centrosome and microtubule dynamics during meiotic ... · centrosome and microtubule dynamics...

Centrosome and microtubule dynamics during meiotic progression in the

mouse oocyte

SUSAN M. MESSINGER and DAVID F. ALBERTINI*

Program in Cell, Developmental and Reproductive Biology, Tufts University Health Sciences Schools, 136 Harrison Ave, Boston,MA 02111, USA

* Author for correspondence at Department of Anatomy and Cellular Biology, Tufts University Health Sciences Schools,136 Harrison Ave, Boston, MA 02111, USA

Summary

The disposition, function and fate of centrosomeswere analysed in mouse oocytes undergoing in vitromeiotic maturation, using multiple-label fluor-escence microscopy. Oocytes fixed at various pointsduring meiotic progression were double labeled witheither human centrosome-specific antibody, 5051,and anti-tubulin antibodies or 5051 and MPM-2antibodies in order to evaluate the microtubulenucleation capacity and phosphorylation status ofcentrosomes during this process. Double labelingwith anti-tubulin antibodies revealed two popu-lations of centrosomes that undergo stage-specificchanges in number, location and microtubule nu-cleation capacity in relation to spindle assembly andcytoplasmic events. Specifically, one population wasconsistently associated with chromatin throughoutmeiotic maturation whereas a second population ofcytoplasmic centrosomes exhibited maximal num-

bers and nucleation capacity at prometaphase andanaphase of meiosis-I. Quantitative evaluation ofcytoplasmic centrosomes indicated increased num-bers during the transition from diakinesis to pro-metaphase and metaphase to anaphase and totaldisappearance during telophase. Colocalizationstudies with MPM-2 revealed that centrosomes werealways phosphorylated. However, at metaphase ofmeiosis I and II the microtubule nucleation capacityof centrosomes was diminished. These resultssuggest the existence of two discrete populations ofcentrosomes in the mouse oocyte that are coordi-nately regulated to subserve aspects of microtubuleorganization relative to both nuclear and cytoplas-mic events.

Key words: centrosome, microtubule, oocyte, MPM-2, meiosis.

Introduction

The centrosome is the major microtubule organizingcenter and focal point for microtubule growth within thecell. By virtue of their microtubule nucleating capacity,centrosomes perform many functions that include organiz-ing the interphase cytoskeleton and cytoplasm anddirecting the formation of the mitotic spindle. Changes inthe function of the centrosome have been associated with achange in the organization of microtubules during specificphases of the cell cycle. During the G2 to M transitionthere is a rapid disassembly of cytoplasmic microtubulesand an increased nucleation capacity associated with thecentrosome (Kuriyama and Borisy, 1981). Many studiesclearly indicate that centrosomes are essential for theformation of the mitotic spindle (Vandre and Borisy, 1990)and are known to influence the location of the cleavagefurrow during cytokinesis (Rappaport, 1986). In thiscontext, it is important to note that many types of proteinsare associated with spindle pole centrosomes, includingmotor molecules such as kinesin (Neighbors et al. 1988)and dynein (Pfarr et al. 1990), micro tubule-associatedproteins such as MAPI (Sherline and Mascaro, 1982) andCa/calmodulin kinase II (Ohta et al. 1990). Recently, cellcycle control proteins such as the cdc2 kinase have beenlocalized to the spindle pole (Bailly et al. 1989; Riabowol et

Journal of Cell Science 100, 289-298 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

al. 1989; Rattner et al. 1990). It has now been shown in invitro systems that cdc2 can regulate changes in centro-some-directed microtubule dynamics during the inter-phase to metaphase transition (Verde et al. 1990). Exactlyhow cdc2 kinase regulates the dynamics and lengths ofmicrotubules nucleated from centrosomes during the cellcycle remains to be resolved, but in vitro systems such asthose described above offer great promise in this direction.

In general, mitotic cells possess two centrosomes, one ateach spindle pole, consisting of a pair of centriolessurrounded by osmiophilic material called pericentriolarmaterial (PCM; Wheatly, 1982). It is the PCM that hasbeen shown to contain the microtubule nucleating ca-pacity of the centrosome (Kuriyama and Borisy, 1981).Most dividing cells contain centrioles in their spindle polecentrosomes; however, plant cells, yeast and fungi, and,most notably, mammalian oocytes are known to dividewithout centrioles (Maro and Karsenti, 1986). In themouse, centrioles are present in oogonia and oocytes butare eventually lost at pachytene of meiosis-I and theabsence of centrioles has been reported in both the firstand second meiotic spindles (Szollosi et al. 1972). Unlikemitotic cells, metaphase-II-arrested mouse oocytes con-tain, in addition to spindle pole-associated centrosomes, apopulation of non-spindle-associated microtubule organiz-ing centers (MTOCs; Maro et al. 1985; Schatten et al.

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1986). After fertilization, these MTOCs nucleate micro-tubules and participate in pronuclear movement and inthe formation of subsequent mitotic spindles (Maro et al.1985; Schatten et al. 1986). In addition, it is not until thelate morulae and early blastocyst that centrioles reappearduring preimplantation development in the mouse (Szol-losi, 1972).

