Sorting and reorganization of centrosomes during oocyte maturation in the mouse

Sorting and Reorganization of Centrosomes During OocyteMaturation in the MouseMARY JO CARABATSOS, CATHERINE M.H. COMBELLES, SUSAN M. MESSINGER, AND

DAVID F. ALBERTINI*Program in Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Department of Anatomyand Cell Biology, Tufts University School of Medicine, Boston, Massachusetts

KEY WORDS microtubule organizing centers; meiosis; oocyte

ABSTRACT In animal oocytes, the centrosome exists as an acentriolar aggregate of centroso-mal material that is regulated in a dynamic manner throughout the process of meiotic maturation.Recently, it has been demonstrated that in female meiotic systems spindle assembly is likelyregulated by chromosomal and microtubule/microtubule-associated influences. The purpose of thisstudy was to analyze the distribution of the integral centrosomal protein, pericentrin, during thecourse of meiotic maturation. The function of the centrosome during meiotic progression wasevaluated by exposing oocytes to pharmacological agents that perturb cytoplasmic homeostasis(cycloheximide, nocodazole, cytochalasin D, taxol, and vanadate). Pericentrin was localized to thespindle poles during metaphase of meiosis-I as O- and C-shaped structures. At anaphase, thesestructures fragment, become displaced from the spindle poles, and associate with the lateral spindlemargin. The metaphase spindle at meiosis-II had incomplete pericentrin rings at both spindle poles.Vanadate treatment, a known inhibitor of dynein-ATPase, resulted in meiotic arrest, constrictionof the spindle pole, and an aggregation of pericentrin at the spindle poles. After taxol exposure,pericentrin incorporation into both spindle poles and cytoplasmic centrosomes was increased.Treatment of oocytes with cycloheximide, nocodazole, and cytochalasin D, influenced early eventsassociated with chromosome capture and spindle assembly and altered the number and distributionof cytoplasmic centrosomes. Thus, although pericentrin incorporation is not required for meioticspindle formation, the dynamic reorganization of pericentrin and changes in centrosome microtu-bule nucleating capacity are involved in critical cell cycle transitions during meiotic maturation.Microsc. Res. Tech. 49:435–444, 2000. © 2000 Wiley-Liss, Inc.

INTRODUCTIONCentrosomes function as dynamic regulators of spa-

tial organization in dividing and nondividing cells. Asthe major microtubule organizing center (MTOC) ofsomatic cells, their duplication, positioning, and com-position have become prominent subjects of study withrespect to cell cycle control (Vandre and Borisy, 1989),mitotic spindle bipolarity (Maniotis and Schliwa, 1991;Mazia, 1984; Zhang and Nicklas, 1995) and the sym-metric (mitosis) or asymmetric partitioning of variousorganelles (Albertini, 1987; Schliwa, 1984) and chro-mosomes (Doxsey, 1998; Zhou et al., 1998). In animaloocytes, centrosomes have been investigated as deter-minants of specialized duties in the female germ celllineage, and differ from their somatic counterparts inlacking centrioles (Szollosi et al., 1972). Thus, for thepurposes of this study, we refer to centrosomes as thecentriole-lacking aggregates of centrosomal materialcapable of supporting microtubule nucleation at vari-ous stages of oogenesis.

Centrosomes presumably subserve the function oforganizing cytoplasm during the growth and differen-tiation of animal oocytes (Albertini, 1992a). In prepa-ration for, and during the final stages of, oogenesiscentrosomes are associated with meiotic spindles invarious species but their exact contribution to spindlemorphogenesis and cell cycle progression remains un-

clear. Thus, while important cell cycle regulators arelocalized to the meiotic spindle (Kubiak et al., 1992;Verlhac et al., 1993), mounting evidence suggests thatg-tubulin, dyneins, and chromatin are necessary andsufficient determinants of meiotic spindle formationand function (Heald et al., 1996; Tavosanis et al., 1997;Vernos and Karsenti, 1995; Zhang and Nicklas, 1995;reviewed in Tournebize et al., 1997; Waters andSalmon, 1997) to the exclusion of centrosomes.Whether spindle-associated centrosomes in mamma-lian oocytes contribute to spindle stability and chromo-somal positioning functions to limit aneuploidy re-mains an interesting subject for study (Albertini,1992b; Mailhes et al., 1998).

As oocytes contribute the bulk of cytoplasm to theembryo following fertilization, it has widely been rec-ognized that the oocyte centrosomes are partial or com-plete sources of embryonic MTOCs. That maternal cen-trosome contributions can support embryonic cleavageis well documented in many species, from Drosophila tomice (reviewed by Schatten, 1994). However, it is im-

Contract grant sponsor: NIH; Contract grant numbers: HD20068, HD07403.*Correspondence to: David F. Albertini, Department of Anatomy and Cell

Biology, Tufts University School of Medicine, 136 Harrison Ave., Boston MA02111. E-mail: [email protected]

Received 20 August 1999; accepted in revised form 29 November 1999

MICROSCOPY RESEARCH AND TECHNIQUE 49:435–444 (2000)

© 2000 WILEY-LISS, INC.

portant to point out that for many species a paternalcentrosome, complemented by maternal stores of cen-trosomal material, is necessary for development to pro-ceed (Simerly et al., 1995). Thus, there is little doubtthat oocyte centrosomes contribute maternal stores ofmaterials that are utilized during embryonic mitoses.What remains unresolved is whether oocyte centro-somes participate directly in the processes associatedwith meiotic cell cycle completion and oocyte matura-tion or represent a soluble pool that will activate dur-ing embryonic development (Riparbelli and Callaini,1996).

