Behavior of Centrosomes During Fertilization and Cell Division In
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Proc. Nati. Acad. Sci. USA Vol. 83, pp. 105-109, January 1986 Cell Biology Behavior of centrosomes during fertilization and cell division in mouse 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 in antibodies 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 cleavage 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. Staining of indeed "flexible" (1). They reproduce during interphase and the same preparations with tubulin antibody 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 microtubule 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 cytoskeletons 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 anaphase, 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 sperm lack centrosomal antigen. In the described (5). Centrosomes, microtubules, and DNA in the unfertilized mouse oocyte, 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 Cell Biology, Baylor College of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medicine, Houston, TX 77030. 105 106 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 centrioles. (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 Cell Biology: Schatten et al. Proc. Natl. Acad. Sci. USA 83 (1986) 107 Table 1. Centrosomal foci during the first cell cycle 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.