J. Cell Sci. 24, 203-216 (1977) 20

Printed in Great Britain

STUDIES ON THE DE NOVO FORMATION OF

CENTRIOLES: ASTER FORMATION IN THE

ACTIVATED EGGS OF SEA URCHIN

TAIKO MIKI-NOUMURAInstitute of Molecular Biology, Nagoya University, Nagoya, Japan*

SUMMARY

Aster formation was studied in sea-urchin eggs artificially activated by Loeb's double method.The number of asters found in an activated egg ranged from 15-40. Observation by electronmicroscopy revealed the presence of a centriole in a high percentage of isolated asters, stronglysuggesting that these centrioles are formed de novo in the activated eggs. Using negativelystained specimens, which were isolated with 5 % hexylene glycol solution containing buffer andEGTA, the whole aster could be examined by electron microscopy. A microtubular frameworkin the aster, extending radially from the astral centre was observed. The effect of mitoticinhibitors on aster formation was found to be similar to that on cell division.

INTRODUCTION

Studies on the division of parthenogenetically activated eggs always present theproblem of the origin of centrioles in the egg, since it has been generally believed thatthe centrioles in the fertilized egg originate from the sperm centriole.

In 1961, Dirksen reported the existence of centrioles in the centre of asters whichdeveloped in sea-urchin eggs activated by Loeb's double method (Loeb, 1913).Following Dirksen's work (1961), Sachs & Anderson (1970) found further that thecentrioles were present in the activated eggs at each developmental stage. Bothexperiments indicate that the centrioles cannot be derived from a sperm centriole andseems to suggest that they are formed de novo. However, a possibility remains that thenew centrioles were formed from a pre-existent centriole present in a 'masked' or'inactivated' state in the eggs. Kato & Sugiyama (1971) demonstrated the presence ofcentrioles in both nucleated and non-nucleated egg halves which were previouslyseparated and then activated artificially by the method of Harvey (1936). Their resultsuggests de novo formation, because even if a centriole existed in a masked or inacti-vated state in the whole egg, it could not be present in both halves and form newcentrioles in them at the same time.

In the work presented here, we tried to obtain evidence from another point of viewfor the de novo formation of centrioles in the activated egg. Observations on the micro-tubular structure of asters and the effect of mitotic inhibitors on aster formation arealso reported.

• Present address: Department of Biology, Ochanomizu University, Otsuka, Bunkyo-Ku,Tokyo, Japan.

204 T. Miki-Noumura

MATERIALS AND METHODS

Materials

Eggs of sea urchins, Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Antiwcidariscrassispina were used as materials. Eggs were obtained by injection of 0 5 M KC1 into the bodycavity. Before use, each batch was checked for fertilizability, and for artificial activation.Batches showing over 98 % elevation of the fertilization membrane were employed for theexperiments.

Methods

Artificial activation of eggs to induce aster formation. In accordance with Loeb's double method(1913) for artificial activation, unfertilized eggs were treated with 4 5 mN butyric acid in sea-water for 80 8, and then washed with ordinary seawater. After 10-20 min, they were treated for50-55 min with hypertonic seawater (8 ml of 2-5 N NaCl + 50 ml of seawater). The eggs werestirred gently during this treatment. Then they were removed to ordinary seawater andmaintained with gentle stirring until asters had formed in them. Sufficient precautions weretaken against contamination with sperm during the experiments, all of which were done at18-22 °C.

Isolation of asters. Unfertilized eggs were treated with o-i % trypsin for 4 min, to preventelevation of the fertilization membranes. After washing with seawater, the eggs were immersedin 4-5 mN butyric acid-seawater for 80 s, and then returned to Ca-free seawater for 10—20 min.Then they were maintained in hypertonic, Ca-free seawater (8 ml of 2-5 N NaCl + 50 ml ofCa-free seawater) for 50-55 min, with gentle stirring. The activated eggs were transferred toordinary seawater, in which they were maintained for 20—135 min.