This article addresses the function of oocyte centro-somes by examining the distribution and organizationof the intrinsic protein pericentrin (Doxsey et al., 1994)to meiotic spindles and cytoplasmic centrosomes(Messinger and Albertini, 1991; Maro et al., 1985;Wickramasinghe and Albertini, 1992). The question ofcytoplasmic organization is approached by investigat-ing cytoskeletal and biosynthetic determinants of cen-trosome organization after treatment of in vitro ma-tured mouse oocytes with selective pharmacologicalagents. Towards resolving centrosome functions inmeiosis, pericentrin organization has been elucidatedby confocal and conventional fluorescence microscopyat various times during meiotic progression. The re-sults suggest that chromatin and the cell cortex exertprofound influences on centrosome reorganization dur-ing meiosis that may confer spindle stability and coor-dinate cytoplasmic partitioning and asymmetric cyto-kinesis.

MATERIALS AND METHODSCollection and Culture of Mouse Oocytes

For all experiments, oocytes were obtained from 20–25-day-old CF-1 mice injected 44–48 hours earlier with5 i.u. of equine chorionic gonadotropin (Sigma Chemi-cal Co., St Louis, MO). Cumulus-enclosed oocytes wereexpressed in collection medium (Eagle’s MEM withHanks salts and Hepes (Life Technologies Gibco/BRL,Gaithersburg, MD) supplemented with 100 i.u./ml pen-icillin, 100 mg/ml streptomycin and 0.3% bovine serumalbumin). Oocytes were cultured for various periods oftime in Eagle’s MEM (Life Technologies Gibco/BRL)supplemented with Earle’s salts, 2 mM glutamine, 0.23mM pyruvate, 100 i.u./ml penicillin, 100 mg/ml strep-tomycin and 0.3% bovine serum albumin (IVM-me-dium) in a humidified atmosphere of 5% CO2 at 37°C(Schroeder and Eppig, 1984). After removal of cumuluscells by gentle pipetting, oocytes were fixed and ex-tracted for 20 minutes at 37°C in a microtubule stabi-lizing buffer containing 2% formaldehyde, 0.5% Triton-X-100, 1 mM taxol, 10 units/ml aprotinin and 50% deu-terium oxide (Herman et al., 1983). Samples were thenwashed three times in a blocking solution of PBS con-taining 2% BSA, 2% powdered milk, 2% normal goatserum, 0.1M glycine, and 0.01% Triton-X-100 (PBS-blocking solution) and stored at 4°C in blocking solu-tion containing 0.2% sodium azide until processing.

Drug Treatment of In Vitro Matured OocytesTo evaluate the role of dynein during spindle assem-

bly, oocytes (n 5 40) were exposed to a continuoustreatment of 500 mM sodium ortho vanadate preparedfrom a 50 mM frozen stock in IVM-medium. Oocytes

were cultured for 12–14 hours in IVM-medium with/without vanadate and fixed as described above.

To examine the effect of taxol on the organization ofcentrosomes, pulse-experiments were performed at themetaphase–anaphase transition of meiosis-I. Oocytes(n 5 40) were cultured for 6–8 hours in IVM-medium.Taxol (1 mM) was applied to oocytes for 10 minutes andfixed for immunofluorescence microscopy.

For drug treatments designed to examine the role ofother cytoskeletal determinants in centrosome organi-zation, groups of oocytes (n 5 20–40) were liberatedinto collection medium and transferred to IVM-me-dium both of which contained test agents. Drugs wereprepared from a dimethyl sulfoxide frozen stock, exceptfor cycloheximide, which was made fresh prior to eachexperiment. The following concentrations were used:100 mg/ml cycloheximide (Sigma), 10 mg/ml nocodazole(Aldrich Chemical Co., Milwaukee WI) and 1 mg/mlcytochalasin D (Sigma). These concentrations havebeen shown to be effective in inhibiting protein synthe-sis, microtubule assembly, and disrupting actin micro-filament organization, respectively, in cultured mouseoocytes (Maro et al., 1986; Hashimoto and Kishimoto,1988). In the first series of experiments, aimed at de-termining the effects of continuous drug treatment oncentrosome organization, microtubule assembly, andmeiotic progression, oocytes were cultured for 3 and 8hours beginning at the time of isolation. Matching con-trols were taken at each time point. The second seriesof experiments was performed to determine the effectsof drug exposure on centrosome and microtubule dy-namics at specific stages of meiosis. To study the mei-osis-1 metaphase–anaphase transition, oocytes werecultured for 0–7 hours and then transferred to drug-containing medium for an additional 1 or 2 hours andfixed. For studying anaphase–telophase and telo-phase–metaphase II transitions, oocytes were culturedfor 0–8 hours and 0–10 hours, respectively, then trans-ferred to drug-containing medium for an additional 2hours before fixation. In all drug treatment experi-ments, similar results were obtained for either cumu-lus-enclosed or cumulus-free oocytes.

