Motility and centrosomal organization during sea urchin and mouse fertilization

Cell Motility and the Cytoskeleton 6:163-175 (1986)

Motility and Centrosomal Organization During Sea Urchin and Mouse Fertilization

Heide Schatten and Gerald Schatten

Department of Biological Science, Florida State University, Tallahassee

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.

Key words: centrosomes, fertilization, mice, microfilaments, microtubules, mitosis, pericentriolar material. sea urchins

INTRODUCTION

Fertilization is an ideal model in which to investi- gate the activity of microfilaments and microtubules as well as to explore the role of centrosomes in specifying microtubule arrays. This article reviews motility and centrosomes during mouse and sea urchin fertilization. Microfilament activity during sperm incorporation is studied with a novel microfilament inhibitor, Latrunculin A. Microtubule-mediated motility and the inheritance and behavior of centrosomes are considered through the entire process of fertilization and cell division in both mice and sea urchins.

Microfilaments are active throughout sea urchin fertilization [reviewed by Vacquier, 198 I] including during the formation of the acrosomal process [Tilney et al. , 19731, sperm incorporation [Longo, 1980; Schatten and Schatten, 1980, 1981; Tilney and Jaffe, 1980; Cline et al., 19831, microvillar elongation [Eddy and Shapiro,

0 1986 Alan R. Liss, Inc.

1976; Schroeder, 19781 and cytolunesis [reviewed by Schroeder, 19811. Changes in the state and organization of actin in the sea urchin egg cortex has been studied by several investigators [Burgess and Schroeder, 1977; Spu- dich and Spudich, 1979; Begg and Rebhun, 1979; Otto et al., 19801. During mammalian fertilization, microfilaments have been reported in some [Clarke and Yanagi- machi, 1978; Clarke et al., 1982; Flaherty et al., 19831, but not all [Halenda et al., 19841 sperm, and implicated during the formation of the second polar body and incorporation cone and for pronuclear apposition [Shalgi et al., 1978; Mar0 et al., 1984; Battaglia and Gaddum- Rose, 19841. Latrunculin is shown here to inhibit the

Address reprint requests to Dr. Heide Schatten, Department of Biological Science, Florida State University, Tallahassee, FL 32306-3050.

164 Schatten and Schatten

microfilament-mediated events during fertilization and early development [Schatten et al., 1986al.

Centrosomes specify the configurations of microtubules, which in turn direct mitosis [Mazia, 19841, the orientation of cellular movements [Albrecht-BueNer, 19851, the organization of the interphase cytoskeleton [Brinkley et al. , 198 13, nuclear migrations at fertilization [Schatten, 19841, and a variety of other intracellular processes including the maintenance of cell shape and structure [reviewed by Wheatley, 1982 and McIntosh, 19831. While the molecular composition of centrosomes is not understood, classical cytologists such as Boveri [1904; reviewed by Wilson, 1928 and by Mazia, 19841 recognized their critical importance. Laser oblation ex- periments by Berns and coworkers [Berns et al., 1977; Peterson and Berns, 1978; Koonce et al., 19841 have demonstrated the importance of the centrosomal region in directing mitotic spindle formation and cellular migrations. Ultrastructural analyses have resolved clouds of osmiophilic material surrounding centrioles termed “pericentriolar material” (PCM) or “microtubule organizing centers” (MTOC) in a variety of animal cells [Gould and Borisy, 1977; Rieder and Borisy, 1982; Paweletz et al., 19841. Indeed similar osmiophilic material is observed at expected organizing centers in plant cells [Pick- ett-Heaps, 1969; Wick et al., 1981; Bajer and Mole- Bajer, 19821. Recently autoimmune antibodies have been shown to be reliable markers for centrosomal detection [Calarco-Gillam et al., 19831 providing a new avenue for their exploration and characterization.