Asters were isolated according to Kane's method for isolating the mitotic apparatus (Kane,1962), with some modifications. After asters were produced in the eggs, the eggs were washedtwice with NaCl-KCl solution (19:1 mixture of isotonic NaCl and KC1) or 1 M dextrose. A12 or 5 % hexylene glycol solution containing 10 raM phosphate buffer pH 6-2 and 5 mM EGTA(ethyleneglycol-bis(2-aminoethylether)-Ar,Ar,Ar',Ar'-tetraacetic acid) was added to 10 times thevolume of the eggs. After 30-60 s, the eggs were sedimented with low-force centrifugation, thesupernatant was discarded, fresh hexylene glycol solution added and the suspension thoroughlystirred. This procedure broke up the eggs, liberating asters, which were separated from thelysate by low-force centrifugation.

Electron microscopy. A Hitachi electron microscope (HU-11D-S) was used to examine thespecimens at magnifications of 5000, 19000, and 50000 times.

For negatively stained specimens, the isolated asters were suspended in distilled water or10 mM phosphate buffer pH 62 , and picked up by touching the surface of a drop of the sus-pension with a carbon-coated grid. The grid was rinsed with distilled water and then with 1 %uranyl acetate. The excess uranyl acetate was immediately removed with filter paper and thegrid was dried in air.

For thin-sectioned specimens, the isolated asters were prefixed with 2 5 % glutaraldehydesolution containing 100 mM phosphate buffer pH 6'2 for 45-60 min and postfixed with 1 %OsOj for i h at o °C. After dehydration, the sample was embedded in Epon 812. Thin sectionswere cut on a Porter-Blum MY-i microtome, and stained with uranyl acetate before observation.

RESULTS

Aster formation after artificial activation

Unfertilized eggs were activated by Loeb's double method. During culture inordinary seawater after activation, many clear spots appeared in the egg cytoplasm.With light microscopy, the clear spots seem not to contain any structures or largegranules and to have an appearance similar to that of the centrosphere in the mitotic

De novo formation of centrioles 205

Fig. 1. Aster (clear spot) formation during culture in ordinary seawater (Antlwcidariscrassispina). A, B, c, D, after o, 20, 30, and 50 min, respectively, in ordinary seawater.Phase-contrast, x 200.

206 T. Miki-Noumura

apparatus of dividing cells. According to the electron-microscopic observations ofKato & Sugiyama (1971), very few yolk granules and oil droplets are present in theastral centres of activated eggs. The size of clear spots increased and their shapebecome more precise during culture in ordinary seawater, as shown in Fig. 1. Whenthe activated eggs are slightly compressed between coverslip and slide, the spotsbecome more distinct, making it easier to count the number of asters in the eggs.

Table 1. Number of asters (clear spots) in an activated egg during culture inordinary seawater following artificial activation*

Time,minf

3°

60

95

135

H. pidcherrimus(in winter)*

2 0 4

1 9 2

17-617-8

2 6 82 6 32 1 0

2 1 0

23-42 0 4

1 7 2

18-4

1 8 0

16-41 9 71 9 4

P . depressus(in winter)*

3i-325-234-033-629-42 6 0

30-727-525-228-5

28-326-33°"425-527-22 9 9

29-33 1 3

31-4

Time,minf

2 0

4 0

60

9 0

A. crassispina(in summer)*

1 6 71 6 515-71 6 0

15-814-817-01 5 0

1 6 9

I7-51 6 71 6 0

1 4 9

1 9 418-814-1

312

• Each value represents the mean no. (per egg) of asters formed in 50 activated eggs,f Time of culture in ordinary seawater after artificial activation.

Table 1 shows the change in number of asters during culture in ordinary seawaterafter artificial activation, indicating that in P. depressus it reaches around 25-35during 30 min of culture in ordinary seawater. A similar tendency was observed in theother 2 species of sea urchin, as shown in Table 1. Since there is no clear indicationthat the number of asters increases during the period of observation, it may be con-cluded that many asters are produced at the beginning of culture in ordinary seawater.

The formation of asters was prevented by mitotic inhibitors such as colchicine andcolcemide, which were added to the ordinary seawater in various concentrations.Fig. 2 shows that the clear spots in the cytoplasm were less distinct in the presence ofthese mitotic inhibitors. The minimum concentrations necessary to prevent asterformation were io~3 M colchicine and io~6 M colcemide (Table 2).