Evidence of MTOCs in maturing mouse oocytes suggeststhat alterations in the spatial organization of centrosomesis closely coupled to follicular development in vivo(Mattson and Albertini, 1990). In addition, several groupshave reported the appearance of MTOCs in taxol-treatedmammalian oocytes (Rime et al. 1987; Albertini, 1987).Taxol treatment results in the formation of subcorticalasters that nucleate microtubules and are always excludedfrom the spindle area over which an actin-rich microvil-lus-free domain exists. Furthermore, cytoplasmic tubulin-containing foci have been observed in mouse oocytesmatured in vitro using fluorescence microscopy (Wassar-man and Fujiwara, 1978). Despite the fact that previousstudies have addressed the organization and function ofcentrosomes during embryonic development, the origin ofcentrosomes in respect of the growth phase and maturatio-nal stages of oogenesis is unknown. In this paper we haveevaluated the organization and function of centrosomesduring meiotic progression in synchronous populations ofmouse oocytes to gain insight into the relative contri-bution of centrosomal material in the formation of themeiotic spindle and in cytoplasmic events during meiosis.Here we report the existence of two populations ofcentrosomes in the mouse oocyte, one group associatedwith the nucleus and the other located in the cytoplasm,which are differentially regulated with respect to micro-tubule dynamics during meiotic cell cycle progression.

Materials and methods

Collection and culture of mouse oocytesIn all experiments oocytes were obtained from 21- to 25-day-oldCF-1 mice injected 44-48 h earlier with 5 i.u. of pregnant mare'sserum gonadotropin (Sigma Chem. Co., no. G-4877) to stimulatefollicular development. Cumulus-enclosed oocytes were collectedby follicular puncture and immediately fixed or cultured for1,2,4,6,8,10,11,12 or 14 h in Phenol Red-free Eagle's MEM (Gibco,Grand Island, NY) supplemented with Earle's salts, 2mMglutamine, 0.23 mM pyruvate, 100 i.u. ml"1 penicillin,100 jiigml"1 streptomycin and 0.3 % bovine serum albumin (BSA)in a humidified atmosphere of 5% CO2 at 37 °C. Mouse oocytescultured under these conditions are developmentally competent(Schroeder and Eppig, 1984). After removal of cumulus cells bygentle pipetting, the oocytes were fixed and extracted, for 20 minat 37°C, in a microtubule stabilizing buffer (0 .1M Pipes, pH6.9,5HIM MgCl2.6H2O, 2.5mM EGTA) containing 2.0% formal-dehyde, 0.5% Triton X-100, 1/un taxol, 10 units ml"1 aprotininand 50% deuterium oxide (Herman et al. 1983), washed threetimes in a blocking solution of phosphate-buffered saline (PBS)containing 2 % BSA, 2 % powdered milk, 2 % normal goat serum,0.1 M glycine and 0.01 % Triton X-100 and then stored at 4°C for3-4 days until processed.

Processing of oocytes for fluorescence analysisIn order to define systematically centrosome and microtubuledynamics in mouse oocytes, multiple fluorescence labeling usingtriple-stain analysis was performed. Adjustments to our standardprotocols with respect to fixation techniques and incubation timewere necessary in order to localize centrosomes. Specifically, itwas found that a higher concentration of Triton (0.5 %) in thefixative combined with an increase in the incubation time of theprimary antibody to 24 h enhanced the immunodetection of

antibody 5051-reactive foci without altering overall microtubuleand chromatin displays. These conditions were employed for all5051 experiments described herein. Primary and secondaryantibodies were diluted in PBS containing 0.02% sodium azideand 0.1% BSA. Oocytes were incubated with the humancentrosome-specific antibody, 5051 (Calarco-Gillam et al. 1983),for 24 h at 4°C, washed for 2h in blocking solution and thenincubated in a 1:50 dilution of fluoresceinated goat anti-humanIgG (Cooper Biomedical, lot no. 24269) or Texas Red-conjugatedgoat anti-human IgG (Accurate Chemical Co., lot no. F2077) for45 min at 37 °C. Oocytes were then stained using one of thefollowing protocols: (1) for localization of phosphoproteins oocyteswere incubated in a 1:50 dilution of a mouse (ascites fluid)monoclonal antibody (MPM-2) (Davis et al. 1983) specific formitotic phosphoproteins for 60 min at 37 °C, washed three times inblocking solution and further incubated in a 1:50 dilution of TexasRed-conjugated goat anti-mouse IgG (Accurate Chemical Co., lotno. E9286) for 45 min at 37 °C. (2) For localization of tubulin,optimal results were obtained using a 1:1 mixture of a 1:50dilution of a rat monoclonal antibody, YOL34 (Kilmartin et al.1982), specific for alpha tubulin and a 1:50 dilution of a mousemonoclonal anti-beta tubulin antibody (Accurate Chemical Co.,lot no. N122020), washed for 1 h in blocking solution and furtherincubated for 45 min in a 1:50 dilution of fluorescein-conjugatedgoat anti-mouse IgG (Accurate Chemical Co., lot no. 29046) thatcrossreacts with rat IgG. Oocytes were mounted in glycerol/PBScontaining 25mgml sodium azide as an antifading reagent(Bock et al. 1985) and ljugml"1 of Hoechst 33258 (PolysciencesInc.) to evaluate chromatin patterns at specific steps in meioticprogression. Meiotic stages were characterized on the basis ofchromosome configuration, spindle organization and location, andpresence or absence of polar bodies. Several control experimentswere performed to confirm the specificity of labeling. For allreagents, oocytes were incubated with secondary antibody aloneand no staining was observed under these conditions. Anadditional control for MPM-2 localization was performed byalkaline phosphatase treatment of oocytes under conditions usedby Vandre and Borisy (1989) prior to treatment with MPM-2 andsecondary antibody. No staining was observed under theseconditions. MPM-2 staining of metaphase figures in primarycultures of mouse granulosa cells showed staining at the spindlepoles as reported in other somatic cells (data not shown).