During fertilization, centrosomes are thought to be paternally inherited and are contributed along with the incorporated sperm centriole. Indeed Boveri’s theory of fertilization postulated that the centrosome was the “fer- tilizing element” that once imported into the egg by the sperm established the future mitotic poles [reviewed by Wilson, 19281. This scheme mandates extranuclear con- tributions by both parents. Since centrosomes establish the precise configurations of assembling microtubules,

they define mitotic axes, unequal cell divisions, and cytoskeletal patterns for development, differentiation, and direction. During the cell cycle centrosomes must reproduce and subsequently separate so that each sibling cell receives a full complement. This investigation provides experimental evidence demonstrating that centrosomes are indeed flexible structures, as recently proposed by Mazia [ 19841, which directly predict the observed microtubule configurations. They reproduce during interphase and aggregate and separate during mitosis. Sea urchins and probably most animals obey Boveri’s rules and the centrioles and centrosomes are paternally inherited. The mouse centrosomes appear to be of maternal origin.

MATERIALS AND METHODS Mouse and Sea Urchin Fertilization In Vitro and Embryo Culture

Mouse and sea urchin gametes were obtained and fertilized in vitro as described by Schatten and co-workers [ 1985al.

Latrunculin Application

Latrunculin A was isolated by Spector and coworkers [1983] and used at 2.6 pM with dimethyl sulfoxide (DMSO) as a solvent as reported by Schatten et al. [ 1986al. Dimethyl sulfoxide (DMSO) never exceeded 0.1 % , and 0.1 % DMSO alone does not produce any of the effects reported here. Sea urchin sperm were induced to undergo the acrosome reaction by exposure to egg jelly. Samples were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy [Schatten and Schatten, 19801.

Microfilament, Microtubule, Centrosome, and DNA Detection

Microfilament detection was performed by fluorescence microscopy with rhodaminyl phalloidin (courtesy of Prof. Th. Wieland, Max Planck Institut, Heidelberg)

TABLE I. Effects of Latrunculin on the Abilitv of SDerm to Fertilize Sea Urchin and Mouse Ems

Percentage of fertilization Control Latrunculin

Sea urchin fertilization Low sperm concentration (ca. 3 sperm/egg)

At 5 min postinsemination 58.0 + 16.4 11.9 1.5 At 10 min postinsemination 80.2 f 8.9 18.6 k 2.8

At 5 min postinsemination 98.0 + 0.2 28.7 f 6.4 At 10 min postinsemination 98.7 + 0.5 35.3 f 2.8

lo5 sperm/oocyte 67.8 k 26.4 58.4 k 15.9

Successful fertilization is assayed in sea urchin eggs by the elevation of the fertilization envelope and the centering of both pronuclei. Successful fertilization in mouse oocytes is determined by the introduction of the spermtail into the egg and the decondensation of a male pronucleus. From Schatten et al. I1986aI.

High sperm concentration (ca. 50 spermlegg)

Mouse fertilization

Motility and Centrosomes During Fertilization 165

Fig. 1 . Latrunculin effects on sea urchin sperm. (A) Acrosome reacted sperm. (B) Latruncu- lin-treated acrosome reacted sperm. Unlike control sperm, latrunculin-treated sperm do not extrude the acrosomal process, though secretion of the acrosomal vesicle occurs. Bars = I fim.

on lysolecithin-permeabilized, formaldehyde-fixed cells [Barak et al., 1981; Cline et al., 19831. For antitubulin [Balczon and Schatten, 19831 and anticentrosomal [Ca- larco-Gillam et al., 1983; autoimmune serum generously provided by Dr. P. Calarco] immunofluorescence, sea urchin eggs and mouse oocytes were extracted, affixed to polylysine-coated coverslips [Mazia et al., 19751, fixed and stained for antigen detection [Schatten et al., 1986bl. DNA was localized with Hoechst 33258 (American Hoechst Corp., San Diego). Zeiss epifluorescence microscopy equipped for Hoechst dye, fluorescein, or rhodamine was used; cells were photographed with Tri-X film at an effective ASA of 1600, which was developed in Diaphine.

RESULTS Microfilaments Are Active During Sperm Incorporation in Sea Urchins

Latrunculin, a novel microfilament inhibitor, appears to prevent the normal elongation of the acrosomal process in sea urchin sperm, but apparently not the secretion of the contents of the acrosomal vesicle. Since cytochalasin has been shown by Sanger and Sanger [1975] not to interfere with the formation of the sea urchin sperm acrosome process, latrunculin has been used to explore the requirements for sperm microfilaments during sea urchin fertilization. As detailed in Table I, sperm treated with latrunculin are inhibited in their ability to fertilize untreated eggs suggesting that assembled microfilaments in the sperm are necessary for sperm incorporation.