De novo formation of centrioles

Fig. 2. Aster (clear spot) formation during culture in ordinary seawater containingcolchicine or colcemide (Anthocidaris crassispina). A, B, C, D, after 50 min in io~5,and io"8 colcemide, and io~' and io~* M colchicine in seawater; respectively. Phase-contrast, x 200.

208 T. Miki-Noumura

Isolation of asters

Having observed that many asters are formed in activated eggs, it was of interest todetermine their structure and the degree of similarity between them and the normallyformed asters of the mitotic apparatus and, in particular, to discover whether allasters have centrioles in their centre. To answer these questions, isolation of the asterswas attempted, using Kane's method for isolation of the mitotic apparatus.

Table 2. Inhibition of aster {clear spot) formation in Anthocidaris crassispina

Inhibitor concentration, AsterInhibitor M formation

Colcemide

I O -

IO"

Colchicine \ io~IO-6-IO-7 +

— indicates no formation; ± indicates very little or doubtful formation; + indicatesformation.

Fig. 3 A shows eggs in 12% hexylene glycol solution containing 10 mM phosphatebuffer, pH 6-2. About 40 asters are visible in the partially dispersed cytoplasm. Thelarger number of asters in this experiment, as compared with the data in Table i,may depend on the effect of trypsin, which was used to digest the vitelline membrane.Since it is known that trypsin has an activating effect on sea-urchin eggs (Runnstrom,1948), the increase in the number of asters might be due to a double activating effectby butyric acid and trypsin, although the mechanism of such activation is as yetunknown. The asters liberated from the dispersed eggs were collected by centri-fugation. Observation with phase-contrast microscopy showed that the isolatedasters had a clear centre, surrounded by a region with a radial structure (Fig. 3 B). Thediameter of the isolated asters was from 10 to 20 fim. A suspension of asters sedi-mented by centrifugation is shown in Fig. 3 c.

Observation by electron microscopy

Activated eggs were cultured in ordinary seawater after artificial activation. After35, 60, 95 and 135 min, they were sedimented by centrifugation and treated with theisolation medium containing 12% hexylene glycol, buffer and EGTA. The astersisolated at each culture time were fixed and thin-sectioned for observation by electronmicroscopy.

A survey image of thin-sectioned asters revealed that the isolated aster is a bodycomposed of a mass of components containing vesicles and small granules, having acentriole at its centre, from which microtubules radiate as a framework (Fig. 4A).After 35 min in culture, the aster already had at its centre a completely formedcentriole, which showed a typical cylindrical structure, about 0-4 /tin in length and

De novo formation of centrioles 209

Fig. 3. Isolation of artificially induced asters with hexylene glycol (Pseudocentrotusdepressus). Phase contrast, A, partially dispersed eggs in 12 % hexylene glycol contain-ing buffer and EGTA; x 200. B, isolated asters; x 900. c, asters precipitated withlow-force centrifugation; x 300.

CEL 24

2 1 0 T. Miki-Noumura

1-2 (im

B

J

0 5 //m

Fig. 4. Isolated asters (Hemicentrotus pulcherrimus). Electron micrographs. A, thin-sectioned aster, formed during 35 min culture in ordinary seawater. The centriole isalready located in the astral centre, B, transversely sectioned centriole at highermagnification, c, longitudinally sectioned centriole at higher magnification, D, 2centrioles formed separately in an aster.

De novo formation of centrioles 211

0-21 /im in diameter, composed of 9 triplet microtubules. The diameter of the micro-tubules was around 25 nm. Fig. 4B, c show such centrioles at higher magnification. Abrief survey of sectioned specimens or serial sections of the asters isolated from sea-urchin eggs of the 3 species (H. pulcherrimus, A. crassispina and P. depressus) sug-gests that a high percentage of asters observed in this experiment have at least onecentriole at the centre. This suggestion is supported by the ease with which centriolescould be found in these specimens. Furthermore, some asters had two or threeseparate centrioles (Fig. 4D). When two or three centrioles were present, theirarrangement was sometimes different from that seen in the mitotic apparatus, in whichthe 2 centrioles are oriented perpendicularly to each other, forming a pair.