Fluorescence microscopyLabeled oocytes were photographed using a Zeiss Photomicro-scope II or an IM-35 equipped with fluorescein (Zeiss 487709),Texas Red (Zeiss 487714) and Hoechst (Zeiss 487702) selectivefilter sets and a 50 W mercury arc lamp using a 40 x Neofluar(0.9NA) or 63x Neofluar (1.3NA) objective lens. Images wererecorded on Tri-X-pan film using uniform exposure times andprocessed with Acufine developer for 5.25 min at 25°C.

Results

In order to define the functional status and organization ofcentrosomes in mouse oocytes, triple-staining techniqueswere used to evaluate synchronous populations of mouseoocytes. A kinetic analysis on meiotic progression wasperformed on oocytes triple-stained for localization ofchromatin, centrosomes and microtubules in order toascertain optimal times for analyzing all stages of meioticprogression including those of extremely brief duration.The data from a representative experiment involving 282oocytes cultured for various periods of time are shown inFig. 1. Within 2 h after isolation from the follicle, 100% ofthe oocytes undergo germinal vesicle breakdown (GVBD).During this 2h period, oocytes rapidly transit throughboth early and late phases of diakinesis. Prometaphaseencompassed a 4h period ranging from 2-6h followingisolation. Metaphase-I is most apparent at approximately5 h after isolation and this step is distinguished cytologi-

290 S. Messinger and D. F. Albertini

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4 5 6 7

Hours in culture

10 12

GVMI

DK(E)ANA

DK(L)• TL

PMMil

Fig. 1. Kinetics of meioticprogression during in vitromaturation of mouse oocytes.The percentage of oocytes ineach of the stages indicated isshown for a representativeexperiment involving a total of282 oocytes. GV, germinalvesicle stage; DK(E), earlydiakinesis; DK(L), latediakinesis; PM, prometaphase;MI, metaphase-I; ANA,anaphase; TL, telophase; Mil,metaphase-II.

cally in having bivalents that lie equatorially between thetwo spindle poles. Although all oocytes isolated at 5hpossess a bipolar spindle, not all the bivalents are tightlyaligned at the spindle equator, with one bivalent typicallyobserved approaching the spindle equator suggestingasynchronous alignment of chromosomes as they movetoward the metaphase plate. Oocytes possessing a tightlyaligned metaphase plate are rare (3 %) when seen at 5 h,suggesting that the transition between late prometaphaseand early anaphase is very brief in duration. Anaphasecovers a 3- to 5-h time span with maximal numbers (76 %)of anaphase figures seen at 8 h after isolation. By 10 h ofculture, 62 % of the oocytes are in telophase and secondmetaphase figures initially appear at this time, with themajority of oocytes (96 %) reaching metaphase-II by 12 hfollowing isolation. Thus, these results demonstrate therelative synchrony of oocyte maturation under theseculture conditions and establish the optimal times formonitoring even transient meiotic states.

To determine the position and organization of centro-somes relative to spindle assembly and cytoplasmicevents, centrosomes were localized by using the 5051antibody in conjunction with anti-tubulin antibodies toestablish the microtubule nucleation status of centro-somes. As shown in Fig. 2B and C, germinal vesicle (GV)stage oocytes consistently display two populations ofcentrosomes from which short microtubules radiate; thosein close proximity to the nucleus (1.7 per oocyte, TI=25)and those located in the periphery near the cortex (6.2 peroocyte, 7i=25). A dramatic rearrangement of centrosomalmaterial occurred upon GVBD, highlighted in earlydiakinesis by the presence of two discrete centrosomal foci,one consistently associated with the condensing chromatinand the other located in the cytoplasm distinct from theregion occupied by the condensing chromatin (Fig. 2E,F).At this stage, the large cytoplasmic centrosome consist-ently displays radiating microtubules whereas thechromatin-associated centrosome does not; however, afocus of anti-tubulin staining is observed within the centerof the nucleus-associated centrosome. During later diakin-esis (Fig. 2G,H,I), numerous centrosomes are associatedwith the thin chromatin strands from which a fewmicrotubules radiate and several distinct MTOCs arelocated in the cytoplasm away from the chromatin region.

By prometaphase, there is a dramatic increase in themicrotubule nucleating capacity of the centrosomes associ-ated with both the newly developing spindle and cytoplas-mic foci as evidenced by the increase in anti-tubulinlabeling (Fig. 2J,K,L). In addition, the number of cytoplas-mic centrosomes increases and they are notably excludedfrom the spindle area.

Fig. 3A,B and C illustrates the chromatin, tubulin- andcentrosome labeling pattern, respectively, for an oocyteapproaching the first metaphase. The position of thechromosomes, with one bivalent in close proximity to thespindle, indicates that the bivalents are almost completelyaligned at the equator. Late in prometaphase or atmetaphase of meiosis-I, oocytes are observed with multiplecytoplasmic centrosomes that lack microtubules. In con-trast to the staining pattern of oocytes approachingmetaphase-I, those in anaphase show numerous cytoplas-mic centrosomes from which microtubules radiate(Fig. 3D,E,F). Shorter microtubules were located oncentrosomes at this stage, compared to prometaphase.Oocytes at telophase consistently show an absence ofcentrosomes in both the meiotic spindle and the cytoplasm(Fig. 3G,H,I). Only upon reaching metaphase-II do thecentrosomes reappear; however, these do not nucleatemicrotubules (Fig. 3J,K,L). The polar body containedbarely detectable centrosomal material observed as smallfoci of immunoreactivity. In a few instances, large polarbodies were formed that contained a spindle with 5051-reactive, material at each pole. These results demonstratethat both the microtubule nucleation capacity associatedwith the centrosome and their immunodetection appear todiffer at specific stages during the cell cycle. Sincecentrosome phosphorylation in mitotic cells is associatedwith an increased nucleation capacity, as shown using theM-phase-specific phosphoprotein antibody MPM-2 (Cen-tonze and Borisy, 1990), we employed this probe toevaluate directly the phosphorylation status of centro-somes by indirect immunofluorescence microscopy.