Sea urchin sperm do not seem to assemble acrosomal processes properly in the presence of latrunculin. Sperm treated with egg jelly elongate the acrosomal process, which appears as a well-defined fiber extending from the tip of the sperm head in Figure 1A. However, when induced to undergo the acrosome reaction in the

presence of latrunculin they do not assemble normal acrosomal filaments though some material appears to have been released (Fig. 1B). This is interpreted to indicate that the secretion of the acrosomal vesicle has oc- curred independently of the assembly of the acrosomal process.

To determine whether this impairment of the formation of the acrosomal process interferes with the ability of latrunculin-treated sea urchin sperm to fertilize eggs, sperm were induced to undergo the acrosome reaction with or without latrunculin. As shown in Table I, treated sea urchin sperm are impaired in their ability to fertilize eggs, although mouse sperm are not greatly affected.

Sperm incorporation in sea urchins has been studied by Longo and Anderson [1968] and Tilney and Jaffe [ 19801 with transmission electron microscopy and by Schatten and Mazia [1976] and Schatten and Schatten [ 19801 with scanning electron microscopy. These studies demonstrate an eruption of the egg surface around the entering sperm.

The appearance of polymerized actin around the sperm during incorporation has been traced with rhodamine-labeled phalloidin, a fluorescence marker for microfilaments [Barak et al., 1981; Cline et al., 19831. In Figure 2, a regional accumulation of polymerized actin surrounds the entering sperm. This information strength- ens the conclusions based on electron microscopy and studies with microfilament inhibitors that microfilaments at the egg cortex regionally assemble around the successful sperm and anchor it to the egg surface during sperm incorporation.

Latrunculin also prevents sperm incorporation in sea urchin eggs if added prior to insemination. Time- lapse video microscopy (not shown) demonstrates that untreated sperm bind to the egg surface and trigger a localized cortical secretion that develops into an exagger- ated concavity, but sperm incorporation is arrested. The egg cortex develops a highly abnormal convoluted ap-


Fig. 2. Rhodamine phalloidin detection of microfilaments in fertilization cones during sperm incorporation. Microfilaments accumulate rapidly around the entering sperm during incorporation and the fertilization cone continues to enlarge as the male pronucleus develops. Figure parts represent: (A) 3 min postinsemination, (B) 5 min, (C) 10 min. Left panels: Rhodamine phalloidin detection of microfilaments. Right panels: Hoechst fluorescence of sperm DNA. Bars = 10pm

pearance; the partially activated egg is unable to move the egg nucleus to the cell center or progress to other events during the first cell cycle, which is unlike the effects of cytochalasins [Schatten and Schatten, 19811.

Egg Microfilaments Are Not Required During Mouse Sperm incorporation but are Necessary for Pronuclear Apposition

Pronuclear apposition, but not sperm incorporation, is completely arrested by latrunculin and cytochalasin during mouse fertilization (Fig. 3). Cytochalasin was shown to produce similar effects by Mar0 and co-

workers [1984]. The second polar body does not form after sperm incorporation, though meiosis is completed as assessed by the appearance of the two maternal pronuclei corresponding to a female pronucleus and a second polar body nucleus. Sperm incorporation and pronuclear formation still occurs at a reduced level, but the sperm and egg nuclei are unable to move into apposition at the egg center. Pseudocleavage is noted at about 8 hr postinsemination. Cytochalasin B has been shown to induce pseudocleavage in preovulatory oocytes [Wassarman et al., 19771. Bundles of cortical actin are found along the egg cortex and in the pseudocleavage furrow, but the

Motility and Centrosomes During FertiIization 167

Microfilament Inhibitors

40 :I 20

Control -

‘111 Cytochalosin Lotrunculin A

&-Sperm lwcrpomlmn 0-Pronwkor Formalion c -RonulLor Cent,OIct*n D-Cleo*a)c E-Reudcdeovoge

Fig. 3. Effects of microfilament inhibitors during mouse fertilization. Microfilament inhibitors do not completely arrest the incorporation of the spermhead, but prevent pronuclear apposition.

pronuclei, unlike the meiotic chromosomes are unable to induce a regional accumulation of microfilaments [Schat- ten et al., 1986al.