Processing of Oocytes for FluorescenceMicroscopy

Oocytes were incubated with a series of primary andsecondary antibodies (see below) for 1 hour/antibody at37°C with shaking followed by three 15-minute washesin a PBS-blocking solution between each antibody in-cubation. Microtubules were monitored using a mix-ture of monoclonal anti-a-tubulin and anti-b-tubulin(Sigma Biosciences) at a 1:100 final dilution, followedby a 1:50 dilution of an affinity-purified fluoresceinateddonkey antimouse IgG (Jackson ImmunoResearchLaboratories, West Grove, PA). Centrosomes were la-beled with the following antibodies: a human centro-some specific antiserum 5051 (Callarco-Gillam et al.,1983) and a polyclonal antibody directed against peri-centrin (4B) (Dictenberg et al., 1998) at a final dilutionof 1:100, followed respectively by either a 1:50 dilutionof an affinity-purified Texas red conjugated goat anti-human IgG (Accurate Chemical Co., Westbury, NY) ora Texas red conjugated donkey antirabbit IgG (Jack-son ImmunoResearch Laboratories). Oocytes weremounted in 50% glycerol/PBS containing sodium azide

436 M.J. CARABATSOS ET AL.

as an antifading reagent (25 mg ml-1; Bock et al., 1985).Labeled oocytes were photographed using a ZeissIM-35 inverted scope, equipped with fluorescein, Texasred, and Hoechst selective filter sets and a 50 W mer-cury arc lamp using a 63X Neofluor objective lens.Images were recorded on Kodak Tri-X-pan film usinguniform exposure time and processed with Acufine(Chicago, IL) developer for 4.5 minutes at 25°C.

Laser Scanning Confocal Microscopy Analysisof Mouse Oocytes

Samples prepared for fluorescence analysis as de-scribed above were analyzed using the Odyssey XLconfocal system (Noran Instruments, Middleton, WI)mounted on a Nikon Diaphot inverted microscopeequipped with a 60X and 100X fluorobjectives. The 488-and 528.7-nm lines of a krypton-argon laser were usedto excite fluorescein and Texas red markers. Dual-fluorimages were collected using Intervision acquisitionmodules software (Noran Instruments) running on aSilicon Graphics Indy 500 workstation (MountainView, CA). The acquisition module software allows foradjustments of scan mode, sample time, and imageprocessing parameters. All images were collected usingslow scan, with a sample time of 400 ns, and real-timeimage processing with jump averaging (n 5 32). Aseries of Z-axis image stacks was acquired using .5 mmsteps and spindle reconstructions were rendered in the

Z-axis using 3-D Intervision software (Noran Instru-ments).

RESULTSThree-Dimensional Reconstructions

of Meiotic SpindlesThree-dimensional reconstructions of prometaphase,

metaphase-I, anaphase-I, and metaphase-II spindleswere generated using immunofluorescence and laserscanning confocal microscopy (Fig. 1). Spindles wereevaluated for microtubule (MTs) and pericentrin orga-nization. In late prometaphase, long, interdigitatingMTs that coalesce into distinct spindle poles were ob-served (Fig. 1A). Pericentrin was distributed along mi-crotubules as individual foci (Fig. 1A, arrow), and clus-ters of foci accumulated at the future spindle poles.Note the overlap patterns (yellow) that indicate closeapposition of MT ends and pericentrin aggregates. Asmeiosis proceeded, the spindle acquired a bipolar bar-rel-shape with chromosomal bivalents tightly arrangedat the metaphase plate and condensed aggregates ofpericentrin appeared at the spindle poles (Fig. 1B).Pericentrin was consistently organized in an asymmet-ric manner at the two spindle poles: as a C-shapedstructure at one end (Fig. 1B, asterisk) and as anO-shaped structure at the opposite pole (Fig. 1B). Mi-crotubule interactions with pericentrin were also pole-specific. Microtubules were associated with pericentrin

Fig. 1. Three-dimensional organization of meiotic spindles. Dual-labeled confocal images of three-dimensional meiotic spindles stainedfor tubulin (green) and pericentrin (red), with codistribution (overlap)depicted in yellow. A representative prometaphase spindle (A) withmicrotubules radiating from the chromosomes (not shown) and peri-centrin foci coalescing at the spindle pole. The spindles at metaphaseof meiosis-I (B) had microtubules arranged in a barrel configurationwith pericentrin asymmetrically distributed at the spindle poles (C-shaped vs. O-shaped structure). Note that the spindle pole with aC-shaped structure represents the spindle attachment site and isdenoted with an asterisk. During anaphase (C), there was a length-ening of the microtubule fibers and pericentrin was organized as

centrosomal aggregates that had been displaced to the sides of thespindle. The metaphase spindle in meiosis-II (D) was shorter inlength than observed at metaphase of meiosis-I, microtubules wereagain arranged into a barrel configuration, but pericentrin wasequally distributed to both spindle poles as incomplete O-shapedstructures. Vanadate exposure during the formation of the metaphasespindle in meiosis-I (E) resulted in a spindle with densely bundledmicrotubules and solitary pericentrin aggregates at the spindle poles.The metaphase-II spindle (F) formed in the presence of vanadate hadloosely arranged microtubules, but pericentrin was now displaced tothe sides of the spindle and no longer appeared to interact withspindle microtubules. Scale bar 5 10 mM.

437REORGANIZATION OF CENTROSOMES IN THE OOCYTE

and overlap was complete at the C-shaped spindle pole,as indicated by yellow (Fig. 1B, asterisk), in contrast tothe O-shaped structure that showed incomplete MTand centrosome overlap (Fig. 1B, arrow). The asymme-try of pericentrin and tubulin codistribution at thespindle poles may reflect either structural differencesto accommodate the requirements of spindle docking ordifferences in the degree of microtubule nucleation atthe spindle poles. Analysis of three-dimensional recon-structions of oocytes (603) was used to determinewhich end of the spindle was anchored to the cortex(data not shown). In all cases, the attached pole corre-sponded to the C-shape pericentrin complex (Fig. 1B,asterisk) while the O-shape complex was oriented to-wards the cytoplasm.