To determine whether the differences in microtubuledynamics associated with the centrosome observed duringmeiotic progression were correlated with phosphorylationstatus, oocytes were cultured and fixed at variousintervals and then stained with MPM-2 and 5051. Fig. 4Aillustrates the MPM-2 staining pattern of an oocyte in

Centrosome dynamics during meiosis 291

Fig. 2. Correlative Hoechst 33258 (A,D,G,J), anti-tubulin (B,E,H>K) and anti-centrosome (C,F,I,L) staining patterns in mouseoocytes during meiotic progression from GV to prometaphase. (A-C) GV stage oocyte (time zero culture) containing 1 microtubuleaster located near the nucleus and several others in the cytoplasm (arrowheads, B) all of which stain with the anti-centrosomeantibody, 5051 (arrowheads, C). (D-F) Early diakinesis ( lh culture) in which the anti-tubulin staining pattern (E) illustrates twoMTOCs, one of which is associated with the condensing chromatin (white arrow, E) and the other that assumes a cytoplasmicposition. Both MTOCs react with the anti-centrosome antibody (F). (G-I) Oocyte in late diakinesis (1 h culture) illustratingchromatin (G), tubulin (H) and centrosome (I) staining pattern. Note the multiple MTOCs in the cytoplasm (arrowheads, H) thatcolocalize with the 5051 antibody (arrowheads, I). (J-L) Oocyte in prometaphase-I (4h culture) is shown with condensedchromosomes (J) associated with microtubules of the newly forming meiotic spindle. Note the ring of centrosomes surrounding thechromosomes and multiple centrosomes located in the cytoplasm away from the spindle area (arrowheads, L). Bar, 20 ;/m.


Fig. 3. Correlative Hoechst 33258 (A,D,G,J), anti-tubulin (B,E,H,K) and anti-centrosome staining patterns in mouse oocytes duringmeiotic progression from metaphase I to metaphase II. (A-C) Staining pattern in metaphase I stage oocyte (5 h culture) containinga brightly stained spindle, stained with anti-tubulin (arrow, B) and cytoplasmic centrosomes (arrowheads, C) that do not nucleatemicrotubules (B). (D-F) Staining pattern of an oocyte in anaphase (8h culture) in which the anti-tubulin staining patternillustrates a brightly stained spindle (arrow, E) and several microtubule asters (arrowheads, E) which colocalize with anti-centrosome reactive foci (arrowheads, F). (G-I) Staining pattern of an oocyte in telophase (10 h culture) revealing a typicalmidbody tubulin pattern (H) that is devoid of centrosomes (I). (J-L) Metaphase-II-arrested oocyte (12 h culture) containing spindle(arrow, K) with tight centrosomal foci at each spindle pole (arrow, L) and cytoplasmic centrosomes (arrowheads, L) that do notnucleate microtubules (K). pb, polar body. Bar, 20 <̂m.


Fig. 4. Correlative MPM-2 (A,B,D,E,G,H) and anti-centrosome (C,F,I) staining patterns of foci in mouse oocytes at various stagesof maturation. (A-C) Late diakinesis oocyte (2 h culture) containing MPM-2-reactive foci (arrowheads, A; enlarged, arrowheads inB) that colocalize with anti-centrosome staining foci (arrowheads, C). (D—F) Anaphase oocyte (8h culture) containing MPM-2-reactive foci (arrowheads, D and E) which colocalize with anti-centrosome staining foci (arrowheads, F). (G-I) Metaphase 2 oocyte(12 h culture) containing cytoplasmic aster that labels with MPM-2 (G,H) and anti-centrosome antiserum (I). Bars: D and G, 20 fan;I, 5 fan.

prometaphase. As described earlier, numerous brightlystained foci were observed throughout the cytoplasm of theoocyte that were excluded from the spindle area (Fig. 4A).Colocalization established these foci to be labeled by boththe MPM-2 and 5051 (Fig. 4B,C). At this stage, foci wereheterogeneous in size and some appeared to be fusion orfission products in view of the proximity of the large andsmall foci (Fig. 4B, arrows). The spindle poles stainedintensely with MPM-2 during anaphase as well as apopulation of non-spindle-associated cytoplasmic foci thatcolocalize with the 5051 antibody (Fig. 4E,F). Attelophaseno cytoplasmic foci were detected by MPM-2 or 5051. Atmetaphase-II, in addition to spindle staining, MPM-2-reactive foci were detected in the cytoplasm that colocal-ized with the anti-centrosome antibody (Fig. 4H,I). Thus,these results indicate that, regardless of meiotic stage or

microtubule status, whenever centrosomal foci weredetectable with 5051, they have been observed to bephosphorylated on the basis of their immunoreactivitywith MPM-2.