Latrunculin also has a remarkable affect on unfertilized mouse oocytes. When the microtubules of the meiotic spindle in the unfertilized oocyte are disrupted with microtubule inhibitors, the meiotic chromosomes are dispersed along the oocyte cortex [Schatten et al., 1985bl. Recently Longo and Chen [ 19851 and Mar0 and co-workers [ 1985al have noted regional accumulations of cortical actin adjacent to each dispersed chromosome mass.

In Figure 4 the regional accumulation of cortical actin (Fig. 4B) are depicted adjacent to each mass of dispersed chromosomes (Fig. 4A) in colcemid-treated unfertilized oocytes. Latrunculin interferes with this dispersion of the chromosomes (Fig. 4C) and cortical microfilaments are restricted to the region that would have formed the second polar body constriction. This experi- ment indicates that microtubules normally hold the meiotic chromosomes together in the unfertilized oocyte and that there is a counterforce of cortical microfilaments pulling the chromosomes apart.

Centrosomes and Microtubules During Sea Urchin Fertilization and First Division

The behavior of centrosomes during sea urchin and mouse fertilization and cell division has recently been reported by Schatten et al. [1986b]. Centrosomes are detected at the bases of sea urchin spermheads but not in the unfertilized egg. After sperm incorporation, the centrosome is found in the egg initially as an accumulation at the posterior face of the male pronucleus. At this stage the sperm aster is a radially symmetric array of microtubules. The centrosome appears split after the migration of the female pronucleus and is found at the junction between the two pronuclei when the sperm astral microtubules extend as an asymmetric crescent from the pronuclei. Following syngamy, the centrosome spreads

further into an arc over the zygote nucleus as the sperm aster widens (Fig. 5) . The incorporated sperm axoneme is also apparent in Figure 5 as is a punctate tubulin- containing site probably corresponding to one of the separating centrioles. At the streak stage two discrete centrosomes are observed and their fluorescence inten- sity has increased considerably; the microtubules ema- nate from these regions and extend from the nuclear surface.

During first division, the centrosomes are initially compact but later flatten and enlarge. At prophase, the centrosomes have moved into the cytoplasm from the chromosome mass and the microtubules appear as two asters. At metaphase (Fig. 6), the centrosomes remain spherical and now the spindle microtubules can be distin- guished from those in the asters. During anaphase, the centrosomes begin to flatten (Fig. 7) as the microtubules at the center of the asters begin to disassemble. The spindle poles widen and the asters continue to elongate. At telophase the centrosomes enlarge into ellipses with centrosomal antigen concentrated in more or less compact masses (Fig. 8). The interiors of the now prominent asters are free of microtubules with a corresponding increase in the size of the centrosomes. The axes of the centrosomal ellipses are perpendicular to the first mitotic axis and parallel with the second; frequently the centrosomes appear oriented perpendicular to each other in the third dimension. At cleavage the centrosomes are detected around the decondensing karyomeres forming crescents along the poleward faces of the reconstituted nuclei. The microtubules correspondingly are organized into partial monasters at cleavage.

Centrosomes During Mouse Fertilization The configurations of microtubules observed dur-

ing mouse fertilization [Schatten et al., 1985bl indicate an unusual organization. To explore the behavior and inheritance of centrosomes in this mammal further,


Fig. 4. Latrunculin inhibits colcemid-induced chromosome dispersion. The meiotic chromosomes of colcemid-treated unfertilized mouse oocytes disperse along the egg cortex (A, Hoechst DNA fluorescence) and the dispersed chromosomes induce regional accumulations of cortical actin (B, Rhodaminyl-phalloidin microfilament fluorescence). This dispersion is prevented when latrunculin is added along with the colcemid (C, Hoechst DNA fluorescence). Latrunculin alone does not affect chromosome distribution (D, Hoechst DNA fluorescence). Bar = 10 pm. From Schatten et al. [1986a].

