Proc. Nati. Acad. Sci. USAVol. 83, pp. 105-109, January 1986Cell Biology

Behavior of centrosomes during fertilization and cell division inmouse oocytes and in sea urchin eggs

(mitosis/cytoskeleton/maternal Inheritance/microtubules)

HEIDE SCHATTEN*, GERALD SCHATTEN*, DANIEL MAZIAt, RON BALCZON*t, AND CALVIN SIMERLY**Department of Biological Sciences, Florida State University, Tallahassee, FL 32306-3050; and tHopkins Marine Station, Stanford University,Pacific Grove, CA 93950

Contributed by Daniel Mazia, September 5, 1985

ABSTRACT The forms and locations of centrosomes inmouse oocytes and in sea urchin eggs were followed through thewhole course of fertilization and first cleavage by immu-nofluorescence microscopy. Centrosomes were identified withan autoimmune antiserum to centrosomal material. Staining ofthe same preparations with tubulin antibody and with the DNAdye Hoechst 33258 allowed the correlation of the forms of thecentrosomes with the microtubule structures that they generateand with the stages of meiosis, syngamy, and mitosis. Theresults with sea urchin eggs conform to Boveri's view on thepaternal origin of the functional centrosomes. Centrosomes areseen in spermatozoa and enter the egg at fertilization. Initially,the centrosomes are compact, but as the eggs enter the mitoticcycle the forms of the centrosomes go through a cycle in whichthey spread during interphase, apparently divide, and con-dense into two compact poles by metaphase. In anaphase, theyspread to form flat poles. In telophase and during reconstitu-tion of the daughter nuclei, the centrosomal material isdposed as hemispherical caps around the poleward surfacesof the nuclei. Mouse sperm lack centrosomal antigen. In theunfertilized mouse oocyte, the meiotic spindle poles are dis-played as broad-beaded centrosomes. In addition, centrosomalmaterial is detected in the cytoplasm as particles, about 16 innumber, which are foci of small aster-like arrays of microtu-bules. The length and number of astral microtubules correlatewith the size of the centrosomal foci. After sperm incorpo-ration, as the pronuclei develop and more cytoplasmic micro-tubules assemble, a few of the foci associate with the peripheriesof the nuclei. The number of foci multiplies during the first cellcycle. At the end of interphase, all of the centrosomal foci haveconcentrated on the nuclear peripheries and the cytoplasmicmicrotubules have disappeared. At prophase, the centrosomes areseen as two irregular clusters, marking the poles which, atmetaphase and anaphase, appear as rough bands with foci, andthe spindle is typically barrel-shaped. At telophase, thecentrosomes are seen as arcs that lie on the nuclear peripheriesafter cleavage. The ordering of microtubules in all the stagesreflects the shapes of the centrosomes. The findings on the seaurchin confirm the classical theory of the paternal origin ofcentrosomes and contrast with observations tracing the mitoticpoles of the mouse egg to maternal centrosomal material. Thisevidence strengthens the conclusion that mouse centrosomesderive from the oocyte.

Centrosomes, recently proposed by Mazia to be "flexiblebodies" (1), have been thought to be of paternal origin sincethe early studies of Boveri (ref. 2, reviewed in ref. 3).However, evidence that microtubules are organized bycenters within the unfertilized egg during mouse fertilization(4) has raised the question whether mammalian centrosomesmight be maternally inherited. With the recent discovery of

antibodies to centrosomal material (5), the origins and be-havior of centrosomes during fertilization and division cannow be explored. This investigation provides experimentalevidence supporting the hypothesis that centrosomes areindeed "flexible" (1). They reproduce during interphase andaggregate and separate during mitosis. Sea urchins andprobably most animals obey Boveri's rules and thecentrosomes are paternally inherited. Surprisingly, mousecentrosomes are of maternal origin.-

MATERIALS AND METHODSMouse and sea urchin fertilization was as described (6). Seaurchin eggs were extracted in a microtubule-stabilizationbuffer (7), and mouse egg cytoskeletons were stabilized witha similar mixture (4). The cells were affixed to polylysine-coated coverslips (8). Sea urchin eggs were fixed in methanolat - 10TC and mouse eggs were fixed in 10mM ethylene glycolbis(succinimidyl)succinate (9). Autoimmune centrosomal an-tiserum 5051 was derived from a patient with scleroderma asdescribed (5). Centrosomes, microtubules, and DNA in thesame egg were detected by first labeling with centrosomalantibodies followed with antitubulin (10) and then staining theDNA with Hoechst dye 33258. Epifluorescence microscopyand photography were as described (6).