The onset of anaphase is characterized by elongationof the meiotic spindle, chromosomal movement fromthe spindle equator, and a change in pericentrin orga-nization (Fig. 1C). Pericentrin was observed as multi-ple protein aggregates offset from both spindle polesand along the sides of the spindle (Fig. 1C). Althoughthe pericentrin aggregates were retained at the spindlepole attachment site (Fig. 1C, asterisk), a solitary ag-gregate offset from the “anchored” pole was observed atthe cortex attached site, consistent with the idea thatspindle rotation commences with anaphase onset(Maro et al., 1986). The displacement of centrosomalmaterial from the nonattached end may ensure thatcentrosomal proteins are excluded from the first polarbody and retained in the oocyte. Although pericentrinwas displaced at anaphase onset, centrosomal materialreappears as incomplete O-shaped structures at bothspindle poles in a symmetrical fashion in metaphase-IIstage oocytes (Fig. 1D).

Effects of Vanadate on CentrosomalOrganization in Metaphase-I and

Metaphase-II SpindlesMicrotubule motors have been implicated in playing

a role during the formation of the bipolar spindle for-mation during both mitosis and meiosis (Walczak et al.,1998; reviewed in Waters and Salmon, 1997). In orderto evaluate the role of dynein, a minus-end directedmicrotubule motor, in the organization of centrosomesduring meiosis, oocytes were exposed to sodium orthovanadate (500 mM), a known inhibitor of the dynein-ATPase (Gibbons et al., 1978; Nechay, 1984; Winkel-haus and Hauser, 1997). Vanadate exposure to mouseoocytes resulted in an irreversible metaphase-I block in70% of oocytes (unpublished data). Metaphase spindleswere bipolar with densely bundled microtubules (Fig.1E). Pericentrin was associated with both spindle polesas a solitary condensed aggregate (Fig. 1E), in contrastto the relaxed configuration seen in control oocytes(Fig. 1B). Microtubules were tightly associated withpericentrin at both spindle poles as evidenced by signaloverlap (Fig. 1E). Metaphase-II spindles were observedin 15% of the oocytes treated with 500 mM vanadateand displayed condensed pericentrin aggregates atboth spindle poles (Fig. 1F). Furthermore, microtubuleand pericentrin interactions were not observed at ei-ther spindle pole (Fig. 1F) (loss of overlap), suggestingdetachment of pericentrin from spindle MTs. The con-densation of the centrosomal material observed in bothmetaphase-I and metaphase-II spindles in response to

vanadate exposure implicates minus-end directed mo-tor proteins in the maintenance of microtubule spacingand centrosome organization.

Effects of Taxol on Spindle Pole andCytoplasmic Centrosomes

To evaluate the contribution of microtubule assem-bly and polymerization on the organization of centro-somal proteins, oocytes were exposed to a brief pulse of1 mM taxol (10 minutes) during metaphase of meiosis-Ifollowed by fixation. Immunofluorescence and laserscanning confocal microscopy were used to evaluatepericentrin and microtubule organization in responseto taxol exposure and a typical response is shown inFigure 2. Taxol treatment resulted in enlargement andelongation of the spindle, as evidenced by increasedspindle length and width (Fig. 2A). Pericentrin wasassociated with both spindle poles but a large aggre-gate appeared at the anchored end (Fig. 2B, arrow),whereas pericentrin foci were associated with the sidesof the spindle at the opposite pole (unattached) (Fig.2B, arrowhead). This pericentrin profile is similar tothe asymmetric organization observed in control an-aphase spindles (Fig. 1C) but shows that taxol causes arapid increases in the amount of both tubulin andpericentrin associated with the enlarged spindle (Fig.2A,B).

Taxol treatment also resulted in the formation ofcytoplasmic asters, consistent with previous observa-tions in the rat (Albertini, 1987). Large cortical micro-tubule asters (Fig. 2A,C) with associated pericentrin(Fig. 2B,D) were observed in response to taxol treat-ment. The microtubules were focused to discrete nucle-ation sites (Fig. 2C) and pericentrin was organized aseither C-shaped structures (Fig. 2D, arrow) or as in-complete-ring structures (Fig. 2D, arrowhead). Cyto-plasmic asters resembled monopolar spindles and peri-centrin organization was reminiscent of the centroso-mal profiles observed in control metaphase-I spindles(Fig. 1B). These data suggest that microtubule poly-merization and assembly plays a critical role in theorganization of centrosomal material. Furthermore,the increased association of pericentrin to both cyto-plasmic asters and the metaphase spindle suggests thepresence of a large soluble pool of pericentrin that isnot utilized during normal spindle assembly or cyto-plasmic centrosome formation.

Effects of Cycloheximide, Cytochalasin D, andNocodazole on Centrosome Organization

As previously illustrated in mouse oocytes, cytoplas-mic centrosomes undergo a series of dynamic alter-ations in their position, number, and microtubule nu-cleation capacity during the course of meiotic matura-tion (Messinger and Albertini, 1991). To begin toaddress further possible mechanisms regulating cen-trosomal dynamics, studies were conducted to evaluateprotein synthetic and cytoskeletal requirements.