Schatten et al. [1986b] have traced centrosomal antigen throughout the entire course of fertilization and first division. Centrosomal material is found at the spindle poles in unfertilized oocytes (Fig. 9) as reported by Calarco-Gillam et al. [1983] and as 16 punctate concentrations scattered throughout the cytoplasm (Fig. 9; Maro et al., 1985b).

At sperm incorporation when the meiotic spindle rotates, centrosomal material is found throughout the egg cytoplasm [Schatten et al., 1986bl. As the pronuclei develop, centrosomal foci and asters begin to associate with the peripheries of both pronuclei. Later numerous foci are found adjacent to the apposed pronuclei, which are embedded within an array of microtubules (Fig. 10).

Towards the end of first interphase the number of detectable foci increases and they all migrate toward the pronuclear surfaces with several between the pronuclei. At the completion of first interphase, at the first signs of chromosome condensation within the intact pronuclear envelopes, the centrosomal antigen aggregates centrally forming bright foci that circumscribe each pronucleus .

Centrosomes During First Division in the Mouse Zygote

The centrosomal foci move as two broad clusters to opposing cytoplasmic regions at prophase (Fig. 11) as an array of microtubules extends from the centrosomes towards the chromosomes. The chromosomes align between the centrosomes at prometaphase as the mitotic spindle becomes apparent. At metaphase (Fig. 12) the centrosomes condense and the spindle is well defined. During anaphase and telophase (Fig. 13) the centrosomes remain closely associated as a band or plate composed of several foci from which microtubules extend. The arrangements of the microtubules at the various stages conform well to the shapes of the centrosomes. As cleavage starts the centrosomes decondense and multiple foci are observed. After cleavage they are found as crescents associated with the poleward faces of the blastomere nuclei. The interzonal microtubules are prominent and typically a partial monaster of microtubules extends from the nuclei.

Microtubules Are Required for Pronuclear Union in Both Mice and Sea Urchin Fertilization

The effects of microtubule inhibitors in arresting the pronuclear migrations during sea urchin fertilization have been well studied [reviewed by Schatten, 1982, 19841 and recently Schatten et al. [1985b] have demonstrated that microtubule inhibitors will prevent pronuclear apposition during mouse fertilization. As shown in Figure 14, colcemid, griseofulvin, and nocodazole arrest the migrations leading to the union of the pronuclei at the mouse egg center. It is noteworthy that if unfertilized


Fig. 5 . Microtubules, DNA fluorescence, and centrosome detection during sea urchin fertilization. During pronuclear fusion the centro- soma1 antigen spreads over the zygote nucleus and organizes a partial monaster of microtubules. Immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 6. Microtubules, DNA fluorescence, and centrosome detection at first mitotic metaphase in sea urchin eggs. At metaphase the centrosomes are compact spheres and the microtubules of the mitotic apparatus extend from these sites with asters. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 7. Microtubules, DNA fluorescence, and centrosome detection at anaphase in sea urchin eggs. During anaphase the centrosomes enlarge and flatten as the microtubules at the center of the asters disassemble. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 8. Microtubules, DNA fluorescence, and centrosome detection at telophase in sea urchin eggs. The centrosomes expand into hemi- spheres with axes parallel with the axis of the next mitotic spindles. Microtubules continue to elongate at the astral peripheries and are lost at the regions of centrosomal expansion. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.


Motility and Centrosomes During Fertilization

loot

ao

60

4 0

20

0 -

L

-

-

-

-

I C

COLCEM ID GRISEOFULVIN

B C B C

NOCODAZOLE

I A

- B C

A -sperm Incorporofion 8-Pronudeor Formofion C-Pronudeor Centrotion

Fig. 14. Effects of microtubule inhibitors during mouse fertilization. Colcemid, griseofulvin, and nocodazole prevent pronuclear formation and the movements leading to pronuclear centration. Sperm incorporation is not inhibited. From Schatten et al. [1985b].

oocytes are treated with these inhibitors of microtubule assembly, the pronuclei are unable to develop (Fig. 14) [Schatten et al., 1985bl.