To determine whether numerical alterations in cyto-plasmic centrosomes occurred during meiotic progression,the number of cytoplasmic centrosomes per cell wasquantitated in 489 oocytes at specific meiotic stages usingthe MPM-2 antibody in conjunction with the 5051antiserum. The results (shown in Fig. 5) indicate thatthere was a direct correlation between the meiotic stage ofthe oocyte and the number of cytoplasmic foci. Germinalvesicle stage oocytes contained a mean number of 5.5cytoplasmic centrosomes per cell. At early diakinesis,centrosome number decreased to a mean of 1.1 per cell. Asthe cell cycle progressed, there was an increase in the


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UK(L-) DK(Lj I'M MI ANA(E) ANA(L)

Stage of oocyte

TL Mil

Fig. 5. Quantitation ofcytoplasmic centrosomes atspecific stages of the meiotic cellcycle. Mouse oocytes wereincubated for various timeperiods then fixed and stainedwith 5051 and MPM-2. Graphsdisplay the mean number ofcytoplasmic centrosomes per cell.The number of oocytes examinedat each stage is indicated abovethe bars in parentheses. GV,germinal vesicle stage; DK(E),early diakinesis; DK(L), latediakinesis; PM, prometaphase;MI, metaphase-I; ANA(E), earlyanaphase; ANA(L), lateanaphase; TL, telophase; Mil,metaphase-II.

number of centrosomal foci during late diakinesis (7.7 percell) and throughout prometaphase (11.8 per cell). Duringmetaphase-I, the number of centrosomes decreased to amean of 1.8 per cell and increased to a maximum of 14 percell during anaphase. At telophase, the foci disappearedcompletely. By metaphase-II centrosomes reappeared;notably, there were approximately half the number foundat anaphase (7.7 per cell). We next examined spindle-associated centrosomes to determine whether there werestage-specific alterations in the organization of thiscentrosome population during meiotic progression.

Oocytes were fixed at various stages of maturation andwere found to exhibit alterations in the organization ofspindle-associated centrosomes that were stage specific.For example, during late diakinesis, small aggregates ofcentrosomal material were observed, in close associationwith the condensing chromatin, from which short micro-tubules radiate (Fig. 6A,B,C). At prometaphase, centro-somal material is distributed as small aggregates at eachspindle pole (Fig. 6D,E,F). Oocytes at metaphase-Iexhibited a tight focus of centrosomal material at each ofthe spindle poles (Fig. 6G,H,I). At anaphase, the ends ofthe spindle were capped by centrosomal material andlarge aggregates were found at the periphery or within thespindle itself (Fig. 6J,K,L). As shown earlier (see Fig. 31),no centrosomal material was detected at telophase,whereas by metaphase-II each spindle pole displayed asingle focus of centrosomal material (Fig. 6M,N,0). Theseresults show that spindle-associated centrosomal ma-terial, as recognized by the 5051 antibody, undergoeschanges in organization that are similar to the changesobserved for non-spindle-associated centrosomes inexhibiting aggregation and dispersion from diakinesis toanaphase, disappearance at telophase and reappearanceat metaphase-II. This suggests that while differences inthe two populations of centrosomes exist with respect totheir location and/or association with chromosomes, thesetwo populations are comparable with regard to the cyclicalchanges in organization that occur at specific meioticstages.

Discussion

The major finding of this study is that two discrete

populations of centrosomes exist in mouse oocytes, one ofwhich appears to function in the formation of the meioticspindle poles and a second that retains a cytoplasmiclocation in the oocyte. Dynamic alterations in thedistribution, phosphorylation status and microtubulenucleation capacity of oocyte centrosomes occur at specificstages of meiotic progression and are presumably subjectto cell cycle regulation. Although the significance of theseobservations remains to be determined, it is likely that thecentrosomal behavior observed reflects strict cell cyclecontrol of the coordinated nuclear and cytoplasmic eventsthat establish normal meiosis in the mouse oocyte.

Cytoplasmic centrosomes were previously observed byMaro et al. (1985) and Schatten et al. (1986) in metaphase-II-arrested mouse oocytes. Following fertilization, thesecentrosomes have been shown to participate in theformation of the first mitotic spindle (Maro et al. 1985;Schatten et al. 1986). The studies reported here weredesigned to examine the origin of these structures duringthe course of meiotic maturation and clearly establishthat, even at the GV stage, two discrete populations arepresent. These results confirm earlier reports of thepresence of perinuclear centrosomes (Calarco et al. 1972;Mattson and Albertini, 1990), but show an additionalsubset of centrosomes located near the oocyte cortex,which exhibit differences in microtubule nucleationcapacity that correlate to specific stages of cell cycleprogression. The total absence of radiating microtubulesfrom cytoplasmic centrosomes at metaphase-I and -II maybe due to differences in the nucleation capacity ofcentrosomes or a total suppression of assembly at thesespecific stages of meiosis. Our ability to detect thesestructures and their dynamic alterations may be due totechnical modifications in specimen processing, sincehigher detergent concentrations and prolonged labelingsteps have repeatedly validated our results. This approachhas further allowed us to establish the dynamic nature ofcentrosomes within mouse oocytes throughout the courseof meiotic maturation.