Specific inhibitors were used to assess possible de-pendence of protein synthesis (cycloheximide, CHX),microtubules (nocodazole, NOCO) or microfilaments(cytochalasin D, CCD) on centrosome dynamics. Thefirst set of experiments was designed to determine theeffects of continuous drug exposures on the earlyevents of maturation, which include the reinitiating of


meiosis, centrosomes/MT reorganization, and spindleassembly (Fig. 3, Fig. 4). Oocytes cultured in the pres-ence of CHX for 3 hours arrested at prometaphase ofmeiosis-1 (Fig. 3D–F). Microtubules were observed ra-diating from the condensed chromosomes (Fig. 3D), butbipolar spindle assembly was not observed (Fig. 3E).Centrosomal foci appeared to be randomly associatedwith the chromosome/spindle region (Fig. 3F). This wasin contrast to the normal spindle pole distribution ofcentrosomes observed at prometaphase (Fig. 3A–C;Fig. 1A) that included an accumulating mass of peri-centrin at the future spindle poles. Pericentrin re-mained associated with the condensed chromatin fol-lowing 8 hours exposure to CHX (Fig. 4D–F); however,cytoplasmic centrosomes were not detected (Fig. 4F),unlike that observed in control oocytes (Fig. 4C).

Oocytes cultured in the presence of NOCO were ableto resume meiosis but arrested prematurely, subse-quent to germinal vesicle breakdown. Oocytes con-

tained a centrally located aggregate of chromatin (Fig.3A,G); pericentrin remained in the cortex (Fig. 3I) andnever associated with condensed chromatin (Fig. 3G,I).This condition persisted through 8 hours of continuoustreatment (Fig. 4G–I). Furthermore, with an 8-hourNOCO exposure cytoplasmic centrosome number de-creased (Table 1, NOCO) in contrast to what was ob-served in control oocytes (Table 1, Control).

After 3 hours in CCD, prometaphase oocytes exhib-ited a recruitment of centrosomal material to the con-densing chromosomes from which microtubules radi-ated (Fig. 3J–L). However, there was less centrosomalmaterial recruited to the condensing chromatin whencompared to controls (Fig. 3C). By 8 hours of culture inCCD, the oocytes reached early anaphase and spindleswith broad poles were located in the center of theoocyte (Fig. 4K). The majority of oocytes exhibited faintstaining of centrosomal material in association withthe first meiotic spindle (Fig. 4L). Interestingly, a char-

Fig. 2. Taxol treatment of mouse oocytes at metaphase of meio-sis-I. Correlative confocal images of antitubulin (A,C) and antiperi-centrin (B,D) staining patterns in mouse oocytes exposed to a pulsetreatment of taxol (1 mM). Microtubule staining indicates an enlarge-ment and lengthening of the meiotic spindle with numerous largecytoplasmic asters associated with the cortex (A). Pericentrin is orga-nized as a large aggregate at one spindle pole, and appears as discrete

individual aggregates associated with both the spindle pole and thesides of the spindle (B). Pericentrin is also observed associated witheach cytoplasmic aster (B). Images generated from three-dimensionalreconstructions of a smaller region of the oocyte cortex demonstratethat the pericentrin (D) within the cytoplasmic asters (C) is organizedas either C- or O-shaped structures. Scale Bar 5 10 mm (A,B) and 2mm (C,D).


acteristic feature of CCD-treated oocytes was the pres-ence of an increased number of cytoplasmic centro-somes (Fig. 4K,L) relative to control oocytes (Table 1).These centrosomes were distributed equidistant fromone another and assumed a subcortical position (Fig.4K,L); this pattern differed from the typical restrictionof centrosomes to an area of the cortex that excludesthe spindle (Fig. 3A,C; Messinger and Albertini, 1991).The lack of pericentrin associated with the spindlesuggests that the bipolar meiotic spindle can be main-tained in the absence of centrosomes. In addition, whenthis material does not become incorporated and/or re-tained at the spindle poles, it may then be recruited tocortical attachment sites, as illustrated by the in-creased numbers of cytoplasmic centrosomes (Table 1).

A second set of experimental manipulations was per-formed to determine the effects of pulsed drug expo-

sures on centrosome, microtubule, and microfilamentorganization during later stages of meiotic maturation.The three intervals chosen for study were 1) the meta-phase to anaphase I transition when the reappearanceof centrosomes occurs, 2) the anaphase to telophasetransition when centrosome loss occurs, and 3) thetransition from telophase to metaphase II when cen-trosomes reappear (Messinger and Albertini, 1991).Collectively, our experiments indicated that thechanges in centrosome number normally associatedwith transitions between metaphase I and metaphaseII were not influenced by brief treatments (1–2 hours)with CHX, NOCO, or CCD (data not shown), suggest-ing that unlike early aspects of centrosome organiza-tion, once metaphase of meiosis-I is achieved subse-quent alterations in centrosome assembly and position-ing follow a programmed course.