Fig. 9. Unfertilized mouse oocyte: Microtubules, DNA fluorescence, and centrosome detection. Unfertilized mouse oocytes retain centrosomes, unlike sea urchin eggs. In addition to the broad beaded spindle poles, there are about 16 concentrations of centrosomal antigen. Each centrosomal focus organizes an aster. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 10. Pronucleate mouse egg: Microtubules, DNA fluorescence, and centrosome detection. The cytoplasmic centrosomal foci increase in number during the first cell cycle and nucleate a cytoplasmic array of microtubules responsible for pronuclear apposition. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 11. Mitotic prophase in a mouse egg: Microtubules, DNA fluorescence, and centrosome detection. The centrosomal particles cluster into two irregular mitotic poles which, in the absence of a functional centriole, organize a barrel-shaped anastral spindle. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 12. Metaphase in a mouse egg: Microtubules, DNA fluorescence, and centrosome detection. The regional concentrations of centrosomal antigen can be detected at the blunt broad mitotic poles at metaphase. Triple labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

Fig. 13. Telophase in a mouse egg: Microtubules, DNA fluorescence, and centrosome detection. At telophase the particulate nature of the centrosomes is still apparent and interzonal microtubules accumulate between the separating chromosome masses. Triple-labeled immunofluorescence detection of microtubules (left) and centrosomes (right) and fluorescence microscopy of DNA (middle). Bar = 10 pm.

171

Summary: Motility Mechanisms at Fertilization Differ Between Sea Urchins and Mice, and Centrosomes are Maternally Inherited in the Mammal

Motility and centrosomal organization is summa- rized in Figure 15 and Table 11. Microfilament inhibitors prevent sperm incorporation in sea urchins but not completely in mice. Microtubule inhibitors inhibit pronuclear apposition in both mice and sea urchins. In addition microfilament inhibitors block pronuclear apposition in mice. Centrosomes are contributed by sperm in sea urchins and may be of maternal origin in mice.

DISCUSSION

Microfilaments are essential for sperm incorporation in sea urchins and for pronuclear apposition in mice. The ability of sea urchin sperm to fertilize eggs is reduced by latrunculin, providing evidence that acrosomal microfilaments are central to the fertilization process. In light of the uncertainty regarding the presence of microfilaments in various mammalian sperm, it is of interest that latrunculin does not markedly affect the ability of mouse sperm to fertilize oocytes. This might well indicate that, unlike sea urchin sperm that undergo a pro- nounced actin-mediated change in cell shape during the acrosome reaction, the acrosome reaction of mouse sperm may not require microfilament activity.

The effects of latrunculin on mouse fertilization differs considerably from its effects on sea urchin fertilization. When the meiotic spindle of ovulated unfertilized mouse oocytes is disrupted with colcemid, the chromosomes disperse along the egg cortex [Longo and Chen, 1985; Mar0 et al., 1985a; Schatten et al., 1985bl. Latrun-


4.l Colcemid

Colcemid

Tom1

Aphidicolin

Colcemid

Fig. 15. Motility and centrosomes during mouse and sea urchin fertilization. Microfilament inhibitors block sperm incorporation in sea urchins and pronuclear apposition during mouse fertilization. Microtubule inhibitors prevent pronuclear union in both systems. The mouse centrosome is probably maternally inherited and frequently organizes a plant-like spindle at first division, unlike the typical pattern in animals in which centrosomes derive from the sperm.

TABLE 11. Motility During Mouse and Sea Urchin Fertilization

Sea urchin Mouse

Sperm motility MT MT Sperm acrosome reaction MF None Sperm incorporation Egg MF None Completion of meiosis NA Egg Mt Second polar body formation NA Egg MF Pronuclear formation None Pronuclear migrations MT

MT MT and MF

First spindle Fusiform, with asters Barrel, anastral Probable centrosome source Paternal Maternal

Abbreviations: MT, Microtubule activity; MF, microfilament activity; NA, not applicable.

culin will prevent this scattering of the meiotic chromosomes, suggesting that microfilaments are active during this dispersion, and indeed regional accumulations of cortical actin are found adjacent to each chromosome mass.

Sperm incorporation during mouse fertilization in vitro is unaffected by treatment of either the sperm or oocyte by latrunculin, suggesting that microfilament ac-

tivity is not required in either gamete during this initial phase of mammalian fertilization, unlike sperm incorporation in sea urchins. However latrunculin will prevent the apposition of the pronuclei during mouse fertilization, as does cytochalasin [Maro et al., 19841. This finding indicates that, also unlike the events in sea urchins, pronuclear apposition in this mammal requires microfilament function.