To obtain a more exact image of the aster, negatively stained specimens of wholeasters were prepared, as described in Methods. Fig. 5 A shows a low-power view of awhole aster, which was isolated with 12 % hexylene glycol containing 10 mM phosphatebuffer (pH 6-2) and 5 mM EGTA. The EGTA was added to the isolation medium toprevent depolymerization of the microtubules during the isolation procedure. How-ever, as shown in Fig. 5 A, it is difficult to observe the detailed structure of astersisolated with this medium: the amount of hexylene glycol was therefore reduced to5%, with the result shown in Fig. 5B. The appearance of asters isolated with 5%hexylene glycol medium was different from that of asters isolated with 12% hexyleneglycol medium. In the former case the microtubules appear more clearly, and thereare fewer granular components around them (Fig. 5 c, D). Although the centriolescould not be observed clearly, the microtubules radiate from the astral centre. Thediameter of the microtubules is around 25 nm, which is the same that observed in theisolated mitotic apparatus (Kiefer, Sakai, Solari & Mazia, 1966). Fig. 6 shows a regionin which the microtubules are well separated. Side-by-side arrays of protofilaments,consisting of a linear arrangement of globular units about 4 nm in diameter, areclearly evident.

The centriole in the astral centre could not be clearly observed in these micrographs.Since the existence of a centriole in the central region was observed in the thin-sectioned specimens, it appears that negative staining seems not to be an appropriatemethod for investigating the fine structure of centrioles of the isolated asters.

DISCUSSION

It has been hitherto supposed that the centrioles in fertilized eggs originate from thesperm centriole. Dirksen (1961) showed the presence of centrioles in the asters ofartificially activated sea-urchin eggs and pointed out that the origin of the centriolesin activated eggs poses a question that is still to be answered. She suggested the possi-bility that the cytoplasm contains materials capable of producing many centrioles,that is, de novo formation of centrioles. Harvey's work (1936) on artificially activatedenucleated half and quarter eggs suggested that asters formed under these conditionsmay be produced around centrioles originating de novo. Kato & Sugiyama (1971) usedelectron microscopy to establish the presence of centrioles in both nucleated and non-nucleated egg halves after artificial activation, giving support to the idea that centrioles

14-3

2 1 2 T. Miki-Noumura

De novo formation of centrioles 213

may be so formed. Weisenberg & Rosenfeld (1975) have recently succeeded in induc-ing the assembly of tubulin, in homogenates of surf clam eggs that were previouslyactivated artificially, into asters and spindles. The existence of centrioles in the centreof the assembled asters was also confirmed.

Concerning the origin of centrioles, there is another possibility, namely, that theymight be derived from pre-existing centrioles, present in a masked or inactive state,even though the existence of such elements in the unfertilized egg has not been shown.However, even if 'procentrioles' or 'masked centrioles' exist in the unfertilized eggand are activated for further development by the double method for parthenogenesis,it seems very difficult to explain the formation of up to 34 or more asters which havecentrioles in their centre after only 30 min of culture in ordinary seawater, as des-cribed in this study. After trypsin treatment and artificial activation, the number ofasters in an egg increased surprisingly to around 40. Whether or not all the astersformed in this experiment have centrioles at their centre, cannot be stated at present.Proof on this point would necessitate analysis of serial sections of isolated asters.However, a brief survey of sectioned specimens or serial sections of asters isolatedfrom the sea-urchin eggs of 3 species, and the ease with which centrioles can be foundin these specimens suggest that a high percentage of asters have at least one centrioleat the centre. Furthermore, an aster had sometimes two or three separate centrioles.

If procentrioles or masked centrioles exist, they must duplicate in very unusualmanner, to exist in so high a percentage of asters. Such an extraordinary mechanism,for centriole production was reported by Mizukami & Gall (1966) in the spermatids ofa fern, Marsilea and a cycad, Zamia. In these cases, a mass of procentrioles known as ablepharoplast is first formed. The wall of the blepharoplast consists of closely packedimmature centrioles, or procentrioles. These are short cylinders which progressivelylengthen during differentiation of the spermatids. The hollow sphere of the blepharo-plast wall begins to degenerate at the end of mitosis, the centrioles migrate to the cellsurface, and change to basal bodies, from which flagella are formed. However, nounusual structure such as a blepharoplast has been found during aster formation inartificially activated eggs. The absence of such a structure supports the idea that thede novo formation of centrioles takes place here.

