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Home , Centrosome cycle, Cytoplasm, Daniel Mazia, Pronucleus

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 105-109, January 1986 Behavior of during fertilization and in mouse and in sea urchin eggs (//maternal Inheritance/) HEIDE SCHATTEN*, *, DANIEL MAZIAt, RON BALCZON*t, AND CALVIN SIMERLY* *Department of Biological Sciences, Florida State University, Tallahassee, FL 32306-3050; and tHopkins Marine Station, , Pacific Grove, CA 93950 Contributed by Daniel Mazia, September 5, 1985

ABSTRACT The forms and locations of centrosomes in to centrosomal material (5), the origins and be- mouse oocytes and in sea urchin eggs were followed through the havior of centrosomes during fertilization and division can whole course of fertilization and first by immu- now be explored. This investigation provides experimental nofluorescence microscopy. Centrosomes were identified with evidence supporting the hypothesis that centrosomes are an autoimmune antiserum to centrosomal material. of indeed "flexible" (1). They reproduce during and the same preparations with tubulin and with the DNA aggregate and separate during mitosis. Sea urchins and dye Hoechst 33258 allowed the correlation of the forms of the probably most animals obey Boveri's rules and the centrosomes with the structures that they generate centrosomes are paternally inherited. Surprisingly, mouse and with the stages of meiosis, syngamy, and mitosis. The centrosomes are of maternal origin.- results with sea urchin eggs conform to Boveri's view on the paternal origin ofthe functional centrosomes. Centrosomes are MATERIALS AND METHODS seen in spermatozoa and enter the egg at fertilization. Initially, Mouse and sea urchin fertilization was as described (6). Sea the centrosomes are compact, but as the eggs enter the mitotic urchin eggs were extracted in a microtubule-stabilization cycle the forms of the centrosomes go through a cycle in which buffer (7), and mouse egg were stabilized with they spread during interphase, apparently divide, and con- a similar mixture (4). The cells were affixed to polylysine- dense into two compact poles by metaphase. In , they coated coverslips (8). Sea urchin eggs were fixed in methanol spread to form flat poles. In telophase and during reconstitu- at - 10TC and mouse eggs were fixed in 10 mM ethylene glycol tion of the daughter nuclei, the centrosomal material is bis(succinimidyl)succinate (9). Autoimmune centrosomal an- dposed as hemispherical caps around the poleward surfaces tiserum 5051 was derived from a patient with scleroderma as of the nuclei. Mouse lack centrosomal antigen. In the described (5). Centrosomes, microtubules, and DNA in the unfertilized mouse , the meiotic spindle poles are dis- same egg were detected by first labeling with centrosomal played as broad-beaded centrosomes. In addition, centrosomal antibodies followed with antitubulin (10) and then staining the material is detected in the cytoplasm as particles, about 16 in DNA with Hoechst dye 33258. Epifluorescence microscopy number, which are foci of small aster-like arrays of microtu- and photography were as described (6). bules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorpo- RESULTS ration, as the pronuclei develop and more cytoplasmic micro- The arrangements ofthe microtubules at the various stages of tubules assemble, a few ofthe foci associate with the peripheries fertilization and cell division conform well to the shapes of of the nuclei. The number of foci multiplies during the first cell the centrosomes in both sea urchins and mice. In sea urchins, cycle. At the end of interphase, all of the centrosomal foci have centrosomes are found at the base ofthe sperm head (Fig. LA) concentrated on the nuclear peripheries and the cytoplasmic but are not detected in the unfertilized egg. After sperm microtubules have disappeared. Atprophase, thecentrosomes are incorporation, they are introduced into the egg, appearing as seen as two irregular clusters, marking the poles which, at a spot (CENTR, Fig. 1B) from which the microtubules of the metaphase and anaphase, appear as rough bands with foci, and sperm aster extend (MTs, Fig. iB). During the pronuclear the spindle is typically barrel-shaped. At telophase, the migrations (Fig. 1C) and syngamy (Fig. ID), the centrosomes centrosomes are seen as arcs that lie on the nuclear peripheries into an arc over the and microtubules from after cleavage. The ordering of microtubules in all the stages spread pronuclei, reflects the shapes of the centrosomes. The findings on the sea these crescents form partial monasters. At the streak stage urchin confirm the