Fig. 3. Effects of 3-hour continuousdrug exposure on nuclear organization,spindle, and centrosomes. CorrelativeHoechst 33258 (A,D,G,H), antitubulin(B,E,H,K), and anticentrosome (C,F,I,L)staining patterns in mouse oocytes cul-tured for 3 hours in control medium(A–C), or medium containing cyclohexi-mide (D–F), nocodazole (G–I), and cytocha-lasin D (J–L). A–C: Staining pattern of anoocyte in prometaphase in which the cen-trosomal staining pattern indicates remod-eling of centrosomal material to the poles ofthe developing spindles. Note several cyto-plasmic centrosomes that nucleate micro-tubules. D–F: An oocyte cultured in thepresence of cycloheximide illustrates cen-trosomal foci associated with the con-densed chromosomes and several cytoplas-mic centrosomes nucleating microtubules.Note the absence of a spindle. G–I: Stain-ing pattern of an oocyte cultured in nocoda-zole illustrating a dense chromatin massand several cytoplasmic centrosomes. Notethe lack of centrosomal material associatedwith the condensed chromatin. J–L: Stain-ing pattern of an oocyte cultured in cy-tochalasin D demonstrating condensedchromosomes with little centrosomal asso-ciated material as compared to controls.Note several cytoplasmic centrosomes thatsupport microtubule nucleation. Scalebar 5 20 mm.


DISCUSSIONThese studies extend earlier observations on the or-

ganization of centrosomes in mouse oocytes (Maro etal., 1985; Messinger and Albertini, 1991; Schatten etal., 1986; Wickramasinghe and Albertini, 1992). Withrespect to the organization of spindle-associated cen-trosomes, we demonstrate that spindle pole pericentrinis assembled into O- and C-shaped aggregates (spindleattachment site) that are asymmetrically disposed inspindles at metaphase of meiosis-I. In contrast, peri-centrin is symmetrically displayed as incomplete O-shaped structures at metaphase-II spindle poles. More-over, under conditions promoting microtubule assem-bly (taxol exposure), spindle-associated pericentrin was

enhanced and O- and C-shaped centrosomal aggre-gates were observed in the ooplasm. Therefore, mouseoocytes contain a substantial pool of pericentrin that isrecruited by taxol to both spindle and cytoplasmic (sub-cortical) sites and is capable of generating closed oropen ring configurations independent of the presence ofchromatin. The role of microtubule assembly and/ordynein-mediated bundling on pericentrin recruitmentand organization into centrosomes remains unre-solved.

In addition, the organization of the centrosomal poolduring meiotic cell cycle progression was addressedwhen protein synthesis, microtubule assembly, and ac-tin polymerization were inhibited. Under conditions of

Fig. 4. Effects of 8-hour continuous drugexposure on nuclear organization, spindle,and centrosomes. Correlative Hoechst 33258(A,D,G,H), antitubulin (B,E,H,K), and anti-centrosome (C,F,I,L) staining patterns inmouse oocytes cultured for 8 hours in controlmedium (A–C), or medium containing cyclo-heximide (D–F), nocodazole (G–I), and cy-tochalasin D (J–L). A–C: Staining pattern ofan oocyte in anaphase with a brightly stainedspindle and several cytoplasmic centrosomesthat radiate microtubules. D–F: Staining pat-tern of an oocyte cultured in cycloheximidewith radial growth of microtubules from thecondensed chromosomes. Centrosomal mate-rial is seen associated with the condensedchromatin. G–I: An oocyte cultured in no-codazole demonstrating a dense mass of chro-matin and only a few cytoplasmic centro-somes relative to control oocytes. Note thatno centrosomal material is associated withthe condensed chromatin. J–L: Staining pat-tern of an oocyte cultured in cytochalasin Dillustrating a brightly stained spindle and anincreased number of cortical centrosomes.Note the decrease in centrosomal stainingassociated with the spindle as compared tocontrols. Scale bar 5 20mm.


protein synthesis inhibition, GVBD centrosome aggre-gation and pericentrin-spindle interactions proceededbut later events involving the reappearance of corticalcytoplasmic centrosomes were impaired. Microtubulesare required for the organization of pericentrin at thespindle poles, although not required for the initial re-cruitment of centrosomal material to the condensingchromatin. Actin microfilaments are implicated in theproper sorting of the pericentrin to either the spindle orcortical centrosome compartments. Given these re-sults, several fundamental questions arise regardingthe regulation of centrosome organization and functionduring oocyte maturation. Is the association of spindlepole centrosomes critical to the morphogenesis or func-tion of meiotic spindles? To what extent is the sorting ofcentrosomes to either spindle or cortical sites coupledto specific stages or characteristics of meiotic cell cycleprogression in oocytes? What functions do subcorticalcentrosomes subserve?

Since the original descriptions of MTOC rearrange-ment during oocyte maturation, the notion that centro-somes play a primary role in meiotic spindle assemblywas generally accepted (Szollosi et al., 1972). Given themovement of MTOCs to the germinal vesicle duringmeiotic resumption, their tendency to coalesce at nas-cent poles, and apparent persistence throughout thecompletion of meiosis (Messinger and Albertini, 1991),centrosome involvement as a major MT nucleator ap-peared likely. More recently, however, evidence thatchromatin itself exerts a dominant influence on bipolarspindle formation during female meiosis has emerged(Brunet et al., 1999; Heald et al., 1996; Woods et al.,1999). This notion has derived from observations ofspindle assembly in vitro using Xenopus oocyte ex-tracts where chromatin beads and functional dyneinwere identified as sufficient (Heald et al., 1996). More-over, g-tubulin localization to condensing chromatin ininsect oocytes seems to preclude direct requirementsfor female centrosomes (Wolf and Joshi, 1996). Whiledetailed studies on g-tubulin distribution in mouse oo-cytes undergoing meiotic maturation have not beenconducted, the possibility that g-tubulin is preferen-tially sequestered within condensing chromatin, and

excluded from centrosomes, receives support from sev-eral observations. Perinuclear centrosomes exhibit lit-tle microtubule nucleation during diakinesis (Mattsonand Albertini, 1990) and taxol treatment during GVBDresults in enhancement of MT assembly only in thenucleus, not cytoplasm (Albertini, 1992a). Coupledwith recent findings, these data suggest that oocytecentrosomes may undergo changes in their MT nucle-ation capacity during meiotic cell cycle progression,due in part to a loss or gain of g-tubulin.

