepericentriolar and centriolar centrosomal g-tubulin,some g-tubulin antibodies recognize only the centriolarg-tubulin (Dibbayawan et al., 1995). Our results fromthe double-label immunocytochemistry analyses carriedout using the pericentrin and g-tubulin antibodiesindicate that the g-tubulin monoclonal antibody usedin our studies recognizes the centriolar g-tubulin andnot the pericentriolar g-tubulin. These results also


Fig. 7. Centrin localizes to the MTOC in S. drobachiensis oocytes.Double-label immunocytochemistry on S. drobachiensis oocytes ofvarying sizes using the centrin and a-tubulin antibodies. A, C: Largeand small oocytes, respectively, reacted with the a-tubulin antibody(green) showing the cortical MTOC (A) and the distribution ofmicrotubules (C). B, D: Same oocytes as in A and C, respectivelyreacted with the centrin antibody (red) showing centrin localization tothe MTOC in large oocytes (B) and to a large structure in small oocyteswhere a MTOC is not clearly visible by a-tubulin staining (D, singlefocal plane shown). A–D: 1,200�.


support the proposal that the centrosome found in seaurchin oocytes contains centrioles.

As with g-tubulin, centrosomal centrin has beenreported to be present in the pericentriolar materialand in centrioles (Baron et al., 1992; Paoletti et al.,1996). However, unlike g-tubulin, centrosomal centrinaccumulates primarily in the distal lumen of the cen-trioles making it a good marker for centrioles (Paolettiet al., 1996; Manandhar et al., 1999). The centrin anti-body used in our studies has been shown to recognizeproteins of the appropriate molecular weight forcentrins in both starfish and sea urchins (Middendorpet al., 1997). The localization of centrin to a substructurewithin the centrosome in medium and large premeioticoocytes indicates that the sea urchin oocyte centrosomecontains centrioles. Furthermore, our results are inagreement with previous studies showing that thestarfish cortical centrosome contains a centriole-likestructure (Kato et al., 1990) and that the starfish meioticapparatus is centriolar (Sluder et al., 1989; Kato et al.,1990). Thus it appears that in echinoderms oocytes donot disassemble their centrioles during oogenesis. These

results provide some evidence in support of the argu-ment put forward by Boveri and Van Beneden whostated that the centrosome is not lost during oogenesis(Wilson, 1925). However, our results seem to contradictwhat has been reported for other systems. Xenopusoocytes disassemble their centrosome, including cen-trioles, during early oogenesis. During GV migration astructure that is capable of assembling a microtubuleaster appears, but it does not exhibit any of the morpho-logical characteristics of a centrosome (Gard et al.,1995). Likewise, as concluded by Conklin, Crepidulaoocytes seem to disassemble their centrosome duringearly oogenesis, but during meiosis they are able toreassemble it from structures described as procentro-somes, including what appears as the formation ofcentrioles de novo (Conklin, 1902; Palazzo et al., 2000).In both cases, in Xenopus and Crepidula, the distribu-tion of centrosomal proteins in the oocyte during itsmaturation process is not known fully. What on thesurface may seem like a profound difference betweenthese two systems and sea urchins may in fact justreflect a difference in how oocyte MTOCs and centro-


Fig. 8. Model of how the basal-apical polarity of PGCs can translateinto the A/V axis of mature sea urchin eggs. PGCs differentiate intooocytes and the oocytes subsquently mature in the gonadal epithelium.During early oocyte maturation, the flagellar apparatus, composed of astriated rootlet (blue) and basal bodies (red) and located on the side ofthe cell in contact with the gonadal wall, begins to degenerate. Thedisassembly of the flagellar apparatus results in the completedegeneration of the striated rootlet structure but not the centrioles.The centrioles are maintained at this site from which they organizea centrosome as the oocyte continues maturing. The centrosome

nucleates microtubules (green) that contact the GV and aid in directingand moving the GV from the center of the oocyte to the cortical areawhere the centrosome is located (depicted by purple arrows). After thebreakdown of the GV the centrosomes organize the meiotic apparatus.Both medium and large oocytes have been depicted in green toillustrate the complex cortical microtubular organization that oocytesexhibit in these two stages. The polarity of the large microtubulesemanating from the MTOCs is illustrated by the direction of the arrow(from � to þ).


somes have been defined in past studies. Thus we mustconclude that the possible differences on centrosomaldynamics between sea urchins and other invertebrateand vertebrate systems remain currently open. Ourresults provide a temporal framework and a moleculardefinition of the centrosome during oogenesis that couldbe helpful in directing future studies that attempt toanswer similar questions in these and other systems.