classical theory of the paternal origin of (Fig. 1E), two discrete centrosomes are observed and two centrosomes and contrast with observations tracing the mitotic microtubule arrays extend from the nuclear surface. poles of the mouse egg to maternal centrosomal material. This During first division, the centrosomes are initially compact evidence strengthens the conclusion that mouse centrosomes but later flatten and enlarge. At prophase (Fig. 1F) and meta- derive from the oocyte. phase (Fig. 1G) the centrosomes are compact spheres from which the asters and spindle extend. During anaphase (Fig. 1H) Centrosomes, recently proposed by Mazia to be "flexible the centrosomes flatten and microtubules are lost at the astral bodies" (1), have been thought to be of paternal origin since centers. At telophase (Fig. 11) the centrosomes enlarge into the early studies of Boveri (ref. 2, reviewed in ref. 3). ellipses with regional concentrations of antigen. The micro- However, evidence that microtubules are organized by tubules continue to elongate at the astral peripheries and centers within the unfertilized egg during mouse fertilization disassemble at the aster centers. At cleavage the centrosomes (4) has raised the question whether mammalian centrosomes condense along the poleward faces ofthe karyomeres (Fig. LI) might be maternally inherited. With the recent discovery of and daughter nuclei (Fig. 1K), with microtubules correspond- ingly organized into partial monasters. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *Present address: Department of , Baylor College of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medicine, Houston, TX 77030. 105 Downloaded by guest on September 25, 2021 106 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 83 (1986) El= -I

FIG. 1. Centrosomes during sea urchin fertilization and division. Centrosomes are found at the base of sperm heads (arrows, A) but not in unfertilized eggs (not shown). After sperm incorporation (B), they appear as a spot (CENTR, left panel) on the male pronucleus (DNA, center panel) at the center of the microtubules comprising the sperm aster (MTs, right panel). Following the pronuclear migrations (C) and during pronuclear fusion (D), the centrosomes spread into crescents from which microtubules are organized. Two centrosomes are observed at the streak stage (E), when the bipolar microtubule array extends from the nucleus. At prophase (F) the centrosomes condense and are at the center of a pair of asters. At tnetaphase (G) they remain as compact spheres from which the astral and spindle microtubules emanate. They flatten at anaphase (H) while the microtubules at the astral peripheries elongate and those at the astral centers disassemble. During telophase (I) the centrosomes expand in the direction of the next mitotic plane and there is a corresponding loss of microtubules at the astral interiors. The centrosomes aggregate on the poleward surfaces of the decondensing karyomeres (J) and reconstituting nuclei (K) during cleavage. In G and H, eggs are triple-stained for centrosomes (CENTR), microtubules (MTs), and DNA. Others are double-stained for centrosomes and DNA, with an antitubulin image at the same stage. M, male pronucleus; F, female pronucleus. Arrows in C and D point to . (Bars = 10 ,um.) Centrosomes -are not detected in mouse sperm, and the organizing centers in mouse oocytes. Microtubules radiate unfertilized mouse oocyte displays an unusual pattern of from each focus (MTs, Fig. 2A). At sperm incorporation (Fig. centrosomal material, as predicted by earlier observation of 2 C and D) and the pronuclear movements (Fig. 2 E and F), the arrangements ofmicrotubules (4). Centrosomal antigen is asters extend from the centrosomal foci. Foci with asters detected at the meiotic spindle poles (ref. 5; Fig. 2 A and B) associate with the pronuclei (Fig. 2 C and E; Table 1). Later, and as 16 cytoplasmic concentrations (CENTR, Fig. 2A; numerous foci are found and the pronuclei are embedded Table 1). Maro et al. (29) also find non-spindle microtubule- within an array of microtubules (Fig. 2 F and G). All Downloaded by guest on September 25, 2021 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 83 (1986) 107 Table 1. Centrosomal foci during the first in mouse eggs Stage No. of foci (mean ± SEM, n = 95) Unfertilized oocyte 16.3 ± 5.6* Oocyte during sperm incorporation 15.5 ± 6.0 Oocyte during pronucleus 14.8 ± 3.1 formation (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 ± 8.7 but 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 ± 16.1 at end of first interphase (11.6 ± 8.7 with F pronucleus; 14.4 ± 6.7 with M pronucleus) Prophase 38.8 ± 12.2 Metaphase 