RESULTSThe arrangements ofthe microtubules at the various stages offertilization and cell division conform well to the shapes ofthe centrosomes in both sea urchins and mice. In sea urchins,centrosomes are found at the base ofthe sperm head (Fig. LA)but are not detected in the unfertilized egg. After spermincorporation, they are introduced into the egg, appearing asa spot (CENTR, Fig. 1B) from which the microtubules of thesperm aster extend (MTs, Fig. iB). During the pronuclearmigrations (Fig. 1C) and syngamy (Fig. ID), the centrosomesspread into an arc over the pronuclei, and microtubules fromthese crescents form partial monasters. At the streak stage(Fig. 1E), two discrete centrosomes are observed and twomicrotubule arrays extend from the nuclear surface.

During first division, the centrosomes are initially compactbut later flatten and enlarge. At prophase (Fig. 1F) and meta-phase (Fig. 1G) the centrosomes are compact spheres fromwhich the asters and spindle extend. During anaphase (Fig. 1H)the centrosomes flatten and microtubules are lost at the astralcenters. At telophase (Fig. 11) the centrosomes enlarge intoellipses with regional concentrations of antigen. The micro-tubules continue to elongate at the astral peripheries anddisassemble at the aster centers. At cleavage the centrosomescondense along the poleward faces of the karyomeres (Fig. LI)and daughter nuclei (Fig. 1K), with microtubules correspond-ingly organized into partial monasters.

*Present address: Department of Cell Biology, Baylor College ofMedicine, Houston, TX 77030.

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Proc. Natl. Acad. Sci. USA 83 (1986)

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FIG. 1. Centrosomes during sea urchin fertilization and division. Centrosomes are found at the base of sperm heads (arrows, A) but not inunfertilized eggs (not shown). After sperm incorporation (B), they appear as a spot (CENTR, left panel) on the male pronucleus (DNA, centerpanel) at the center of the microtubules comprising the sperm aster (MTs, right panel). Following the pronuclear migrations (C) and duringpronuclear fusion (D), the centrosomes spread into crescents from which microtubules are organized. Two centrosomes are observed at thestreak stage (E), when the bipolar microtubule array extends from the nucleus. At prophase (F) the centrosomes condense and are at the centerof a pair of asters. At tnetaphase (G) they remain as compact spheres from which the astral and spindle microtubules emanate. They flattenat anaphase (H) while the microtubules at the astral peripheries elongate and those at the astral centers disassemble. During telophase (I) thecentrosomes expand in the direction of the next mitotic plane and there is a corresponding loss of microtubules at the astral interiors. Thecentrosomes aggregate on the poleward surfaces of the decondensing karyomeres (J) and reconstituting nuclei (K) during cleavage. In G andH, eggs are triple-stained for centrosomes (CENTR), microtubules (MTs), and DNA. Others are double-stained for centrosomes and DNA, withan antitubulin image at the same stage. M, male pronucleus; F, female pronucleus. Arrows in C and D point to centrioles. (Bars = 10 ,um.)

Centrosomes -are not detected in mouse sperm, and theunfertilized mouse oocyte displays an unusual pattern ofcentrosomal material, as predicted by earlier observation ofthe arrangements of microtubules (4). Centrosomal antigen isdetected at the meiotic spindle poles (ref. 5; Fig. 2 A and B)and as 16 cytoplasmic concentrations (CENTR, Fig. 2A;Table 1). Maro et al. (29) also find non-spindle microtubule-

organizing centers in mouse oocytes. Microtubules radiatefrom each focus (MTs, Fig. 2A). At sperm incorporation (Fig.2 C and D) and the pronuclear movements (Fig. 2 E and F),asters extend from the centrosomal foci. Foci with astersassociate with the pronuclei (Fig. 2 C and E; Table 1). Later,numerous foci are found and the pronuclei are embeddedwithin an array of microtubules (Fig. 2 F and G). All

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Proc. Natl. Acad. Sci. USA 83 (1986) 107

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FIG. 2. Centrosomes during mouse fertilization. Centrosomes(CENTR, left panels) are found as cytoplasmic foci (A) and at themeiotic spindle poles (A and B) in unfertilized oocytes. Microtubules(MTs, middle panels) extend from the centrosomal material, formingthe meiotic spindle and cytoplasmic asters; each focus organizes anaster (arrows) with brighter ones associated with larger asters(triangles). Centrosomes are not detected in mouse sperm or with theentering sperm during incorporation (C and D). They associate withthe developing pronuclei (C-G) as microtubules fill the cytoplasm.