With respect to cytoplasmic centrosomes, the consistentreduction in number from the germinal vesicle stage toearly diakinesis coupled with the increase in size of thecentrosomes observed at this stage suggest that centro-somal material aggregates at this early step in matu-ration. This was followed in prometaphase by increased


Fig. 6. Correlative Hoechst 33258 (A,D,G,J,M), anti-tubulin (B,E,H,K,N) and anti-centrosome (C,F,I,L,O) staining patterns ofspindles in mouse oocytes fixed at various stages of meiosis. (A-C) Chromatin, tubulin and centrosome staining patterns,respectively, of an oocyte in late diakinesis; note the aggregates of centrosomal material, some of which nucleate microtubules(arrowheads, C). (D-F) Prometaphase-I stage oocyte showing aggregates of centrosomal material at the spindle poles (arrowheads,F). (G-I) An oocyte in metaphase-I illustrating a tight centromal focus at each spindle pole (arrowheads, I). (J-L) Anaphase stageoocyte containing centrosomal material capping poles of spindle as well as aggregates (arrowheads, L) overlying ends of spindle.(M-O) Metaphase-II stage oocyte; note the tight focus of centrosomal material at each spindle pole (arrowheads, 0). Bar, 10/un.


numbers of smaller centrosomes that were not detectablein metaphase of meiosis-I but reappear at anaphase ofmeiosis-I. Coupled with the total absence of immunodetec-table centrosomes during telophase and re-emergence ofcentrosomes at metaphase-II, these results stronglysuggest that cycles of aggregation, dispersion and re-assembly occur at specific cell cycle transitions. Similarchanges in centrosomal organization have been observedin taxol-treated oocytes. Rime et al. (1987) showed taxol-induced asters in mouse oocytes during meiotic pro-gression in numbers comparable to those reported here. Inaddition, Albertini (1987) demonstrated taxol-inducedaster formation in rat oocytes only when the drug wasadministered after GVBD. In conjunction with the recentobservations that taxol-induced aster formation requiresphosphorylation in M-phase extracts of Xenopus oocytes(Verde et al. 1991), these studies suggest that the ability toform asters is temporally correlated with an increase inmaturation promoting factor (MPF) activity in the oocytethat occurs upon meiotic resumption.

The role of centrosome phosphorylation in the localcontrol of microtubule assembly was further studied inthese experiments. In the mouse oocyte prior to theresumption of meiosis, characteristic changes in bothcytoskeletal and nuclear components occur that aresimilar to the G2 to M transition seen in mitotic cells.During this time an elaborate interphase-like network ofmicrotubules gradually disappears and is replaced bymicrotubule asters that form at the nuclear periphery(Mattson and Albertini, 1990). These transitions inmicrotubule organization in growing mouse oocytes havebeen shown to be associated with the developmentalexpression of meiotic competence, and are furthercorrelated with the phosphorylation of centrosomes(Wickramasinghe et al. 1991). Recent experiments per-taining to the regulation of centrosome and microtubuledynamics in mitotic cells have demonstrated an importantrole for MPF in the modulation of centrosome andmicrotubule dynamics during the G2/M transition. Verdeet al. (1990) snowed that when exogenous cdc2 kinase isadded to isolated centrosomes, both phosphorylation andM-phase microtubule growth dynamics result. Further-more, investigations by Alfa et al. (1990) have shown thatcdc2-cyclin acts locally to regulate spindle formation andthat regulation of cdc2 itself is dependent on microtubules,and recently a microtubule associated protein (MAP)kinase from Xenopus oocytes was shown to phosphorylateisolated centrosomes directly (Gotoh et al. 1991). Thepresent work demonstrates that immunodetectable cen-trosomes (5051) are always phosphorylated at cell cyclesteps characterized by high levels of MPF; conversely, attelophase, when centrosomes are not immunodetectable,phosphorylated foci are absent when MPF levels areknown to be low (Hashimoto and Kishimoto, 1988). Thissuggests that the phosphorylation status of centrosomalmaterial determines the apparent assembly and disassem-bly dynamics of centrosomes that we have correlated withspecific cell cycle stages during meiotic maturation.Moreover, the nucleation capacity of cytoplasmic centro-somes was shown to vary at specific stages, despite the factthey were always phosphorylated, as noted above. Forexample, GV stage, prometaphase and anaphase oocyteshave centrosomes with associated microtubules whereasthe centrosomes of metaphase-I and -II stage oocytes donot. Although subtle changes in phosphorylation cannotbe eliminated, owing to the limited resolution afforded byour technique, the results support the argument that

phosphorylation of the centrosome does not adequatelyexplain the differential regulation of microtubule dy-namics observed. What mechanisms could be responsiblefor these differences in microtubule nucleating capacity?

The suppression of centrosome-nucleated microtubuleassembly observed at metaphase-I and -II occurs whenboth maturation promoting factor (MPF) and cytostaticfactor (CSF) levels are thought to be elevated (Sagata et al.1989). In the mouse oocyte, it has been shown that MPFlevels peak at metaphase of meiosis-I and -II, with lowlevels detectable between these stages as noted earlier(Hashimoto and Kishimoto, 1988). Karsenti et al. (1984)demonstrated that purified centrosomes injected intounactivated metaphase-II-arrested Xenopus oocytes do notsupport microtubule growth. However, when these eggswere artificially activated, microtubule growth occurred.Since the increase in nucleation capacity appeared inconjunction with the loss of CSF, it is possible that CSF isinvolved in the differential regulation of microtubuledynamics of the centrosome during both metaphase-I and-II. Recently, CSF has been identified as the c-mos proto-oncogene product and its destruction at fertilization isdependent on the calcium-activated enzyme, calpain(Watanabe et al. 1989; Sagata et al. 1989). In addition,recent biochemical studies showing the interaction be-tween c-mos protein and tubulin (Zhou et al. 1991) suggestthat c-mos protein may bind to tubulin subunits andprevent assembly or it may act directly on the centrosometo alter microtubule dynamics. The possibility that c-mosprotein may be responsible for the differences in micro-tubule nucleating capacity associated with the centrosomeobserved in these studies deserves further attention.