15.4 ± 4.1 Anaphase and telophase 15.6 ± 3.0 Cleavage 20.5 ± 3.3 The number of detectable aggregates of centrosomal antigen U increases 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 the pronuclear surfaces at the end of first interphase as the cytoplasmic microtubules disassemble, leaving pronuclear sheaths of microtubules (Fig. 2I). During mitosis in the mouse egg, the centrosomes form blunt irregular poles (5) and the barrel-shaped anastral spindle is organized in the absence of functional centrioles (4). At prophase (Fig. 3 A and B) the centrosomal foci aggregate as two broad clusters from which microtubules extend towards the . At prometaphase (Fig. 3C) the centrosomes become more compact and the mitotic spindle becomes apparent. At metaphase the centrosomes remain condensed and the spindles are well-defined (Fig. 3 D and E). Foci not included in the spindle poles (arrows, Fig. 3E) organize small asters. During anaphase (Fig. 3F) the centrosomes remain as a plate composed of several foci from which microtubules extend. During cleavage (Fig. 3 G and H) the centrosomes decondense into multiple foci as interzonal microtubules become prominent and typically partial monasters extend from the reconstituting nuclei. Comparison Between Centrosomes During Fertilization, the First Cell Cycle, and the First Division in Sea Urchins and Mice. In sea urchins, the centrosomes are contributed by the sperm (Fig. 4A-1) and organize the sperm astral microtubules (Fig. 4A-2). After the pronuclear migrations (Fig. 4A-3) the centrosomes spread and separate over the nucleus, organizing the bipolar streak (Fig. 4A-4). Mouse fertilization depends on centrosomes derived from

FIG. 2. Centrosomes during mouse fertilization. Centrosomes The foci aggregate (H) and condense (I) around the apposed (CENTR, left panels) are found as cytoplasmic foci (A) and at the pronuclei at the completion of first interphase as the cytoplasmic meiotic spindle poles (A and B) in unfertilized oocytes. Microtubules microtubules disassemble leaving perinuclear sheaths. In A-E, eggs (MTs, middle panels) extend from the centrosomal material, forming are triple-labeled for centrosomes, microtubules, and DNA. In F-I, the meiotic spindle and cytoplasmic asters; each focus organizes an eggs are double-labeled for centrosomes and DNA, with antitubulin aster (arrows) with brighter ones associated with larger asters images at the same stage. MC, meiotic chromosomes; M, male (triangles). Centrosomes are not detected in mouse sperm or with the pronucleus; F, female pronucleus; arrows, centrosomal foci and entering sperm during incorporation (C and D). They associate with small asters; triangles, corresponding bright centrosomal foci and the developing pronuclei (C-G) as microtubules fill the cytoplasm. larger asters. (Bars = 10 Mim.) Downloaded by guest on September 25, 2021 108 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 83 (1986) 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- U3E pear as spheres in sea urchins (Fig. 4A-5) and as irregular clusters in mice (Fig. 4B-5). At metaphase (Fig. 4A-6 and Fig. 4B-6) the centrosomes remain compact and widen during anaphase and telophase in seaurchins (Fig. 4A-7 and -8) but not always in mice (Fig. 4B-7). After cleavage the centrosomes decondense into crescents on the poleward faces ofthe nuclei, both in sea urchins (Fig. 4A-9) and in mice (Fig. 4B-8). DISCUSSION Fertilization in sea urchins depends on the paternal contri- bution of centrosomes, which are introduced into the egg surrounding the sperm , and this pattern is expected to be confirmed in most all other animals. However, in the mouse and perhaps in other mammals, the sperm does not have centrosomes and the egg retains them during oogenesis; their first mitotic spindles are organized in the absence of centrioles. These differences are not ascribable to the relative states of oocyte maturation (11), the mature pronucleate egg in sea urchins vs. the oocyte arrested at second meiotic metaphase in the mouse, since many other eggs such as those of amphibians (12) are fertilized at stages identical to that in the mouse and are expected to have paternally derived centrosomes as judged by the appearance of a monastral sperm aster adjacent to the incorporated sperm nucleus. The ease and success rates with which parthenogenesis occurs in mammals (13), in contrast with its relative difficulty and vanishingly small percentage in sea urchins (14, 15), under- scores this finding. Indeed, parthenogenetically activated mouse eggs undergo duplication similar to that observed during normal fertilization (unpublished results). The retention of the centrosome