Table 1. Centrosomal foci during the first cell cycle inmouse eggs

Stage No. of foci (mean ± SEM, n = 95)Unfertilized oocyte 16.3 ± 5.6*Oocyte during sperm

incorporation 15.5 ± 6.0Oocyte during pronucleus 14.8 ± 3.1formation (0.8 ± 0.5 with F pronucleus;

1.8 ± 0.8 with M pronucleus)Pronucleate eggs 16.5 ± 4.4

(2.2 ± 1.3 with F pronucleus;4.0 ± 2.3 with M pronucleus)

Eggs with adjacent 17.3 ± 8.7but eccentric pronuclei (1.9 ± 1.5 with F pronucleus;

4.1 ± 2.8 with M pronucleus)Eggs with apposed centered 14.5 ± 1.7

pronuclei (1.3 ± 0.5 with F pronucleus;3.8 ± 2.1 with M pronucleus)

Pronucleate eggs 54.0 ± 16.1at end of first interphase (11.6 ± 8.7 with F pronucleus;

14.4 ± 6.7 with M pronucleus)Prophase 38.8 ± 12.2Metaphase 15.4 ± 4.1Anaphase and telophase 15.6 ± 3.0Cleavage 20.5 ± 3.3

The number of detectable aggregates of centrosomal antigenincreases during first interphase and then condenses during mitosis.F, female; M, male.*Exclusive of the meiotic spindle poles.

centrosomal foci migrate (Fig. 2H) and aggregate to thepronuclear surfaces at the end of first interphase as thecytoplasmic microtubules disassemble, leaving pronuclearsheaths of microtubules (Fig. 2I).During mitosis in the mouse egg, the centrosomes form

blunt irregular poles (5) and the barrel-shaped anastralspindle is organized in the absence of functional centrioles(4). At prophase (Fig. 3 A and B) the centrosomal fociaggregate as two broad clusters from which microtubulesextend towards the chromosomes. At prometaphase (Fig.3C) the centrosomes become more compact and the mitoticspindle becomes apparent. At metaphase the centrosomesremain condensed and the spindles are well-defined (Fig. 3 Dand E). Foci not included in the spindle poles (arrows, Fig.3E) organize small asters. During anaphase (Fig. 3F) thecentrosomes remain as a plate composed of several foci fromwhich microtubules extend. During cleavage (Fig. 3 G and H)the centrosomes decondense into multiple foci as interzonalmicrotubules become prominent and typically partialmonasters extend from the reconstituting nuclei.Comparison Between Centrosomes During Fertilization, the

First Cell Cycle, and the First Division in Sea Urchins andMice. In sea urchins, the centrosomes are contributed by thesperm (Fig. 4A-1) and organize the sperm astral microtubules(Fig. 4A-2). After the pronuclear migrations (Fig. 4A-3) thecentrosomes spread and separate over the zygote nucleus,organizing the bipolar streak (Fig. 4A-4).Mouse fertilization depends on centrosomes derived from

The foci aggregate (H) and condense (I) around the apposedpronuclei at the completion of first interphase as the cytoplasmicmicrotubules disassemble leaving perinuclear sheaths. In A-E, eggsare triple-labeled for centrosomes, microtubules, and DNA. In F-I,eggs are double-labeled for centrosomes and DNA, with antitubulinimages at the same stage. MC, meiotic chromosomes; M, malepronucleus; F, female pronucleus; arrows, centrosomal foci andsmall asters; triangles, corresponding bright centrosomal foci andlarger asters. (Bars = 10 Mim.)