In this study, confocal microscopy has revealed thatspindle centrosomes change in their MT binding undernormal and experimental conditions. For example, cor-tex attached pole centrosomes exhibit overlap with MTminus-ends, whereas those facing the cytoplasm haveincomplete microtubule interactions unless exposed tovanadate. Under conditions of vanadate-induced cellcycle arrest, pericentrin is condensed and closely asso-ciated with both spindles poles (Fig. 1E). These datasuggest that meiotic spindle pole centrosomes are non-nucleating and are more likely to capture preformedspindle microtubules. Moreover, consistent with otherstudies (Gaglio et al., 1997; Heald et al., 1997), dyneinwould mediate minus-end MT spacing that may dictatepericentrin organization (Fig. 1). Thus, these data areconsistent with the idea that spindle-associated centro-somes are not primary determinants in spindle assem-bly, but play a stabilizing and/or anchoring role oncechromatin directed MT-assembly has occurred andspindle bipolarity is established (reviewed in Compton,1998). It is also possible that spindle-associated centro-somes serve as reservoirs for important cell cycle reg-ulatory factors that would be released at anaphase as aresult of centrosome dispersion, as previously sug-gested by evidence of MPM-2 positive vesicle formationat anaphase (Messinger and Albertini, 1991).

Why extra centrosomes? While spindle pole centro-somes are not unexpected participants in the process ofmeiotic nuclear maturation (the process of genomicreductive division), the dynamics of cytoplasmic cen-trosome organization has remained perplexing. Earlierreports on the existence of these structures in maturemetaphase-II mouse oocytes suggested that they rep-resent a maternal store of centrosomes that supportsearly embryonic cleavages (Maro et al., 1985; Schattenet al., 1986). Consistent with this is the notion thatmice and other rodents represent oddities in the gen-eral scheme of centrosome inheritance, since paternalcentrosomes are not involved in the formation of zy-gotic mitotic spindles (Schatten, 1994). It is noteworthyto point out that other than rodent oocytes (Albertini,1987; Maro et al., 1985; Plancha and Albertini, 1994),cytoplasmic centrosomes have not been observed.Thus, answers to any inquiry into the role of cytoplas-mic centrosomes or species representation will awaitfurther study into their dynamics or relative contribu-tions to oocyte meiosis and embryonic development.

What is known about extra centrosomes in mouseoocytes derives from previous studies on meiotic cellcycle progression and data reported here. Messingerand Albertini (1991) demonstrated cell cycle stage-spe-cific changes in cytoplasmic centrosome number andMT nucleation during oocyte maturation. Specifically,conserved numbers (mean number of 8) of corticallypositioned centrosomes, with associated MTs, are

TABLE 1. Experimental manipulations of centrosome dynamicsduring in vitro maturation of mouse oocytes

Treatment AggregationSpindlecapture

Dispersion ofcytoplasmiccentrosomes

Range ofcentrosome

numbers/cellat T8 (%) N

Control 1 1 1 3–14 (82) 52CHX 1 1 2* 0–2 (88) 52NOCO 1 2 2* 0–2 (69) 35CCD 1 2 11 9–.20 (91) 25

*Denotes cell cycle block at prometaphase and post-GVBD for CHX and NOCOtreatments, respectively.Mouse oocytes were incubated for various time periods as described in Materialsand Methods. The initial decrease of cytoplasmic centrosomes (“aggregation”)occurred in the presence of all three inhibitors. The interaction of centrosomeswith chromosomes at GVBD is microtubule-dependent, and insensitive to micro-filament disruption; however, CCD impaired the reorganization of centrosomalmaterial to the spindle pole (“spindle capture”). Typical centrosome numberswere not maintained in the presence of NOCO, and disappearance of cytoplasmiccentrosomes was observed in the presence of CHX. In addition, CCD treatmentresulted in increased numbers of centrosomes relative to controls during laterincubation times (“dispersion of cytoplasmic centrosomes”). Data representspercentage of oocytes with centrosome number in each category. The number (N)of oocytes examined is indicated, and data is pooled from three experiments.


noted in germinal vesicle and anaphase stages of mei-osis-1; metaphase of both meiosis-1 and meiosis-2 havecentrosomes but show no evidence of MT association.Since centrosomes at all stages exhibit MPM-2-positivestaining, phosphorylation alone cannot account for ap-parent differences in microtubule nucleation capacity.Rather, nucleation reactivation is restricted to an-aphase onset during both meiotic divisions. This tem-poral and spatial cell cycle regulation of MT nucleationraises immediate questions regarding cause and effect.

One factor that would explain the onset of nucleationat anaphase would be recruitment and integration ofg-tubulin, which has been shown to be preferentiallyincorporated into meiotic spindles in other species(Wolf and Joshi, 1996). Thus, a likely cause of differ-ential nucleation by cytoplasmic centrosomes would beg-tubulin transport from the meiotic spindle, or a cyto-plasmic pool, to cortical centrosomes. Increased MTcapture is also a formal possibility to explain the MTdisplays seen at anaphase but no evidence of cytoplas-mic MTs at this or subsequent meiotic stages has beenforthcoming. Studies are under way to determine ifg-tubulin sorting underlies this dramatic alteration incentrosome nucleation capacity.