In addition to the expression of CSF, vertebrate oocytesare also unique in exhibiting asymmetric cleavage at thetime of polar body formation. The polarity exhibited by themammalian oocyte at the time of polar body formation hasbeen thought to reflect the effect of the meiotic spindle onthe oocyte plasma membrane and subcortical cytoplasm.Documented examples of this polarity include changes inthe cortical organization of the oocyte membrane duringmaturation with the establishment of an actin-richmicrovillus-free domain overlying the spindle (Longo andChen, 1985) that exhibit differences in lipid and proteinmobility (Wolf and Ziomek, 1983) and the exclusion ofcortical granules (Ducibella et al. 1988). It is interesting tonote that taxol treatment of rat oocytes results in theformation of multiple subcortical asters excluded from thecortex overlying the meiotic spindle (Albertini, 1987) andthat cytoplasmic centrosomes are excluded from thespindle area in mouse oocytes (Fig. 3F and L). Whether afunctional relationship exists between cytoplasmic centro-somes and polarity determination in the oocyte remains tobe established. Since the mouse oocyte appears tosegregate its centrosomes into spindle organizing andcytoplasmic components, it is possible that this structuralsegregation is used to coordinate the unique demands ofkaryokinesis and cytokinesis for asymmetric cleavageduring meiotic progression. Further studies will be aimedat characterizing the functional roles of both centrosomepopulations during the meiotic cell cycle.

This work was supported by NIH grant HD20068. We thankMarc Kirschner, John Kilmartin and Potu Rao, respectively, fortheir provision of anti-centrosome, anti-tubulin and MPM-2antibodies; and Nancita R. Lomax of the Drug Synthesis andChemistry Branch of the National Cancer Institute for kindlyproviding taxol. It is a pleasure to acknowledge the steady input


from Dineli Wickramasinghe and Peter Sherline during thecourse of this work.

References

ALBERTINI, D. F. (1987). Cytoplasmic reorganization during theresumption of meiosis in cultured preovulatory rat oocytes. Devi Biol.120, 121-131.

ALFA, C. E., DUCOMMU, B., BEACH, D. AND HYAMS, J. S. (1990). Distinctnuclear and spindle pole body populations of cyclin-cdc2 in fissionyeast. Nature 347, 680-682.

BAILLY, E., DOREE, M., NURSE, P. AND BORNENS, M. (1989). p34cdc2 islocated in both nucleus and cytoplasm; part is centrosomallyassociated at G2/M and enters vesicles at anaphase. EMBO J. 8,3985-3995.

BOCK, G., HILCHENBACH, M., SCHAUSENSTEIN, K. AND WICK, G. (1985).Photometric analysis of antifading reagents for immunofluorescencewith laser and conventional illumination sources. J. Histochem.Cytochem. 33, 699-705.

CALARCO, P. G., DONAHUE, R. P. AND SZOLLOSI, D. (1972). Germinalvesicle breakdown in the mouse oocyte. J. Cell Sci. 10, 369-385.

CALARCO-GILLAM, P. D., SIEBERT, M. C, HUBBLE, R., MITCHISON, T. ANDKIRSHNER, M. (1983). Centrosome development in early mouseembryos as denned by an autoantibody against pericentriolarmaterial. Cell 35, 621-629.

CENTONZE, V. E. AND BORISY, G. G. (1990). Nucleation of microtubulesfrom mitotic centrosomes is modulated by a phosphorylated epitope. J.Cell Sci. 95, 405-411.

DAVIS, F. M., TSAO, T. Y., FOWLER, S. K. AND RAO, P. N. (1983).Monoclonal antibodies to mitotic cells. Proc. natn. Acad. Sci. U.S.A.80, 2926-2930.

DUCIBELLA, T., ANDERSON, E., ALBERTINI, D. F., AALBERG, J. ANDRANGARAJAN, S. (1988). Quantitative studies of changes in corticalgranule number and distribution in the mouse oocyte during meioticmaturation. Devi Biol. 130, 184-197.

GOTOH, Y., NISIHIA, E., MATSUDA, S., SHIINA, N., KOSAKO, H.,SHIOHAWA, K., AKIYAMA, T., OHTA, K. AND SAKAI, H. (1991). In vitroeffects on microtubule dynamics of purified Xenopus M phase-aactivated MAP kinase. Nature 349, 251-254.

HASHIMOTO, N. AND KISHIMOTO, T. (1988). Regulation of meioticmetaphase by a cytoplasmic maturation-promoting factor duringmouse oocyte maturation. Devi Biol. 126, 242-252.

HERMAN, B., LANGEVIN, M. A. AND ALBERTINI, D. F. (1983). The effectsof taxol in the organization of the cytoskeleton in cultured ovariangranulosa cells. Eur. J. Cell Biol. 31, 34-45.

KARSENTI, E., NEWPORT, J., HUBBLE, R. AND KIRSCHNER, M. (1984).Interconversion of metaphase and interphase microtubule arrays, asstudied by the injection of centrosomes and nuclei into Xenopus eggs.J. Cell Biol. 98, 1730-1745.

KILMARTIN, J. V., WRIGHT, B. AND MILSTEIN, C. (1982). Rat monoclonalanti-tubulin antibodies derived by using a new non-secreting rat cellline. J. Cell Biol. 93, 576-582.

KURIYAMA, R. AND BORISY, G. G. (1981). Microtubule-nucleating activityof centrosomes in CHO cells is independent of the centriole cycle, butcoupled to the mitotic cycle. J. Cell Biol. 91, 822-826.

LONGO, F. J. AND CHEN, D. Y. (1985). Development of cortical polarity inmouse eggs: Involvement of the meiotic apparatus. Devi Biol. 107,382-394.

MARO, B., HOWLETT, S. K. AND WEBB, M. (1985). Non-spindlemicrotubule organizing centers in metaphase II-arrested mouseoocytes. J. Cell Biol. 101, 1665-1672.