Kato & Sugiyama (1971) observed a pericentriolar mass in artificially activated eggsduring the process of centriole formation. Pericentriolar masses of densely stainedmaterial were also observed at the periphery of completely formed centrioles. Analysisof this densely stained material, of the process by which it is incorporated into thecentrioles and the relationship between the postulated materials capable of producingmany centrioles (Dirksen, 1961) and the densely stained materials remain as interest-ing problems to be solved in future.

Fig. 5. Isolated asters (Hemicentrotus pulcherrimus). Electron micrographs. A, nega-tively stained aster isolated with 12 % hexylene glycol solution containing 5 mM EGTAand 10 mM phosphate buffer pH 6-2. B, negatively stained aster isolated with 5 %hexylene glycol solution containing 5 mM EGTA and 10 mM phosphate bufferpH 6'2. Arrows indicate regions shown at higher magnification in Figs. 5 C, D. c, D, theregions indicated in Fig. SB, at higher magnification.

2 I 4 T. Miki-Noumura

50 nm

"A1'

V••-<.

Fig. 6. Negatively stained microtubules in aster isolated in 5 % hexylene glycol solu-tion containing buffer and EGTA (Hemicentrotus pulcherrinrns). Electron micrograph.

Dirksen (1964) isolated the asters from activated eggs, applying Kane's isolationmethod for the mitotic apparatus (Kane, 1962), and then analysed the nature of theirprotein. She concluded that the protein forming the asters is similar to that of themitotic apparatus. Electron microscopy in the present study also revealed a structuralsimilarity between the asters in the activated eggs and those of the mitotic apparatus,

De novo formation of centrioles 215

as reported by Kiefer et al. (1966). Asters isolated from activated eggs with 12%hexylene glycol containing phosphate buffer pH 6-2 and EGTA did not present aclear image. The asters were covered with many granular and vesicular components.However, decreasing the hexylene glycol concentrations produced a more distinctimage of the aster, permitting observation of the astral fine structure in whole,negatively stained specimens.

It should be pointed out that only one centriole was usually present in most astersobserved in thin-sectioned specimens, although two or three centrioles were some-times present in an aster. Paired centrioles oriented perpendicular to each other havebeen found in the mitotic apparatus of normally fertilized eggs. The asters of theactivated eggs are different in this respect from those produced in sperm-fertilizedeggs. The difference might reflect a different mode of centriole production.

On the other hand, naturally and experimentally induced asters are not onlystructurally similar, but also their microtubular structure is found to be similarlyaffected by mitotic inhibitors. The same concentration of these inhibitors, colchicineand colcemide, suppressed the spindle and asters, and arrested cell division (Inoue" &Sato, 1967; Swann & Mitchison, 1953). The inhibitory effect of these reagents ontubulin polymerization in vitro (Borisy, Olmsted & Klugman, 1972) is consistent withthe in vivo inhibition reported here. It can be said therefore, that these reagentsdirectly inhibit microtubule formation in the eggs, which would bring about arrestof cell division or aster formation.

As mentioned above, Weisenberg & Rosenfeld (1975) succeeded in obtaining invitro formation of aster and spindle, using homogenate of surf clam eggs which hadbeen artificially activated. Although tubulin molecules, presumably present in thehomogenates, assembled into the microtubules of aster and spindle, no success hasyet been achieved using a more purified tubulin fraction. Further study on this pointis awaited, to determine the precise conditions controlling the in vitro or in vivo poly-merization of aster and centriole, in order to define more accurately the role ofcentrioles during the assembling of asters.

The author wishes to express her gratitude to Mr Koichi H. Kato of Nagoya City Universitywho kindly thin-sectioned specimens for electron microscopy, to Dr Jean C. Dan who revisedthe manuscript, and to Drs Masao Sugiyama and Manabu K. Kojima of Nagoya Universitywho gave valuable advice. Thanks are also due to the Director and Staff of the SugashimaMarine Biological Station of Nagoya University and of Misaki Marine Biological Station ofTokyo University for their kind help during the course of this work.

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(Received 15 June 1976)