As to possible consequences of varying cortical MTdisplays during meiosis, it is interesting to note thatnucleation coincides with anaphase onset at a timewhen the asymmetry of cytokinesis is established. Wepropose that cortical centrosomes anchor organellesand the oocyte cortex during the asymmetric cleavagethat results in polar body formation. The fact thatthese structures are absent in oocytes of other speciessuggest that different mechanisms exist to retain “cy-toplasmic components” or these structures are labile toavailable means for their preservation and detection.

Finally, the fact that multiple centrosomes exist inmouse oocytes, and that they are dynamically regu-lated with respect to location and cell cycle state, im-plies that, as a whole, the oocyte centrosome complexserves to sort and coordinate the multiple activities ofnuclear and cytoplasmic maturation.

Accumulating large pools of centrosomal proteinssuch as pericentrin and g-tubulin would be an expectedconsequence of the growth phase of oogenesis given theneed to support centrosome functions in embryonic mi-toses, independent of parental inheritance patterns(Schatten, 1994). As pointed out by Schatten (1994),even paternal centrosome function must draw on ma-ternal reserves. Just as other maternal mRNAs or pro-teins must be regulated for their involvement at spe-cific stages of meiosis or embryogenesis, so too would itbe beneficial to carefully modulate centrosome functionprior to and during meiotic maturation. The proposaloutline below addresses the aforementioned patterns ofcentrosome distribution and nucleation capacity basedon a model involving spatial sorting of key cell cycleand structural proteins. The applicability of this modelto all vertebrate oocytes is unknown but it may explainhow the special qualities of female meiosis that ensuremeiotic chromosome segregation and volume regula-tion during cytokinesis are accomplished.

Stable, nucleation competent, perinuclear centro-somes are present in small growing mouse oocytes(Wickramasinghe and Albertini, 1992). Once oocytegrowth is completed, a solitary perinuclear centrosome

persists and multiple cortical centrosomes assemble inresponse to hormones that influence the meiotic com-petence (ability to enter M-phase) of oocytes. In fact,exogenous FSH stimulation both increases the numberand MT nucleation in cortical centrosomes (Messingerand Albertini, unpublished). Since the total MT nucle-ating activity of a cell may be influenced by MTOCnumber (Zimmerman et al., 1999) or availability ofg-tubulin/pericentrin stores, any spatial and temporalconstraint on MT nucleation would require strict sort-ing or sequestration of rate-limiting factors for MT-assembly. Oocytes have an enormous pool of availablea/b tubulin that is accessible for assembly in responseto taxol (Fig. 2A,C; Albertini, 1987). Moreover, we showthat taxol readily recruits a substantial pool of pericen-trin that is assembled into aggregates not unlike thosetypically seen at meiotic spindle poles. Thus, given theexistence of a large pericentrin and tubulin pool, how isit that spindle MT assembly is associated with chroma-tin at meiotic resumption (and maintained through-out), whereas cytoplasmic centrosomes exhibit limitedbursts of nucleation at anaphase of both meiotic divi-sions?

This spatially restricted pattern of MT nucleationduring the meiotic cell cycle is best explained by sortingof a nucleation rate-limiting factor like g-tubulin. Wepropose that at the onset of meiotic maturation, inresponse to rising MAP kinase and MPF levels, g-tu-bulin is released from cytoplasmic centrosomes so thatit can interact directly with germinal vesicle chroma-tin. Loss of centrosome g-tubulin ensures that limited“extranuclear” MT assembly occurs and, in conjunctionwith dynein, chromatin directs bipolar meiotic spindleassembly through metaphase of meiosis-1 (Brunet etal., 1999). Upon spindle anchoring to the cortex, medi-ated by an actin-based remodeling of spindle associatedcentrosomes, anaphase onset occurs resulting in therelease of spindle associated cell cycle factors (MAPkinase, proteasome, cyclin) and g-tubulin that are rap-idly incorporated into and sequestered by cytoplasmiccentrosomes. g-Tubulin confers MT nucleation to cor-tical centrosomes and, concurrently, through dynein orother motors, stabilizes both the cell cortex and or-ganelles to prevent their displacement during polarbody extrusion.

A predictable sequelae to this transient period of MTnucleation is transport of the proteasome that de-grades cyclins, g-tubulin, and pericentrin (Zimmermanet al., 1999). This explains the total loss of centrosomesduring telophase (Messinger and Albertini, 1991) andthe subsequent release of components needed to reca-pitulate assembly of the spindle and the reformation ofcortical centrosomes during metaphase of meiosis-II.

While this model brings focus to explaining the ob-served dynamics of centrosomes in oocytes, it also lendsperspective on the shifting balance of function thatleads on the one hand to normal segregation of chro-mosomes (nuclear maturation) and on the other toasymmetric cytokinesis (cytoplasmic maturation). Justas defects in male centrosomes have been implicated incertain human infertility conditions, it seems likelythat comparable deficits in female gametes will findtheir basis in centrosome dysfunction during oogenesis.


ACKNOWLEDGMENTSThis work was supported by NIH Grant HD20068 to

D.A. Mary Jo Carabatsos and Catherine Combelles aresupported in part by the NIH Training Program inDevelopmental Biology HD07403. We thank SteveDoxsey for the generous gift of pericentrin antibodyand Elizabeth Benecchi for continued support and en-couragement.

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Sorting and reorganization of centrosomes during oocyte maturation in the mouse

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