in sperm and its loss in eggs appears as a common strategy for ensuring biparental inher- itance. It is unclear why the mouse and perhaps other mammals violate this scheme though genomic contributions by each parent are required for normal fetal development (16). Understanding the nature of the centrosome is critical because various shapes of centrosomes specify different configurations of microtubules (1). In the past, there was some uncertainty as to the significance ofcentrioles for these activities, but increasing evidence assigned the microtubule- initiating function to osmiophilic material within which centrioles were observed (17-19). The same sort of material is found in cells, which lack centrioles (20-22). By the use of autoimmune antibodies, which have been shown to be 'FIG. 3. Centrosomes during first division in mouse eggs. reliable for centrosome detection (5, 23, 24), the present Centrosomes (CENTR, left panels) move as two clusters into the study provides further evidence distinguishing centrioles cytoplasm at prophase (A and B), as an irregular mass of from centrosomes, since the mouse sperm has centrioles but microtubules (MTs, middle panels) forms around the aligning mitotic lacks centrosomal antigen, whereas the mouse egg lacks chromosomes (DNA, right panels). At prometaphase (C) the centrioles (25) but contains centrosomes (ref. 5; Fig. 2). centrosomes appear as broad clusters on opposing sides of the the this serum have not mass as a barrel-shaped anastral spindle becomes Though components recognized by apparent. At metaphase the centrosomes aggregate into either loose been identified and might recognize only a subset ofantigens, irregular bands (D) or more tightly focused sites (E). Centrosomal its crossreactivity with somatic (5, 23), embryonic (5), and foci not associated with spindle poles organize microtubules (arrows, germ cells (ref. 5; Figs. 3 and 4) in mammals, with inverte- E). During anaphase (F) the centrosomes continue their separation. brate eggs (Fig. 1), and even with plant cells (26, 27) indicates At cleavage (G and H) the centrosomes are found along the poleward the well-conserved nature of this particular antigen. surfaces of the blastomere nuclei and the midbody becomes appar- Strong parallels between the chromosome cycle and the ent. All eggs were triple-labeled for centrosomes, microtubules, and centrosome cycle are emerging. Centrosomes as well as DNA. (Bar = 10Lm.) chromosomes are in expanded states during interphase. Although we know little about the replication of centro- the oocyte (Fig. 4B-1), which are found at the meiotic spindle somes, it is clear that they double in number during inter- poles and as 16 dispersed foci; each focus organizes an aster. phase. Both chromosomes and centrosomes attain their most Some of the foci associate with the developing male and compact state at metaphase. As cells enter interphase, chro- female pronuclei (Fig. 4B-2), and during pronuclear apposi- mosomes and centrosomes resume their associations with tion the number of foci and the density of microtubules nuclear envelopes. The phosphorylation ofboth nuclear lam- increases (Fig. 4B-3). At the end of first interphase, all ins (28) and centrosomes (24) might regulate the transition Downloaded by guest on September 25, 2021 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 83 (1986) 109 A B

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FIG. 4. Centrosomes during fertilization and cell division. (A) Sea urchin eggs. The unfertilized egg lacks centrosomes, and they are introduced along with the sperm centriole during incorporation (A-1). As the microtubules of the sperm aster assemble, the centrosomes spread around 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 nucleus at the streak stage (A-4). At prophase, when the has disintegrated, they are displaced into the cytoplasm and nucleate the formation ofthe bipolar mitotic apparatus (A-5). They enlarge by metaphase but retain their spherical configurations (A-6), and at anaphase they begin to flatten and spread with axes perpendicular to the mitotic axis (A-7). At telophase (A-8) the centrosomes have expanded into hemispheres as the astral microtubules disassemble within the asters and continue to elongate at the astral peripheries. Centrosomes are found on the poleward faces of the blastomere nuclei in cleaving eggs (A-9). (B) Mouse eggs. Mouse sperm lack centrosomes and the unfertilized oocyte has 16 cytoplasmic aggregates ofcentrosomal antigen as well as centrosomal bands at the meiotic spindle poles (B-1). Each centrosomal focus organizes an 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 between the adjacent pronuclei (B-3). Toward the later halfofthe first cell cycle, the number offoci increases. At the end ofinterphase all the foci condense on the pronuclear surfaces and sheaths of microtubules circumscribe the adjacent pronuclei (B4). At prophase the centrosomes detach from the nuclear regions, appearing as two broad clusters (B-5) that aggregate into irregular bands at metaphase (B-6); the first mitotic spindle is typically barrel-shaped, anastral, and organized in the absence ofcentrioles. 