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Proc. Natl. Acad. Sci. USA 83 (1986)

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'FIG. 3. Centrosomes during first division in mouse eggs.Centrosomes (CENTR, left panels) move as two clusters into thecytoplasm at prophase (A and B), as an irregular mass ofmicrotubules (MTs, middle panels) forms around the aligning mitoticchromosomes (DNA, right panels). At prometaphase (C) thecentrosomes appear as broad clusters on opposing sides of thechromosome mass as a barrel-shaped anastral spindle becomesapparent. At metaphase the centrosomes aggregate into either looseirregular bands (D) or more tightly focused sites (E). Centrosomalfoci not associated with spindle poles organize microtubules (arrows,E). During anaphase (F) the centrosomes continue their separation.At cleavage (G and H) the centrosomes are found along the polewardsurfaces of the blastomere nuclei and the midbody becomes appar-ent. All eggs were triple-labeled for centrosomes, microtubules, andDNA. (Bar = 10Lm.)

the oocyte (Fig. 4B-1), which are found at the meiotic spindlepoles and as 16 dispersed foci; each focus organizes an aster.Some of the foci associate with the developing male andfemale pronuclei (Fig. 4B-2), and during pronuclear apposi-tion the number of foci and the density of microtubulesincreases (Fig. 4B-3). At the end of first interphase, all

centrosomal foci associate with the pronuclei (Fig. 4B-4).Mitosis in both systems is similar except that the mouse egg

lacks functional centrioles. At prophase the centrosomes ap-pear as spheres in sea urchins (Fig. 4A-5) and as irregularclusters in mice (Fig. 4B-5). At metaphase (Fig. 4A-6 and Fig.4B-6) the centrosomes remain compact and widen duringanaphase and telophase in sea urchins (Fig. 4A-7 and -8) but notalways in mice (Fig. 4B-7). After cleavage the centrosomesdecondense into crescents on the poleward faces of the nuclei,both in sea urchins (Fig. 4A-9) and in mice (Fig. 4B-8).

DISCUSSIONFertilization in sea urchins depends on the paternal contri-bution of centrosomes, which are introduced into the eggsurrounding the sperm centriole, and this pattern is expectedto be confirmed in most all other animals. However, in themouse and perhaps in other mammals, the sperm does nothave centrosomes and the egg retains them during oogenesis;their first mitotic spindles are organized in the absence ofcentrioles. These differences are not ascribable to the relativestates of oocyte maturation (11), the mature pronucleate eggin sea urchins vs. the oocyte arrested at second meioticmetaphase in the mouse, since many other eggs such as thoseof amphibians (12) are fertilized at stages identical to that inthe mouse and are expected to have paternally derivedcentrosomes as judged by the appearance of a monastralsperm aster adjacent to the incorporated sperm nucleus. Theease and success rates with which parthenogenesis occurs inmammals (13), in contrast with its relative difficulty andvanishingly small percentage in sea urchins (14, 15), under-scores this finding. Indeed, parthenogenetically activatedmouse eggs undergo centrosome duplication similar to thatobserved during normal fertilization (unpublished results).The retention of the centrosome in sperm and its loss in eggsappears as a common strategy for ensuring biparental inher-itance. It is unclear why the mouse and perhaps othermammals violate this scheme though genomic contributionsby each parent are required for normal fetal development(16).Understanding the nature of the centrosome is critical

because various shapes of centrosomes specify differentconfigurations of microtubules (1). In the past, there wassome uncertainty as to the significance of centrioles for theseactivities, but increasing evidence assigned the microtubule-initiating function to osmiophilic material within whichcentrioles were observed (17-19). The same sort of materialis found in plant cells, which lack centrioles (20-22). By theuse of autoimmune antibodies, which have been shown to bereliable for centrosome detection (5, 23, 24), the presentstudy provides further evidence distinguishing centriolesfrom centrosomes, since the mouse sperm has centrioles butlacks centrosomal antigen, whereas the mouse egg lackscentrioles (25) but contains centrosomes (ref. 5; Fig. 2).Though the components recognized by this serum have notbeen identified and might recognize only a subset ofantigens,its crossreactivity with somatic (5, 23), embryonic (5), andgerm cells (ref. 5; Figs. 3 and 4) in mammals, with inverte-brate eggs (Fig. 1), and even with plant cells (26, 27) indicatesthe well-conserved nature of this particular antigen.