MARO, B. AND KARSENTI, E. (1986). Centrosomes and the spatialdistribution of microtubules in animal cells. Trends biochem. Sci. 11,460-463.

MATTSON, B. A. AND ALBERTINI, D. F. (1990). Oogenesis: Chromatin andmicrotubule dynamics during meiotic prophase. Molec. Reprod. Dev.25, 374-383.

NEIGHBORS, B. W., WILLIANS, R. C, JR AND MCINTOSH, J. R. (1988).Localization of kinesin in cultured cells. J. Cell Biol. 106, 1193-1204.

OHTA, Y., OHBA, T. AND MIYAMOTO, E. (1990). Ca/calmodulin-dependent

protein kinase II: Localization in the interphase nucleus and themitotic apparatus of mammalian cells. Proc. natn. Acad. Sci. U.S.A.87, 5341-5345.

PFARR, C. M., COVE, M., GRISSON, P. M., HAYS, T. S., PORTER, M. E. AND

MCINTOSH, J. R. (1990). Cytoplasmic dynein localizes to kinetochoresduring mitosis. Nature 345, 263-265.

RAPPAPORT, R. (1986). Establishment of the mechanism of cytokinesis inanimal cells. Int. Rev. Cytol. 105, 245-281.

RATTNER, J. B., LEW, J. AND WANG, J. H. (1990). p34cdc2 kinase islocalized to distinct domains within the mitotic apparatus. Cell MotilCytoskel. 17, 227-235.

RIABOWOL, K., DRAETTA, G., BRIZUELA, L., VANDRE, D. AND BEACH, D.(1989). The cdc2 kinase is a nuclear protein that is essential formitosis in mammalian cells. Cell 57, 393-401.

RIME, H., JESSUS, C. AND OZON, R. (1987). Distribution of microtubulesduring the first meiotic cell division in the mouse oocyte: Effect oftaxol. Gamete Res. 17, 1-13.

SAGATA, N., WATANABE, N., VANDE WOUDE, G. F. AND IKAWA, Y. (1989).The c-mos proto-oncogene product is a cytostatic factor responsible formeiotic arrest in vertebrate eggs. Nature 342, 512-518.

SCHATTEN, H . , SCHATTEN, G., M A Z I A , D. , B A L C Z O N , R. AND SlMERLY, C.

(1986). Behavior of centrosomes during fertilization and cell divisionin mouse oocytes and in sea urchin eggs. Proc. natn. Acad. Sci. U.S.A.83, 105-109.

SCHROEDER, A. C. AND EPPIG, J. J. (1984). The developmental capacity ofmouse oocytes that matured spontaneously in vitro is normal. DeviBiol. 102, 493-497.

SHERLINE, P. AND MASCARO, R. N. (1982). Epidermal growth factorinduces rapid centrosomal separation in HeLa and 3T3 cells. J. CellBiol. 93, 507-511.

SZOLLOSI, D. (1972). Changes of some cell organelles during oogenesis inmammals. In Oogenesis (ed. J. D. Biggers and A. W. Schuetz), pp.47-64. Baltimore, Maryland: University Park Press.

SZOLLOSI, D., CALARCO, P. AND DONAHUE, R. P. (1972). Absence ofcentrioles in the first and second meiotic spindles of mouse oocytes. J.Cell Sci. 11, 521-541.

VANDRE, D. D. AND BORISY, G. G. (1989). Anaphase onset anddephosphorylation of mitotic phosphoproteins occur concomitantly. J.Cell Sci. 94, 245-258.

VANDRE, D. D. AND BORISY, G. G. (1990). The centrosome cycle inanimal cells. In Mitosis (ed. J. S. Hyams and B. R. Brinkley), pp.39—75. New York, London: Academic Press.

VERDE, F., BERREZ, J., ANTONY, C. AND KARSENTI, E. (1991). Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs:Requirement for phosphorylated factors and cytoplasmic dynein. J.Cell Biol. 112, 1177-1187.

VERDE, F., LABBE, J., DOREE, M. AND KARSENTI, E. (1990). Regulation ofmicrotubule dynamics by cdc2 protein kinase in cell-free extracts ofXenopus eggs. Nature 343, 233-238.

WASSARMAN, P. M. AND FUJIWARA, K. (1978). Immunofiuorescent anti-tubulin staining of spindles during meiotic maturation of mouseoocytes in vitro. J. Cell Sci. 29, 171-188.

WATANABE, I., VANDE WOUDE, G. F., IKAWA, Y. AND SAGATA, N. (1989).Specific proteolysis of the c-mos proto-oncogene product by calpain onfertilization of Xenopus eggs. Nature 342, 505-511.

WHEATLY, D. N. (1982). The Centriole: A Central Enigma of CellBiology. New York: Elsevier Biomedical Press.

WICKRAMASINGHE, D., EBERT, K. AND ALBERTINI, D. F. (1901). Meioticcompetence acquisition is associated with the appearance of M-phasecharacteristics in growing mouse oocytes. Devi Biol. 143, 162-172.

WOLF, D. E. AND ZIOMEK, C. A. (1983). Regionalization and lateraldiffusion of membrane proteins in unfertilized mouse eggs. J. CellBiol. 96, 1790-1796.

ZHOU, R., OSKARSSON, M., PAULES, P. S., SCHULZ, N., CLEVELAND, D. ANDVANDE WOUDE, G. F. (1991). Ability of the c-mos product to associatewith and phosphorylate tubulin. Science 251, 671-675.

(Received 28 May 1991 - Accepted 11 July 1991)


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