Establishment of OocytePolarity in Echinoderms

Centrioles are part of the basal flagellar apparatus ofearly oocytes. We have observed that the striated rootletdegenerates (J. A. Boyle and S. G. Ernst, unpublishedresults), but the centrioles remain. Centrioles are foundin the centrosome that assembles prior to GV migrationand is present throughout GV migration. Although it ispossible that centrioles migrate from one site to anotherduring oocyte maturation, the fact that they are asso-ciated with microtubules in all stages examined and thatin starfish the centrosome is anchored to the cortex(Miyazaki et al., 2000), suggests to us that the centriolesare firmly anchored to the oocyte’s cortex, at the sitewhich marks the animal pole. Thus the apical-basalpolarity exhibited by the precursor germinal cells in thegonad translates into the animal-vegetal axis of thematuring oocyte. The proposal that the basal-apicalpolarity of the precursor germinal cells contributes toand translates into the animal/vegetal (A/V) axis of thefuture embryo is not new. Based on studies of gameto-genesis in sea cucumbers Frick et al. (1996) proposedthat the apical-basal polarity of the epithelial oocytegives rise to the A/V axis of the oocyte. They observedthat gonial cells and small oocytes contain flagella orcentrioles in their apical side. The apical side corre-sponds to the future animal pole of the developing seacucumber embryo. Based on their observations and theresults we present here we can conclude that in at leasttwo classes of echinoderms the position of the flagellaand centrioles in the maturing oocyte appears to markthe future animal pole of the embryo.

It is interesting that the polarity of echinoid andholothuroid oocytes seems to be reversed. Here wereport that the striated rootlet/centriole complex islocated on the side of the oocyte attached to the gonadalwall, while in sea cucumbers the flagellum protrudesfrom, and the centrioles are present on, the side of theoocyte that is facing the lumen. It is also noteworthythat the five classes of echinoderms seem to fall intotwo categories in terms of the relationship betweengrowing oocytes and the gonad. Holothuroid, crinoid,and ophiuroid oocytes grow into the hemal sinus andonly maintain contact with somatic cells at their apicalside where the future animal pole is located. However,asteroid and echinoid oocytes grow into the lumen andget surrounded by somatic cells (nutritive phagocytes)on all sides except for the site of contact with the gonadalwall (reviewed in Frick et al., 1996) where the jelly canalwill form (Schroeder, 1980). These two different modes

of growth in relation to the gonadal compartment may bea reflection of the opposite location of the future animalpole, as marked by the opposite location of the striatedrootlet and centrioles in sea cucumber and sea urchinoocytes. Based on comparative studies with cephalo-chordata (lancelet) and cnidaria, it has been proposedthat of these two modes of oogenesis the one exhibitedby holothuroids, ophiuroids, and crinoids seems to beplesiomorphic to echinoderms, to all deuterostomes, andpossibly to all metazoans (reviewed in Frick et al., 1996;Frick and Ruppert, 1997).

ACKNOWLEDGMENTS

We acknowledge and thank Dr. J. L. Salisbury forgenerously providing us with the 20H5 centrin anti-body. We also extend our sincere thanks to members ofthe HVEM Facility at the University of Colorado whoassisted us with the high-pressure fast freezing techni-que and the use of their equipment, specially MaryMorphew and Dr. Kent McDonald. This work wassupported by a Grant-In-Aid of Research from theNational Academy of Sciences through Sigma Xi toJ.A.B. and by the Betty Vernon and Charles TaylorVernon Fund for Biological Research and a TuftsUniversity Faculty Research Award to S.G.E.

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Strongylocentrotus drobachiensis oocytes maintain a microtubule