19821 is particularly noteworthy because if the entering sperm had functional kinetochores like the second meiotic chromosomes, they too might induce the formation of a meiotic spindle and the ejection of some of its chromatin.

Centrosomes mirror chromatin during the cell cycle. At interphase they are dispersed and duplicate. At prophase both the chromatin and the centrosomes condense and loose their associations with the nuclear envelope. At metaphase chromosomes and centrosomes are at their most compact state. During anaphase and telophase both separate, but in different directions; the chromosomes move to the centrosomes while the centrosomes flatten into plates with their axes predicting the next mitotic planes. As the cells enter the next interphase, both the chromosomes and centrosomes decondense and again resume their association with the reconstituted nuclear envelope. Phosphorylation of both nuclear lamins [Gerace and Blobel, 19801 and centrosomes [VandrC et al., 19841 at prophase and dephosphorylation of both at telophase, and the association of a cyclic AMP-dependent protein kinase with centrosomes [Nigg et al., 19851, may provide clues of the modifications necessary for these interconversions from interphase structure during mitosis.

The organization and arrangements of centrosomes during fertilization, the first cell cycle, and mitosis in sea urchins and mice solves some essential problems in cell biology but raises a number of questions. The appearance of centrosomes in sea urchin sperm but not sea urchin eggs and in mouse eggs but not sperm predict centroso- ma1 retention during oogenesis in this mammal rather than the typical pattern of centrosome fidelity during spermatogenesis. Typically the mitotic centrosomes appear organized perpendicular to one another in the third dimension. While this organization does not affect the next mitotic axis, it may have important consequences during the following division. This shifting in centroso- ma1 axes may prove critical in the embryo’s ability to organize future division axes and unequal cleavage planes that may generate pattern.

Comparisons of these fertilization systems provide insights into the interactions between centrosomes and nuclei or chromosomes. Nuclei attract and associate with centrosomes. This is observed in the mouse after sperm incorporation when both pronuclei acquire centrosomes. The mature sea urchin sperm has a tightly affixed centriole and centrosomes, which after incorporation spread around the decondensing male pronucleus but always remain associated with it. In the absence of centrosomes in the unfertilized sea urchin egg, the female pronucleus can only associate with them after contact with the male pronucleus and from that point on is always found in association with centrosomes. It is of interest that centrosomes always reside between the tightly apposed male and female pronuclei. After first cleavage each blastomere nucleus in both systems is associated with centrosomes .

Chromosomes appear to repel centrosomes. Cen- trosomes remain associated with the nuclear regions until the breakdown of the nuclear envelopes when they are displaced into the cytoplasm. In sea urchin eggs the centrosomal particles remain tightly packed around the centrioles while in mouse eggs they are only loosely associated. This repulsion of centrosomes from chromosomes may explain the requirement for kinetochores to anchor the opposing microtubule ends. Microtubules organized by centrosomes as asters at interphase may be organized into spindles at meiosis or mitosis by the bun- dling together of opposing microtubule ends. The strin- gent requirement for microtubule assembly in meiotic or mitotic cytoplasm and the regional influence of chromosomes in promoting localized microtubule assembly from centrosomes [Karsenti et al., 19841 explains the loss of cytoplasmic microtubules and the appearance of spindles. The absence of functional lunetochores during interphase is interpretable since centrosomes will interact with the nuclear surface. The loss of detectable kinetochores during mammalian spermatogenesis [Brenner and Brinkley,

ACKNOWLEDGMENTS

The research reviewed in this article is the result of several investigations and it is our pleasure to acknowl- edge our collaborations with Drs. Ron Balczon, Pat Ca- larco, Christi Cline, Daniel Mazia, Ilan Spector and Mr. Cal Simerly. The support of this research by grants from the National Institutes of Health (Research Career Devel- opment Award HD363, Research Grant HD12913, and Training Grant HD7098 awarded to the Embryology Course at the Marine Biological Laboratory, Woods Hole) and the National Science Foundation (PCM83- 15900) is acknowledged gratefully.

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Motility and centrosomal organization during sea urchin and mouse fertilization

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