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 a 4. Schatten, G., Simerly, C. & Schatten, H. (1985) Proc. Natl. Acad. Sci. monoclonal antibody to phosphoproteins (24) detects centro- USA 82, 4152-4156. 5. Calarco-Gillam, P. D., Siebert, M. C., Hubble, R., Mitchison, T. & somes in sea urchin eggs (D. Vandrd, R. Kuriyama, and Kirschner, M. (1983) Cell 35, 621-629. G. G. Borisy, personal communication). 6. Schatten, G., Maul, G. G., Schatten, H., Chaly, N., Simerly, C., This investigation provides experimental proof for the Balczon, R. & Brown, D. L. (1985) Proc. Natl. Acad. Sci. USA 82, "flexible-centrosome" hypothesis (1). The description of 4727-4731. 7. Balczon, R. & Schatten, G. (1983.) Cell Motil. 3, 213-226. centrosomal foci in this paper does not imply that the latter 8. Mazia, D., Schatten, G. & Sale, W. (1975) J. Cell Biol. 64, 198-200. are discrete. If unitary centrosomes can exist in linear form, 9. Miake-Lye, R. & Kirschner, M. W. (1985) Cell 41, 165-175. our methods might only detect nodes ofhigher concentration 10. Brinkley, B. R., Fistel, F. S., Marcum, J. M. & Pardue, R. L. (1980) ofthe antigen. The tubulin images may delineate centrosomal Int. Rev. Cytol. 63, 59-95. 11. Austin, C. R. (1968) Ultrastructure ofFertilization (Holt, New York). structures that are not resolved by centrosomal antibody. 12. Gerhart, J. C. (1980) in Biological Regulation and Development, ed. Centrosomes, which organize microtubule configurations, Goldberger, R. F. (Plenum, New York), Vol. 2, pp. 133-316. undergo replication and division like chromatin: duplication 13. Kaufmann, M. K. (1983) Early Mammalian Development: Parthenoge- during interphase when they are decondensed and separation netic Studies (Cambridge Univ. Press, Cambridge, U.K.). 14. Loeb, J. (1913) Artificial Parthenogenesis and Fertilization (Univ. during mitosis when they condense. The sea urchin centro- Chicago Press, Chicago). some is traced to the sperm and this pattern is expected in 15. Kallenbach, R. J. & Mazia, D. (1982) Eur. J. Cell Biol. 28, 68-76. most all animals (other than mammals). In contrast, the 16. Surani, M. A. H., Barton, S. C. & Norris, M. L. (1984) Nature (Lon- mouse centrosome is maternally inherited. don) 308, 548-550. 17. Gould, R. R. & Borisy, G. G. (1977) J. Cell Biol. 73, 601-615. 18. Rieder, C. L. & Borisy, G. G. (1982) Biol. Cell. 44, 117-132. We thank Dr. Patricia Calarco-Gillam for the generous contribu- 19. Paweletz, N., Mazia, D. & Finze, E.-M. (1984) Exp. Cell Res. 152, 47-65. tions of centrosomal antiserum; Drs. G. Borisy, P. Calarco-Gillam, 20. Pickett-Heaps, J. D. & Northcote, D. H. (1966) J. Cell Sci. 1, 109-120. S. Inoue, M. Kirschner, M. Johnson, B. Maro, N. Paweletz, G. 21. Wick, S. M., Seagull, R. W., Osborn, M., Weber, K. & Gunning, Sluder, and D. Szollosi for helpful discussions and sharing unpub- B. E. S. (1981) J. Cell Biol. 89, 685-690. lished results; and Mr. R. Golder (Marine Biology Laboratory, 22. Bajer, A. S. & Mole-Bajer, J. (1982) Cold Spring Harbor Symp. Quant. Woods Hole) for the drawing (Fig. 4). This research was supported Biol. 46, 263-283. by grants from the National Institutes of Health (HD12913 to G.S.; 23. Brenner, S. L. & Brinkley, B. R. (1982) Cold Spring Harbor Symp. Quant. Biol. 46, 241-254. RCDA HD363 to G.S.; T35-HD7098 to Embryology Course, Marine 24. Vandre, D. D., Davis, F. M., Rao, P. N. & Borisy, G. G. (1984) Proc. Biology Laboratory, Woods Hole) and the National Science Foun- Natl. Acad. Sci. USA 81, 4439-4443. dation (PCM81-04467 to D.M. and PCM83-15900 to G.S.). 25. Szollosi, D., Calarco, P. & Donahue, R. P. (1972) J. Cell Sci. 11, 521-541. 1. Mazia, D. (1984) Exp. Cell Res. 153, 1-15. 26. Wick, S. M. (1985) Cell Biol. Int. Rep. 9, 357-371. 2. Boveri, Th. (1904) Zellen-Studien IV: Ueber die Natur der Centrosomen 27. Clayton, L., Black, C. M. & Lloyd, C. W. (1985) J. Cell Biol. 101, (Fischer, Jena, Germany). 319-324. 3. Wilson, E. B. (1928) The Cell in Development and Hereditary, (Macmil- 28. Gerace, G. & Blobel, G. (1980) Cell 19, 277-287. lan, New York), 3rd Ed. 29. Maro, B., Howlett, S. K. & Webb, M. (1986) J. Cell Biol., in press. Downloaded by guest on September 25, 2021