Strong parallels between the chromosome cycle and thecentrosome cycle are emerging. Centrosomes as well aschromosomes are in expanded states during interphase.Although we know little about the replication of centro-somes, it is clear that they double in number during inter-phase. Both chromosomes and centrosomes attain their mostcompact state at metaphase. As cells enter interphase, chro-mosomes and centrosomes resume their associations withnuclear envelopes. The phosphorylation ofboth nuclear lam-ins (28) and centrosomes (24) might regulate the transition

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Proc. Natl. Acad. Sci. USA 83 (1986) 109

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FIG. 4. Centrosomes during fertilization and cell division. (A) Sea urchin eggs. The unfertilized egg lacks centrosomes, and they areintroduced along with the sperm centriole during incorporation (A-1). As the microtubules of the sperm aster assemble, the centrosomes spreadaround the male pronucleus (A-2). Following the migration of the female pronucleus, they reside at the junction between the pronuclei (A-3)and separate around the time for syngamy. The centrosomes have increased in intensity and are found at opposing poles of the zygote nucleusat the streak stage (A-4). At prophase, when the nuclear envelope has disintegrated, they are displaced into the cytoplasm and nucleate theformation of the bipolar mitotic apparatus (A-5). They enlarge by metaphase but retain their spherical configurations (A-6), and at anaphase theybegin to flatten and spread with axes perpendicular to the mitotic axis (A-7). At telophase (A-8) the centrosomes have expanded into hemispheresas the astral microtubules disassemble within the asters and continue to elongate at the astral peripheries. Centrosomes are found on the polewardfaces of the blastomere nuclei in cleaving eggs (A-9). (B) Mouse eggs. Mouse sperm lack centrosomes and the unfertilized oocyte has 16cytoplasmic aggregates ofcentrosomal antigen as well as centrosomal bands at the meiotic spindle poles (B-1). Each centrosomal focus organizesan aster and, after sperm incorporation, some foci along with their asters begin to associate with the developing male and female pronuclei (B-2).When the pronuclei are closely apposed at the egg center, several foci are found in contact with the pronuclei and typically a pair reside betweenthe adjacent pronuclei (B-3). Toward the later halfofthe first cell cycle, the number offoci increases. At the end ofinterphase all the foci condenseon the pronuclear surfaces and sheaths of microtubules circumscribe the adjacent pronuclei (B4). At prophase the centrosomes detach fromthe nuclear regions, appearing as two broad clusters (B-5) that aggregate into irregular bands at metaphase (B-6); the first mitotic spindle istypically barrel-shaped, anastral, and organized in the absence of centrioles. At anaphase and telophase the centrosomes widen somewhat (B-7),and at cleavage the centrosomes appear on the poleward nuclear faces (B-8). Triangles, centrosomal foci; lines, microtubules.

from interphase structure to that during mitosis, and amonoclonal antibody to phosphoproteins (24) detects centro-somes in sea urchin eggs (D. Vandrd, R. Kuriyama, andG. G. Borisy, personal communication).

This investigation provides experimental proof for the"flexible-centrosome" hypothesis (1). The description ofcentrosomal foci in this paper does not imply that the latterare discrete. If unitary centrosomes can exist in linear form,our methods might only detect nodes of higher concentrationof the antigen. The tubulin images may delineate centrosomalstructures that are not resolved by centrosomal antibody.Centrosomes, which organize microtubule configurations,undergo replication and division like chromatin: duplicationduring interphase when they are decondensed and separationduring mitosis when they condense. The sea urchin centro-some is traced to the sperm and this pattern is expected inmost all animals (other than mammals). In contrast, themouse centrosome is maternally inherited.

We thank Dr. Patricia Calarco-Gillam for the generous contribu-tions of centrosomal antiserum; Drs. G. Borisy, P. Calarco-Gillam,S. Inoue, M. Kirschner, M. Johnson, B. Maro, N. Paweletz, G.Sluder, and D. Szollosi for helpful discussions and sharing unpub-lished results; and Mr. R. Golder (Marine Biology Laboratory,Woods Hole) for the drawing (Fig. 4). This research was supportedby grants from the National Institutes of Health (HD12913 to G.S.;RCDA HD363 to G.S.; T35-HD7098 to Embryology Course, MarineBiology Laboratory, Woods Hole) and the National Science Foun-dation (PCM81-04467 to D.M. and PCM83-15900 to G.S.).

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